THE EFFECT OF PHARMACOLOGICAL AND DIETARY MODULATORS OF LIPID METABOLISM ON GENE EXPRESSION IN A PORCINE MODEL Except where reference is made to the work of others, the work described in this dissertation is my own or was done in collaboration with my advisory committee. This dissertation does not include proprietary or classified information. Ling Tang Certificate of Approval: Kevin W. Huggins Werner G. Bergen, Chair Assistant Professor Professor Nutrition and Food Science Animal Sciences Douglas C. Goodwin Russell B. Muntifering Associate Professor Professor Chemistry Animal Sciences Stephen L. McFarland Dean Graduate School THE EFFECT OF PHARMACOLOGICAL AND DIETARY MODULATORS OF LIPID METABOLISM ON GENE EXPRESION IN A PORCINE MODEL Ling Tang A Dissertation Submitted to the Graduate Faculty of Auburn University in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy Auburn, Alabama December 15, 2006 THE EFFECT OF PHARMACOLOGICAL AND DIETARY MODULATORS OF LIPID METABOLISM ON GENE EXPRESSION IN A PORCINE MODEL Ling Tang Permission is granted to Auburn University to make copies of this dissertation at its discretion, upon request of individuals or institutions and at their expense. The author reserves all publication rights. Signature of Author Date of Graduation iii DISSERTATION ABSTRACT THE EFFECT OF PHARMACOLOGICAL AND DIETARY MODULATORS OF LIPID METABOLISM ON GENE EXPRESION IN A PORCINE MODEL Ling Tang Doctor of Philosophy, December 15, 2006 (M.S., China Agriculture University, 2000) (B.S., Xinjiang Agriculture University, 1996) 322 Typed Pages Directed by Werner G. Bergen Modifying the size and/or carcass distribution of adipose tissue in meat producing animals by nutritional and pharmacological means has long been of interest to animal scientists. In pigs, as in other meat animals, a more complete understanding of lipid metabolism and its regulation at the molecular level will be necessary to develop more effective strategies to modify fat deposition in different tissues of animals to improve muscle food quality and production efficiency. Studies in this dissertation describe an experimental model system to determine gene expression responses to dietary catecholamine analog in porcine adipose tissue and to a sudden change of dietary fat content in adipose, skeletal muscle, liver and intestinal epithelium in finishing pigs. Studies described herein were designed to test the hypothesis that: 1) Ractopamine (a catecholamine) modulates lipid metabolism in adipose tissue of pigs in vivo through iv transcriptional control of genes involved in fatty acid synthesis, fatty acid oxidation transcription factors, and regulatory pathways. 2) Feeding a high fat diet modulates transcription of genes involved in nutrient metabolism pathways, especially those involved in lipid and carbohydrate metabolism in the liver, adipose and muscle tissues of the pig. 3) A sudden shift from a typical high carbohydrate, low fat diet to a fat- supplemented diet in finishing pigs results in metabolic adaptations and changes in transcription of genes associated with triacylglycerol and cholesterol trafficking in the pig. Collectively, results of the first two studies established that microarray can be used as a tool with which to detect transcription changes in the porcine tissues. The first study clearly indicated that long-term (28 days) exposure to the cAMP-elevating agent ractopamine was negative for the expression of genes in fatty acid synthesis in porcine adipose tissue. The second study showed that the short-term (14 days) of a fat enriched diet affected the transcription of lipid metabolism genes in different tissues of the pig. The third study determined the distribution pattern of genes ACAT, LCAT, apoB and HL in the porcine liver, subcutaneous adipose, gut and skeletal muscle tissues, and found that high fat diet depressed the transcription of ACAT in porcine liver. v ACKNOWLEDGEMENTS The author would like to thank Dr. Werner Bergen for his dedication, mentoring, support, unending patience, and encouragement of critical thinking. The author thanks members of her doctoral advisory committee for their teaching, assistance, guidance, and patience during the process; Dr. Douglas Goodwin, Dr. Kevin Huggins and Dr. Russell Muntifering, with special thanks to Dr. Russell Muntifering, for his dedication as an outstanding graduate program officer. The author also wishes to express her gratitude to Dr. Anthony Moss for his encouragement, patience and assistance during the dissertation writing and final oral exam. The author would also like to especially acknowledge the members of Dr. Frank Bartol?s lab, especially Anne Wiley for her unending technical advice in the microarry analysis. Special thanks go to all the members of the Bergen lab, especially Julia Bartosh, whose technical assistance was dedicated and helpful. She also wishes to thank all the faculty and graduate students in the Department of Animal Sciences for making my graduate study enjoyable. The author would further like to thank Dr. Meng-min Liu, her boyfriend for his encouragement and help with data analysis and dissertation writing. Finally, the author wishes to thank her parents and her sisters and brother for their love and support during her entire academic career. vi Style manual or journal used____________Journal of Nutrition Computer software used_______________Microsoft Word 2003 vii TABLE OF CONTENTS LIST OF TABLES................................................................................................... x LIST OF FIGURES................................................................................................. xi I. INTRODUCTION........................................................................................ 1 II. LITERATURE REVIEW............................................................................. 6 Lipid metabolism overview?????????????..?..?....?. 6 Fatty acid biosynthesis and triacylglycerol synthesis????. .??....... 7 TAG mobilization and fatty acid oxidation ?????????. 9 Fat deposition in pig??????????????????. 11 Regulation of gene expression by dietary fat?????????....?.. 14 PPAR??????????????????????........ 16 SREBP???????????????????????... 18 Lipoprotein metabolism ??????????????..?.??....... 19 Lipoprotein formation and transportation??????...?.?..... 19 Effects of dietary fat on lipoprotein metabolism???????... 22 Cyclic AMP and lipolysis???????????????.???... 25 Cyclic AMP system???????????.????..??... 25 ? adrenergic agonists????????????.???.......... 27 Ractopamine?????????????????..?.??. 29 Efect of Ractopamine on the adipose tissue metabolism???? 29 Microarray technology in pig genome research??????????... 31 Gene expression analysis????????????.?.?....... 31 Microarray technology??????????.???.???... 32 III. EFFECTS OF DIETARY RACTOPAMINE ON PORCINE ADIPOSE GENE EXPRESSION ??????????????...????? 38 Introduction??????????????...??????... 38 Materials and methods?????????????????. 41 Results and discussion...?????????????.............. 67 Conclusion??????????????????.???... 94 IV. OLIGOMER ARRAY ANALYSIS OF TRANSCRIPTION RESPONSE OF PORCINE TISSUES TO A SUDDEN DIET SHIFT FROM LOW FAT DIET TO HIHG FAT DIET???????? ???????... 106 viii Introduction?????????????????????.. 106 Materials and methods?????????????????. 109 Results and discusison?????????????.???? 113 Conclusion?????????????????????... 138 V. EXPRESSION OF PORCINE GENES RELATED TO FATTY ACID AND CHOLESTEROL METABOLISM IN DIFFERENT PORCINE TISSUES?????????????????????????.. 152 Introduction??????????????????.???. 152 Materials and methods??????????????.??? 155 Results?????????????????.??????. 164 Discussion?????????????????.????... 168 Conclusion????????????????.?.??......... 172 VI. CONCLUSIONS AND PERSPECTIVES?............................................... 180 BIBILIOGRAPHY?????????????????..???????. 186 APPENDICES......................................................................................................... 210 Appendix A. Images of total RNA resolved on the agarose gel????.. 210 Appendix B. Microarray Analysis Protocol?????????.??? 216 Appendix C. Images of fluorescent dye Cy5-labeled cDNA probe for slides hybridization in microarray analysis????????????.. 221 Appendix D. Pig Genome Oligo Set and Pig Genome Oligo Extension Set 1.0?????????????????. 229 Appendix E. Microarray images of Ractopamine experiment ????... 238 Appendix F. M-A plots of pro- and post- LOWESS normalization for Ractopamine experiment (Chapter III)????????. 241 Appendix G . Microarray images of diet shifting experiment (Chapter IV) 245 Appendix H. M-A plots of pro- and post- LOWESS normalization for diet shifting experiment (Chapter IV)???????? 256 Appendix I. List of transcripts highly up/down regulated (top 200 highest/lowest Log2ratio value) by dietary ractopamine supplement or dietary shifting (including Log2ratio value for each replication)???.????????????...................................... 268 Appendix J. An example of searching tentative biological information for one unknown transcript (gene)???????????????.. 308 ix LIST OF TABLES III. 1. An example of the information recorded for MIAME compliance???????? 42 2. Pig identification number, length of treatment, amount of treatment, and the date of sample collection for the pigs in the Eli Lilly feeding trial?... 44 3. Part of GenePix Arrya List (GAL) file. It presents genes information corresponding to each spot????????????????????.. 58 4. Part of GenePix Result (GPR) file. The head of GPR file describes all the parameters used when scanning the array. The main body of GPR file list the determined and calculated values for each spot (gene)???????? 60 5. Interesting gene list used to filter results??????????????? 95 6. Filtered SAM result???????????????????????. 103 IV. 1. Composition of feed given to the pigs in the dietary shifting from low fat diet (LFD) to high fat diet (HFD) experiment???????????.. 110 2. Pig identification number, diet treatment, and the date of sample collection for the pigs??????????????????????. 110 3. Differential transcription responses in liver to feeding LFD and HFD to pigs.. 143 4. Differential transcription responses in adipose tissue to feeding LFD and HFD to pigs??.??..???????????????????.. 146 5. Differential transcription responses in skeletal muscle to feeding LFD and HFD to pigs???????????????????????? 149 6. Number and percentage of detected (present) and undetected (absent) out of 13297 genes among tissues studied??????????????. 115 7. Number and percentage of differently expressed transcripts by SAM???.. 115 V. 1. Composition of feed given to the pigs in the dietary shifting from low fat diet (LFD) to high fat diet (HFD) experiment?????????... 156 2. Identification numbers, assigned dietary treatment, and the date of sample collection for experimental pigs??????????????????. 156 3. DNA sequence of the primers, annealing temperature used, and number of PCR cycles performed for the semi-quantitative RT-PCR??????? 159 4. Final body weights at slaughter and plasma cholesterol and triglycerate concentration??.??????????????????.. 164 x LIST OF FIGURES II. 1. Overview of lipoprotein metabolism????????????????. 20 2. Overview of the steps involved in oligonucleotide microarray experiments? 35 III. 1. Cy5-labeled RNA on a 2% agarose gel at 130V for 45 minutes, the gel was imaged on Typhoon 9410 at PMT 600 using emission filter for Cy5 (laser 633 nm). ?..???????????????????... 52 2. An example of M-A plot before and after LOWESS normalization???? 64 3. RNA integrity categories???????????????????? 72 4. Electropherograms of microcapillary electrophoresis from four RNA samples 73 5. An example of boxplot of different arrays before and after LOWESS normalization????????????????????????? 81 6. Effect of feeding 0, 20, 60 ppm of ractopamine on mRNA abundance of FAS, SREBP, GLUT4, SCD-1 and housekeeping gene (actin, ) in porcine adipose tissues from corssbred pig. ?....????????????. 93 V. 1. Intact total RNA resolved on a 1.0% agarose gel at 120V for 30 minutes?? 158 2. Tissue distribution patterns of expression the four genes????????.. 166 3. Gel image of relative RT-PCR in porcine liver, adipose and gut tissues??.. 166 4. Relative gene expression in pigs fed HF diet (n=4) vs. the HCHO diet (n=4).. 167 5. Comparison of LCAT sequences (cds) between pig (AY349156) and human (BC014781.1) by LASTn???????????????? 174 6. Comparison of ACAT sequences (5?UTR) between pig (AY676347) and human (L21934.2) by BLASTn??????????????????. 175 7. Comparison of LCAT protein sequences between pig (AAQ24609.1) and human (AAA59500.0) by BLASTp????????????????.. 176 8. Multiple sequence alignment of human apoB 100 protein (NP_000375.1), apoB48 (AAA51741.1) and translated fragment of pig apoB mRNA???.. 177 xi I. INTRODUCTION Consumer acceptance of pork products is a major factor for the sus consumption has been associated with an overall increase in diabet cardiovascular disease. Because animal products are higher in SFAT, research has focused new strategies to lower carcass SFAT. Fat depositio tainability of the pork industry. Elevated intakes of saturated fatty acids are associated with an increased incidence of heart disease, diabetes and obesity. Increased saturated fatty acids (SFAT) es, obesity, animal production n in meat animals is affected by genetics (breed) and nutritional conditions (1). An animal has to process a large number of different nutrients and other diet components, but nutrients can so bind to es are major tools to reduce fat content in pork. These include changes in feed components during specific growth phases and administration of exogenous agents such as ? adrenergic agonists (2). ances in providing acceptable pork products for today?s market, overall regulatory mechanisms of porcine lipid metabolism are not well characterized. Deeper understanding of the effects of dietary and hormonal factors on regulation of lipid metabolism will aid in developing future production strategies to provide juicier and healthy pork products. reach high concentrations without becoming toxic. Each nutrient can al numerous targets with different affinities and specificities. Genetic selection, pharmacological agents and production strategi Despite adv 1 Essentially every metabolic process represents regulated interactions of a large number of proteins encoded by their respective mRNA molecule in given cells, organs and organisms (3). Alterations of mRNA abund consequently the corresponding protein amounts are critical i s as they are expressed ances and n controlling the flux of metabolites or nutrients through a biochemical pathway (4). Protein, fat and carbohydrates in feed/food may affect every successive step in the flow of genetic protein kinase A via a beta-adrenergic receptor-G protein-adenylyl cyclase-protein kinase A cascade in porcine adipose tissue (2). Catecholamines and ?-adrenergic agonists are compounds that bind to 0% ?2, and 7% y-alpha-[[[3-(4- ol) is a ?1, ?2 -sensitive- and increase lean growth (9). Ractopamine in porcine adipose tissue binds to ?-adrenergic agonist receptors and activates adjacent G proteins which catalyze adenosine triphosphate (ATP) pamine decreased relative f acetyl Co-A carboxylase (ACC), fatty acid synthase (FAS), malic enzyme (ME) and glycerol phosphate dehydrogenase (GPDH) in adipogenic cell line TA1(2). In this dissertation, (Chapter III), the effect of ractopamine on the gene transcription response in adipose tissue in finishing pigs was determined using microarray technology. information, thereby altering metabolic functions (5). The cyclic AMP system regulates lipolysis in fat cells by activating and hormone sensitive lipase. cAMP-elevating agents regulate lipid metabolism membrane ?-adrenergic receptors. Porcine adipocytes contain 73% ?1, 2 ?3 adrenergic receptors. Ractopamine ((1R*, 3R*), (1R*, 3S*)-4-hydrox hydroxyphenyl)-1-methylpropyl]-amino] methyl]benzenemethan adrenergic agonist used in livestock production to decrease fat accretion s conversion to cyclic adenosine-monophosphate (cAMP). Racto mRNA abundance o 2 Comprehensive understanding of porcine genome function is critical to understanding how dietary nutrients affect complex metabolic proces deposition. However, presently little is known how genomic/molecul lipid metabolism in pigs is coordinated across liver, skeletal muscle nutrient composition have not been evaluated. Thus, assessment of the underlying metabolic adaptation of pigs switched from a typic ses and fat ar regulation of and adipose tissue during the growing and finishing phases of production. Coordinated gene expression responses to a sudden change of lipid metabolism, brought about by changing dietary molecular events al corn-based high ) should provide a model for exploring differential gene expression for lipid metabolism in pigs. Fat concentration may vary from 2% to more than 40% of dry matter in diets of long-chain fatty acids are s (? and ?) and ays (6). The up of transcription factors regulated by fatty acids. SREBP-1c regulates expression of a number genes involved in de novo lipogenesis (7). In rodents, polyunsaturated fatty acids (PUFA) ity in depressing deposition (9), but the molecular mechanism is still not clear. In modern swine industry, domestic pigs consume a large proportion of carbohydrate and relatively low- fat (4%) from the diet. Therefore, the rate of de novo synthesis of long-chain fatty acids is rapid in well-fed pigs (10). In this study a high-dietary-fat intake model was specifically applied to study lipid carbohydrate, low-fat diet (LFD) to a tallow-based high-fat diet (HFD animals and humans. Besides providing energy for the animal, ligands for nuclear receptors, peroxisome proliferators activated receptor liver X receptor (PPAR and LXR), which in turn control metabolic pathw sterol regulatory element binding protein (SREBP) family is another gro depress SREBP and lipogenesis, but saturated fatty acids have little abil SREBP (8). In the pig, saturated fat has similar effect in depressing fat 3 metabolism in pigs (see Chapters IV and V). In one set of experiments in this dissertation, I used a shift from a control high-carbohydrate, low-fat d saturated-fat diet to study effects of fat on gene expression pattern in tissues. The adaptation to a sudden diet shift requires rapid and sus iet to a high- different porcine tained coordinated responses in gene expression and specific regulation signals to enzymes and other proteins across all tissues in pigs. poprotein synthesis xists among ucidate the control of fatty acid metabolism at the molecular level in pigs. Such knowledge of pig lipid biology will be the basis for further utilizing pigs as an animal model in biomedical ality in the future. fat in pork over the last wer propensity to ulgate a new production strategy that will result in relatively low subcutaneous and visceral fat accumulation coupled with some intramuscular fat deposition. Based on what is known igs, such new temporal and tissue-specific regulation of fat deposition in pigs. Dietary nutritents and pharmacological agents are strategies used to modify fat deposition in meat animals. Therefore, experiments presented in this dissertation were designed to determine: 1) Long-term effects (28 days) of cAMP-elevating agent Paylean TM (ractopamine hydrochloride) on the Research of triacyglycerol and cholesterol metabolism, and li and export have been studied in rodent liver (11, 12), but differences e animals(13). It is thus necessary to collect gene transcription data and el research and for developing new techniques to improve pork qu During industry-wide programs to significantly lower total 25 years, finishing programs had been modified and pigs with much lo deposit fat were utilized. Today the industry is attempting to prom about the biology of fat deposition in storage depots and muscle in p strategies will not emerge without a more complete understanding of the 4 5 gene expression profile in porcine adipose tissue; 2) transcription response of the pig acyglycerol and icking associated genes after shifting from a high-carbohydrate, low-fat diet to a high-fat diet. genome to a sudden diet shift from high-carbohydrate, low-fat diet (LFD) to high-fat diet (HFD).in porcine liver, adipose and muscle tissues; 3) expression of tri cholesterol traff II. LITERATURE REVIEW As a major fuel source in the animal, fat is stored as triacylglycerol (TAG) in the adipose tissue and mobilized in the form of plasma free/ nonesterified fatty acids (FFAs). ts as parts of phospholipid d to monoglycerides and free fatty acids (FFA) by the action of specific lipases. FFA or non- esterified fatty acids (NEFA) readily aggregate to form micelles until they are taken up esterified into microns. e circulatory system via the thoracic duct. In the adipose and muscle, TAG of chylomicrons is hydrolyzed to release FFA by lipoprotein lipase, an enzyme attached to the endothelial cells primarily e transported to d and stored as TAGs; in other organs, such as muscle and liver, small amounts of TAG are stored intracellularly. The stored TAG is mobilized in the form of plasma FFAs when energy is required by the animal. In human and rodents, the primary sites of FFA oxidation are cardiac skeletal muscle and liver. The liver and muscle oxidizes FFA to help fuel their LIPID METABOLISM OVERVIEW Long chain fatty acids are also critical structural componen molecules of cellular membranes. After dietary TAG enters the small intestine, TAGs are hydrolyze individually by the enterocytes. The FFAs inside the enterocytes are re TAGs and packaged with lipoproteins and phospholipids to form chylo Chylomicrons enter the lymphatic system and eventually pass into th lining the capillaries. Released FFAs bind with serum albumin and ar peripheral tissues. In adipose tissue, the FFAs are primarily reesterifie 6 various metabolic activities, through sequential formation of acetyl-CoA through ? tyl-CoA in TCA cycle and transferring electrons nts and humans because intake of a high level saturated fat from the diet is linked to the development of cardiovascular disease, insulin resistance, diabetes and obesity. For example, increased istance. In meat ulation underlies t tissues of animals and improving meat quality. In the review below, multiple lipid metabolism pathways will be discussed. Most of metabolic pathways will be described based on rodent and d on the limited ing fatty acid chains esterified to acyl-carrier protein (14). Long-chain fatty acid synthesis occurs in two stages: First, the conversion of acetyl-CoA to malonyl-CoA is catalyzed by acetyl-CoA verted to palmitate in the presence of NADPH, a process catalyzed by fatty acid synthetase (FAS) (15). Malonyl-CoA is the immediate precursor in fatty acid biosynthesis. Fatty acid precursor, acetyl-CoA is transferred from the mitochondrion to the cytosol as citrate via the tricarboxylate transport system and citrate cleavage. Palmitate is the primary product oxidation, completely oxidation of ace into oxidative phosphorylation to produce ATP. Regulation of fat metabolism has been extensively studied in rode muscle accumulating TAG contributes to the development of insulin res animals, understanding lipid metabolism and the mechanism of its reg the development of strategies for modifying fat deposition in differen humans studies; specific research in the pig will be emphasized base literature. Fatty Acids Biosynthesis and Triacylglycerols Synthesis Fatty acids biosynthesis occurs in the cytosol with the grow carboxylase (ACC) and, secondl, acetyl-CoA and malonyl-CoA are con 7 of fatty acid biosynthesis in animals. Longer chain fatty acids and unsaturated fatty acids actions (16). A synthetase to m triacylglycerols (17). Glycerol kinase catalyzes the activation of glycerol to glycerol-3-phosphate in the liver, and glycerol-3-phosphate dehydrogenase catalyzes the formation of glycerol-3- hate acyltransferase A esters and ty acyl-CoA from undergoing lipid oxidation and leads fatty acyl-CoA to triacylglycerol synthesis (10). Triacylglycerols synthesized in the liver are transported via VLDL to adipose tissue e in adipose- and long-term regulation (18). is pathway. ACC is inhibited by palmitoyl-CoA and by a glucagon-stimulated cAMP-dependent increase in phosphorylation, and it is activated by citrate and by insulin-stimulated term regulation, with etary poly unsaturated long-chain fatty acids (PUFA) decrease the concentration of liver ACC and FAS (20). De novo fatty acid synthesis or lipogenesis (DNL) is inhibited by free or non- esterified fatty acids by inhibiting cytosolic acetyl-CoA carboxylase activity (21). For are synthesized from palmitate (16:0) by elongation and desaturation re After fatty acids are synthesized, they must be activated by acyl-Co produce acyl-CoA, and then combine with glycerol-3-phosphate to for phosphate from dihydroxyacetone phosphate (17). Glycerol-3-phosp (GPAT) catalyzes the synthesis of triacylglycerols from fatty acyl-Co glycerol-3-phosphate. Glycerol-3-phosphate acyltransferase prevents fat and then hydrolyzed by lipoprotein lipase (LPL) before fatty acid storag synthesized TAG. Fatty acid synthesis is controlled by both short-term Acetyl-CoA carboxylase (ACC) catalyzes the first committed step of th dephosphorylation (19). Lipid synthesis is also controlled by long- insulin via SREBP-1c stimulating the synthesis of ACC and FAS. Di 8 fatty acid synthesis in the rodent liver, PUFA are highly effective DNL inhibitors, but . PUFA reduce mRNA levels of lipogenic and rodents, liver is the primary site of de novo lipogenesis (DNL), with adipose tissue is a secondary DNL site. However, in pigs, DNL takes place primarily in the adipose tissue while the tion (13). When ergy expenditure, the fatty acid and TAG synthesis. In domestic pigs, daily intake of energy is usually provided from dietary carbohydrates. Fatty acids are synthesized from glucose in the adipose tissue of swine zes 40% of the (24). Glycolysis is the fate of glucose in the adipose tissue. te, is then converted to acetyl-CoA by pyruvate de novo fatty acid synthesis in the adipose tissue (17). TAG Mobilization and Fatty Acid Oxidation obilized to provide energy. The hydrolysis of TAG to FFA is catalyzed by hormone-sensitive lipase (HSL), which is the rate-limiting enzyme in lipolysis (25, 9). The free fatty acids arising from lipolysis bind with serum albumin and are transported to various tissues for further oxidation by fatty acid binding proteins (26). saturated NEFA have a lesser effect (22) enzymes via gene expression or mRNA decay (23). However, species differences exist in lipid metabolism. In humans liver synthesizes relatively low amounts of fatty acids for metabolic func animals ingest carbohydrates exceeding the amount required for en excess carbohydrates are stored as TAG in the adipose tissue through (13). Adipose tissue is the major glucose-utilizing tissue and metaboli daily glucose uptake in pigs The end product of glycolysis, pyruva dehydrogenase. Acetyl-CoA from glycolysis is the precursor for the During states of negative energy balance, TAG in adipose tissue is m 9 Uptake of FFA by tissues for subsequent oxidation occurs via the plasma membrane, ase on entering rough the inner ne and the enzyme carnitine palmitoyltransferase I (CPT-I) and carnitine palmitoyltransferase II (CPT-II). Once inside the mitochondrial matrix, the fatty acyl?CoA enters ?-oxidative pathway and are successively xidative pathway includes FAD-dependent dehydrogenation of an alkyl group, hydration of the resulting trans 2,4 enoyl CoA , NAD -dependent oxidation of this hydroxy acid to a ketone, and C- o fewer carbon ation of fatty acids in the blood, which is controlled by the hydrolysis rate of TAG in adipose tissue by hormone- sensitive lipase (29). HSL is regulated by phosphorylation and dephosphorylation in hrine and 30). This second messenger activates cAMP-dependent protein kinase (cPKA), which in turn increases phosphorylation of perilipin and HSL. The phosphorylation of perilipin by PKA facilitates a large increase in the rate of lipolysis (31-33). HSL is both an abundant intracellular triacylglycerol lipase in adipocytes and a substrate for PKA; the and FFAs are esterified to coenzyme (CoA) via fatty acyl?CoA synthet the cell. The resulting fatty acyl?CoA is then transported to the matrix th membrane of the mitochondrion. This transport is mediated via carniti sequentially yields acetyl-CoA. During ? -oxidation of even chain fatty acyl-CoA, 2-carbon units removed as the acyl-CoA is reduced to multiple acetyl CoA (17). This o + C bond cleavage to form acetyl-CoA and a new fatty acyl-CoA with tw atoms (27). Complete oxidation of the acyl-CoA, NADH and FADH 2 is achieved by the Krebs cycle and oxidative phosphorylation (28). Fatty acid oxidation is regulated largely by the concentr response to hormonally controlled cAMP levels. Glucagon, epinep norepinephrine increase adipose tissue cAMP concentration ( 10 phosphorylation of PKA sites in HSL triggers the translocation of the lipase from the urs. A recent d droplets is required HSL-binding proteins. After HSL is phosphorylated, it catalyses lipolysis in adipose tissue, elevating blood fatty acids levels, and finally activating the beta-oxidation pathway in other tissues iency, ATP s results in activation of ivity and enhances fatty acid oxidation (37). Here, cAMP-dependent PKA, acting in concert with AMP- dependent protein kinase (AMPK), causes the inactivation of ACC; thus, cAMP- on and inhibits is responsive to a , somatotropin, adrenocorticotropin, thyrotrophin, thyroid hormones, and glucocorticoids. Adipocytes from other mammalian species exhibit less breadth of endocrine control with meager or sulin stimulates c receptor (?AR) agonists have the opposite effects (40). Fat Deposition in Pigs The literature on the effects of the type of dietary fat on lipogenesis is dominated by studies in rodent liver tissue. The degree of inhibition between saturated and cytosol to the surfaces of lipid droplets (33-35) where lipolysis then occ study has found that the presence of perilipin on the surfaces of lipi to dock HSL onto lipid droplets (33), suggesting that perilipin may be such as liver and muscle (36). When cells experience an energy insuffic becomes depleted and cellular AMP concentration rise (37). Thi AMP-dependent protein kinase (AMPK), which lowers ACC act dependent phosphorylation simultaneously stimulates fatty acid oxidati fatty acid synthesis (38). According to Bergen and Mersmann (13): ? The rodent adipocyte wide variety of endocrine entities including adrenergic hormones, insulin no demonstrable response to many of these hormones (39)?. In general, in fat deposition and inhibits lipid catabolism, whereas ?-adrenergi 11 unsaturated fatty acids showed little difference in rodent adipose tissue, and the effects of that the 42). The overall DNL ctivity (43, 44). The pig has a very high capacity for synthesis and storage of fatty acids. The amount of fattening depends on the age, sex and genetic lineage of the animal. Pigs from 0% of daily energy tored as fat (45). In fed pigs, fat is readily deposited in peripheral adipose rbohydrates to fatty acids (46-47). Dietary fat alters triacylglycerol deposition in the pig based on dietary fat source he intake of here are reports e tissue of the pig is he nature of the dietary fats thus affects lipid homeostasis and body fat deposition. Ding et al. (53) found fatty acid composition in the plasma and adipose tissue was similar to the dietary fatty fat or fish oil-based, high-fat diet for 2 weeks. They also found fatty acid profiles of liver and muscle reflected dietary specific fatty acids to a greater degree than plasma free fatty acids and adipose tissue. In pig, the major changes of fatty acid profiles in adipose tissue TAG fatty acids were observed only after a dietary fat source had been consumed for 4-5 weeks (54). degree of unsaturation are specific to liver (41). The difference indicates promoter regions of fatty acid synthase may differ between tissues ( rates are low in the liver of pigs because of low lipogenic enzyme a 5-7 months of age can deposit large amounts of fat with as much as 5 intake being s tissue and body subcutaneous adipose tissue of the leg by converting ca (saturated or unsaturated), fat content, and duration of fat ingested (48). T feed and feed quality may also affect the fat deposition in the pig (49). T from three independent groups that inhibition of lipogenesis in adipos greater with saturated fat sources than with unsaturated sources (50-52). T acid profile after young pigs were fed with either a tallow-based high 12 Porcine adipocytes are distinct from rodent adipocytes in a number of ways. Rodent less sensitive (55). The ay be different t porcine adipocyte differentiation has been reviewed by Hausman (57) and is beyond the scope of this dissertation. ly on data from adult chondrial matrix scribed by rodent models of beta-oxidation that emphasize accelerated ketogenesis concomitant with enhanced mitochondrial flux of fatty acids (58). In neonatal rabbits (59) as well as mature from new born tion in piglets n in baby pigs can hondria contribute significantly to acetogenesis. The low rate of fatty acid oxidation in swine liver might be related to a lower tissue-specific metabolic rate (O 2 consumption per unit mass) resulting from lower energy need to meet cellular ATP requirements (65). Thus, low rates of ?- oxidation and ketogenesis infer that alternative, nonketogenic routes of carbon flow may predominate in swine liver (62). adipocytes are highly insulin-sensitive, but porcine adipocytes are hormonal and growth factor-driven differentiation of adipocytes m between rodent and porcine adipocytes (56). Extensive information abou Models of mammalian hepatic lipid catabolism are based large rats, emphasizing formation of ketone bodies as primary adjuncts to mito ?-oxidation. Clearly, lipid metabolism in swine liver is not adequately de rats (60-61), in vitro rates of liver ?-oxidation were higher than in liver pigs (60, 62-63). Adams (64) observed that in vitro hepatic O 2 consump was only 50% of that noted in rats. The relatively low rate of ?-oxidatio be explained in part by a lower overall metabolism and that the mitoc 13 REGUALTION OF GENE EXPRESSION BY DIETARY FAT l environment. In cells to metabolize of the mechanisms involve conditional transcription of genes encoding enzymes specific to a metabolic pathway in response to an appropriate nutrient. The best-characterized examples are the gulon of the individual f the adaptations to environment are controlled by hormonal or neuronal signals. Major dietary components (protein and energy) or lesser (trace mineral, vitamin) dietary constituents may regulate Cells regulate gene expression in response to changes in the externa unicellular organisms, specific mechanisms have evolved to allow the fuels according to their availability in the external milieu (23). Most nutritional regulation of the lac operon of Escherichia coli and the gal re Saccharomyces cerevisiae (23). In multicellular organisms, the needs of cell and of the whole organism must be managed. In mammals, most o gene expression in a hormonal-independent manner (66). In preadipocyte, hormone-like effects have been attributed derivatives in the regulation of gene expression and consequent preadipo proliferation and differentiation (67). Regulation of expression of trans to fatty acids and their cyte cription factors, such as CCAAT/enhancer binding protein and peroxisome proliferator activated receptors, during early adipocyte development has been attributed to long-chain saturated and unsaturated fatty acids (68). Some fatty acids or their metabolites ac to control the activity or abundance of specific transcription factors. Th t like hormones ese transcription factors interact with specific target genes through cis-regulatory elements and interface with common components of the transcriptional apparatus (69). Activities of lipogenic genes are principally regulated at the transcription level (20- 21, 70). Recent research results with rodents and humans showed that dietary fatty acids 14 had clearly enhanced gene expression, resulting in changes in metabolism, growth and turated fat (>45% n resistance, and xpression of fatty acid translocase and beta-hydroxyacyl-CoA dehydrogenase genes, but did not change the mRNA concentration of epithelial membrane fatty acid binding protein, mitochondrial etal muscle (73). cell differentiation (71). Storlien (72) found ingestion of diets high in sa as calories) for several weeks increased serum TAG and promotes insuli obesity in rodents. High-fat diets (>65% of energy as lipids) increased e carnitine palmitoyltransferase I, and uncoupling protein 3 in human skel The domestic pig can easily consume dietary energy in excess of its needs and hence is prone to deposit excess fat as indicated above. Domestic pigs expression, leading to changes in metabolism and growth. The effe gene expression reflect an adaptive response to changes in the quantity a ingested. Regulation of gene transcription by fatty acids seems to be due activity or abundance of at least 4 transcription factor families: PPAR typically are given a small amount of fat from the diet (2%-4%), except in a production setting where fat replaces some dietary corn to lower feed cost. Dietary fat has profound effects on gene cts of dietary fat on nd type of fat to changes in the (peroxisome proliferator-activated receptor), LXR (liver x receptor), HNF-4? (hepatic nuclear factor 4) and SREBP (sterol regulatory element binding protein) (74). Except for SREBP, all these uclear receptors ily (75). Several mechanisms have been proposed to clarify the molecular basis for fatty acid regulation of gene transcription. Below, more detail is presented as to how fatty acids regulate gene expression through two families of transcription factors, the PPARs and SREBPs. transcription factors are members of the steroid and thyroid hormone n superfam 15 PPAR (Peroxisome Proliferator-Activated Receptors) members of the e active oid X receptor (RXR). This heterodimer binds to specific DNA recognition sequences, PPAR response elements (PPRE), in the promoter region of target genes (77). In rodents, three PPAR s and PAR? is expressed cytes (69). PPAR? is expressed in more tissues than PPAR?, but most importantly in skeletal muscle (78); PPAR? is expressed in adipose tissue, spleen, retina, hematopoietic cells, and epithelial ary gland (79). In pigs, PPAR? is abundant in the ose tissue; while rms, PPAR?1 and ?2, (80). PPARs are activated by numerous fatty acids including: palmitic (16:0), oleic (18:1, n-9), linoleic (18:2 n-6), arachidonic (20:4 n-6) acids and eicosanoids. These nuclear d by one of the three members of the PPAR family (82). It has been shown that PPAR? regulates pathways of fatty acid oxidation, while PPAR? modifies fatty acid synthesis and storage in adipose tissues (83). PPAR?2 is involved in the induction of genes encoding enzymes involved in lipid storage in adipocytes. Fatty acids and some prostanoids induce adipocyte Peroxisome proliferator-activated receptors (PPARs) are superfamily of ligand-activated nuclear transcription factors (76). Th transcriptional form of PPARs are heterodimeric complexes with retin subtypes have been identified, which are encoded by three separate gene demonstrate tissue specificity in expression. The transcription factor P in hepatocytes, cardiomyocytes, renal proximal tubule cells, and entero cells of the colon, prostate, and mamm porcine liver and muscle. PPAR? and PPAR? are both expressed in adip PPAR? is the predominant form in adipose tissue (56). PPAR? isofo result from different splicing of same gene receptors appear to act as sensors for fatty acids (81). Many genes involved in fatty acid metabolism are regulate 16 differentiation. In the process of adipocyte differentiation, the mRNA abundance of ty acid transport, fatty , as well as various lipogenic and lipolytic hormone receptors are up PPARs are among several nuclear receptors that requireRXR as a heterodimer partner for DNA binding (78). The effect of fatty acid on RXR abundance or activity may 5). tty acid oxidation nd mitochondrial carnitine palmitoyltransferase (CPT-1) (86). PPAR?, AOX and CPT-1 are expressed at high levels in rodent liver; while PPAR? is not expressed in rodent adipose with a modest on has led to the that underscores s however, 7), suggesting that swine adipose tissue may oxidize fatty acids. However, Sundvold et al. (88) demonstrated PPAR? was mainly expressed in kidney and liver but not in adipose in mature swine. PPAR? is also expressed in skeletal muscle, and promotes fatty acid oxidation, ketone body synthesis, and glucose sparing when activated by a fatty acid ligand in the rodent and human (86). enzymes in the lipogenic and triglyceride synthesis pathways, fat acid binding regulated (84). impact other signaling systems utilizing RXR as a heterodimer partner (8 PPAR? is proposed to regulate peroxisomal and mitochondrial fa by modulating the expression of peroxisomal acyl coA oxidase (AOX) a extent expression of AOX and CPT-1 in rodent adipose. This observati conclusion that fatty acid oxidation is unlikely in adipose tissue; an idea the primary function of adipose tissue as a TAG storage depot. In pig expression of PPAR? appears higher in adipose tissue than in liver (8 17 SREBP (Sterol Regulatory Element-Binding Proteins) ctors in lipid and -helix-leucine the endoplasmic reticulum (ER) (90). Three ancillary proteins, SREBP cleavage- activating protein (SCAP), site-1 protease (S1P) and site-2 protease (S2P) are required lycerides, and s are overexpressed under special experimental circumstances, each SREBP isoform can activate genes involved in synthesis of fatty acids, triglycerides and cholesterol (93). SREBP-1a is a mes that mediate ription of genes not for cholesterol P-1c include in order: ATP citrate lyase, acetyl-CoA carboxylase (ACC), fatty acid synthase (FAS), stearoyl- CoA desaturase (SCD), glycerol-3-phosphate acyltransferase(GPAT) (95-96). l activation domain, SREBP-2 preferentially stimulates cholesterol biosynthesis (97). SREBP-1c and SREBP-2 activate three genes required to generate NADPH, malic enzyme, glucose-6-phosphate dehydrogenase (G6PD), and 6-phosphogluconate dehydrogenase (PGDH). NADPH is required for reduction steps in lipid biosynthetic pathways (96). SREBP transcription factor family has emerged as regulating fa cholesterol metabolism (70, 89). SREBPs belong to the basic helix-loop zipper family of transcription factors and are synthesized as inactive precursors bound to for SREBP maturation (91). SREBPs directly targets expression of more than 30 genes involved in the synthesis and uptake of cholesterol, fatty acids, trig phospholipids, as well as the NADPH coenzyme (92). When SREBP ubiquitous activator of all SREBP-responsive genes. These include enzy the synthesis of cholesterol, fatty acids, and triglycerides (94). Transc needed for DNL are preferentially regulated by SREBP-1c but synthesis under physiological conditions. The target genes of SREB Characterized by a longer transcriptiona 18 Regulation of SREBPs occurs at transcriptional and posttranscriptional levels. The n , and liver X- EBP-1c expression is ). One of the properties of insulin is to stimulate DNL during carbohydrate excess. This action of insulin is counteracted by glucagon, which increases cellular cAMP . Many researhers lated by SREBP-1c (100-101). In es the Although cholesterol biosynthesis depends almost entirely on SREBPs, fatty acid synthesis is only partially dependent on the expression of SREBPs. In liver, the gene y upstream AS promoter LXR ligands even merges to be quite complex, and SREBP-1c is highly sensitive to dietary polyunsaturated fatty acids (PUFA) in rodents and pigs (105-106). The SREBP-1 processing includes tethered nascent P-1c mRNA egradation (106). LIPOPROTEIN METABOLISM In animals and humans, three major pathways of lipoprotein metabolism are tightly interrelated and interdependent: (1) the transport pathway of dietary or exogenous fat; (2) the transport pathway of hepatic or endogenous fat; and (3) the reverse cholesterol transcription of SREBP-1c is selectively regulated by insulin, glucago activated receptors (LXRs),.In the presence of LXR agonists, SR activated via a LXR binding site in the SREBP-1c promoter (98, 99 suggest that insulin?s stimulation of DNL is modu isolated rat hepatocytes in vitro, insulin treatment simultaneously increas abundance of mRNA for SREBP-1c and its target genes (102). encoding fatty acid synthase (FAS) can be transcriptionally activated b stimulatory proteins (USP) that act in concert with SREBPs (103). The F also contains an LXR element that permits a low-level response to when SREBPs are suppressed (104). Regulation of liver SREBP-1c e SREBP-1c processing by SCAP and SP1 and SP2, degradation of SREB (107) and ubiquitination of nSREBP and subsequent proteasomal d 19 transport pathway. Similar to the work on the effects of dietary fatty acid on lipid lism and its control by dietary factors has rodents and humans. An overview of lipoprotein physiology is depicted in Figure 1 and then described below. In the figure, A-1, B, C-II, and E represent the various apolipoproteins. Lipid is ed by oteins. Lipoprotein particles are the aggregates of proteins and lipids (23). Dietary fat is secreted from intestinal cells as chylomicrons, a process that requires apolipoprotein B (apoB). metabolism, research on the lipoprotein metabo been almost exclusively conducted in Lipoprotein Formation and Transportation insoluble in aqueous media, and its transportation in the animal is perform lipopr Fig 1. Overview of lipoprotein metabolism (108) Chylomicrons are synthesized in the intestine and found in lymph and plasma after a fat-containing meal (109). The TAG within the chylomicrons is hydrolyzed by lipoprotein lipase (LPL) with apolipoprotein C-II (apo C-II) as cofactor, producing a 20 chylomicron remnant that is taken up by the low-density lipoprotein-like receptor protein the liver, and LPL and apo C-II to e IDL particles through the interaction of apo E with the LDL receptor; other IDL particles produce low- density lipoprotein (LDL) after hydrolysis by hepatic lipase (HL). If LDL is oxidized, it ascent HDL s containing ripheral cells such as macrophages and removes unesterified cholesterol through the ATP-binding cassette 1 (ABC1) transporter protein. The cholesterol in the nascent HDL is then esterified to a (LCAT) and its e cholesteryl ester in f HDL with the cholesteryl ester transfer protein (CETP) (Fig 1). In the liver, Acyl-CoA:cholesterol acyltransferase (ACAT) catalyzes the transfer of fatty acid from coenzyme A to the hydroxyl group of Cholesteryl esters are transported in secreted lipoprotein particles to other tissues that use cholesterol, or are stored in the liver. A severe reduction in the cholesteryl ester content of hepatoma cells reduces apoB secretion (110). Description of above lipoprotein pathways are based on research in rodents and guinea pigs. The lipoprotein pathways in domestic pigs are not (LRP) in the liver. Very low-density lipoprotein (VLDL) is secreted from the TAG in the core of the VLDL is hydrolyzed in various tissues by produce intermediate-density lipoprotein (IDL). The liver takes up som can enter macrophages via the scavenger receptors, CD36 and SR-A. N particles are made in the liver and intestine. They are secreted as particle mainly phospholipid and apo A-I. The nascent HDL interacts with pe fatty acid derived from lecithin by lecithin cholesterol acyl transferase co-factor, apo A-I, producing a spherical, mature HDL particle. Th the core of HDL is then returned to the liver, either by the interaction o SR-B1 receptor, or transferred to the apo B-containing lipoproteins by cholesterol, converting the cholesterol into a more hydrophobic from. 21 completely similar. For example, it is still not clarified if gene CETP exists in domestic t on the chain length and degree of saturation. A saturated fatty acid diet (31% coconut oil) alone caused elevations of total, IDL, and LDL cholesterol when normolipidemic adult males were showed that diets ed serum cholesterol levels guinea pigs dietary fat saturation influences secretion rate of VLDL from the liver and the composition of nascent VLDL (114). Saturated fat-enriched diets increased secretion rate aturated fatty acid- of chain length and 15). Guinea pigs sterol (LDL-C) levels, followed by those fed palmitate rich palm oil. The lowest plasma LDL-C concentrations were seen upon feeding coconut oil (114,116-117). In addition, significant lard or CO diets. inea pigs fed a saturated fatty acids (SAT) diet have cholesterol ester (CE) ?enriched LDL that are associated with higher circulating concentrations of LDL-C. However, when guinea pigs are given PUFA diets, LDL particles are CE-poor, exibit 1.5 times faster turnover in plasma than the larger LDL particles from guinea pigs fed the SAT diet (117). pig (111). Effects of Dietary Fat on Lipoprotein Metabolism Effects of fatty acids on lipoprotein metabolism are dependen supplied with a high-fat diet for 9 days (112). Goldberg et al. (113) supplemented with C-12 and C-14 saturated fatty acids increas compared with polyunsaturated fatty acid containing diets in humans. In of smaller VLDL particles rich in cholesterol compared with a polyuns enriched diet in guinea pigs (114). Using guinea pigs the effects saturation of fatty acids on hepatic VLDL synthesis have been studied (1 fed a palm kernel oil (PK) diet exhibited the greatest plasma LDL-chole differences in LDL composition have been noted in guinea pigs fed PK, Gu 22 ApoB-100 is required for the assembly and secretion of VLDL by the liver. Dashti position and 2 cells. They poA-I and apoB, while monounsaturated fatty acids decreased plasma apoA-I level. Hepatic microsomal TAG transfer protein (MTP) mRNA levels have been shown to be influenced by dietary ing increased by saturated fatty acids compared with monounsaturated Feeding high-saturated-fat diets affected apoE levels and distribution among the lipoproteins in plasma. Cole et al. (121) found an increase in total apoE and apoE (112) found that t apoE in VLDL, r HDL fractions. racellular enzyme responsible for catalyzing cholesterol esterification in liver and other organs (37). In the human, there are two isoforms of ACAT. ACAT1 is expressed in all tissues while T2 is responsible for supplying cholesteryl ester for VLDL (122). Saturation of dietary fat and cholesterol content change liver ACAT activity in guinea pigs (123). Monounsaturated fatty acids increased ACAT activity in contrast to saturated fatty acids or PUFA. Increasing cholesterol concentration in the diet resulted in matching increases in liver ACAT activity. et al. (118) compared the effects of different fatty acids on the com concentration of apoA-I and apoB containing lipoproteins using HepG observed that saturated fatty acids increased plasma concentration of a fats, with levels be and n-6 PUFAs (119). For example, guinea pigs fed the PK diet exhibit the highest apoB secretion rate (120). containing lipoprotein in rats fed a SAT diet for 2 weeks. Fisher et al. apoE was redistributed on the saturated fat diet in adult males, such tha IDL and LDL were increased while apoE was decreased in heavie Acyl coenzyme A:cholesterol acyltransferase (ACAT) is the int ACAT2 is present mainly in liver and intestine. ACA 23 Lecithin:cholesterol acyltransferase (LCAT) is responsible for cholesterol ester inea pig is mostly tivity may also . Fernandez et al. (125) found higher LCAT activity in guinea pigs fed high-fat diets (35 or 45% total dietary energy from fat) than those fed low-fat diets (10 or 19% total energy from fat), dipocytes and AG. Apolipoprotein C-II (apoC-II), a protein constituent of human VLDL, is the activator for lipoprotein lipase (126). ApoC-III depress the action of of LPL. The regulation of LPL is tissue- role in directing VLDL TAG to muscle tissues during ed, while supply fatty acids to Substituting dietary carbohydrates for fat affects the metabolism of all the lipoprotein fractions, especially for VLDL-TAG and apoB metabolism because of carbohydrate- takes (128-129). ith a higher-fat diet) on LDL apoB metabolism are conflicting and likely depend on total energy intake (130). Various animal models have been employed to access the role of diet on lipoprotein metabolism and concentration. A big problem in interpreting these studies is that animal formation in the plasma compartment (37). VLDL cholesterol in gu derived from LCAT activity, as is also the case in humans. LCAT ac contribute to the formation of CE (cholesterol ester) in VLDL (124) which correlated with the higher concentration of VLDL cholesterol. Lipoprotein lipase (LPL) is present within intracellular pools in a muscle cells and acts on TAG-rich lipoprotein and hydrolyzes the T specific. LPL has an important fasting. Under condition of scarcity, adipocycte LPL expression is reduc expression in muscle cells is increase which is related to the need to muscle for oxidative metabolism (127). induced hypertriglyceridemia, but this required excess dietary energy in Observations on the effects of a high-carbohydrate diet (as compared w 24 species can vary greatly in their distribution of plasma cholesterol among lipoproteins iew on the latest dates on mechanism by which fatty acids modulate plasma lipids has been published CYCLIC AMP AND LIPOLYSIS Cyclic AMP System n of lipolysis in fat MP promotes osphorylates a serine residue on hormone sensitive lipase (HSL) and promotes its activation and translocation towards the lipid droplet (37, 133). HSL catalyzes the rate-limiting step in TAG an antilipolytic tivation. The final of HSL involves a rol (135). Adenylyl cyclase which catalyzes the formation of cAMP, is the key enzyme for activating the lipolytic cascade. The adenylyl cyclase system is composed of three major classes of g proteins 36). Most of the physiological regulators of adenylyl cyclase interact with membrane-bound stimulatory or inhibitory receptors. These ligands through the receptor modulate the activity of effector units of adenylyl cyclase through signal transducing proteins; i.e. the guanine nucleotide-sensitive coupling proteins, (G-proteins) that bind and hydrolyze guanosine that may not be comparable to men and women (131, 13). A recent rev up (132). The cyclic AMP system plays an important role in the regulatio cells. The receptor-controlled incremental production of intracellular cA activation of cAMP-dependent protein kinase A (PKA) which ph breakdown or lipolysis in adipose tissues (134). Insulin, functionally hormone, causes dephosphorylation of HSL which results in its deac breakdown of the monoacylglycerols that appear after the activation monoacylglycerol lipase that is not directly under hormonal cont membrane proteins; i.e., receptors (beta adrenergic receptors), couplin (heterotrimeric G proteins) and effector units of the cyclase enzyme (1 25 triphosphate (GTP) (137). Several forms of G-proteins exit in fat cell membranes and are e form of G proteins, ors (138). The oteins belong to a G protein, seven transmembrane protein domain superfamily referred to as G protein coupled receptors (GPCR) (139). s are cGMP- osphorylase kinase, noceptors (140). These enzymes are controlled by allosteric and covalent regulation. The variety of potential phosphorylation sites in these enzyme proteins may actually control the sequential tensity of the tes from cAMP increments (141). Further research is needed modulate/determine gulate lipid metabolism enzymes in the animal. Lipid metabolism in adipose tissues is regulated by catecholamines that bind to MP ?PKA cascade, genes by transcriptional regulation (32). Agents that enhance cAMP content (cAMP elevating agents) generally induce lipolysis in fat cells but cAMP-elevating agents may also decrease expression of lipogenic enzymes during differentiation of adipocytes (142). Cyclic AMP regulates the transcription of its target genes through cAMP response referred to as Gs?,?,? or Gi?,?,? (17). Gs (stimulatory G protein), on activates adenylyl cyclase when coupled with various beta-adrenocept membrane complex consisting of the adrenergic receptor and G pr In addition to HSL, the best-identified substrates for PKA in fat cell inhibited, low Km cAMP-phosphodiesterase, glucose transporter, ph glycogen synthase, acyl-CoA carboxylase, and the ?1 and ? 2-adre phosphorylation of sensitive sites in an order that depends on the in stimulatory signal that initia to better understand how putative physiological substrates or ligands priority of PKA sensitive sites in some sort of hierarchical fashion to re membrane GPCR (?-adrenergic receptors) to activate through the cA adipose HSL (covalent modification; lipolytic response) and other 26 elements (CRE) that are distinct DNA sequences present in the promoter regions of target element binding protein, a en it is ulation of gene expression was extended by the discovery of a family of CREB proteins and of a cAMP- responsive element modulator (CREM) which affects CREs in a negative fashion (144). own that may bind e promoter region of various lipogenic and lipid oxidative lay an important role in the control of expression of fat cell- specific genes containing CREs (146). ?-Adrenergic Receptors (?-AR) e adipose enzymes (13). This is GPCR) present in c antagonists, it was confirmed in numerous studies that in pigs, ?-AA act specifically by binding to porcine adipose GPCR or ?-AR (13). The ?-AR is cell surface receptors that belong to e ?-AR belong (147). Three subtypes of ?-adrenergic receptors (?1 AR, ?2 AR and ?3 AR) have been identified in adipose tissue and other mammalian tissues. All three ?-AR subtypes have been cloned in several species including swine (148-150). The ?-AR subtypes share approximately 50% sequence homology with the highest homology in the trans genes. These elements are recognized by the cAMP response transcription factor that is able to activate target gene transcription wh phosphorylated by PKA (143). The complexity of cAMP-dependent reg CREB is a substrate for PKA, and it is the only transcription factor kn cAMP response elements in th genes (145). CREB may p Beta adrenergic agonists /catecholamines (?-AA) stimulate porcin lipolysis and attenuate fatty acid synthesis and activity of lipogenic accomplished via binding of ?-AA on transmembrane beta receptors ( membranes of porcine adipose tissues. By strategic use of beta adrenergi the large superfamily of 7-transmembrane domain proteins. A subset of these proteins is a large family of G-protein coupled receptors (GPCR) to which th 27 membrane domains associated with ligand binding pockets. The subtype distribution, ipose, the almost all other ied by mRNA abundance determinations (151) and receptor binding studies (152). In pigs, while ?3AR mRNA is detectable in several tissues including skeletal muscle, heart and adipose tissue, es. The ?1AR g nearly 80% in adipose, 3). In rodents, ?- AR subtypes exhibit ligand selectivity such that the rank order of affinity for norepinephrine is ?1-AR> ?2-AR> ?3-AR. Subtypes ?1-AR and ?2-AR are co-expressed , the major is ?2AR. In re are species -AR and porcine adipocytes have less than 10% ?3-AR) (154). In addition, the amino acid sequence of all ?-AR subtypes varied among species, Hence, it may be expected that some ?AR agonists would have divergent effects in the same tissue among species because of different ?AR subtype distributions and (or) amino acid sequences and consequently ligand binding affinity. Subtypes also differ in G-protein selectivity, and regulation by gene expression and phosphorylation (147). however, in adipose and other tissues is species specific. In rodent ad predominant subtype is ?3-AR, but this subtype is basically absent in species (13). The distribution of ? AR subtypes in pigs has been quantif it represents a small number of all subtypes present in these major tissu subtype is the predominant subtype in most tissues, comprisin 72% in heart, 65% in lung, 60% in skeletal muscle, and 50% in liver (15 in most tissues of the body, but the ratio of each can vary. For example isoform of the ?-AR in the rat heart is ?1AR, while in guinea pig lung it contrast, ?3AR is found mainly in rat adipose tissue as noted above. The variations in tissue subtype distributions (e.g., human heart has 65% ?1 28 Ractopamine techolamines adipose and muscle tissue beta receptor/GPCR-cAMP-protein kinase A-dependent mechanism, fat accumulation and expression of lipogenic genes (2,154). Ractopamine contains two chiral centers and a is the most selectivity for ?2AR expressed in Chinese hamster ovary cells (157). The RR isomer has the highest affinity for each subtype. Experimental receptor activation and adenylate cyclase stimulation indicated ine/ catecholamine efore, it was expected that administration of these types of pharmacological agents to pigs would result in lowered adipose tissue accretion. Direct ligand activation of ??AR in adipose -sensitive lipase to phosphorylation and inactivation of GLUT4 and acetyl-CoA carboxylase, and reduced expression of other lipogenic genes (2, 145, 151). Pigs fed ractopamine in the 1980s had a lower carcass fat than control pigs (159); however, a lowered rate of fat accretion has not been consistently observed in all animal trials by other workers using more contemporary, leaner pigs The chemical structure of ractopamine is similar to the natural ca epinephrine and norepinephrine, and it binds to ? AR in pig with high affinity. In cultured fat cells, ractopamine suppressed via a possible four sterioisomers, RR, RS, SR, and SS (155). The RR isomer lipolytic and accounts for enhanced growth in rats (154). Pig ? AR ractopamine stereoisomers has been studied using cloned ?1AR and ? that the ?2AR was a more functional target than the ?1AR (158). Effect of Ractopamine on the Lipid Metabolism in the Pig In the development of ractopamine, a family of phenethanolam ligands for ??AR was first screened for their anti-obesity properties. Ther tissue enhanced PKA and causes activation and translocation of hormone and TAG hydrolysis (151) Activation of PKA is anti-lipogenic due 29 (160). In addition, Purdue workers reported that expression and activity attenuation of el et al. (9) were attenuated 2) and Reiter (163) found that mRNA abundance of FAS was significantly lowered in pigs fed ractopamine for 42 to 54 days. Other results indicated that administration of ractopamine to pigs ma non-esterified ffect of ogenesis in finishing pigs. The response to ractopamine may be short-lived because adipose membrane ??AR is desensitized by nearly 50% within the first 7 days of administration (165). retion in pigs. cids, Bergen et al. ractopamine fed ers have reported that protein accretion responses were maximal within the first week and declined toward zero over 4 to 6 weeks (25). Clearly, there are divergent views from different laboratories and lower fat ommercial ractopamine recommends a treatment period of up to 28 days to optimize effects both on lean and fat deposition (167). The mechanism whereby ractopamine isomers specifically down-regulating porcine ??AR has not been delineated; however, there is consensus in the literature (160-161) that classic receptor down-regulation as described in rodents may lipogenic genes may only be present for up to a week (161), while Merk showed that adipose fatty acid synthesis and lipogenic enzyme activities up to 42 days by ractopamine in finishing pigs. In addition, Halsey (16 resulted in an acute (short time spike), but not chronic, increase in plas fatty acids in vivo (164). Such results have cast doubt on any long term e ractopamine on lipolysis and lip Ractopamine, however, consistently increased muscle protein acc Utilizing in vivo protein synthesis procedures with radiolabeled amino a (166) reported that fractional protein synthesis rate in skeletal muscle of pigs was significantly elevated after 14 and 28 days over controls. Oth on the long-term effectiveness of ractopamine to enhance lean deposition accretion. Based on recent translational studies, the manufacturer of c 30 limit the effectiveness of ractopamine because of variable results in effects on fat n-regulation of the actopamine to reduce fat accretion more In this dissertation, the cAMP-elevating agent/ catecholamine Paylean? (commercial name for ractopamine) was used to explore its effects on transcription of oduct of Elanco d for finishing n 1999. The active ingredient in Paylean , ractopamine hydrochloride, is a member of beta-adrenergic agonist family. The commercial product contains four ractopamine stereoisomers. G GENOME ife; more specifically, to characterize the genetic programs which the genome employs to manifest a multitude of different cell functions and physiological characteristics (169). Genomic to RNA, particularly pts that are expressed or transcribed from genomic DNA, also named ?transcriptome?, is a major determinant of the cellular function and phenotype (169). The process of transcription affects the subsequent process of protein synthesis, thus alteration of gene expression reflects phenotypic differences and cellular responses to environmental stimuli (17). deposition. Implementing feeding strategies to circumvent dow receptors may enhance the ability of r consistently (168). lipid metabolism genes in porcine adipose tissue. Paylean? is the pr Animal Health, a division of Eli Lilly and Company, and was approve swine by the Food and Drug Administration (FDA) i TM APPLICATION OF MICROARRAY TECHNOLOGY IN PI RESEARCH Gene Expression Analysis The ultimate goal of genomics is to understand how DNA encodes l DNA becomes biologically/functionally ?active? when transcribed in mRNA. The collection of mRNA transcri 31 The transcriptome is highly dynamic and changes rapidly and dramatically in m types of nction, knowing nderstand the activity and biological roles of its encoded protein. Furthermore, changes in the multi-gene expression patterns can provide clues about regulatory mechanisms and broader cellular the change of rentiation of cells (172). Diet is possibly the most important environmental factor that organisms encounter that has a long-lasting effect on the genome. The relationship between specific nutrients cts of nutrients or increasing or be utilized to investigate the molecular events by which the genomes perceive nutritional signals and mobilize the organism to respond (173). Dietary nutrients can affect gene expression at lic pathways and Microarray Technology Microarrays are a technology for simultaneously measuring the number of copies of many distinct DNA or RNA fragments in a complex mixture. Microarrays work by exploiting the ability of complementary DNA strands to selectively bind (hybridize) to response to exogenous and endogenous stimuli ranging, for example, fro nutrients consumed to endocrine secretions (170). To understand gene fu when, where and to what extent a gene is expressed is essential to u functions and biochemical pathways (171). It is well known that cells respond with altered gene expression to environments and that nutrition can influence the proliferation and diffe or diet composition and gene expression could help to identify effe dietary ingredients components at the molecular level. Genomes can respond in a rapid and specific manner by selectively decreasing the expression of specific genes, and these responses can the molecular level through the interactions with receptors, metabo signals (174). 32 each other, even in the presence of a large background of non-complementary competing ), longer base pairs) as array in binding complementary target gene fragments with high fidelity. Oligonucleotides (40-80 bases in length) may be treated as ?short cDNAs? and there is much less chance of spurious rt oligonucleotides agments or oligonucleotides are deposited on a surface in a way that keeps distinct fragments spatially separated, and a labeled (fluorescent or radioactive) probe mixture of (single t strands. The amount of rom the probe labels er of provide simultaneous quantity measurements for tens of thousands of target DNA fragments. The simplest realization of this technology is the spotted cDNA or oligonucleotides is the approach used y, synthesized oligos are now used much more frequently than cDNA fragments in microarray platform design and construction. The oligonucleotides (typically 70 mer) can be printed onto the same slides as cDNAs, with the same printing device, the hybridization and washing conditions are similar, and no new analytical programs for expression analysis is required fragments. Microarrays primarily use short oligonucleotides (15-25nt oligonucleotides (50-120nt) or PCR-amplified cDNAs (100-3,000 elements (175) but such short oligos are not sufficiently discriminatory cross-hybridization with unrelated sequences compared with sho (176). In microarrays, typically specific target (single strand) cDNA fr strand) fragments is applied and allowed to hybridize to the targe each distinct probe fragment is measured by detection of the signals f bound at each target site on the surface. This accomplishes a large numb measurements in a small area, so that a single hybridization reaction can microarray, combined with two-color fluorescent detection, and this in this study. With the progress of oligonucleotide synthesis methodolog 33 (177). In this approach, distinct long oligonucleotide (70 mer) fragments are printed as an ligomers are RNA) mixtures - green labels and are combined in solution and applied to the array. After hybridization, slides are scanned to generate fluorescent images from the two channels as depicted in Fig 2 (178). The om each spot reflect the relative numbers of red and green labeled fragments conjugated at the spot, and hence the relative numbers of fragments in the control and experimental samples. array of distinct spots on a suitably treated glass microscope slide. The o spotted with a mechanical robotic system. Two distinct probe DNA (or the control and the experimental sample - are given fluorescent red and fluorescent intensities of red and green detected fr 34 Fig 2. Overview of the steps involved in oligonucleotide microarray experiments (178) 35 The design of the oligonucleotides to be spotted on the microarray slides is the logy. Basically, -C content, have ined within an exon, and have no repetitive- or hairpin sequences (179). When every oligo in a spotted microarray is of the same length and has almost the same melting temperature and G-C e array (180). Since o not require a denaturation step as with the m of renaturation, which can decrease hybridization efficiency (181). A highly useful application of microarray technology is to assess global gene umbers of genes re of changes in mplex regulatory interactions (182). The search may provide new insights into the effects of nutrition and food ingredients like fats, carbohydrates, proteins, vitamins, minerals at the molecular level (183). een cloned, lotting for many genes are not available in the pig. Furthermore, there is limiting coding fragment sequence data in the porcine genome to design primers and develop a qRT-PCR approach on a gene-by-gene basis to exploit differential gene expression to diet or pharmacological agents in pigs. Thus, development of the 70mer oligonucleotide spotted slide platform most important for success in utilizing this functional genomics techno the oligonucleotides should have very similar melting temperature or G very little homology with other oligonucleotides, be entirely conta content, hybridization conditions are consistent for every gene on th 70 mer oligos are single stranded, they d cDNA format during the procedure; this also eliminates the proble expression (mRNA abundance). Microarray technology enables large n to be screened simultaneously, giving a comprehensive, detailed pictu gene expression, thereby shedding light on co integration of microarray analysis into basic and applied nutrition re Because only a small portion of genes in the porcine genome have b cDNA probes for standard mRNA abundance studies with Northern b 36 37 designed by the Iowa State Porcine Genomics Center provides researchers interested in xpression analysis ine oligonucleotide orcine lipid metabolisms experimental models (such as feeding catecholamines and high-fat diets) in a much more favorable time frame and comprehensiveness than a long tedious gene by tterns of known genes uscle, we will also odels for genes hitherto not recognized as participants in lipid metabolism. Once global expression patterns have been established, more specific analytical platforms and confirmatory f what genes to study in igs. This knowledge is critical if we want to minimize fat accumulation in pigs during production (fattening, per se, is inefficient use of feed and other economic resources) while enhancing intramuscular fat deposition to provide tasty and juicy porcine muscle foods. pigs a front-end analytical resource for across-tissues-integrated gene e for various physiological and nutritional states. The Iowa State porc array was used in this research to establish gene expression patterns in p gene approach. Not only will we be able to determine expression pa involved in lipid metabolism in liver, adipose tissue and skeletal m likely encounter expression responses to our experimental porcine m procedures can be developed or applied with a clearer focus o order to better understand whole-animal lipid metabolism in p III. EFFECTS OF DIETARY RACTOPAMINE (PAYLEAN TM ) ON PORCINE ADIPOSE GENE EXPRESSION INTRODUCTION The long-term goal of pork industry is to maximize the production efficiency and minimize the pollution to surrounding environment, to ensure that pig production is sustainable. Genetics, nutrition and facility management strategies have been implemented to increase pork quality and decrease the effects of animal production on the environment. In food animals, BAA depresses fat deposition by increasing lipolysis and depressing lipogenesis and triacylglycerol synthesis. Increased lipolysis is dependent on increased activity of triacylglycerol lipase (9, 191). Decreased lipogenesis in adipose tissue is due to lowered activity of lipogenic enzymes, partly attributable to allosteric regulation through phosphorylation by cAMP-dependent kinase (191). In rat liver, cAMP also directly lowers expression of fatty acid synthase (37). In addition, BAA diverts dietary energy from fat deposition toward enhancement of muscle fiber hypertrophy characterized by increased deposition of skeletal muscle protein in the ham, belly, and shoulder in meat animals (184). Duration of BAA treatment also affects the magnitude of growth response because of the putative down regulation phenomenon of GPCR, particularly in pig adipose tissues (192-193). Dunshea et al (24) found that strongest response to RAC occurred during the 38 first week of treatment and then declined. Others investigated the temporal response to RAC over a seven-week period and found that the greatest growth response to RAC occurred from day 6 to 22, after which time there was a linear decline in the magnitude of the response (194). Ractopamine (RAC), a beta-adrenergic agonist (BAA), marketed as Paylean TM , is an epinephrine analogue that binds to beta adrenergic receptors in the meat animals. Researches demonstrated that RAC improves average daily gain, stimulates muscle growth, decreases fat content in pigs and several other species (185-187), and reduces days to market. (184). Merkel et al (188) found that RAC lowered subcutaneous adipose depot size in finishing pigs nearly 20% and lowered carcass total fat percentage by 20 to 24%. Besides modifying carcass of meat animals, the use of RAC in animal production can potentially reduce the environment pollution caused by excretion. In a study by Sutton et al (189), barrows were used to determine effects of RAC and crude protein (CP) level on nitrogen (N) and phosphorus (P) retention, and excretion in urine and feces of finishing pigs. Average excreta output was reduced by 3.9% in pigs fed RAC, and N excretion was reduced by 10.7 to 34.2%. During anaerobic storage of manure arising from RAC-fed pigs, reduced levels of N, NH 3 emissions, and volatile fatty acids were observed (190). There is still incomplete understanding of putative molecular mechanisms whereby RAC or other BAA affect fat deposition and muscle growth in pigs. The Bergen laboratory has shown that RAC lowered mRNA-abundance of lipogenic genes in the adipogenic cell line TA1 (2). Furthermore, results indicate that cAMP is directly involved because exogenously administrated, non-metabolizable cAMP analogs attenuated 39 lipogenic gene expression in cultured TA1 cells (2). Several studies (162-163) by the Bergen lab observed lower mRNA abundance of FAS, SREBP-1 and GLUT4 in adipose tissue when pigs were fed a 60-ppm RAC-supplemented diet for 28 days (162-163). In addition Thacker (Reiter) (163) found that RAC attenuated adipose SCD expression in RAC fed pigs, while in a separate study RAC increased expression of lipoprotein lipase (LPL) in porcine muscle (195). While only limited studies on the effect of RAC on regulation of lipogenesis have been conducted, the role of BAA in adipose lipolysis has been more clearly delineated. Bergen and Merkel (9) proposed that BAA modulates lipid metabolism via the beta receptor-G protein-coupled adenylyl cyclase-PKA cascade in porcine adipose tissues. BAAs stimulate cAMP production and induce protein kinase A (PKA) activity, which stimulates hormone-sensitive triacylglycerol lipase in adipose tissue and subsequent mobilization of storage triacylglycerol. As noted previously, limited work with cultured fat cells have shown that BAA also inhibits lipogenesis (2). Thus, combining anti- lipogenic and pro-lipolytic effects of BAA, the overall net effect of BAA feeding is a depression of fat accretion, increase in lipolysis and possibly increased fatty acid oxidation. This effect would redirect energy from fat deposits toward tissue (skeletal muscle) to promote increased protein deposition. Based on these previous findings and interpretations about the effect of RAC on the expression of a few key genes in the fatty acid metabolism, I proposed that RAC modulates fat deposition in pigs at the transcription level through regulating expression of genes in the lipid metabolism and regulation pathways. The work in this study thus emphasized the expression of genes associated with lipid metabolism in adipose tissue 40 and was designed to explore the global mRNA esponse to ractopamine treatment in porcine adipose tissue. We utilized an oligonucleotide microarry analysis platform to quantify the expression of 13,297 porcine transcripts simultaneously. this study was designed to explore the effect of RAC over a 28-day period on the relative abundance of mRNA of 13,297 transcripts in the middle layer of subcutaneous adipose tissue of pigs. MATERIALS AND METHODS In the area of global gene expression analysis, certain guidelines have been established for conducting and reporting such extensive data sets. In this study, Minimum information about a microarray experiment (MIAME), was used to guide the development of describing the experimental methods and the analysis and subsequent reporting of data sets. MIAME is a standard designed to contain the minimum information required to ensure that microarray data can be interpreted and that the resulting derived data can be independently ratified (196). MIAME can be broken down into four different areas: experimental design, sample description, software and techniques for the analysis and interpretation of data, and the array design (197). Table 1 shows an example of the information recorded for MIAMI compliance (198).The microarray data in this study will be deposited at the NCBI Gene Expression Omnibus (GEO) that is data repository of high-throughput gene expressions and hybridization arrays. In this study, the pig array was obtained through U.S. Pig Genome Coordination Program (http://www.animalgenome.org/ pigs/resources/ array_request.html). Information about oligo probe design, location of each spot in the array and gene information of each spot are described below. 41 Table 1. An example of needed information to be recorded for MIAME compliance (198) Title and type of experiment Time course, control vs treatment, mutant vs wildtype? Authors Names and addresses of researchers Background Important publications justifying the experimental use of arrays Experimental design Species used Worm, fly, plant, animal? Strain/genotype of species What, if any genetic manipulations have the species being used undergone? Tissue/sample type Whole organism or specific tissue type? muscle, nervous tissue? Sex of species used Male, female or hermaphrodite? Maintenance of organism Conditions on which that organism is routinely maintained Method of sample collection Dissection method used Dates of collection References to lab book Sample storage Temperature (?80?C), location Sample manipulations RNA extraction method How was the RNA extracted, what chemicals/kits were used? RNA storage Temperature (?80?C), location? Gel/bioanalyser Gel images or Agilent bioanalyser output demonstrating quality of RNA Amplification protocol Was an amplification step required? Labeling protocol How was the RNA labeled? Hybridization protocol How was the hybridization performed? Chip identification number Has the ID of the array used been included? Data analysis Raw data Are the raw data readings included and how were they produced? Normalized data Is the normalized data included and how were they produced? Analysis methods What analysis methods or programs were used? Results Final list of genes with changed expression values 42 Animal Feeding Trial in Eli Lilly & Co (Greenfield, IN) This study used porcine adipose tissues collected from an animal feeding trial in Eli Lilly & Co conducted in 1996. The feeding trial was originally designed to study the effect of ractopamine on gene expression with gene-by-gene methods such as Northern blots. Previous graduate students in our lab conducted Northern blot analyses on the same tissue samples. Microarray analysis was applied in this study to the same samples. Six finishing pigs (white large composite castrated males) were provided ad libitum access to a 16% crude protein corn-soybean meal diet, supplemented with 0 or 60 ppm Paylean? for 28 days. The diet met all nutrient requirements for finishing pigs (199). Pigs did not fast prior to slaughter and were slaughtered at 28 days of the trial. The middle layer subcutaneous adipose tissue was collected. The pig feeding, slaughter, and sampling protocols were approved by the Lilly Research Laboratory Institutional Animal Care and Use Committee. Tissue samples from 0 and 60 ppm Paylean? treated pigs for 28 days were used in the following oligonucleotide microarray analysis. Pig identification number, length of treatment, amount of Paylean? treatment, and the date of sample collection are presented in Table 2. 43 Table 2. Pig identification number, length of treatment, amount of treatment, and the date of sample collection for the pigs in the Eli Lilly feeding trial. Finishing diets were supplemented with 0 or 60 ppm Paylean? for 28 days. Pig ID# Length of Treatment (days) Amount of Paylean? (ppm) Date of Samples Collection 787 28 0 9/24/1996 807 28 0 9/24/1996 826 28 0 9/24/1996 779 28 60 9/24/1996 784 28 60 9/24/1996 796 28 60 9/24/1996 Animal Slaughter and Tissue Collection Pigs were slaughtered by electrical stunning followed by exsanguination. Middle- layer subcutaneous adipose tissues were removed immediately, snap-frozen in liquid nitrogen, and stored at minus 80?C after samples were collected. Experimental Design of Oligomer Microarray Analysis of Gene Expression In this experiment, microarray analyses were performed with a pool of control RNA isolated from the adipose tissues of three pigs fed 0 ppm Paylean? (control) and individual adipose RNA preparations from each pig fed 60 ppm Paylean? diet (n=3; experimental). The labeling dyes Cyanine 3 (Cy3) and Cyanine 5 (Cy5) were randomly assigned between pooled control RNA and experimental RNA from each 60 ppm Paylean? treated pig (200). RNA isolation Total RNA was isolated by using the one-step guanidinium-phenol-chloroform extraction method (201). One-half gram of frozen adipose tissue from each pig was 44 powdered using a hammer-driven, stainless steel mortar and pestle that was constantly cooled with liquid nitrogen. The tissue was then placed in a 50 ml conical tube containing 10 ml of TriZol reagent (Invitrogen Corp, Carlsbad, CA), and RNA was isolated according to the instructions provided. The RNA concentration was determined using an Ultrospec 3000 UV/ visible spectrophotometer (Amersham Pharmacia Biotech; Piscataway, NJ) by reading optical density (OD) at 260 nm (by using an OD 260 unit equivalent to 40 ?g/ml). Quality of the RNA was evaluated by gel electrophoresis of ~1 ?g RNA on an 1% agarose 0.5X tris(hydroxymethyl)aminomethane (Tris)-borate-EDTA buffer (TBE) gel containing ethidium bromide at 120V for 30 minutes using 0.5X TBE as the running buffer. Images of the gels were taken under ultraviolet (UV) light using Polaroid instant film number 55 (Polaroid Corporation, Waltham, MA) to generate printed image of the gel. The RNA quality was evaluated by observing smearing at 18s and 28s bands, band intensity of 18s and 28s, and DNA contamination. No analysis was performed on any sample with degraded RNA appearance. RNA samples were stored at 80?C in 1?l of RNA Secure/ ?g of RNA (Ambion; Austin, TX) until subsequent analyses. Gel images of RNA used in the microarray analysis were present in Appendix A. RNA purification DNA contamination can seriously affect the validity of reverse transcription (RT) reaction. To remove genomic DNA in the RNA sample, RNA samples were purified using DNA-free TM kit (Ambion, Austin, TX) according to the manufacturer?s protocol. 0.1 volume 10X DNase I buffer and 1?l rDNase I (2U/ ?l) were added to each RNA sample followed by the addition of RNase free water to bring the total volume to 50 ?l. 45 The solution was mixed and incubated at 37 o C for 20-30 min. After incubation, 5 ?l DNase inactivation reagent was added and incubated at room temperature for 2 min. Then, the resultant solutions were centrifuged at 10,000 x g for 1.5 min in a 16M Microcentrifuge (Labnet International Inc, Edison, NJ), and the RNA was transferred to a new tube. The purified RNA was analyzed by measuring UV absorption at 260nm and 280nm. To assess the RNA quality, 1 ?g purified RNA sample was processed, stained with ethidium bromide and resolved on a 1.0% agarose gel. After DNaseI treatment, control RNA samples from three pigs were pooled, and another UV absorbance reading was taken at 260nm and 280 nm to determine the pooled RNA concentration and assess the pooled RNA quality. Besides checking RNA quality by agarose gel electrophoresis, DNA contamination was also assessed by attempting PCR amplification of the RNA with primers targeting at a fragment of intron of stearoyl CoA desaturase (SCD) using a PTC100 programmable thermal controller (MJ Research, Waltham, MA). The primer was designed based on pig SCD complete mRNA sequence (GenBank accession number: AY487830) using on-line software Primer3 (http://frodo.wi.mit.edu/cgi-bin/primer3/primer3_www.cgi). The sense primer sequence was: CAGCCACCTTTTTCGAGTTGT, and the antisense primer sequence was: AAATTGGGGAGGAGGGTGAAA .The expected PCR amplicon is 219 basepair (bp). 20 ?l of resulting PCR amplicon was loaded on a 1.0% agarose gel to check if there was amplified molecule from DNA. Since the SCD primers targeted at a fragment of intron sequences, the primers could not anneal on the RNA and no amplicon 46 was produced by PCR. Therefore, the PCR amplicon was observed only when RNA was contaminated by genomic DNA. Synthesis of Experimental Probes for Porcine Purified RNA Using Reverse Transcription First strand cDNA synthesis with aminoallyl dNTP The first strand of cDNA for each sample was synthesized using the Superscript II cDNA synthesis kit (Invitrogen Corp., Carlsbad, CA) according to the manufacturer?s protocol. 18 ?g of total RNA which had been purified with DNase I treatment was mixed with 2 ?l oligo dT18-20 primer (0.5 ?g/?l) (Invitrogen Corp., Carlsbad, CA) and the final volume was brought up to 18.5 ?l with RNase-free water. Then the solution was incubated at 70 o C in a dry bath incubator (Fisher Scientific, Pittsburgh, PA) for 10 min. After this incubation, this solution was mixed with 6 ?l 5X first strand buffer, 3 ?l 0.1M DTT, 0.6 ?l 50X aminoallyl-dNTP mix (Appendix B), 2 ?l SuperScript IIRT, and 0.5 ?l RNAse inhibitor (Ambion Incorporation; Austin, TX). The final reaction cocktail was gently mixed and incubated overnight at 42 o C in a micro-hybridization incubator (Robbins Scientific, Sunnyvale, CA). RNA hydrolysis and neutralization after reverse transcription 47 After incubation overnight at 42 o C, 10 ?l NaOH (1M) and 10 ?l EDTA (0.5 M) were added to the RT reaction tubes (cDNA solution) and incubated at 65 o C for 15 minutes. After incubation, 10?l HCl (1 M) was added to neutralize the solution. This is an important step since DNA binds to Qiagen column at pH below 7.5 in the next purification step. Immediately after neutralization, the cDNA mixture was purified by the steps described below. Purification of aminoallyl labeled cDNA The purification protocol was modified from the Qiagen QIAquick PCR purification kit (Qiagen Inc, Valenica, CA) protocol. The phosphate wash and phosphate elution buffers (Appendix A) were substituted for the Qiagen-supplied buffers because the Qiagen buffers contain free amines that compete with the Cyanine (Cy) dye coupling reaction in the down stream steps. First, 300 ?l (5X reaction volume) buffer PB was added to the cDNA and transferred to a QIA-quick column. Then the column was placed in a 2-ml collection tube and centrifuged at 10,000g for 1 min. The collection tube was emptied after centrifugation. Continuing the process, 750 ?l phosphate wash buffer was then added to the column, and the column was centrifuged at 10,000g for 1 minute. After emptying the collection tube, the wash and centrifugation steps were repeated once. Then the flow-through in the collection tube was discarded and the column was centrifuged at 10,000g for another 1 min at maximum speed. Thereafter, the column was transferred to a new 1.5-ml microfuge tube, and 30 ?l phosphate elution buffers were carefully added to the center of the column membrane. Then the collection tube was incubated for 1 minute at room temperature, and the cDNA was eluted by centrifugation at 10,000g for 1 min. The elution step was repeated in the same tube with another 30ul phosphate elution buffer. Finally the eluted cDNA sample was dried to completion about 45 min in a Savant SC100 Speed Vac concentrator (Savant, San Joes, CA). Coupling aminoallyl-cDNA to Cyanine (Cy) dye ester The coupling procedure was conducted under light-safe conditions. The completely dried aminoallyl-labled cDNA was resuspended in 4.5 ?l 0.1M sodium carbonate buffer (Na2CO3). Then 4.5 ?l of the appropriate Cy dye (Amersham Pharmacia Biotech, 48 Piscataway, NJ) was added to each tube and mixed. To prevent photobleaching of the Cy dyes, all reaction tubes were wrapped in foil and kept away from light. After dye addition, the reaction mixture was incubated for 1 hr at room temperature. Thereafter, the reaction mixture was purified in the following steps to remove uncoupled Cy-dye. Purification of Cy-dye coupled cDNA The purification steps were performed under light-safe conditions. The Qiagen QIAquick PCR purification kit was used. First, 35 ?l 3M sodium acetate was added to the Cy-dye coupled cDNA solution. Then, 250 ?l buffer PB (DNA binding buffer, supplied by Qiagen) was added and the mixture was applied to a QIA quick spin column that was placed in a 2 ml collection tube. The column then was centrifuged at 10,000g for 1 min, and the flow-through was discarded. Then, 0.75 mL buffer PE (washing buffer, supplied by Qiagen) was added to the column and centrifuged at 10,000g for 1 minute. After discarding the flow-through, the column was centrifuged for an additional 1 minute at maximum speed. Then, the column was placed in a clean 1.5-mL microfuge tube and 40 ?L Buffer EB (elution buffer,supplied by Qiagen) was carefully added to the center of the column membrane. After incubation for 1 min at room temperature, the column was centrifuged at 10,000g for 1 min. The elution step was repeated another time in the same tube. The final elution volume should be around 80ul. Then, the tube was wrapped with aluminum foil, and the Cy dye-coupled cDNA containing solution from porcine adipose RNA was analyzed employing the following steps. Analysis of labeling reaction This step was conducted in the dark. An Ultraspec 300 UV/Visible spectrophotomer and a 50?l Beckman quartz MicroCuvette (Beckman Coulter Inc, Fullerton,CA) was used 49 to analyze the entire undiluted Cy-dye coupled cDNA solution. The cuvette was washed with MilliQ water and blow dried with a compressed air duster. For each sample the absorbance at 260 nm and either 550 nm for Cy3 or 650 nm for Cy5 were measured for each sample. Finally, the dye-coupled cDNA solution was pipetted back into the original sample tube. To determine concentration and purity of dye labeled aminoallyl-cDNA, the following formulas were used to calculate total picomoles of cDNA synthesized as described by Hedge et al. (202). pmol nucleotides = [OD260 * volume (?L) * 37 ng/?L * 1000 pg/ng] 324.5 pg/pmol = OD260 * volume (?L) *114.02 1 OD260 = 37 ng/?L for cDNA; 324.5 pg/pmol is the average molecular weight of dNTP For each sample the total picomoles of dye incorporated (Cy3 or Cy5 as appropriate) and the nucleotides/dye ratio were calculated using formula (195): pmol Cy3 = OD550 * volume (?L) /0.15 pmol Cy5 = OD650 * volume (?L) /0.25 nucleotides/dye ratio = pmol cDNA/pmol Cy dye For the formula, 0.15and 0.25 are correction factors for Cy3 and Cy5 respectively, which are corrected by extinction factors of Cy3 (? = 150000 M -1 cm -1 ) and Cy5 (? = 250000 M- 1cm-1) molecules (202). Fragmentation of Cy-labeled cDNA will cause low hybridization of cDNA with the oligo probes on the array. It is important to check the quality of Cy-labeled cDNA at this step. Cy5-labeled cDNA 2 ?g was resolved by electrophoresis on 2% agarose gel at 50 130V for 45 minutes in the dark. Then the gel was wrapped with aluminum foil and imaged on Typhoon 9410 (Amersham Pharmacia Biotech, Piscataway, NJ) at photomutiplier tube (PMT) 600 using emission filter for Cy5 (excitation at 633nm). An example of Cy5-labeled cDNA is presented in Fig 1. Gel images of Cy5-labeled cDNA used in each slide bybridization are presented in Appendix C. 51 2,072 bp Over-saturated 1,500 bp fluorescence 600 bp 100 bp Fig 1. Cy5-labeled cDNA on a 2% agarose gel (right) at 130V for 45 minutes, the gel was imaged on Typhoon 9410 (Amersham Pharmacia Biotech, Piscataway, NJ) at PMT 600 using emission filter for Cy5 (laser 633nm). DNA ladder was resolved on another 2% agarose gel (left) under identical electrophoresis condition to identify the size of the Cy5-labeled cDNA. The fluorescently labeled cDNA population was mainly distributed from 600 bp to 2,000 bp with weak distribution in the range smaller than 400 bp. These data exhibited no fragmentation of cDNA and reflected the intactness of RNA. 52 Only purified unfragmented Cy labeled aminoallyl-cDNA probes were used for hybridization on the spotted slides. Then, equal pmol of Cy3 and Cy5 labeled cDNA solutions were mixed together, and the mixed cDNA solution was completely dried in a aluminum foil covered Savant SC100 Speed Vac concentrator for 2 hr. Gene Expression Analyses Utilizing the Pig Genome Center pig Microarray Platform The pig microarray In this study, a pig-based 70mer oligomicroarray was used. The pig microarray was distributed by U.S. Pig Genome Coordination Program (Iowa State University, Ames, Iowa), produced with the QIAGEN Array-Ready Oligo Set for the Pig Genome (version 1.0) and the Pig Genome Oligo Extension Set (version 1.0) (Qiagen, Valencia, CA). The Pig Genome Oligo set contains 10,665 70-mer probes representing Sus scrofa gene sequences with a hit to human, mouse, or pig gene transcript. Some sequences contain a 3' expressed sequence tag (EST). The Pig Genome Oligo Extension Set contains 2,632 Sus scrofa gene sequences with at least one 3' EST. In total, 13,297 transcripts probes are included in this pig microarray. The gene-lists, data sheets, and product profiles of the Pig Genome Oligo Set Version 1.0 and Pig extension Genome Oligo Set Version 1.0 are available from Qiagen (http://omad.operon.com/download/index.php ). All spotted probes were designed from TIGR Gene Index SsGI (Sus scrofa Gene Index) Release 5.0 (http://www.tigr.org/tigr-scripts/tgi/T_index.cgi?species=pig). Details and criteria for selecting these particular 70-mer probes are given in Appendix D. Each 13,297 element oligo set was printed onto one single slide at the Microarray Printing 53 Facility at the University of Minnesota, Minneapolis. The pig microarrays (slides) were stored under dry and dark conditions before their use. Pre-hybridization of slides Briefly, the oligo array slides were plunged into a Coplin jar (Fisher Scientific) filled with boiling MilliQ water and immediately shaken vigorously for 2 minutes. Then slides were transferred to 95% ethanol for 15 seconds and dried by spinning at 500g for 1 minute in RT6000B refrigerated centrifuge (Sorvall, Thermo Electron Corporation, Asheville, NC). Thereafter, slides were put in a Coplin jar filled with pre-hybridization buffer [5X SSC (0.75M NaCl and 0.075M NaCitrate), 0.1% SDS, 1% BSA] and incubated in a Precision Model 181water bath (Precision Scientific. Inc, Chicago, IL) for 45 min at 42 o C. After pre-incubation, slides were washed with MiliQ water 5 times and then washed in isopropanol. Finally, slides were dried by centrifugation at 500g for 1 minute and put into a Corning Hybridization chamber (Corning, Corning, NY) with the spotted side upward. Preparation of the hybridization chamber Dry LifterSlips (Fisher Scientific) were cleaned with clean dry air, and put on the slide gently with the white-edged side downward to the slide. To prevent arrays from drying during the following hybridization process, two thin strips 6.25cm of Whatman filter paper (Whatman Inc, Florham Park, NJ), were placed into the wells located on either end of the hybridization chamber, and the filter paper was saturated with 20 ?l MilliQ H 2 O. Hybridization The hybridization steps were conducted under light-safe conditions. Twenty ?l of human Cot 1 (Invitrogen Corp), 2 ?l poly A (Invitrogen Corp) and 13.6 ?l H 2 O were 54 added to the completely dried cDNA mixture. The solution (hybridization mixture) was mixed by pipetting up and down and heated at 95 o C for 5 min in a dry bath incubator (Fisher Scientific). The hybridization mixture was centrifuged at 10,000 g for 1 minute to pellet any particle material. The upper clear solution was carefully transferred to a new tube avoiding any sediment material. The hybridization mixture was carefully pipetted onto the oligo array while it rested within the hybridization chamber. The hybridization mixture was slowly pipetted to spread the hybridization solution evenly across the array surface. When the hybridization mixture had diffused out completely under the cover slip, the hybridization chamber was quickly sealed to keep the moisture within the chamber. Thereafter, the hybridization chamber was wrapped with aluminum foil without touching the top of hybridization chamber. Finally, the wrapped hybridization chamber was placed overnight in the 42 o C serological water bath (Boekel Scientific, Feasterville, PA). Post hybridization array washing procedure The following washing steps were conducted in the dark. After hybridization overnight in the water bath at 42 0 C, the wrapped slide was removed from the water bath with the spotted side facing upwards. The foil was carefully removed without touching the slide. Then, the exterior of the hybridization chamber was dried with a Kimwipe (Kimtech Science, Erie, PA), and was unsealed carefully to avoid water entering the chamber. The slide was removed from the chamber and quickly put into a Coplin jar filled with 1X SSC, 0.2% SDS buffer (Appendix D). To make the LifterSlip fall away from the slide, the Coplin jar was incubated for 4 min at room temperature with gentle mixing on a BellyDancer with speed 4 (Strovall, Greensboro, NC ). The slide without 55 cover slips was transferred to another Coplin jar filled with 0.1X SSC, 0.2 % SDS buffer and incubated for 4 min at room temperature with gentle mixing on the BellyDancer with speed 4. For the third washing, the slide was quickly transferred to a Coplin jar filled with 0.1X SSC, and incubated for another 4 min at room temperature with gentle mixing on an orbital shaker with speed 4. Thereafter, the slide was dried by spinning at 1000 g in RT6000B refrigerated centrifuge for 1 minute. Then, the slide was put into a foil- wrapped 50-ml conical tube, and scanned with a Gene Pix 4000B simultaneous dual wavelength scanner as soon as possible (Axon Instruments, Inc., Union City, CA). Scanning of arrays The scanner was turned on 15 minutes before the slide scanning. Then, the slide was loaded onto the scanner with the spot side downward in the dark. After launching the GenePix Pro 4.0 software, the slide was quickly read by clicking ?Prescan? button in order to locate the scan zone of the slide. Several scans at various level of photomultiplier tube (PMT) were conducted to establish the PMT offset. After optimizing PMT level, separate images were acquired for each fluorophore at a resolution of 10 ?m per pixel. For scaling of the two channels with respect to signal intensity, PMT and laser power settings were adjusted to achieve a signal ratio of channel 635 nm/channel 532 nm as close to 1.0 as possible. According to the GenePix Pro manual, the acceptable signal ratio ranges from 0.8~1.2. All along the scan, the ?histogram? panel was observed to verify that the image was correctly equilibrated; the two curves corresponding to the two channels should be superimposed for intensities greater than the background. After finishing scanning, two single-channel (channel 635 and channel 532) images and one ratio image were automatically created by GenePix Pro and were saved with tagged 56 image file format (TIFF). All array images of this experiment are presented in the Appendix E. Image analysis The combined ratio image of the two single-channel images (Cy3 and Cy5) was used for image analysis with GenePix Pro 4.0 software. First GenPix Array List (GAL) file, provided by Qiagen, was loaded to the image. The GAL file was used to link the information from the slides printing process to the image analysis. Gene names and identifiers are located to corresponding spots on the image. A partial GAL file is presented in Table 3. In the next step, a grid mask was used to aid align the blocks on the array. Each block was placed exactly over the corresponding spots by adjusting the position of the grid mask. After aligning the blocks, all the spots were flagged to evaluate the spot quality using the standard set by GenePix Pro software and the flagged values were stored in the final results table. Finally, the image analysis process extracted fluorescence intensity data from the validated spots. Results and related calculated values were automatically saved in the GenePix Result( GPR) file. The GPR file was applied in the following data processing and analysis steps. An example of partial GPR file was presented in the Table 4. 57 Table 3. Part of GenePix Array List (GAL) file. It presents genes information corresponding to each spot. Each spot is located by the block, row and column number, and has a unique identification number. 58 Block48=16500, 61160, 160, 18, 240, 17, 240 Supplier=BioRobotics ArrayerSoftwareName=TAS Application Suite (MicroGrid II) ArrayerSoftwareVersion=2.2.2.8 Block Column Row ID Name 1 1 1 SS00013045 unknown 1 1 2 SS00011959 unknown 1 1 3 SS00011151 unknown 1 1 4 SS00000701 heat shock protein 70.2 [Sus scrofa] 1 1 5 SS00010360 unknown 1 1 6 SS00009274 unknown 1 1 7 SS00008466 unknown 1 1 8 SS00007748 unknown 1 1 9 SS00006940 unknown 1 1 10 SS00005854 PIR|A40915|OKHUR2 protein kinase (EC 2.7.1.37) cAMP-dependent type II-beta regulatory chain - human, partial (14%) 1 1 11 SS00005046 homologue to SP|O97704|NPT2_SHEEP Renal sodium-dependent phosphate transport protein 2 (Sodium/phosphate cotransporter 2) (Na(+)/Pi, partial (47%) 1 1 12 SS00004328 similar to SP|P49916|DNL3_HUMAN DNA ligase III (EC 6.5.1.1) (Polydeoxyribonucleotide synthase [ATP]). [Human] {Homo sapiens}, partial (27%) 1 1 13 SS00003520 homologue to GP|14388542|dbj|BAB60792. hypothetical protein {Macaca fascicularis}, partial (44%) 1 1 14 SS00002434 PIR|JE0272|JE0272 low density lipoprotein receptor-related protein 6 - human, partial (12%) 1 1 15 SS00001626 homologue to GP|12654407|gb|AAH01029.1 N- Acetylglucosamine kinase {Homo sapiens}, complete 1 1 16 SS00000908 delayed rectifier potassium channel Kv2.1 [Sus scrofa] 1 1 17 SS00000100 SP|Q99832|TCPH_HUMAN T-complex protein 1 eta subunit (TCP-1-eta) (CCT-eta) (HIV-1 Nef interacting protein). [Human], partial (36%) 1 2 1 SS00013069 unknown 1 2 2 SS00011983 unknown 1 2 3 SS00011175 unknown 1 2 4 SS00000701 heat shock protein 70.2 [Sus scrofa] 1 2 5 SS00010384 unknown 1 2 6 SS00009298 unknown 1 2 7 SS00008490 unknown 1 2 8 SS00007772 unknown 1 2 9 SS00006964 unknown 1 2 10 SS00005878 homologue to GP|14035806|emb|CAC38499. unnamed protein product {Homo sapiens}, partial (59%) 1 2 11 SS00005070 homologue to GP|2078323|gb|AAB54006.1| Ch-1PTPase delta form {Homo sapiens}, partial (19%) 1 2 12 SS00004352 similar to GP|12803737|gb|AAH02705.1 chromosome 22 open reading frame 3 {Homo sapiens}, partial (43%) 1 2 13 SS00003544 similar to GP|12804035|gb|AAH02870.1 Unknown (protein for MGC:11266) {Homo sapiens}, partial (30%) 1 2 14 SS00002458 homologue to GP|11322247|emb|CAC16786. nucleolar protein No55 {Homo sapiens}, partial (36%) 1 2 15 SS00001650 homologue to GP|12804063|gb|AAH02884.1 hypothetical protein similar to beta-transducin family {Homo sapiens}, partial (27%) 1 2 16 SS00000932 protein carboxyl-o-methyltransferase [Sus scrofa domestica] 1 2 17 SS00000124 homologue to GP|1815762|gb|AAB42020.1| gamma-glutamylcysteine synthetase {Mus musculus}, partial (42%) 1 3 1 SS00013093 aromatase type II [Sus scrofa]aromatase cytochrome P450 [Sus scrofa]aromatase cytochrome P450 [Sus scrofa]type I cytochrome p450 aromatase [Sus scrofa]type II cytochrome p450 aromatase [Sus scrofa] 1 3 2 SS00012285 unknown 1 3 3 SS00011199 unknown 1 3 4 SS00001180 glyceraldehyde-3-phosphate dehydrogenase [Sus scrofa]glyceraldehyde-3-phosphate dehydrogenase [Sus scrofa] glyceraldehyde- 3-phosphate dehydrogenase [Sus scrofa]glyceraldehyde 3-phosphate d 1 3 5 SS00010408 unknown 59 Table 4. Part of GenePix Result (GPR) file. The head of GPR file describes all the parameters used when scanning the array including PixelSize, FocusPosition, Temparature, PMTGain, and ScanPower. The main body of GPR file list the determined and calculated values for each spot (gene), such as median/mean spot foreground and background pixel intensity at each channel (532 nm and 635 nm), calculated median/mean of ratios, sum of medians/means, the signal-to-noise-ratio etc. 60 61 Normalization Microarray data normalization is an important step for obtaining data that are reliable and usable for subsequent analysis. In this study, a robust local regression technique, locally weighted scatterplot smoothing (LOWESS) algorithm was used to normalize all the data by GenePixpro package. In experiments where two fluorescent dyes (Cy3 and Cy5) have been used, intensity-dependent variation in dye bias may introduce spurious variations in the collected data. LOWESS normalization assumes that the dye bias is dependent on spot intensity and applies a spot intensity dependent smoothing adjustment to remove dye bias. All samples in the dataset are corrected independently in LOWESS. The adjusted ratio is computed by: log(R/G) = log(R 0 /G 0 ) ? c(A) In the above formula, R and G are normalized fluorescent intensity of Cy3 (red) and Cy5 (green) respectively for a spot; R 0 and G 0 are non-normalized fluorescent intensity of Cy3 (red) and Cy5 (green) respectively for the identical spot; c(A) is the Lowess fit to the log(R/G) vs log(sqrt(R*G)) plot. If green has been chosen as the treatment dye and red as the control dye, then R and G are reversed in the above formula (203). Potential dye intensity biases in the microarray data sets were visualized using M vs. A scatter plots constructed for each array, where log intensity ratios M = log 2 (Cy3/Cy5) = log 2 Cy3 - log 2 Cy5 were plotted against mean log intensities A = (log 2 Cy3 + log 2 Cy5)/2 for each array spot (204). The efficiency of LOWESS normalization was assessed by monitoring M-A plots for data from each array before and after LOWESS normalization. An example of M-A plots before and after LOWESS normalization is presented in Fig 2. All M-A plots of this experiment are presented in Appendix F. The log transformed ratios 62 by LOWESS normalization were used in the following analysis. The log transformation of data reduced skew and produced desirable variance properties. Thus, the distribution of transformed data was closer to the normal distribution. More discussion about choosing normalization methods is in the discussion part of this chapter. 63 Fig 2. M-A plot before and after LOWESS normalization For the X axis, A = (log 2 Cy3 + log 2 Cy5)/2, reflecting the spot intensity; for the Y axis, M = log 2 (Cy3/Cy5) = log 2 Cy3 - log 2 Cy5, reflecting the expression difference between the two channels. In this example, in the M-A plot after normalization, the fluorescence intensity (A value) of spots ranged from 0-16 with a few spot in the lowest and highest part, which means the scanner laser intensity was set properly to detect spots with low fluorescence intensity. Only a few spots were saturated. Before the normalization, The M-A plots exhibited systematic trends which depended on the value of A; therefore, local intensity-dependent regression lines through the data were fitted using the lOWESS fit function to remove the spot intensity dependent dye bias. After LOWESS normalizatrion, The difference between Cy5 and Cy3 labeling spots (M value) were symmetrically spread with the center of M=0, which means dye bias was minimized. 64 Fig 2. A) M-A plot before LOWESS normalization B) M-A plot after LOWESS normalization 65 Statistical Analysis To search differently expressed genes between control (0 ppm Paylean) and treatment (60 ppm Paylean) groups, Significant Analysis of Microarray (SAM) software (http://www-stat.stanford.edu/~tibs/SAM/) was used to conduct statistical analysis in this study (205). The SAM (version 1.12) add-in to Microsoft Excel was used for comparisons of replicate array experiments. SAM assesses the difference between two mean values when taking into account the standard errors of those means. The significance of that difference is estimated by comparing it against the probability of it?s occurrence once. The model of chance occurrence is generated by permutation of the input data, rather than a predetermined model (e.g., a normal distribution) in the standard t-test. The SAM algorithm was used instead of the standard t test because SAM was proved to have a better ability to scale down to small numbers of replicates (n=3 in this study) (205). SAM computes a statistic d i for each gene i, measuring the strength of the relationship between gene expression and the response variable. It uses repeated permutations of the data to determine if the expression of any gene is significantly related to the response. The cutoff value for significance is determined by a tuning parameter delta (?), which is chosen by a user to obtain different false positive rate. More discussion about application of statistical methods is in the discussion part of this chapter. In this study SAM was employed using the one-class response with 1,000 permutations to determine genes whose expression was significantly different from zero. Significant genes were determined by setting the number of falsely called genes to less than one and choosing similar false discovery percentage medians for each biological 66 replicates. At these values, the false discovery rate (FDR) for the positive genes was 0.05 (5%) and the q value (a measure of significance in terms of the false discovery rate) for all biological replicates were less than 0.003. After obtaining a list of significantly differentially expressed genes from SAM, in order to focus our attention on genes related to the hypothesis of the researches in this dissertation, a list including genes of interest (named genes of interest list) was constructed which includes genes for transcription factors, carbohydrate, lipid and cholesterol metabolism, electron transport, oxidative phosphorylation, and the cAMP pathway. The above genes of interest were randomly spotted on the pig array. This gene list was used to filter the SAM results and these filtered results are reported in Table 5. RESULTS AND DISCUSSION Issues in Evaluating RNA Quality from Porcine Adipose Tissue The assessment of RNA integrity can be accomplished by various methods: traditional agarose gel-electrophoresis, innovative lab-on-chip technologies like Bioanalyzer 2100 (Agilent Technologies) and Experion (Bio-Rad Laboratories), and modern OD measurement via NanoDrop. Absorption at 260 nm and 280 nm indicates the presence of nucleic acids and proteins respectively. The ratio of the absorbance at 260nm and 280nm has been used to measure the purity of isolated RNA for a long time. For instance, when using a spectrophotometer, a ratio of absorbances at 260 and 280 nm (A 260 :A 280 ) greater than 1.8 is traditionally considered to be an acceptable indicator of RNA purity (206). However, the accuracy of A 260 :A 280 ratio method has been questioned. The pH and ionic strength of the solution significantly affect the A 260 :A 280 ratio of nucleic acids (207). Warburg and 67 Christian (208) showed that the ratio was a good indicator of contamination of protein preparation by nucleic acid, but the ratio did not reflect the contamination of nucleic acid by protein. Because the extinction coefficient of nucleic acid at 260nm and 280nm are much greater than that of proteins, significant contamination with protein will not greatly change the A 260 :A 280 ratio of a nucleic acid solution (209). In the traditional method of assessing RNA quality, RNA integrity is evaluated using agarose gel electrophoresis (denaturing gel is preferred) stained with ethidium bromide, which produces a well-established banding pattern. Typically, gel images show two bands comprising the 28S and 18S ribosomal RNA (rRNA) species and other bands where smaller RNA species are located (210). Mammalian 28S and 18S rRNAs are approximately 5 kb and 2 kb in size. The theoretical 28S:18S rRNAs is approximately 2.7:1(212). The proportion of the ribosomal bands (28S:18S) has conventionally been viewed as the primary indicator of RNA integrity, with a ratio of 2.0 considered to be typical of ?high quality? intact RNA (211). Total RNAs from mammalian tissues rarely have a 28S:18S rRNA ratio of 2.0 or greater because 28S rRNA structure is unstable relative to the 18S rRNA. The instability of 28S rRNA results from its size and its high degree of secondary and tertiary structures (212). Certainly total RNA with a 28S:18S rRNA ratio of 2.0 denotes high quality. However, the total RNA with lower rRNA ratios (<1.8) is not necessarily of poor quality especially for downstream applications if no degradation products can be observed in the electrophoretic trace (211). Visual assessment of the 28S:18S rRNA ratio on agarose gels is subjective because interpretation of gel images is dependent on individual and the resulting data cannot be processed digitally. Appearance of rRNA bands is affected by 68 electrophoresis conditions, amount of RNA loaded, and saturation of ethidium bromide fluorescence (213). Because of lack of reliability, the 28S:18S rRNA ratios may not be used as a gold standard for assessing RNA integrity. Imbeaud et al. (211) did not find clear correlation between 28S:18S rRNA ratio and RNA integrity in some samples although RNAs with 28S:18S >2.0 were usually of high quality. Moreover, most of the RNAs (83%) displaying a 28S:18S > 1.0, could be considered of good quality. This was determined after those RNAs were applied to determine the expression of four house- keeping genes using real-time quantitative reverse transcription PCR methods. The Agilent 2100 Bioanalyzer provide a framework for the standardization of RNA quality control. RNA samples are electrophoretical separated on a micro-fabricated chip and subsequently detected via laser induced fluorescence detection. A RNA ladder is used as a mass and size standard during electrophoresis, which allows the estimation of the RNA band sizes (214). The integrity of the RNA may be assessed by visualization of the 18S and 28S ribosomal RNA bands. An elevated threshold baseline and a decreased 28S:18S ratio are both indicative of degradation. Degradation of the RNA sample produces a shift in the RNA size distribution toward smaller fragments and a decrease in fluorescence signal as dye intercalation sites are destroyed (215). RNA Integrity Number (RIN) was developed to assess RNA quality for the lab-on- chip capillary gel-electrophoresis used in the Bioanalyzer 2100 (215). The RIN algorithm allows calculation of RNA integrity using a trained artificial neural network based on the determination of the most informative features that can be extracted from the electrophoretic traces out of 100 features identified through signal analysis (215). The selected features which collectively catch the most information about the integrity levels 69 include the total RNA ratio (ratio of area of ribosomal bands to total area of the electropherogram), the height of the 18S peak, the fast area ratio (ratio of the area in the fast region to the total area of the electropherogram) and the height of the lower marker (214). The RIN algorithm allows the classification of total RNA on a numbering system from 1 to 10, with 1 being the most degraded profile and 10 being the most intact (Fig 3). A smearing of either 28S and 18S peaks, or a decrease in their intensity ratio indicates degradation of the RNA sample. In this way, interpretation of an electropherogram is facilitated, comparison of samples is enabled and repeatability of experiments can be assessed (211, 215). While intact RNA obviously constitutes the best representation of the natural state of the transcriptome, there are situations in which gene expression analysis may be satisfactory even on partially degraded RNA. Schoor et al. (216) found gene expression profiles obtained from partially degraded RNA samples with still visible ribosomal bands exhibit a high degree of similarity compared to intact samples. They concluded that RNA samples of suboptimal quality might therefore still lead to meaningful results if used carefully. Moreover, Auer et al. (217) recently concluded that degradation does not preclude microarray analysis if comparison is done using samples of comparable RNA integrity. Imbeaud et al. (211) reported collection of reasonable microarray data and meaningful results from RNA samples of impaired quality. Schroder et al. (218) observed RIN shows a strong correlation to the expression value of house keep genes, while the ribosomal ratio (28S:18S) exhibited weak correlation to the expression value of the housekeeping genes. 70 In this study, RNA concentration and purity were evaluated by UV measurement after resolving on the agarose gel. RNA quality was assessed by visualizing smearing on 28S and 18S bands, DNA contamination, and the relative intensity of bands for 28S and 18S. The gel pictures for RNA samples used in this study are present in Appendix A. Because interpreting an RNA gel image is subjective, remaining four adipose RNA samples from the microarray analysis (totally six RNA samples in the RAC study) were analyzed using Agilent Bioanalyzer 2100 after receiving comments and suggestion. These remaining original RNAs had been stored at -80 o C for 6-8 months when they were analyzed with an Agilent Bioanalyzer 2100. These four adipose RNA samples from different pigs were run in accordance with the manufacturer's instructions (http://www.cbse.ucsc.edu/pdf_library/Bioanalyzer%20protocol071304.pdf). The electropherograms are presented in Fig 4. 71 Fig 3. RNA integrity categories. The figure shows typical representatives of the ten integrity categories. RIN values range from 10 (intact) to 1 (totally degraded). The gradual degradation of rRNA is reflected by a continuous shift towards shorter fragment sizes (218). 72 Fig 4. Electropherograms of microcapillary electrophoresis from four RNA samples. Electropherogram of RNA includes a clearly visible 28S:18S rRNA peak, showing slight degradation with a little elevating of baseline. RNA degradation is progressive: as the area of the 28S rRNA peak decreases, reflecting breakdown, there is first a rise in the baseline between the 18S and 28S rRNA and then a progressive increase in the baseline area below the 18S rRNA that spreads as the 28S rRNA fragments become smaller. 73 Sample 1 (#826) 28S rRNA 18S rRNA 5S rRNA Sample 2 (#779) 28S rRNA 18S rRNA 5S rRNA 74 Sample 3 (#796) 28S rRNA 18S rRNA 5S rRNA Sample 4 (#784) 5S rRNA 18S rRNA 28S rRNA 75 Issues in Choosing Microarry Experimental Design An experiment utilizing a spotted 70mer-oligo microarray is in fact a competitive hybridization between one RNA sample that is labeled with the red-fluorescent dye Cyanine 5 (Cy5) and the other RNA sample that is labeled with the green-fluorescent dye Cyanine 3 (Cy3) to a single spotted oligo probe or vice verse. Hybridization of two dye labeled aminoallyl-cDNA (which represent the mRNA from the preparation on the oligo probes (spotted slides) are inherently comparative. Therefore the pairing of RNA samples (as the labeled cDNAs) for hybridization leads to relative/differential expression data. Each microarray run provides investigators with the relative abundance of two sets of mRNA to each other. When designing a microarray experiment, besides constraints such as the number of slides available, the amount of RNA available and other cost etermine which RNAs are to be on f highly considerations, the most important design issues are to d hybridized together on the same slide, which RNAs are to be labeled with which fluorescent dye, and how many biological replicates are necessary to estimate variati among the biological samples by statistical methods (219). An experimental design should ensure that efficient use is made of the available resources, that obvious inherent experimental biases will be avoided and that the data obtained will provide useful information on treatment-driven differential gene expression. In this study, the primary objective was to identify differentially expressed genes in the adipose tissue upon RAC feeding to pigs for 28 days. Comparison of gene expression o two groups (0ppm Paylean vs. 60ppm Paylean) directly on the same array is the most efficient method. While there is heterogeneity among pigs used here, they were selected and represented an identical genotype. In order to be able to conduct this 76 differential gene expression experiment in a cost effective manner, the RNAs from control group (0ppm Paylean group) were mixed to provide the pooled control RNA. Th RNA from each pig in the 60ppm Paylean group was hybridi e zed with the pooled control n e he aim in ver, other evidence indicates that a RNA in order to get biological replicates (n=3). In essence, replication allows averaging, and averages are less variable than their component terms. Lack of replication greatly restricts the ability to use statistical tests to determine whether a given intensity log ratio value is significantly different from zero (200). In particular, biological replication is essential to estimate the variance of the log ratios across slides. Replication is intimately connected with the statistical extrapolatio from sample to population. Although almost all experiments that use statistical inferenc require biological replication, technical replicates are almost never required when t is to make inferences about populations that are based on sample data, as is the case most microarray studies (200). There is no consensus about the necessary number of biological replicates in microarray analysis. Lee et al. (221) indicates that three replicates are sufficient for robust statistical analysis. Howe minimum of 5 biological cases per group should be analyzed for designs in which two groups of cases are evaluated for differential expression (222-224). Another important design issue is to determine which RNAs are to be labeled with which fluorophore. Most microarray experiments show systematic differences between the red and green intensities because different incorporation efficiencies and quantum efficiencies between dye Cy3 and Cy5 (225). Because labeling dye Cy5 is less efficiently incorporated into nucleotides than Cy3, low expression genes in the Cy5-labeled comparative sample are likely to be incorrectly identified as being down-regulated. 77 Therefore, in the biological replications of miroarray analysis, labeling RNA samples from same treatment group with identical fluorescent dye should be avoided because color bias might persistent and accumulative (226). The dye-labeling bias can be effectively alleviated by two methods. One method is reverse labeling design (dye-swap design). Reverse labeling offers useful protection against the non-lin earity of label . this gs. . For ver, f the other normalization without the need to explicitly model the non-linearity. Reverse labeling increases the experimental cost but does not improve the number of biological replicates Another useful alternative for dye error is performing non-linear normalization which corrects the systematic differences of Cy3 and Cy5, based on the fact that systematic trends by dye labeling are due to the inadequacy of linear normalization (227). In study, besides performing non-linear normalization on all the data, random dye assignments was used for RNA arising from either 0 (pooled) and 60 ppm RAC-fed pi Gene-label interaction is another factor that may influence the accuracy of microarray analysis. Use of two labels may also introduce gene-label interactions example, Cy3-dCTP may be preferentially incorporated into a specific sequence, relative to Cy5-dCTP. Theoretically, some degree of gene-label interaction may exist. Howe this interaction appears to be insignificant in magnitude compared with other sources o variation (228). Issues in Choosing Methods for Microarray Data Normalization After extracting data from image analysis, systematic errors need to be removed before the data are applied to downstream statistical analysis. Any spot with intensity lower than the background plus two standard deviations should be excluded. On hand, the intensity ratios should be log-transformed so that upregulated and 78 downregulated values are on the same scale and comparable (229). Normalizatio data processing tool applied to remove systematic errors by balancing the fluorescence intensities of the two labeling dyes. The dye bias comes from various sources including differences in dye labeling efficiencies, heat and light sensitivities, as well as scanner settings for scanning two channels (230). When microarray analysis initially emerged as a tool for measur n is a ing differences of gene expression on a large scale, three methods are rmalization that developed ed e commonly used to calculate normalization factor including: (i) global no uses all genes on the array (ii) housekeeping genes normalization that uses constantly expressed housekeeping/invariant genes; and (iii) internal controls normalization that uses known amount of exogenous control genes added during hybridization (229). With the development of mciroarray techniques, new normalization methods were to replace above three normalization methods. The shortcoming of the three methods arises from the fact that dye bias may be dependent on spot intensity and spatial location on the array. Furthermore, housekeeping genes are not as consistently expressed has been previously assumed, thus, using housekeeping genes normalization might introduce another potential source of error (231). Dye-swapping experiments are viewed as a plausible solution to reduce the dye bias problem, but may be impractical because of the limited supply of certain precious samples. Global locally weighted scatterplot smoothing (LOWESS) has become a widely us normalization method after it was first utilized in microarray data analysis (232). LOWESS is a non-linear normalization method on the basis of gene intensity and spatial information which experts agree is superior to other methods (229). LOWESS applies a smoothing adjustment to obtain the calibration factor and remove dye bias based on th 79 spot intensity and location (232). Compared with other techniques like housekeeping- based normalization or dye-swap experiments, scatter plot-based normalization is more robust in many types of scenarios where the assumption of constantly expressed gene may break down (226). In this study, all image analysis data were normalized by LOWESS due to the robustness of fit in the presence of a few extreme outliers. An M-A plot was produced to investigate the log-inte s nsity after microarray image was normalized ribution slides by LOWESS. The plots show log 2 of the expression ratio versus average spot intensity. After removing the dye labeling effect, in an ideal M-A plot, the center of the dist of log-ratios should be zero. The log-ratios should be independent of spot intensity, and the fitted line should be parallel to the intensity axis (233). After LOWESS normalization within each microarray slide, normalization between slides may be applied dependent on the dispersion of M-values of all slides study. Boxplots are used for between-slides normalization. Boxplots involve comparing the ranges of the regression-corrected M- values across the slides, and scaling them so that M-values on each slide span symmetrically around zero (234). For example, between-slides normalization of 3 in RAC study is presented in the Fig 5. Notice that there is some variation in the average intensity between hybridizations. There are several factors that can cause this. Perhaps one array got slightly more DNA (the right one), or maybe there are slight variations during the production of the arrays. Maybe there were variations in the laboratory environment (temperature or humidity) during the preparation of these samples that influenced the readings (234). 80 A) Boxplots of the pre-normalization of M-value for three microarray slides B) Boxplots of the post-normalization of M-value for three microarray slides Fig 5. Boxplots of three microarray slides before and after normalization in RAC study 81 Issues in Choosing Method for Statistical Analysis to Identify Significantly Differentially Expressed Genes Since microarry analysis is still developing, scientists working in bioinformatics are trying different statistical methods to analyze microarray data to identify genes whose expression changes across experimental paradigms, such as variants of F-statistics, modified t-test, non-parametric approaches and empirical Bayesian methods (235). A fixed threshold cut off method (i.e. a two-fold increase or decrease) was used to identify differentially expressed genes during early stage of microarry work. However, this method is not efficient statistically, the main reason being that there are numerous systemic and biological variations that occur during a microarray experiment (236). Although some of the systemic variations such as dye bias can be effectively removed by normalization, random biological variations such as sample-to-sample and physiological variations are more difficult to handle (237-238). Because of these underlying variations, merely using a fixed threshold to infer significance might increase the proportion of false positives or false negatives. A better framework of inference of significance includes calculation of a statistic ranking genes according to their possibilities of differential expression (based on replicate array data), and selection of a cut-off value for rejecting the null-hypothesis that the gene is not differentially expressed (239). Setting a cut-off for differential expression is tricky, because one has to balance the false positives (Type I error) and the false negatives (Type II error). Furthermore, performing statistical tests for tens of thousands of genes creates a multiple hypothesis- 82 testing problem. For example, in an experiment with a 10,000-gene array in which the significance level ? is set at 0.05, 10,000?0.05=500 genes would be inferred as significant even though none is differentially expressed (236). Therefore, using a p-value of 0.05 is likely to exaggerate Type I errors. The multiple hypothesis testing problems is conventionally tackled by conservative approaches that control the family-wise error r (FWER). Controlling the FWER limits the probability of making one or more type I errors to less than the -value across the entire experiment, which limits the power to identify significantly differentially expressed genes (240). It is often ac ate biologists to have few false positives if the majority of true positives are chosen, For example, an investigator might specify that it is acceptable for a small proportion o findings (for example, a maximum of 10%) to be wrong. Therefore, it is more practical control the false discovery rate (FDR), the expected proportion of false positives among the number of rejected hypotheses (241). SAM as was used here, a highly preferable new method for microarray data analysis, has been developed to utilize this FDR concept as a tool to assist in determining a cut-off after performing adjusted t-tests (http://www-stat.stanford.edu/tibs/SAM) (242). Overview of Microarray Studies in the Porcine Adipose upon RAC Feeding To the best of my knowledge, this study provides, for the first time with pigs, new insights into gene expression occurring in adipose tissue after 4 week RAC exposure This was performed with the aid of microarray technology. A gene-by-gene approach would be very time consuming and technically challenging in this case because it wo be difficult to obtain a global picture of differential gene expression patterns because there are insufficient individual porcine gene sequence data available to design primers for the many qRT-PCR runs required to tes ceptable for f to . uld t so many genes. In addition, at the present time there are a limited number of appropriate porcine specific cDNA are available for 83 Northern analysis of mRNA abundance. Thus, the decision was made to embark o path using microarray technology which at least theoretically allows for expression analysis of thousands of mRNAs from a given tissue at once, and should provide a n a ental . er e 254 pathways unrelated to the purpose of this research or their functions are comprehensive assessment of multiple gene expression responses to given experim variables. In order to further explore mechanisms associated with RAC supplementation, we conducted a genomic analysis on adipose tissue collected on day 28 of the feeding trial The microarray system returned 8,157 spots suitable for data analysis. Among 8,157 spots, 3,607 spots represented unknown genes. In this dissertation, unknown genes ref to spots whose oligo probe sequences were designed based on EST and whose spotted targets (their transcripts) were never annotated. After SAM analysis, 1,128 transcripts were detected as significantly different in the transcription level. Five hundred sixty-nin transcripts were down regulated, of which 284 out of the 569 transcripts were un- annotated genes. Five hundred fifty-nine transcripts were up regulated, and of those, were non-annotated genes. In this microarray platform, nearly half of spotted targets (transcripts) were not annotated, and a large proportion of known transcripts are either involved in unknown. Based on the objectives of this study, we focused our attention on genes involved in the cAMP pathway, lipid metabolism, glycolysis pathway, TCA cycle and oxidative phosphorylation. The log 2 ratio values of genes of interest were presented in Table 6. In addition, for most up/down regulated top 200 genes, I presented the log 2 ratio value from each microarray analysis (representing each biological replication) in the Appendix I. 84 The expression concentration of PPAR?, a transcription factor associated with regulation of fatty acid oxidation, was higher in the RAC-treated pigs. Expression gene CPTII was up regulated but expression of gene peroxisomal acyl-CoA oxidase was down regulated with RAC treatment (Table 6). The gene for leptin, an adipocyte ?der hormone, had a low expression level in the adipose tissue of RAC-treated pigs (Table 6 In RAC-treated pigs, we observed decreased expression of genes encoding key enzym in lipogenesis such as acetyl-CoA carboxylase (ACC), fatty acid synthase (FAS), stearoyl-CoA desaturase (SCD), glucose transpoter 4 (GLUT4), fatty acid biding protei (FABP), long-chain fatty acid-CoA synthetase, and diacylglycerokinase in the adipose. For genes encoding for ?-oxidation, expression of enoylCoA hydratase and hydroxyacyl- CoA dehydrogenase were down-regulated in RAC feeding pigs, but no difference was observed in k of the ived ). es n etoacyl-CoA thiolase (Table 6). very important for this study such as lipoprotein lipase, aP2 (adipose fatty acid binding This study also found significant decreases in expression of genes participating in electron transport and oxidative phosphorylation in the adipose tissue after RAC treatment. Some of the most notable genes include those that encode pyruvate kinase, isocitrate dehydrogenase, succinate dehydrogenase, NADH dehydrogenease, cytochrome b, and ATPase (Table 6). The expression results for FAS, SCD, ACC, GLUT4 and leptin have been confirmed by independent experiments using Northern-blot in our laboratory (see discussion). Pig has not been a model animal in genetics or genomics analysis, thus, the availability of pig microarray slides is quite restricted. The pig array used in this study is limited by the current state of research in pig genetics. Many genes which are potentially 85 protein), uncoupling protein, PPAR?, protein kinase A, and hormone sensitive lipase ar unfortunately not included in this pig array. e ndria at y or may not be of great physiological importance. PPARs belong to a r. The f PPAR? activating fatty acid ? oxidation ession level of PPAR? Expression of Genes Involved with Fatty Acid Oxidation In rodents and humans, the primary sites of fatty acid ? oxidation are mitocho within skeletal muscle, heart and liver (37), with adipose tissue representing a tissue th does not exhibit extensive fatty acid oxidation or need for ATP synthesis. Hence, the significance of changes in expression of genes involved in ? oxidation, TCA cycle and electron transport pathways needs be further studied from a physiological perspective. These changes ma family of nuclear transcription factors that function in a ligand-dependent manne tissue-specific expression pattern of PPAR isoforms is indication of their functions. In rodents and humans, PPAR? is most abundant in liver although found in kidney, brown adipose tissue and heart (243), and it targets genes involved in fatty acid catabolism (? and ? oxidation pathways). In pigs, the expression of PPAR? appears higher in adipose tissue than in liver (87), and the mechanism o pathways has not been well documented. In this study, the expr (log 2 ratio=1.05) and CPT-II (log 2 ratio=1.17) was significantly increased in RAC-treated pigs, but expression of genes in ?-oxidation were not up-regulated by RAC. PPAR? may act simply as a general sensor of overall tissue bulk fatty acid supply, providing coordinated changes in the capacity for fatty acid oxidation to prevent excessive triacylglycerol accumulation and subsequent development of obesity (72). In a circumstance of chronic stimulation of lipolysis by a catecholamine within a tissue with 86 limited oxidative capacity for fatty acids, the implication of increased PPAR? expressio is not clear. Expression of CPTII (log 2 ratio=1.17) was increased RAC-treated pigs. CPT-II, an enzyme that is associated with inner mitochondrial membrane, catalyzes the transfer of acyl residues from carnitine to CoA to form acyl-CoA thioesters that then enter ? oxidation spiral. Here we have a coordinated expression between PPAR? and CPTII; however, other genes encoding enzymes in the ? oxidation pathway were down regulated in RAC-treated pigs, including short-chain acylCoA dehydrogenase and hydroxyacylCoA n dehydrogenase. Ractopamine binds porcine ?AR and stimulates lipolysis (161, 16 Peterla et al. (244) found lipolysis (release of glycerol and free fatty acids) was st by RAC in porcine adipose tissue explants in vitro. In well-fed pigs, adipose is a energy storage tissue not a major energy mobilizing tissue. From the findings of enhanced lipolysis by BAA in vivo, it is safe to say that arising fatty acids in response to BAA are not 5). imulated major reincorporated into adipose storage TAG or oxidized to produce energy in , AC ) the adipose under chronic RAC influence, but would become available for oxidation in skeletal muscle and liver. The microarray analysis was only conducted on adipose tissue thus there are no data about overall gene expression pattern in other tissues upon R feeding. However, in other studies on RAC-fed pigs, Halsey (162) and Gottschalk (195 presented some preliminary data that RAC increased mRNA abundance of acyl-CoA dehydrogenase (ACDH), CPT1 and lipoprotein lipase (LPL) in porcine longissimus muscle tissue. HSL is the primary enzyme that hydrolyzes triacylglycerol stored in adipose tissue. Agonist stimulation of ?-adrenergic receptors ultimately activates HSL via PKA- 87 dependent phosphorylation (29) and immunoassays have suggested that the activi HSL is primarily regulated by covalent modification via reversible phosphorylation (245). Thus, HSL activity is principally controlled at the post-translational level (246). Because an oligo probe of HSL was not included in the present pig array platform, no information about any putative effect of RAC on gene expression of HSL could be obtained from this study. Peffer et al. (247) found that supplementation of clofibrate, a strong PPAR? expression enhancer, did not result in measurable changes in expression of PPAR? and CPT-1 in the liver of young pig. However, Odle et al. (248) found a 5 fold induction CPT-1 activity ty of of coincident with elevated mRNA abundance for PPAR? upon clofibrate supplementation in young pigs. Thus, work form the same laboratory led to opposite finding in young piglets and the relationship between PPAR? (transcription factor promoting fatty acid oxidation) and CPT-1 (rate limiting enzyme for fatty acid flux into mitochondria) at gene expression level needs be further studied. Peffer et al. (247) also found no changes in rates of hepatic ? oxidation of [1- 14 C]-palmitate and CPT-1 activitie when suckling piglet were treated by isoproterenol (another BAA) for 12 days. Thus, BAA did not activate CPT-1 and ? oxidation in the liver of young pigs. More resear needed to clarify if the mec s ch is hanism of PPAR? regulating ? oxidation exists in porcine adipose tissue and if BAA attenuates fat deposition through regulatory mechanism involving PPAR?. To what degree these dissimilarities in gene expression behavior are related to differences in the age of the pigs and liver versus adipose tissue can not be discerned based on limited literature in the pig study. 88 Expression of Genes Involved in Fatty Acid Synthesis This study showed significant decreases in the expression of genes encoding key enzymes in de novo fatty acid synthesis and triacyglycerol synthesis, such as FAS (log 2 ratio= ?1.16), ACC (log 2 ratio= ?1.28), malate dehydrogenase (log 2 ratio= ?1.04) and fatty acid binding protein (FABP) (log 2 ratio= ?1.45). FAS and ACC are the principal enzymes for de novo fatty acid synthesis, and malate dehydrogenase is involved in NADPH generation for lipogenesis. FABP binds long-chain fatty acids and plays important roles in fatty acid uptake, transport and metabolism. Expression of Stearoyl CoA desaturase SCD (log 2 ratio= ?2.76) was depressed in response to RAC. SCD is the rate-limiting enzyme in the cis-desaturation process of F The oxidative reaction converts saturated FA myristic, A. palmitic and stearic acid into their orresponding delta-9 monounsaturated FA (249). SCD gene expression is activated 50). However, this cAMP lation ts ssion dipose c through cAMP during early preadipocyte differentiation (2 induction of SCD transcripts has only been shown in pre-adipocytes and not in mature adipocytes (250). The induction of SCD expression directly corresponds to accumu of fat droplets. Cyclic AMP response element binding protein (CREB) is the transcription factor often responsible for mediating cAMP induction, but no CREB response elemen have been identified in the SCD promoter (250). In this study, decreased SCD expre by RAC might relate to effect of RAC in attenuating fat deposition in the porcine a tissue, but the mechanism is unknown. Expression of Glucose Transporter 4 (GLUT4) (log 2 ratio= ?0.77) was shown a decreased tendency in response to RAC. The first step of glucose metabolism is the transport of glucose across the plasma membrane of glucose-sensitive tissues by glucose 89 transporters (37). The major isoform of this protein in muscle and adipose tissues is GLUT4. Glucose uptake is an insulin-stimulated process in the adipocyte. Glycolysis the fate of glucose in the adipose tissue. Porcine adipose tissue specifically is the major glucose utilizing tissue and metabolizes 40% of the daily glucose uptake (37). The expression of GLUT4, therefore, can be a direct reflection of lipogenic activity in the pig. Exposure of 3T3-L1 adipocyte to cAMP for 24 h causes a down-regulation of GLUT4, both at mRNA and protein levels, and a decrease in insulin-mediated glucose transport (251). Vinals et al. (252) demonstrated that presence of cAMP analogues repressed GLUT4 protein and mRNA expression is in cultured cells, but the mechanism is not clear from ained s, ), and ATPase (log 2 ratio= ?1.56). Because most of above enzymes are also controlled by allosteric and because of limit information about promoter structure of GLUT4 gene. The down-regulated expression of genes involved in lipogenesis indicated that effect of RAC in modifying pork quality involved controlling expression of genes in lipid metabolism, and this was consistent with my hypothesis for this study. Other studies the Bergen lab using Northern blots, conducted before the present study with the same pig adipose tissue, have showed that expression of genes for FAS, SREBP, GLUT4 and SCD was decreased in response to RAC, indicating that the data for these genes obt from the present oligo array analysis are confirmed (162-163). Decreased expressions of genes encoding enzymes in energy metabolism (glycolysi citric acid cycle and electron transport chain) were observed in the present study in adipose tissue of RAC-treated pigs, including pyruvate kinase (log 2 ratio= ?1.58), isocitrate dehydrogenase (log 2 ratio= ?1.22), succinate dehydrogenase (log 2 ratio= ?1.75), NADH dehydrogenase (log 2 ratio= ?1.36), cytochrome b (log 2 ratio= ?1.31 90 covalent regulation, it is not appropriate to make inference about the activities of rela metabolic pathways based on the transcription response alone. In a tissue not predominantly involved in oxidative metabolism, the physiological implications of these results are unclear. In RAC-treated pigs, I found decreased expression of leptin (log 2 ratio= ?1.5) in the adipose tissue. Thacker (163) also found that RAC lowered leptin expression in the adipose tissue using Northern blotting. Leptin is the product of th ted e ob gene, a protein imary ajority of ncentration of leptin in obese people (260). The connection between es predominately secreted by adipocytes (253). Leptin action is exerted through specific receptors that are highly expressed in many tissues (254). Leptin acts on the brain to control food intake, energy expenditure and endocrine functions (255-256). The pr role of leptin is still unclear; however, it is thought that the protein may serve in the feedback regulation of adipose mass on feed intake. Furthermore, studies in rats have shown that leptin simultaneously induces lipolysis and lipid oxidation (222,257). It has been demonstrated that body fat content correlates with circulating plasma leptin concentration in human (258-259) but Wauters (260) suggested that the vast m human obesity can not be attributed to defects in leptin or its receptors, since they observed elevated co leptin, lipogenesis and energy metabolism is still unclear. Further research is needed to find correlations between RAC, leptin and fat deposition in pigs. Limited Independent Verification of Microarry Analysis Data It is necessary to confirm microarray data using an alternate technology, such as quantitative real-time PCR, Northern blot or in situ hybridization (261). Validation do not necessarily need to be performed for every gene of interest, but should be related to 91 the biological conclusion generated from the data. There is no absolute requirement f the amount of validation that needs to be included, but the more verification has been included, the more reliable the data will be deemed and the more useful any such study will become (262). The microarray data from this study can be confirmed by the previous work in our lab. Previous members of our lab determined the expression of some key genes rela lipid metabolism by Northern blot using same adipose tissue from same pigs. Expression of SREBP-1, FAS, SCD, and GLUT4 was lower (P<0.05) in the adipose tissue when pig were treated by 60ppm ractopamine for 28 days (Fig6). mRNA abun or ted to s dance of leptin in the (237). In this oligo n porcine tissues was also decreased by RAC using Northern blotting array study, the log 2 (60ppm/0ppm) values for SREBP-1, FAS, SCD, GLUT4 and lepti were -0.76, -1.16, -2.76, -0.77 and -1.5 respectively. 92 Fig 6. Effect of feeding 0, 20, 60 ppm of ractopamine on mRNA abundance of FAS, SREBP-1, GLUT4, SCD and housekeeping gene (?-actin) in porcine adipose tissues from crossbreed pig (162-163). mRNA abundances were measured by Northern blotting as described in the thesis of Halsey (162) and Thacker (163). A, SREBP-1; B, FAS; C, GLUT4; D. SCD (day 28). For each gene, blots displayed above the tabulated data in graph from show the gene of interest on the upper and housekeeping gene on the lower portion on day 28 of the study. Lanes of blots are from left to right: 1 and 2, 0ppm; 3 and 4, 20 ppm and 5 and 6, 60ppm ractopamine respectively. For each lane data are from an individual pig. Expression data (normalized using ? actin) for each gene are presented in 93 a graphical format for all sampling times (n=3). Columns not sharing common letter es, n in y to confirm results of and further assays to measure enzyme activities or protein f (within gene/day category) were significantly different (P<0.05). CONCLUSION Feeding RAC to pigs induces changes in the gene expression of adipose tissue. These changes mainly involve increases in the transcription of PPAR? and CPTII gen decreases in fatty acid oxidizing enzymes and decreases in genes encoding enzymes in fatty acid synthesis and electron transport. These data provide an overview of RAC actio on the RNA abundance of genes in the adipose tissue. This research revealed some candidate genes, such as cytosolic phospholipase A2 (log 2 ratio= -1.555), porcine interleukin2 (log 2 ratio=1.899) apolipoprotein precursor (log 2 ratio= - 2.158), that might be useful to elucidate mechanisms underlying the anti-adipogenic effect of RAC in pigs the future research. Other mRNA quantification assays are necessar microarray analysis, concentration of key enzymes are important to determine the physiological changes o pigs in response to RAC supplemention in the diet. 94 Table 5. List of metabolic pathways and genes of interest for this dissertation present on the pig array Genbank/ bl Accession No. Gene Name em fatty acid oxidation and Lipolysis P23786 Carnitine O-palmitoyltransferaseII[Human], partial (34%) NM_213897 long-chain acyl-CoA dehydrogenase [Sus scrofa] NM_213898 short-chain acyl-CoA dehydrogenase [Sus scrofa] NM_001359 mitochondrial 2,4-dienoyl-CoA reductas[human] AY344366 peroxisome proliferator activated receptor alpha [Sus scrofa] NM_213901 Propionyl-CoA carboxylase beta chain precursor [Sus scrofa] AAH08906.1 enoyl Coenzyme A hydratase short chain 1 mitochondrial {Homo sapiens}, partial (50% O02691 3-hydroxyacyl-CoA dehydrogenase type II [Bovine] complete AAB30019.2 peroxisomal acyl-coenzyme A oxidase {Homo sapiens}, partial (52%) AAF12736.1 acyl-Coenzyme A dehydrogenase-8 precursor {Homo sapiens}, partial (54%) EGAD45512 3-hydroxyisobutyryl-coenzyme A hydrolase {Homo sapiens}, partial (49%) AAH12172.1 acetyl-CoA synthetase {Homo sapiens}, partial (33%) Q99424 Acyl-coenzyme A oxidase 2 peroxisomalpartial (15%) AAH11968.1 2 4-dienoyl CoA reductase 2 peroxisomal {Homo sapiens}, partial (58% AAH00286.1 malonyl-CoA decarboxylase {Homo sapiens}, partial (46%) AAF60277.1 carnitine palmitoyltransferase I {Ovis aries}, partial (32%) BAA29057.1 very-long-chain acyl-CoA dehydrogenase {Homo sapiens}, partial (27%) AAH01964.1 acyl-Coenzyme A dehydrogenase family member 8 {Homo sapiens}, partial (42%) AF185048 acyl-CoA oxidase [Sus scrofa] NM_213966 long-chain 3-ketoacyl-CoA thiolase [Sus scrofa] NM_214315 hormone-sensitivelipaseHSL[Susscrofa] CAB76256.1 enoyl coA/acyl coA hydratase/dehydrogenase complete Fatty acid synthesis and triacylglycerate synthesis Z97186 stearoyl-CoA desaturase [Sus scrofa] diacylglycerol acyltrans NM_001004046 liver fatty acid binding protein [Sus scrofa] AY700218 CCAAT/enhancer binding protein alpha [Sus scrofa] U97256 cytosolic glycerol-3-phosphate dehydrogenase [Sus scrofa] NM_214060 esterase D [Sus scrofa] X98558 heart fatty acid-binding protein [Sus scrofa] AJ416020 us scrofa] partial mRNA for adipocyte fatty acid-binding protein [S X94251 glyceraldehyde-3-phosphate dehydrogenase [Sus scrofa] Q06055 ATP synthase lipid-binding protein mitochondrial precurso complete{Homo sapiens} r, NM_214051 ferase [Sus scrofa] 95 AAH01918.1 acetyl-Coenzyme A acyltransferase 2 {Homo sapiens}, partial (20%) P33121 HUMAN Long-chain-fatty-acid--CoA ligase 2 partial (24%) BOVIN BAA00401.1 thiolase precursor {Rattus sp.}, partial (40%) mitochondrial acetoacetyl-CoA P36956 HUMAN Sterol regulatory element binding protein-1 (SREBP-1), partial (21%) G01880 fatty-acid synthase (EC 2.3.1.85) (version 2) - human, partial (7% BAA95446.1 acetyl-CoA transporter {Rattus norvegicus}, partial (41%) AAC50478.1 diacylglycerol kinase zeta {Homo sapiens}, partial (28%) P48201 HUMAN ATP synthase lipid-binding protein mitochondrial precursor, complete EGAD 125291 ATP lipid-binding protein P1 precursor {Sus scrofa}, complete AAH00618.1 elongation of very long chain fatty acids {Homo sapiens}, complete O95573 HUMAN Long-chain-fatty-acid--CoA ligase 3 BAA86054.1 fatty acid coenzyme A li AAA41145.1 ial (6%) fatty acid synthase {Rattus norvegicus}, part BAB47242 ) CREB/ATF family transcription factor {Homo sapiens}, partial (32% Q9TTS3 BOVIN Acetyl-CoA carboxylase 1partial (7%) AAK01477.1 C/EBP-induced protein {Homo sapiens}, partial (25%) adipocyte determination an AF175308 acetyl-CoA carboxylase [Sus scrofa] AF103945 CCAAT/enhancer binding protein beta [Sus scrofa] AF252267 acetyl-CoA carboxylase alpha [Sus scrofa] BAA20097.1 CCAAT/enhancer-binding delta protein {Bos taurus}, partial (54%) L06944 succinyl-CoA synthetase beta-subunit J03489 pyruvate dehydrogenase (lipoamide) [Sus scrofa domestica] A29170 phosphopyruvate hydratase alpha - human, complete AJ251197 pyruvate kinase [Sus scrofa] AF217652 glucose-6-phosphatase catalytic subunit [Sus scrofa] Q16654 HUMAN [Pyruvate dehydrogenase [lipoamide]] kinase isozyme 4 mitochondrial precursor, partial (18%) AF008589 succinyl-CoA synthetase alpha subunit [Sus scrofa] X17058 glucose transport protein [Sus scrofa] M21197 citrate synthase precursor M86719 NADPH-specific isocitrate dehydrogenase NM_213980 UDP glucose pyrophosphorylase [Sus scrofa] M16427 malate dehydrogenase precursor AF061966 ATP-specific succinyl-CoA synthetase beta subunit [Sus scrofa] AJ300475 succinate dehydrogenase {Sus scrofa}, complete P00883 RABIT Fructose-bisphosphate aldolase A [Rabbit], partial (24%) P70404 MOUSE Isocitrate dehydrogenase [NAD] subunit gamma mitochondrial P55052 Fatty acid-binding protein epidermal (E-FABP), complete gase 5 {Homo sapiens}, partial (14%) AAK84175.1 diacylglycerol acyltransferase 2 {Mus musculus}, partial (49%) AY496867 d differentiation-dependent factor 1 [Sus scrofa] Glycolytic/glucogenesis pathway A29170 phosphopyruvate hydratase alpha - human, complete 96 precursor, complete P12382 MOUSE 6-phosphofructokinase liver type, partial (33%) AAH01454.1 o sapiens}, phosphoenolpyruvate carboxykinase 2 (mitochondrial) {Hom partial (23%) AF054835 glucose transporter type 2; GLUT-2 [Sus scrofa] AF141956 GLUT4 [Sus scrofa] typealdolase).[Rabbit].partial(24%) glucokinase regulator - human, partial ( AAB59563.1 0%) glucokinase {Homo sapiens}, partial (4 AJ557236 crofa}, complete pyruvate kinase M2 {Sus s AAH05811.1 nase isoenzyme 2 {Homo sapiens}, partial (53%) pyruvate dehydrogenase ki Cholesterol/sterol metabolism CV876047 ase/delta-5-delta-4 isomerase [Sus scrofa] 3-beta-hydroxysteroid dehydrogen U84399 steroidogenic factor-1 SF-1 [Sus scrofa] NM_213911 steroid membrane binding protein [Sus scrofa] AF414124 11-beta hydroxysteroid dehydrogenase isoform 1 [Sus scrofa] CAB41234.1 piens}, (sterol regulatory element binding transcription factor 2) {Homo sa partial (18%) sterol/retinol dehydro AAH10570.1 3-hydroxymethyl-3-methylglutaryl-Coenzyme A lyase (hydroxymethylglutaricaciduria) {Homo sapiens}, complete Q15738 HUMAN NAD(P)-dependent steroid dehydrogenase, partial (53%) CAC88111 17beta hydroxysteroid dehydrogenase {Homo sapiens}, partial (23%) DQ020476 sterol regulatory element binding protein-2 [Sus scrofa] S79678 3-hydroxy-3-methylglutaryl coenzyme A reductase/HM [Sus scrofa] G-CoA reductase NM_213988 steroid 5-alpha-reductase 2 [Sus scrofa] Q15800 C-4 methyl sterol oxidase. [Human] {Homo sapiens}, partial (43%) NM_214306 17beta-estradiol dehydrogenase [Sus scrofa] AAH00054.1 7-dehydrocholesterol reductase {Homo sapiens}, complete A42912 3alpha(or 20beta)-hydroxysteroid dehydrogenase - pig, complete Lipid transport, Lipoprotein/apolipoprotein M22646 apolipoprotein B NM_001002801 apolipoprotein C-III AJ222966 apolipoprotein A-IV [Sus scrofa] AF467889 high density lipoprotein receptor SR-BI [Sus scrofa] NM_214308 apolipoprotein-E [Sus scrofa] apolipoprotein AF118147 low density lipoprotein receptor [Sus scrofa] ABC-transporter {Gorilla gorilla}, partial (54%) S71363 probable ATP-binding cassette transporter ABC-3 - human, partial (10%) P00883 Fructose-bisphosphatealdolaseA(EC4.1.2.13)(Muscle- S52485 26%) A54113 pyruvate kinase - rabbit, partial (13%) AAC39922.1 genase {Homo sapiens}, complete X59414 A-I [Sus scrofa] AAB36587.1 97 AAH14305.1 iens}, Similar to high density lipoprotein binding protein (vigilin) {Homo sap partial (23%) P18656 Apolipoprotein A-II precursor (Apo-AII). [Crab eating macaque Cynomolgus monkey], complete AAD15748.1 ATP-binding cassette protein M-ABC1 {Homo sapiens}, partial (31%) AF074421 putative ABC-transporter [Sus scrofa] Q07954 HUMANLow-densitylipoproteinreceptor- relatedprotein1precursor(LRP).partial(5%) AAH14305.1 Similartohighdensitylipoproteinbindingprotein(vigilin){Homosapiens}.partia l(23%) JE0272 lowdensitylipoproteinreceptor-relatedprotein6-human.partial(15%) Amino acid/protein metabolism O15371 Eukaryotic translation initiation factor 3 subunit 7 (eIF-3 zeta){Homo sapiens}, partial (72%) Q07205 IF5_RAT Eukaryotic translation initiation factor 5 (eIF-5). {Rattus norvegicus}, partial (32%) P29562 Eukaryotic initiation factor 4A-I (eIF4A-I) (Fragment). [Rabbit] {Oryctolagus cuniculus}, partial (63%) AAG34759.1 amino acid transporter SLC3A1 {Canis familiaris}, partial (20%) P23588 HUMAN Eukaryotic translation initiation factor 4B (eIF-4B). {Homo sapiens}, partial (27%) RAT Hydroxy synthase), partial (28%) PIG Glycine amidinotransferase NM_001005208 albumin [Sus scrofa] NM_214048 arginase I [Sus scrofa] X86791 beta-globin [Sus scrofa] AY013261 glycoprotein [Sus scrofa] ribosom NM_214066 D-amino acid oxidase (EC 1.4.3.3) NM_213927 cytosolic aspartate aminotransferase NM_001001638 0S ribosomal protein L35 [Sus scrofa] 6 NM_214363 40S ribosomal protein S12 [Sus scrofa] D13308 glycine N-methyltransferase [Sus scrofa] non-muscle myosin light ch AF239165 serine hydroxymethyltransferase [Sus scrofa] acylamino acid-releasing enzyme [Sus sc D89497 smooth muscle myosin light chain kinase [Sus scrofa] Z84093 L-aromatic amino acid decarboxylase, pkDDC [swine, kidney] NM_213896 aminoacylase-I [Sus scrofa] probable translation initia partial (61% P04720 HUMAN Elongation factor 1-alpha 1 (EF-1-alpha-1) P17425 methylglutaryl-CoA synthase cytoplasmic (HMG-CoA P50441 AJ429141 al protein S4 [Sus scrofa] AF044259 ain [Sus scrofa] D00524 rofa] T08757 tion factor eIF-2B delta chain - human (fragment), 98 P23396 HUMAN 40S ribosomal protein S3 A39760 ribosomal protein S16 cytosolic [validated] - human, partial (65%) JC2369 ribosomal protein L15 cytosolic [validated] - rat, complete AAG15419.1 tor 3 subunit p42/p44 {Homo sapiens}, eukaryotic translation initiation fac complete AAC84044.1 translation initiation factor eIF3 p40 subunit; eIF3p40 {Homo sapiens}, partial (44%) Q16576 HUMAN Histone acetyltransferase type B subunit 2, partial (19%) P25112 40S ribosomal protein S28. {Rattus norvegicus}, complete AAL31549.1 ns}, complete glutathione transferase T1-1 {Homo sapie AAK20884.1 {Cricetulus longicaudatus}, arginine N-methyltransferase p82 isoform partial (26%) O15372 Eukaryotic translation initiation factor 3 subunit 3 (EIF-3 gamma). [Human], partial (41%) S49172 translation initiation factor eIF-4 gamma - human (fragment), partial (14%) Q28690 Translation initiation factor eIF-2B beta subunit (eIF-2B GDP-G exchange factor). [Rabbit], partial (50%) TP P34897 Serine hydroxymethyltransferase mitochondrial precursor (Serine methylase), partial (31%) eukaryotic tr (33%) GP 5107727 Eif1 Nmr 29 Structures, partial (89%) Human Translation Initiation Factor S18294 translation elongation factor eEF-2 - human, partial (19%) P56537 Eukaryotic translation initiation factor 6 (eIF-6) (B4 integrin interactor), complete translation AAC28633.1 putative nuclear protein {Homo sapiens}, partial (60%) eukaryotic tran (89%) AAH16670.1 (56%) Similar to argininosuccinate lyase {Mus musculus}, partial P49410 Elongation factor Tu mitochondrial precursor.{Bos taurus}, partial (66%) AAF21465.1 kidney and liver proline oxidase 1 {Homo sapiens}, partial (47%) translation ini (29%) Eukaryotic tran (33%) AAK11184.1 histone deacetylase 3 {Rattus norvegicus}, partial (50%) glutamate dehydrogenase {Homo sapiens} Q27991 BOVIN Myosin heavy chain nonmuscle type B (Cellular myosin h chain type B), partial (19% eavy ) Q9QZ81 Eukaryotic translation initiation factor 2C 2 (eIF2C 2) (Golgi ER protein 95 kDa), partial (38%) AAB48437.1 arginine N-methyltransferase 2 {Homo sapiens}, partial (29%) B31486 translation initiation factor eIF-5A [validated] - human, complete AAA62667.1 myosin-IC {Homo sapiens}, partial (28%) translation Q9TSZ7 e system Aminomethyltransferase mitochondrial precursor (Glycine cleavag AAB71410.1 anslation initiation factor XeIF-4AIII {Xenopus laevis}, partial S25432 elongation factor eEF-1 beta chain - human, complete AAH05392.1 slation initiation factor 4E-like 3 {Homo sapiens}, partial A53048 tiation factor eIF-2 gamma chain [validated] - human, partial P23588 slation initiation factor 4B (eIF-4B). {Homo sapiens}, partial AAA52524.1 , partial (53%) S18294 elongation factor eEF-2 - human, partial (25%) 99 T protein), partial (29%) L-serin P26443 Glutamate dehydrogenase mitochondrial precursor{Mus musculus}, partial (32%) Q9XSJ7 Collagen alpha 1(I) chain precursor.{Canis familiaris}, partial (5%) S31212 collagen alpha 1(XIV) chain precursor short form - chicken, partial (12%) Collage BAA01185.1 alanine aminotransferase {Rattus norvegicus}, partial (24%) transla EGAD 136325 fa}, complete neutral and basic amino acid transporter protein {Sus scro AF044969 collagen VIII alpha 1 [Sus scrofa] AF041024 alpha-1 type VII collagen [Sus scrofa] AF222917 myosin light chain kinase[Sus scrofa] myosin heavy chain AF437511 carbamoyl-phosphate synthetase 1 [Sus scrofa domestica] EGAD 4603 collagen type VI alpha 1 {Homo sapiens}, partial (7%) Q04857 Collagen alpha 1(VI) chain precursor. [Mouse]partial (13%) AAC31665.1 partial (10%) Myosin heavy chain (MHY11) (5'partial) {Homo sapiens}, M21683 non-histone protein HMG1 glutamine--phenylpyruva AY372187 beta-lactoglobulin [Sus scrofa] S32425 glutathione transferase - human, complete AAD02563.1 mitochondrial branched chain aminotransferase precursor; BCATm {Ovis aries}, partial (21%) Q28640 (Histidine-proline rich glycoprotein) (HPRG) (Fragment)., partial (34%) CAC22253 alanine:glyoxylate aminotransferase 2 homolog 1 splice form 1 {Homo sapiens}, partial (28%) AJ309014 myosin heavy chain [Sus scrofa] U11771 fast myosin heavy chainmyosin heavy chain [Sus scrofa] AAB65435.1 elongation factor 1 alpha {Bos taurus}, partial (24%) A53019 collagen alpha 1(XVIII) chain - human (fragment), partial (17%) P12234 Phosphate carrier protein mitochondrial precursor (PTP). {Bos taurus}, partial (54%) P52943 Cysteine-rich protein 2 (CRP2) {Homo sapiens}, partial (35%) Electron transport and oxidative phosphorylation S27226 NADH dehydrogenase (ubiquinone) (EC 1.6.5.3) 14.5K chain - bovine, complete P34943 BOVIN NADH-ubiquinone oxidoreductase 39 kDa subunit mitochondrial precursor, partial (37%) AY786556 cytochrome c oxidase subunit I [Sus scrofa] AW574405 NADH2 [Sus scrofa] AW574375 NADH3 [Sus scrofa] cytochrome b [Sus scr AW584050 NADH4 [Sus scrofa P24311 Cytochrome c oxidase polypeptide VIIb mitochondrial precursor. [Human] P20132 e dehydratase{Homo sapiens}, partial (37%) O46392 n alpha 2(I) chain precursor.{Canis familiaris}, partial (12%) A26711 tion initiation factor eIF-2 alpha chain - rat, partial (62%) NM_214136 [Sus scrofa] CAA57702.1 te aminotransferase {Homo sapiens}, complete DQ020119 ofa] 100 {Homo sapiens}, complete AAG28221.1 ) ATPase6{Susscrofa}.partial(71% O14521 HUMAN Succinate dehydrogenase [ubiquinone] cytochrome B small subunit mitochondrial precursor (CybS), complete NM_214291 (H++K+)-ATPase ATPasealpha-subunit(aa1-1021)[Susscrofa] AAH06949.1 SimilartoATPaseclassIItype9A{Musmusculus}.partial(19%) P11019 BOVINVacuolarATPsynthasesubunitE.partial(76%) O75185 robablecalcium-transportingATPase{Homosapiens}.partial(40%) P12953 HUMANVacuolarATPsynthasesubunitd.partial(81%) BOVINVacuolarATPsyn cAMP pathway AF319662 G protein-coupled receptor[Sus scrofa] AJ005981 cAMP-regulated phosphoprotein [Sus scrofa] U12148 5'-AMP-activated protein kinase catalytic alpha-2 subunit U95009 cyclic AMP-responsive element binding protein, delta variant [Sus scrofa] Q01518 Adenylyl cyclase-associated protein 1 (CAP 1). [Human] {Homo sapiens}, complete P18848 HUMAN Cyclic-AMP-dependent transcription factor ATF-4 (Activating transcription factor 4), complete cAMP-dependent protein kinase inhibitor gamma fo piens}, complete [Human] {Homo sa P43250 mo sapiens}, partial (30%) G protein-coupled receptor kinase GRK6 {Ho AAC51339.1 ial (41%) CREB-binding protein {Homo sapiens}, part BAB47242 CREB/ATF family transcription factor {Homo sapiens}, partial (32%) P54619 5'-AMP-activated protein kinase gamma-1 subunit (AMPK gamma-1 chain) (AMPKg). [Human], partial (53%) AJ251728 alpha-1A adrenergic receptor [Sus scrofa] NM_214138 protein kinase A anchoring protein B31927 GTP-binding regulatory protein Gs alpha chain (adenylate cyclase- stimulating) splice form 2 -, partial (54%) BAB47242 CREB/ATFfamilytranscriptionfactor{Homosapiens}.partial(32%) Q9Y2D1 HUMANCyclic-AMP-dependenttranscriptionf 5(Activatingtranscriptionfactor5).partial(24%) actorATF- P18848 HUMANCyclic-AMP-dependenttranscriptionfactorATF-4.complete Miscellanous (transcription factor, hormones, phosphatase) AAH01221.1 nuclear receptor binding protein {Homo sapiens}, partial (53%) retinoid X AF102858 insulin receptor [Sus scrofa] insulin receptor precursor [Sus sc NM_213840 leptin[Susscrofa] AF184172S2 Sus scrofa leptin receptor (LEPR) gene, exon 4 and partial cds O08950 RATTranscriptioninitiationfactorIIAgammachain(TFIIAP12subunit).comple X16951 Ca(2+)-transportATPase(class2)[Susscrofa] CAB96823.1 (novelATPase){Homosapiens}.partial(41%) P79251 thasesubunitG1.complete Q9Y2B9 rm (PKI-gamma). AF053922 receptor beta [Sus scrofa domestica] AF128438 rofa] 101 te S12741 transcriptionfactorATF-a-human.partial(35%) 70)[Rat].partial(59%) Q16594 TranscriptioninitiationfactorTFIID31kDasubunit(TAFII- 32).[Human].partial(41%) HUMANTranscriptioninitiationfactorTFIID 54%) T03829 transcriptionfactorTFII-I-human.partial(9%) HUMANTranscriptioninitiationfactorTFIID20/1 P29053 TranscriptioninitiationfactorIIB(TFIIB).[Rat].partial(80%) AAD46767.1 TFIIDsubunitTAFII55{Musmusculus}.partial(20%) Q92759 HUMANTFIIHbasaltranscriptionfactorcomplexp52subunit(Basictranscriptio nfactor52kDasubunit).partial(44%) AAK28025.1 mosapiens}.partial(11%) proteintyrosinephosphataseTD14{Ho S44454 transcriptionfactorBTF2chainp44-human.partial(41%) S10099 transcriptionfactorITF-1-human(fragment).partial(37%) AAH01454.1 phosphoenolpyruvatecarboxykinase2(mitochondrial){Homosap 23%) iens}.partial( AAC24498.1 phospholipaseD2{Homosapiens}.partial(47%) sim CAC69306 phospholipaseA2{Homosapiens}.partial(38%) AAD32135.1 }.partial(18%) cytosolicphospholipaseA2beta;cPLA2beta{Homosapiens Q15172 HUMANSerine/threonineproteinphosphatase2A56kDareg aisoform.partial(59%) ulatorysubunitalph AAL09472.1 groupXIIIsecretedphospholipaseA2{Homosapiens}.partial(93%) AAD30424.1 sapiens}.partial(13%) calcium-independentphospholipaseA2{Homo Q28653 RABITSerine/threonineproteinphosphatase2A56kDaregulatorysubunitdeltais oform.partial(25%) S28173 phosphoproteinphosphatase(EC3.1.3.16)Xcatalyticchain- human.partial(48%) AB016735 proteinphosphatase-1delta[Susscrofa] AAF64456.1 ELKLmotifkinase2shortform{Musmusculus}.partial(65%) P67776 proteinphosphatase2Aalphasubunit M80709 20-beta-hydroxysteroiddehydrogenase Q63801 TranscriptioninitiationfactorTFIID70kDasubunit(TAFII- Q15544 28kDasubunit(TAFII28).partial( Q16514 5kDasubunits.partial(68%) AAD15617.1 ilartoGilamonsterphospholipaseA2{Homosapiens}.partial(81%) AAK14906.1 phospholipaseCbeta-3{Rattusnorvegicus}.partial(28%) 102 Table 6. Statistic an permutations was u nes whose expression was significantly different from zero (log 2 ratio rol). At these analysis parameters, the false iscovery rate (FDR) for the positive genes was 0.05 (5%); the q value (a measure of nce in term ll biological replicates were less an 0.003. Different from normal t-test, FDR instead of q value was used to control mits of significant analysis as described in the Material and Methods. In this table, the enes with bold log 2 ratio values are differently expresses genes. Positive value means igher expression in treatment group (60ppm Paylean); negative value means lower xpression in treatment group. 2 ratio alysis was performed by SAM. One-class response with 1,000 sed to determine ge =log 2 treatment/cont d significa s of the false discovery rate) for a th li g h e Gene log fatty acid oxidation CarnitineO-palmitoyltransferaseIImitochondrialprecursor (CPTII). {Homosapiens}.partial(34%) 1.17323 mitochondrial2.4-dienoyl-CoAreductase{Homosapiens}. -1.27451 Peroxisomeproliferatoractivatedreceptoralpha(PPAR?)[Susscrofa] 1.04572 enoylCoenzymeA hydrataseshortchain1, mitochondria {Homosapiens}.partial(50%) -2.47947 hydroxyacyl-CoAdehydrogenasetypeII(TypeIIHADH).[Bovine] complete -1.73103 peroxisomalacyl-coenzymeAoxidase{Homosapiens}.partial(52%) -1.81188 very-long-chainacyl-CoAdehydrogenase{Homosapiens}.partial(27%) 0.73684 long-chain3-ketoacyl-CoAthiolase[Susscrofa] -0.66613 fatty acid synthesis stearoyl-CoAdesaturase[Susscrofa] -2.7639 Fattyacid-bindingproteinepidermal(E-FABP). -1.45 (version2)-human.partial(7%) 8 esteraseD[Susscrofa] -1.56863 161 fatty-acidsynthase -1.15948 diacylglycerolkinasezeta{Homosapiens}.partial(28%) -1.09199 ATPsynthaselipid-bindingproteinmitochondrialprecursor -1.99459 HumanLong-chainacyl-CoAsynthetase3(LACS3) -1.77748 fattyacidcoenzymeAligase5 {Homosapiens}.partial(14%) .30171 -1 Acetyl-CoAcarboxylase1(ACC-alpha) [Susscrofa] -1.13074 acetyl-CoAcarboxylase[Susscrofa] -1.28647 103 cAMP pathway Gprotein-coupledreceptor3[Susscrofa] 0.47979 cAMP-regulatedphosphoprotein[Susscrofa] -0.46759 5'-AMP-activatedproteinkinasecatalyticalpha-2subunit3 -0.54501 HuamnCyclic-AMP-dependenttranscriptionfactorATF- 4(Activatingtranscriptionfactor4) complete -1.63231 HUMANcAMP-dependentproteinkinase inhibitorgammaform(PKI-gamma).{Homosapiens}complete 0.46832 proteinkinaseAanchoringprotein3 0.81146 proteinphosphatase-1delta[Susscrofa] -1.46678 Serine/threonineproteinphosphatase2A56kDaregulatorysubunitdeltaisoform. {Homosapiens}.partial(25%) -1.6 lipoprotein apolipoproteinC-III3 1.48956 highdensitylipoproteinreceptorSR-BI[Susscrofa]3 -1.1438 weaklysimilartoPIRS71363S71363probableATP- bindingcassettetransporterABC-3-human.partial(10%)3 1.25436 homologuetoGP15679991gbAAH14305.1Similartohighdensitylipoprotein )3 bindingprotein(vigilin){Homosapiens}.partial(23% -1.13628 Carbohydrate metabolism succinyl-CoAsynthetasebeta-subunit3 .9844 -0 pyruvatekinase[Susscrofa] -1.58221 UDPglucosepyrophosphorylase[Susscrofa] -1.00408 malatedehydrogenaseprecursor -1.04048 HUMANSuccinatedehydrogenase[ubiquinone] -1.95208 succinatedehydrogenase{Susscrofa}. -1.75676 MOUSEIsocitratedehydrogenase[NAD] -1.22769 Phosphopyruvatehydratase alpha-human.complete -2.16842 GLUT4 [Sus scrofa] -0.77164 Electron trnsport NADHdehydrogenase(ubiquinone) -1.36497 NADHdehydrogenasesubunit2[Susscrofa] 052 -2.07 NADH3[Susscrofa] .88885 -1 cytochromeb[Susscrofa] -1.3111 NADH4[Susscrofa]NADH5[Susscrofa]NADH6[Susscrofa] -1.58104 54kDavacuolarH(+)-ATPasesubunit[Susscrofa] -1.56453 Ca(2+)-transportATPase(class2)[Susscrofa] -1.58707 Others leptin[Susscrofa]] -1.50565 transmembraneleptinreceptor[Susscrofa] .52401 1 homologuetotranscriptionfactorBTF2chainp44-human.partial(41%) 1.02749 insulinreceptor[Susscrofa] 1.43410 insulinreceptorprecursor[Susscrofa] 0.70099 CREB/ATFfamilytranscriptionfacto -1.10598 104 r{Homosapiens}.partial(32%) similartoGilamonsterphospholipaseA2{Homosapiens}.partial(81%) 1.0616 cytosolicphospholipaseA2beta;cPLA2beta{Homosapiens}.partial(18%) -1.55553 groupXIIIsecretedphospholipaseA2{Homosapiens}.partial(93%) 1.18925 hydroxymethylglutaryl-CoAsynthasecytoplasmic e). {Homosapiens}.partial(28%) -0.23794 (HMG-CoAsynthas 105 IV. OLIGOMER ARRAY ANALYSIS OF TRANSCRIPTION RESPONSES OF PORCINE TISSUES TO A SUDDEN SHIFT FROM LOW FAT DIET TO HIGH FAT DIET INTRODUCTION Contemporary societies across the globe are facing an ever increasing incidence in diabetes and obesity. Changes in nutrition that often accompany the emergence of populations from subsistence nutrition to plentiful available food commodities as well as unintended life-style changes, such as increased reliance on restaurant/fast foods in the developed world, appear to be among the underlying causative factors of massive weight gains observed in many people. Epidemiological observations indicate that in particular excessive consumption of diets rich in energy, with the nutrition being provided primarily by lipids, are a major contributor to the development of obesity (263). Pigs frequently overeats, therefore our lab has sought to utilize late finishing pigs as a model for the molecular adaptation of liver, adipose tissue and skeletal muscle to a diet containing at least 40% fat energy. In the modern US swine industry, finishing diets are principally composed of corn and soybean meal. Hence, excess energy intake is accompanied by de novo lipogenesis (DNL) from the glucose released from dietary starch coupled with active deposition of triacylglycerol in adipose tissues. In the US, humans do not exhibit high DNL since most consume diets in which energy arising from lipids is greater than 40% and long chain 106 acyl CoAs inhibit DNL by allosteric mechanisms. Allee et al. (46) demonstrated in pigs that 10% dietary corn oil and 10% dietary beef tallow were equally effective in depressing lipogenesis in porcine adipose tissue, suggesting that unsaturated and saturated fatty acids were similar in their effects on DNL. Often a much more pronounced effect by PUFA rather than a saturated FA on DNL is noted in laboratory animals (66). Smith (52) found that both fatty acid chain length and extent of de- saturation are determinants for the effects of dietary fat on the DNL in pigs. Recently it was reported that, when rats were fed a high fat diet for one week, unexpectedly fatty acid oxidation was not enhanced but intramyocellular lipid content was elevated (264). Feeding extra fat to pigs in the finishing phase to enhance intramuscular fat deposition has been tried and was only partially successful. This strategy has been seldom used at the commercial level because of potential lowering of feed intake and diet mixing problems during incorporation of fat (265). Recent studies in vertebrates have identified a number of molecules that regulate nutrient signaling and metabolic activity with respect to lipid metabolism. Such molecules include transcription factors that control a battery of genes involved in lipid metabolism, such as ADD1/SREBP1, PPARs, C/EBPs (266, 267) and adipokines such as leptin, which controls fat homeostasis and feeding behavior (268). Pigs differ in lipid biology from rodents and humans in one key aspect in that the primary site of de novo lipogenesis in pigs is adipose tissue while in humans liver is the primary DNL site with adipose as secondary site. Lipid biology in pigs has been studied to limit excessive fat deposition, while enhancing pork quality. In addition, researchers have attempted to adapt pigs as a model for human lipid metabolism (13). 107 Comprehensive understanding of porcine genome function is critical to understanding how dietary nutrients affect complex metabolic processes and fat deposition. However, presently little is known how genomic/molecular regulation of lipid metabolism in pigs is coordinated across liver, skeletal muscle and adipose tissue during the growing and finishing phases of production. Coordinated gene expression responses to a sudden change of lipid metabolism, brought about by changing dietary nutrient composition have not been evaluated. Thus, assessment of the molecular events underlying metabolic adaptation of pigs switched from a typical corn-based high carbohydrate, low-fat diet (LFD) to a tallow-based high-fat diet (HFD) should provide a model for exploring differential gene expression for lipid metabolism in pigs. The USDA group at Baylor University reported differential expression results of a few genes key to fatty acid metabolism in liver, adipose tissue and muscle in young pigs fed a 40% fat energy diet (48). These results showed an increase in mRNA abundance for acyl-CoA oxidase and CPT-1 in muscle and a decline in SREBP-1 in liver. Expression of four other genes studied was not affected. In the present work, a 13,297 gene array was utilized to obtain a much broader/global expression pattern in pigs fed a high fat diet in liver, adipose and skeletal muscle. In this study I propose that feeding a diet of high fat content will modulate transcription of genes involved in nutrient metabolism pathways, especially those involved in lipid and carbohydrate metabolism. Therefore this study was designed to determine the impact of high dietary fat on the transcription response of genes involved in nutrient metabolism in liver, adipose and muscle tissues of finishing pigs using microarray techniques. 108 MATERIALS AND METHODS Animal Feeding Trial Eight adult castrated crossbred male pigs were provided ad libitum access toeither a corn and soybean-based, low-fat diet [LFD] (n=4) or a tallow/corn oil-supplemented high fat diet [HFD] (n=4) for 14 days. For the LFD, 4.3% diet energy was from fat contributed mainly by the corn, while, in the HFD, 40% diet energy was from a supplemental fat source of 4:1 saturated fat (beef tallow) and a PUFA source (corn oil; added to maintain a constant PUFA to SFA ratio for both diets). The composition of the feed offered for the 14 days before slaughter is presented in Table 1. The calculated crude protein concentration in the experimental diet were 20% and 19%, respectively, and both diets met or exceeded all NRC (199) requirements for finishing pigs. The average body weights on slaughter were 105.1? 5.4 kg and 106.1?2.93 kg for LFD and HFD treated pigs respectively, and they were not significantly different in statistics (P-value=0.34). This experiment was approved by the Auburn University Institutional Animal Care and Use Committee (IACUC #0207-R-2448). The pigs were slaughtered at 14 days and liver, subcutaneous adipose and skeletal muscle tissues were collected. Pig identification number, diet treatment for each pig, and the day the samples were collected are presented in the Table 2. 109 Table1. Composition of feed given to the pigs Ingredient Control (%) High Fat (%) Corn 68.05 51.65 Starch 51.04 38.74 Fat source (Tallow/Sat Fat /corn oil/equiv) 0 13.25 3.25 Soybean Meal 29.00 29.00 Premix Di-Calcium Phosphate 1.00 1.00 Limestone, grd 0.80 0.80 Salt 0.35 0.35 Vit & TM 0.2 0.2 Additive/fiber 0.5 0.5 Calculated analysis Kcal/gm 4.1 5.2 Total Protein 20% 19% Polyunsat to saturated fatty acid 0.2 0.2 *Meets all NRC (1998) requirements for finishing pigs *The formulations are presented as feed ingredients (not dry matter corrected). These diets were not formulated to be iso-energetic. Table2. Pig identification number, diet treatment, and the date of sample collection for the pigs. Pig Length of Treatment (days) Fat Supplemented to diet (%) Date of Samples Collection 4901 14 0 (LFD) 11/19/2003 5504 14 0 (LFD) 11/19/2003 5205 14 0 (LFD) 11/19/2003 6002 14 0 (LFD) 11/19/2003 4905 14 16.5 (HFD) 11/19/2003 5207 14 16.5 (HFD) 11/19/2003 5502 14 16.5 (HFD) 11/19/2003 6001 14 16.5 (HFD) 11/19/2003 110 Tissue Collection All the pigs were killed at the Auburn University Meat Laboratory by electrical stunning followed by exsanguinations under USDA/APHIS inspection. Liver, subcutaneous adipose and skeletal muscle tissues were removed immediately, snap- frozen in liquid nitrogen prior to scalding and dehairing of the carcass. Liver samples were removed from the right lobe, adipose tissue samples were removed from the middle layer of subcutaneous adipose depot near 12 th rib, and skeletal muscle samples were removed from the longissimus muscle between the 10 th and the last ribs. This procedure minimized contamination and eliminated product rejection for the further processing of the carcass for human consumption. Experimental Design Microarray analyses were conducted on the liver, adipose and muscle tissues. For each tissue, I used a pooled control RNA preparation isolated from the four LFD pigs and individual RNA preparations from each HFD pig (n=4). Images of gel for all RNA preparations are presented in Appendix A. The labeling dye Cyanine 3 (Cy3) and Cyanine 5 (Cy5) were assigned randomly between control pool RNA and RNA from each HFD-treated pigs such that there were 2 controls pools with Cy3 and 2 HFD pigs with Cy5, and 2 control pools with Cy5 and 2 HFD pigs with Cy3. Our laboratory did not have the resources to conduct a dye swap for each control RNA (LFD) and each of the 4 individual RNAs from the four HFD pig combinations. Microarray Analysis The experimental procedures for isolating RNA, synthesizing cDNA, aminoallyl labeling cDNA, hybridizing labeled cDNA with oligos on the pig array are described in 111 the Chapter III. Images of fluorescent dye Cy5 labeled cDNA are presented in Appendix C. Similar procedures were also performed to scan the slides and conduct image analysis, normalization and statistics. All array images of this experiment are presented in Appendix G. Normalization To remove systematic error in the experimental analysis, as described in the Chapter 2, LOWESS was used to normalize all the slides. M-A plots for all experimental runs before and after LOWESS normalization are presented in Appendix H. Statistical Analysis SAM was employed using the one-class response with 1,000 permutations to determine genes whose expression was significantly different from zero. Differently expressed genes were determined by setting the number of falsely called genes to less than one and choosing similar false discovery percentage medians for each of the biological replicates. In the SAM analysis, a similar false discovery rate (FDR=5%) was chosen for each tissue analysis. Then, as described in the chapter III, all significantly differently expressed genes from the SAM list for each tissue were filtered by the genes of interest list. The differential expression results for the predetermined genes of interest list (filtered results) for liver, adipose and muscle are reported in Tables 3-5 respectively. In addition, for the top 200 genes in each tissue, I presented the log 2 ratio value from each microarray analysis (representing each biological replication) in the Appendix I. 112 RESULTS AND DISCUSSION General Pattern of Gene Expression and Its Changes in Expression of Porcine Tissues Utilizing the Iowa State Porcine Genomic Center oligo pig array, differential expression of 13,297 70 mer spot targets was measured. The oligo probe (sequence) of each spot was designed based on known gene sequences or expressed sequence tags (ESTs). Six thousand, six hundred and fifty (50.01%) of the oligo probes were designed based on ESTs. Of the total 13,297 specific 70mer spots or genes, 52.47%, 48.59% and 47.73% did not achieve any visible hybridization for liver, adipose and muscle tissues respectively. These are called ?absent genes?, and the variation of total absent genes among tissues was only 4.74%. This result shows that essentially the same spotted gene targets hybridized to the prepared dye-aminoallyl cDNA probes across all three tissues. Genes or spots on slides to which the dyed cDNA did hybridized were 47.5% for liver, 51.4% for adipose and 52.3% for muscle. These genes are called ?present genes?. Out of the ?present genes? 71.3% (liver), 44.5% (adipose tissue) and 41.9% (muscle) were not annotated or based on EST in the Qiagen-provided list of gene spots, and these spots are called ?unknown genes?. A summary of the overall performance results of the spotted arrays is presented in Table 6. The percentage of differentially expressed genes (from a total of 13,297 genes) identified by SAM were 7.9% (liver), 6.4% (adipose tissue) and 8.5% (muscle) in liver, adipose and muscle tissue respectively. Such a finding is consistent with the overall theory (182) about absolute expression data using microarray analysis, in that from any given sample or source most of genes on an array are not differently expressed. As can be 113 seen in Table 7, ?unknown genes? accounted for a large proportion in the differentially expressed genes. Approximately 1000 genes were either up or down regulated by the dietary regime. Numeric details on the up/down regulated genes resulting from the diet shift across three tissues are presented in Table 7. 114 Table 6 Number and percentage of detected (present) and undetected (absent) out of 13,297 genes among tissues studied. Liver Adipsoe Muscle Absent genes out of total 6977 (52.5%) 6461 (48.6%) 6347 (47.7%) Present genes out of total 6320 (47.5%) 6836 (51.4%) 6950 (52.3%) Unknown genes out of present 4504 (71.3%) 3039 (44.5%) 2912 (41.9%) Table 7 Number and percentage of differently expressed transcripts by SAM Liver Adipose Muscle Significantly differentially expressed genes (number) and (% out of total) 1055 (7.9%) 847 (6.4%) 1138 (8.6%) Up-regulated (number) 440 (41.7%) 552 (65.2%) 416 (36.6%) Unknown genes out of up-regulated total 339 (77.1%) 293 (53.1%) 84 (20.2%) Down-regulated (number) 615 (58.3%) 295 (34.8%) 722 (63.4%) Unknown genes out of down-regulated total 407 (66.3%) 102 (34.6%) 207 (28.7%) 115 Microarray Study in the Liver Table 3 shows the effect of comparing LFD and HFD on differential transcription of genes in liver. In spite of a less than primary role of liver in porcine fat anabolism, gene expression in liver was affected by HFD. All results are expressed as log 2 ratio. For each transcript, ratio = normalized pixel intensity labeling HFD RNA normalized pixel intensity labeling LFD RNA The HFD when fed to finishing pigs decreased the mRNA abundance of SREBP- 1(log 2 ratio=?1.15) and other genes in involved in triacylglycerol (TAG) synthesis including diacylglycerol acyltransferase (log 2 ratio= ?1.77), and glycerol-3-phosphate dehydrogenase (log 2 ratio= ?2.4). Gene expressions of enzymes in the fatty acid ?- oxidation pathway were depressed by the fat supplement, including long-chain-fatty acid- CoA synthetase (log 2 ratio= ?1.519), long-chain acyl CoA dehydrogenase (log 2 ratio= ? 1.14), and enoylCoA hydratase (log 2 ratio= ?1.57). HFD did not alter mRNA abundance of PPAR?, a transcription factor regulating fatty acid oxidation. However, expression of acyl CoA oxidase (log 2 ratio= ?2.9), an enzyme in peroxisome fatty acid oxidation pathway and target gene of PPAR?, was down-regulated in response to HFD. In the liver of rodents and humans, mechanism of PPAR? activating expression of genes in fatty acid ? oxidation has been well documented (269-271). As described in chapter 2, PPAR? is highly expressed in the liver and not expressed in the adipose tissue of rodents and human, but PPAR? is expressed more abundant in the adipose tissue than the liver in the pig (44). Peffer et al. (247) did not observe a positive relationship of expression of PPAR? and genes in fatty acid ? oxidation in the porcine liver. Similarly, the data in this study did not show a positive relationship between expression of PPAR? and genes in 116 fatty acid ? oxidation in the liver upon HFD. Effects of PPAR? on fatty acid ? oxidation in the pig need more research to be confirmed. Expression of SREBP-1 (log 2 ratio= ?1.2) in the liver was significantly suppressed by high saturated fat in finishing pigs. When young pigs were fed tallow-supplemented high-fat diet or low-fat corn-soybean meal diets for 2 weeks, Ding et al. (48) found, that liver transcript concentration of SREBP-1 mRNA tended to be decreased in high-fat (tallow) fed pigs compared with the low-fat-fed pigs (P=0.06) in the liver. SREBPs belong to the helix-loop-helix family of transcription factors. SREBP-1 regulates the expression of genes in lipid synthesis while SREBP-2 has been shown to control genes important to cholesterol homeostasis (97, 272-273). However, SREBPs themselves are not very potent activators of transcription and require the actions of ancillary proteins to affect transcription of target genes (274). Therefore, reduced expression of lipogenic enzyme genes (see below) by high fat in our study may be related to the low expression of SREBP-1. Results of this study showed lower mRNA abundance of fatty acid synthase (FAS) (log 2 ratio= ?0.91) and ACC (log 2 ratio= ?1.51 and log 2 ratio= ?1.93 for sus scrofa and bovine ACC respectively) in the liver after diets were shifted to HFD in the pigs. FAS and ACC are rate-limiting enzymes for long-term fatty acid biosynthesis. FAS is a multienzyme complex that synthesizes long-chain, saturated fatty acids (primarily palmitic acid) from acetyl-CoA, malonyl-CoA, and NADPH (275). FAS is not always limited to the production of palmitate; it can also construct longer fatty acids like stearate (but independent elongases appear to be more important in mammalian cells) and shorter 117 fatty acids like myristate, or other shorter fatty acids (276). It should be noted that independent elongases appear to be more important in mammals. Yin et al. (277) found that the abundance of FAS mRNA in porcine liver was responsive to hormonal manipulation as shown with recombinant porcine somatotropin. Irrespective of that fact, less than 20% of total body fatty acid synthesis may be attributed to the liver (13). As indicated previously, most of the fat deposited in pigs under production conditions is derived from de novo fatty acid synthesis in the adipose tissue when typical farm diets, which contain a large proportion of the corn; i.e., dietary starch, are used. In contrast, in rodents, adipose tissue de novo fatty acid synthesis accounts for less than 50% of the total carcass fatty acid synthetic capacity (13). The relative contribution of each tissue to total carcass lipogenesis may also vary somewhat with age of the animal and dietary composition. The expression of lipogenic enzymes is dependent on the nutritional status of the animal and the composition of dietary energy (46, 277-278). Diets high in fat have been reported to suppress the expression of genes coding for lipogenic enzymes in the rodents (279, 280). Similarly, expression of lipogenic genes was down-regulated in response to HFD in porcine liver. While Ding et al. (48) observed a lowered SREBP-1 expression, they found no expression differences in the transcript concentration of FAS in the liver and adipose tissues between young pigs fed corn-based, low-fat diet or tallow-based, high-fat diet for 2 weeks. As noted above, in the present study, decreased transcription of FAS and ACC was observed when diet was shifted from LFD to HFD in pigs. These different findings between Ding et al. and my study may be related with the different developmental phase 118 of the pigs. In this study finishing pigs were used and the final average bodyweights were 104.06?5.37 kg and 105.08? 2.90 kg for LFD and HFD groups, respectively, while the pigs used in the experiment by Ding et al. (48) were young pigs with average initial bodyweights of 6.16? 1.01kg. Like FAS, ACC is also a multi-protein subunit enzyme complex. Acetyl-CoA carboxylase catalyzes the first committed reaction in fatty acid synthesis from acetyl- CoA. This enzyme catalyzes the ATP- and biotin-dependent carboxylation of acetyl CoA to malonyl CoA (17). This response is mediated when the intake of dietary carbohydrates exceeds the amount of energy required by the animal. The activity of ACC is controlled by allosteric regulation (long chain fatty acids) and cAMP ?PKA-directed covalent modification by phosphorylation. In either case, ACC activity may be lowered rapidly via allosteric and covalent regulation, and under such circumstances ACC is the rapid- response, rate-limiting enzyme in lipogenesis. In addition, other dietary and pharmacological factors may also regulate ACC via gene expression (17, 37) In this study, expression of liver fatty acid binding protein (L-FABP) was depressed (log 2 ratio= ?1.18) in the HFD animals, while the mRNA abundance of FABP was increased in the adipose and muscle tissue (see Table 4 & 5). Liver fatty acid binding protein is a member of the genetically related cytosolic FABP family (281). The FABPs are a class of soluble proteins that function by facilitating the intracellular diffusion of fatty acids between cellular compartments and/or enzymes. FABPs reversibly bind hydrophobic ligands, including long-chain fatty acids (LCFA), LCFA-CoA, phospholipids, peroxisome proliferators, and other hydrophobic molecules (282, 283). The transcription rate of the L-FABP gene is tightly regulated and induced by LCFA 119 through a peroxisome proliferator-activated receptor (PPAR)-responsive element located in the proximal part of the promoter in the rodent model (283). Wolfrum (284) proposed that L-FABP is the gateway by which hydrophobic compounds influence gene transcription. L-FABP and PPAR-? exhibit a similar ligand- binding spectrum (285). Wolfrum et al. (282) and Tan et al. (286) convincingly demonstrated that L-FABP was able to activate PPAR-? in hepatoma and COS cells, respectively. Furthermore, numerous reports imply that L-FABP has a critical role in LCFA metabolism by modulating availability of substrate and increasing enzymatic capacity through activation of PPAR-? and possibly other transcription factors (284-286). Hung et al. (287) determined the FABP protein abundance and correlated its levels with the extent of LCFA metabolism in the rats and found that L-FABP was important in hepatic LCFA metabolism. Erol et al. (288) confirmed that L-FABP is a cell-intrinsic stimulator of LCFA oxidation in vivo, but they found that L-FABP effects on fatty acid oxidation might vary with physiological condition. These workers showed that both in vivo and in hepatocyte incubations (in vitro), L-FABP is a limiting factor in the production of ?-hydroxybutyrate, the final product of (mainly) hepatic fatty acid oxidation. They concluded that FABPs might be important for the action of cognate PPARs only under conditions of low lipid metabolism. In this dissertation, the mRNA abundances of FABP (log 2 ratio= ?1.18) and genes in fatty acid ?-oxidation were decreased but PPAR? was not changed in the liver in response to HFD in pigs. Because this study only determined transcription responses, the regulatory mechanism of L-FABP on PPAR? and fatty acid ?-oxidation could not be fully described nor could any definite conclusion be reached about the role of 120 triacylglycerol under the experimental conditions at this time. More research is needed to clarify if the L-FABP has a regulatory role on fatty acid oxidation at the transcription level in pig and if similar response exists for protein concentration and enzyme activity upon high influx of NEFA/LCFA in the porcine liver. Fatty acid supply and cellular uptake of fatty acids has been shown to parallel the level of liver fatty acid binding protein (L-FABP) in rats (289). mRNA abundance of FABP in the liver was decreased ( ratio= ?1.18) in response to the high fat diet. L-FABP is not only way for fatty acid to get in liver. Fatty acids can cross plasma membrane with the help of the protein fatty acid translocase (37). As a non-adipose tissue, liver has limited ability to handle extra fatty acids. When lipids overaccumulate in the liver, they may enter deleterious nonoxidative pathways leading to cell injury and death (290). A single in vitro study in endothelial cells and cardiac myocytes has suggested that accumulation of FFAs may result in lysosomal permeabilization (291) Furthermore, lysosomal breakdown with cathepsin B (ctsb) release into the cytosol is a feature of TNF- ? signaling cascades (292). The reason for decreased expression of FABP in porcine liver upon a large influx of LCFA/NEFA from the diet are not clear. The general down regulation of genes in lipid metabolism may relate to the protective reaction of liver in the condition of excess influx of fatty acids, or may relate to a lesser role of porcine livers in lipid metabolism than has been observed in rodents. Clearly, the LFD-to-HFD induced down-regulated expression of FABP and genes in the fatty acid ?-oxidation was unexpected in the liver. It is not correct to make further inference without assays in histology and proteins on liver tissue, and I will not speculate on the underlying mechanism without more data. 121 The first step of glucose metabolism is the transport of glucose across the plasma membrane of glucose-sensitive tissues aided by glucose transporter (293). Glucose transporter (GLUT) facilitates transporting glucose down the concentration gradient; the major isoform of this protein in the liver is GLUT2. Transcription of GLUT2 (log 2 ratio= ?1.9) was lower in the pigs fed HFD. It has been demonstrated that glucose induces GLUT2 expression due to transcription activation of gene GLUT2 (294). Gremlich et al. (295) demonstrated that palmitic acid induced a decrease in GLUT2 mRNA abundances, but it did not induce consistent changes in GLUT2 protein expression. Therefore, the decreased mRNA abundance of GLUT2 in the liver may be related to the high saturated fat content in HFD. Once inside the cell, the glucose is activated by phosphorylation to form glucose-6-phosphate. This metabolite may be further metabolized via glycolysis and/or the pentose phosphate pathway or utilized for glycogen synthesis in the liver and other tissues (17). Transcription of pyruvate kinase (log 2 ratio=0.9934), a regulatory and irreversible enzyme in glycolysis catalyzing formation of pyruvic acid from phosphenolpyruvic acid, was up regulated in the HFD pigs. The activity of pyruve kinase is also controlled by allosteric and covalent regulation. For example, in the liver, activity of pyruvate kinase is inhibited by cAMP-dependent phosphorylation (17). Correspondingly, expression of key enzymes in gluconeogenesis and glycogen synthesis, glucose-6-phosphatase (log 2 ratio= ? 2.94), phosphoenolpyruvatecarboxykinase 2 (log 2 ratio= ?3.7527), and UDP glucose pyrophosphorylase (log 2 ratio= ?1.612), were down-regulated in HFD pigs. Because pyruvate kinase, phosphoenolpyruvatecarboxykinase 2 and glucose 6 phosphatase are 122 also controlled by allosteric and covalent regulation, the real change of glycolysis and glucogenesis in the liver may be better observed by assaying activities of those enzymes. The mRNA abundance of pyruvate dehydrogenase (PDH) (log 2 ratio= ?1.8422) in the liver was decreased in HFD treated pigs (Table 3). Pyruvate dehydrogenase (PDH) catalyzes the production of acetyl-CoA from pyruvate. Pyruvated dehydrogenase is inhibited by high-energy potential and when fatty acids are being oxidized (37). The pyruvate dehydrogenase is primarily regulated by phosphorylation/dephosphorylation (297). Pryuvate dehydrogenase kinase phosphorylates and inactivates pyruvate dehydrogenase. Expression of pryuvate dehydrogenase kinase (log 2 ratio= ?4.2257) was greatly down-regulated upon HFD. PDH kinase is activated by increases in the [ATP]/[ADP], [acetyl-CoA]/[CoA], and [NADH]/[NAD + ] ratios (297). The great decrease of mRNA concentration of pryuvate dehydrogenase kinase might lead to lower expression of the protein. The decreased expression of Pryuvate dehydrogenase kinase might relate with the decreased energy potential connecting with down-regulated expression of genes in fatty acid oxidation, glycolysis and TCA-cycle in the porcine liver in response to the HFD. The regulation mechanism between pyruvate dehydrogenase and pyruvate dehydrogenase kinase happens by phosphorylation at the protein level. Herein, I can not completely explain the extremely low mRNA abundance of pryuvate dehydrogenase kinase and middle low mRNA abundance of pyruvate dehydrogenase in porcine livers upon HFD. Expression of gene succinyl-CoA synthetase (log 2 ratio= ?1.5813) in the liver was decreased in response to HFD diet, but gene expression of two regulatory enzymes in the TCA cycle, NADPH-specific isocitrate dehydrogenase (log 2 ratio= ?0.3766) and succinate 123 dehydrogense (log 2 ratio=0.3003), was not altered upon the HFD in the liver. No differences were observed in the mRNA abundance of genes involved in electron transport and the oxidative phosphorylation process by HFD, such as cytochrome b, cytochrome c oxidase subunit, NADH dehydrogenase, and H (+) -ATPase. Transcriptions of genes in fatty acid oxidation, fat synthesis and glucogenesis were all lowered in the liver after dietary shifting from LFD to HFD. This may infer some type of negative feedback of the liver in response to high levels of LCFA, particular on lipid and energy metabolism enzymes. It must be stressed that actual activities of glucose and lipid metabolism are not clear based on transcription response alone, because mRNA stability and translation, enzyme phosphorylation and enzyme degradation also have important roles in controlling the overall activity of metabolic pathways. In response to HFD treatment pigs, expression of gene HMG-CoA synthase (log 2 ratio=0.0974) was not changed. However, the mRNA abundance of HMG-CoA reductase (log 2 ratio= ?2.1), rate-limiting enzyme in the overall cholesterol biosynthesis pathway was decreased in HFD pigs. A number of genes involved in regulating cellular cholesterol homeostasis are controlled at the level of transcription by nutrients. For example, addition of cholesterol to the diets of mice led to a rapid 5-10 fold decline in the mRNA abundances for HMG-CoA synthase, HMG-CoA reductase, farnesyl diphosphate and the LDL-receptor (124). HMG-CoA reductase expression is also controlled by changes in mRNA translation and stability and protein stability. In addition, enzyme activity is modulated by phosphorylation, making it one of the most highly regulated enzymes. The decreased mRNA abundance of HMG-CoA reductase may relate to the down regulated genes in fatty acid ? oxidation which provide acetyl-CoA, the precursor 124 of cholesterol biosynthesis. However, nearly no literature is available about the effect of dietary fat on the expression of genes involved in cholesterol biosynthesis in the pig. Harris et al. (256) found the cholesterol content of liver was lower in pigs fed a high-fat, high-cholesterol diet than a low-fat, low-cholesterol diet fed pigs (starting at 12 week of age) for 92 days, but no difference was observed in cholesterol content and percentage of fat in the cerebrum, fat, heart, ileum, kidney, and muscle tissues. Harris et al. (256) concluded that the serum cholesterol or dietary fat and cholesterol content did not influence the cholesterol accretion in most tissues of pigs, and liver is a modulator of cholesterol homeostasis in the pig. In this experiment about 440 genes not involved in lipid, carbohydrate and energy metabolism were up-regulated (significant log 2 ratio; see Appendix G). This included endothelin receptor (log 2 ratio=3.59), ligatin (log 2 ratio=2.73), neuritin (log 2 ratio=2.477), calcineurin catalytic subunit (log 2 ratio=2.42), and Rho-related GTP-binding protein (log 2 ratio=2.139) (Appendix G). This would indicate that the dietary shifting from LFD to HFD broadly affected expression of genes in the liver, not restricted in energy metabolism pathways. In this study, I made no further attempt to identify or work with any genes that were filtered out by our pre-experimentally genes of interest list. Finally, it is worth reiterating that I observed changes in gene expression. However, I have tried to focus these results on metabolism and its regulation with the clear reservation that metabolic pathways are not solely regulated at the gene expression level. Obviously, verifying the above gene expression response through determining protein concentration and enzyme activity is crucial to know the metabolic adaptation of liver 125 upon the HFD, specifically those rate-limiting enzymes and regulatory enzymes in the lipid and carbohydrate metabolism pathways. Microarray Studies in the Adipose Tissue Expression results for adipose tissue are presented in Table 4. In this study, after the diet shift from LFD to HFD there were no changes in the mRNA abundances of genes involved in the long-chain fatty acid mobilization including CPTII (log 2 ratio=0.3925), long-chain acyl-CoA dehydrogenase (log 2 ratio=0.5026), acyl- CoA oxidase ((log 2 ratio=0.6129), enoyl-CoA hydratase (log 2 ratio=0.678) and 2,4- dienoyl-CoA reductase (log 2 ratio=0.5058). No change was observed in genes involved in de novo fatty acid synthesis SREBP- 1 (log 2 ratio=0.0242), ACC (log 2 ratio=0.0847) and FAS (log 2 ratio= ?0.4467), although mRNA abundances of pyruvate kinase (log 2 ratio=0.6373) and pyruvate dehydrogenase (log 2 ratio=0.6096) tended to be higher in HFD pigs. My data are in agreement with previous findings reported by Ding et al. (48). In their study, no changes were observed in the transcription level of FAS and aP2 after young pigs were fed a tallow-based high fat diet for 2 weeks. Allee (46) found that, in porcine adipose tissue, fatty acid synthesis and the total activity of lipogenic enzymes were reduced by dietary fat, and that this reduction was dependent on the amount of fat in the diet. Previously published research indicates that feeding high-fat diets to rodents will modulate adipose lipogenesis. Weaning mice onto a high-fat, low-carbohydrate diet prevented the rise in adipose tissue FAS and ACC mRNA associated with weaning onto a typical high-carbohydrate laboratory chow type diet (278). Pape et al. (279) found that resuming feeding of previously unfed rats with a high-fat diet blocked the induction of 126 ACC mRNA in adipose tissue. Clarke et al. (298) found that when carbohydrate intakes were maintained constant among animals fed diets containing fat, the expression of lipogenic enzymes was not suppressed by dietary fats. Jump et al. (101) in a recent review concluded that the inhibition of de novo fatty acid synthesis by dietary fat likely reflects an acute allosteric feedback inhibition mechanism (suppression of acetyl-CoA carboxylase catalytic efficiency) rather than a primary adaptive change in expression of genes coding for lipogenic enzymes (101). In adipose of our pigs, mRNA abundance of genes involved in TAG synthesis were increased including glycerol-3-phosphate dehydrogenase (G-3PDH; log 2 ratio=0.9255), diacylglycerol acyltransferase (DGAT; log 2 ratio=0.8850), and fatty acid-CoA ligase (log 2 ratio=0.7823). The up-regulation of genes in TAG synthesis may be related to the increased expression of FABP. Transcription of FABP (log 2 ratio=1.1244) was up- regulated in the adipose tissue and this may enhance fatty acid uptake by adipose tissues. Working with a diet-induced obesity rodent model using excessive dietary fat intake, Li et al. (299) noted that after a 1-week exposure to a high-fat diet, several adipogenic genes, in particular those involved with TAG synthesis, such as G-3 PDH, DGAT, were upregulated in adipose tissue as noted in my study. Because genetic regulation of adipogenesis is complex, any putative increase in overall adiposity upon HFD would result in a mixture of new adipocytes at various stages of development. Micorarray results in this study reflected an overall multiple-step process and gene expression profile. This scenario may result in complex and sometimes inconsistent gene expression patterns in the process of expanding adipose tissue. 127 In adipose, expression of fructose-bisphosphate aldolase A (log 2 ratio=0.9142) in finishing pigs was increased by HFD while transcription of other genes in glycolytic and TCA pathways tended to be slightly up-regulated by dietary high fat including pyruvate kinase (log 2 ratio=0.6373), pyruvate dehydrogenase (log 2 ratio=0.6096), succinate dehydrogenase (log 2 ratio=0.6685). Such up regulation of genes in glycolysis may be related to increased expression of TAG synthesis genes in the adipose tissue since adipose is a major storage site for TAG (300). If large amounts of LCFA were taken up by adipose tissue upon action of LPL in HFD fed pigs, then significant amounts of glycerol 3- phosphate would be required for esterification. Porcine adipose tissues lack glycerol kinase, an enzyme which phosphorylates endogenous glycerol arising from hydrolysis of stored TAG to produce glycerol 3-phosphate (301). Since the HFD diet was not really lacking glucose precursors (starch), adequate glucose was likely available to support sufficient adipose glycolysis and ultimately generate glycerol 3-phosphate to enable TAG deposition. The results in this study further showed that the diet shift to HFD did not change the transcription of SREBP-2 (log 2 ratio=0.3377) and genes involved or associated with cholesterol biosynthesis. These results are consistent with the finding by Harris et al. (229) as described above and the known function of SREBP-2 in regulation of cholesterol synthesis (65). In addition, porcine adipose may not be an important site for cholesterol synthesis. This study did not observe changes in the transcription of genes of ?-oxidation pathway in the adipose tissue after the diet shift to HFD. No change was observed in the transcription of CPT-II (log 2 ratio=0.3925) and in the genes encoding different types of 128 acyl-CoA dehydrogenase, such as very-long-chain acyl-CoA dehydrogenase (log 2 ratio=0.4309), long-chain acyl-CoA dehydrogenase (log 2 ratio=0.5026) and short- chain acyl-CoA dehydrogenase (log 2 ratio=0.2494). My CPT-II results are consistent with the report by Ding et al. (48) who found no differences in CPT-1 transcript concentration between control and high fat fed pigs. Acyl-CoA dehydrogenase catalyzes the initial step of the mitochondria fatty acid ?-oxidation pathway. Consistent with the expression results for acyl-CoA dehydrogenase, expression of down-stream genes in ?-oxidation was not changed in porcine adipose tissue by HFD, including 2,4-dienoyl-CoA reductase (log 2 ratio=0.5058), enoyl CoA hydratase (log 2 ratio=0.648) and long-chain-3-ketoacyl- CoA thiolase (log 2 ratio=0.2841). CPT-II catalyzes the release of acyl-CoA from acyl- carnitine for ?-oxidation in the mitochondria matrix, and CPT-II is thought to play an important role in the rapid transfer of activated long-chain fatty acids into mitochondrial matrix for ?-oxidation (302). Acyl-CoA oxidase expression was not enhanced (log 2 ratio=0.6129) by feeding HFD. Acyl-CoA oxidase catalyzes the commitment step in peroxisomal lipid oxidation by converting fatty acyl-CoA to 2-trans-enoyl-CoA and H 2 O 2 (303). Fatty acid substrates for the enzyme include very-long-chain, long-chain, and some medium-chain fatty acids. Various AOX isoforms located in the peroxisome metabolize either straight or branched- chain fatty acids (304). Expression of AOX is regulated by PPAR which binds to PPRE in the AOX promoter region. The peroxisomal proliferator-induction of AOX varies between tissues and among species. In our study, mRNA abundance of AOX was not altered by high fat. This is consistent with the report by Ding et al. (48) who also noted 129 no difference in AOX transcript abundance between young pigs fed a corn-based, low-fat diet and tallow-based, high fat diet for 2 weeks. Detection of altered transcription in tissues in response to feeding HFD to pigs may depend on the timing of the transcripts measurements. Modifications of adipose tissue transcripts concentrations by dietary lipids have been demonstrated after feeding high fat experimental diets for various lengths of time. For example, PPAR? and aP2 mRNA concentrations were decreased when rat were fed a high fat vs. low-fat diet for 8 days, but the two transcripts were increased when the same treatment lasted for 30 Days (305). Pigs fed high fat diets for 12 wk have increased adipose tissue PPAR? mRNA concentrations (306). Unfortunately, only a PPAR? but not a PPAR? probe was spotted on the pig array platform used here. In addition all my results are from a single 14 day experimental HFD feeding period. Future work should include longer experimental periods and should include expression of PPAR? and its target gene aP2, which may help understand how supplemental fat modifies porcine adipose lipid metabolism. Triacylglycerols in adipose tissues are continually being hydrolyzed and resynthesized. The glucose concentration within adipose cells is a major factor determining whether fatty acids are released into the blood after TAG hydrolysis. Fatty acids from TAG hydrolysis are released to the plasma as NEFA if glycerol-3-phosphate is scarce because of low availability of glucose (17). As noted above, glucose was unlikely to be limiting factor of TAG synthesis. An increased TAG accumulation also depends on the concentration of exogenous fatty acids (i.e., the higher the concentration of fatty acids, the more the lipid accumulation will likely occur (307). In this study, it is 130 very possible that increased expression of genes involved in TAG synthesis was the result of the high-fat diet coupled with adequate glucose supplies. In porcine adipose, expression of gene GLUT4 (log 2 ratio = 0.5715) was not altered by HFD. This study did not observe significant alterations in the transcription of genes in the TCA cycle in response to HFD, i.e. succinyl-CoA synthetase (log 2 ratio = 0.494), succinate dehydrogenase (log 2 ratio = 0.6685). The main role of TCA cycle is to completely oxidize acetyl-CoA to provide energy. Considering pigs were well fed in the experimental condition and adipose was not the energy-producing tissue in the feeding and rest states, the lack of change in expressions of genes in TCA cycle upon HFD might relate to the lack of change of expression of genes in ? oxidation and peroxisomal oxidations pathways. No differences were observed for the transcription of genes in the electron transport and oxidative phosphorylation in porcine adipose tissue after HFD treatment including NADH dehydrogenase 4 (log 2 ratio=0.5037), NADH dehydrogenase (log 2 ratio=0.5219) and ATPsynthase subunit (log 2 ratio=0.8818 for bovine subunit E and log 2 ratio=0.2563 for human subunit D). This observation was consistent with no observed changes in the transcription of genes in ?-oxidation and TCA cycle in the adipose tissue after abruptly dietary change from LFD to HFD in pigs. In this study mRNA abundance of leptin tended to be increased (log 2 ratio=0.622) when high fat was fed to pigs. Increased expression of leptin in the HFD fed pigs was likely a response to increased deposition of TAG in adipose depots. The adipocytokine leptin is secreted by adipose tissues and released in proportion to the size of body fat stores. Leptin is detected in the blood at concentrations that directly reflect the adiposity 131 of the animal, but more work in pigs will be necessary to delineate all functions and effects of this adipocytokine (245). Concentrations of leptin in serum from obese swine were 306% higher than concentrations in control, leaner pigs (302). During periods of energy excess, high levels of leptin interact with the hypothalamus to suppress appetite as well as increasing energy expenditure by enhancing fatty acid oxidation in liver and other tissues (308). Studies in rats have shown that leptin simultaneously induces lipolysis and lipid oxidation (309). Finally transcription of a few genes that were included in the filtering process including various types of transcription initiation factors, transcription factors and components of signal pathways were found to be up regulated in adipose after pigs were fed a high fat diet for 14 days (Table 4). Microarray Studies in the Longissimus Muscle Tissue Results for the transcriptional profiling of genes involved in lipid and energy metabolism in skeletal muscle (longissimus muscle) are presented in Table 5. In skeletal muscle, no change was observed in mRNA abundance of some ? oxidation enzymes including long-chain acyl-CoA dehydrogenase (log 2 ratio=0.6594), very-long-chain acyl-CoA dehydrogenase (log 2 ratio=0.4263), short chain acyl-CoA dehydrogenase (log 2 ratio= ?0.1393) in response to HFD in pigs. For other genes associated with fatty acid oxidation, expression of 3-hydroxyacyl-CoA dehydrogenase (log 2 ratio= ?0.8938) was decreased and CPTI (log 2 ratio= ?0.7693) tended to be decreased by feeding HFD. For the genes involved in de novo fatty acid synthesis, no change was observed for FAS (log 2 ratio= ?0.3833), SCD (log 2 ratio=0.282), pyruvate dehydrogenase (log 2 ratio=0.1319) and pyruvate kinase (log 2 ratio= ?0.2641) expression in 132 the muscle after the 14 day shift to HFD. Expression of FABP (log 2 ratio=1.2) was increased after high fat treatment. For the genes of TAG synthesis in muscle from HFD pigs, no changes were observed in the mRNA abundance of acetyl-CoA acyltransferase (log 2 ratio=0.4629), diacylglycerol acyltransferase (log 2 ratio= ?0.09) although glycerol-3- phosphate-dehydrogenase expression was lowered (log 2 ratio= ?1.2937). No differences were observed in the mRNA abundance of genes involved in cholesterol and sterol biosynthesis in HFD pigs including SREBP-2, HMG-CoA reductase, 17 beta-estradiol dehydrogenase, and 20 beta-hydroxysteroid dehydrogenase. Similar to results in adipose tissue, expression of leptin (log 2 ratio=0.711) tended to be elevated after pigs were fed HFD in muscle tissue (not a major site of leptin production). For genes in the glycolytic pathway, based on this micro-array analysis, transcription of fructose-bisphosphate aldolase A (log 2 ratio= ?0.8919) was decreased by HFD. The transcription of glucose transport protein (log 2 ratio=0.0623) was not changed in response to the high fat diet. No differences were observed in mRNA abundance of some genes involved in the TCA cycle such as succinyl-CoA synthetase ? (log 2 ratio=0.0739), ? (log 2 ratio=0.2177), and citrate synthase precursor (log 2 ratio=0.2159). In mature human, skeletal muscle is the predominant tissue for whole body energy substrate oxidation (either as glucose or fatty acids), and oxygen consumption accounts up to 90% of whole body oxygen consumption in total exercising muscle (310). During the resting state the energy requirements of muscle may be met with fatty acid oxidation (311). Typically skeletal muscle accounts for the majority of glucose utilization (312) but when people are obese or people or animals consume diets containing significant 133 amounts of fat, adjustments in the pattern of substrate oxidation are necessary that are not always accompanied by desirable outcomes. Thus, glucose oxidation may be compromised in muscle upon excessive fat intake/or fat deposition that results in hyperglycemia and potentially insulin resistance (312). Multiple sites of metabolic control exist within muscle that govern oxidative flux, although recent evidence indicates that substrate availability regulates the transcription of metabolic genes (313). Ellis et al. (314) found that the expression of genes important for fat oxidation tended to increase in both type I (slow twitch, oxidative) and type II (fast twitch, glycolytic) muscles after an HF dietary intervention for 8 weeks in the rats, but the expression of muscle-type carnitine palmitoyltransferase I was not increased in type II muscle. Excess dietary fat has been implicated in the development of obesity and diabetes. In the skeletal muscle, fatty acid and glucose metabolism pathways are cross linked, and the regulatory mechanism of fatty acid and glucose oxidation by dietary nutrients is still unclear (315). Randle et al. (316) introduced the glucose-fatty acid cycle in muscle suggesting that the availability of FFA determines the rates of fat oxidation, and an increased availability of FFA would then lead to an increased fat oxidation over glucose utilization. This glucose?fatty acid cycle was based on results from in vitro experiments on rat heart and diaphragm muscle metabolism. It may not be surprising that in vivo studies have yielded conflicting results regarding the effect of FFA on glucose oxidation. Some studies showed an inhibitory effect of fatty acids on glucose oxidation in rat skeletal muscle (317), while others found no such effect (318-319). 134 In humans there is no direct support for the glucose-fatty acid cycle as proposed by Randle et al. (316) to account for control of glucose metabolism by fatty acids. Changes were not observed in concentrations of either muscle citrate or glucose-6-phosphate when fatty acid concentrations were altered in various experiments (320-322). When glucose uptake is maintained at a constant high rate, a 10-fold increase in FFA concentration had no affect on glucose oxidation (323-324). In my study, the transcription of genes in the TCA cycle and most genes in glycolysis (except decreased mRNA abundance of fructose-bisphosphate aldolase A) were not changed in the muscle when diet was shifted from LFD to HFD. In this study, corn (starch) content was different between LFD and HFD besides the difference of fat content. The starch content in HFD was 38.74% compared with 51.04% in LFD. Glucose metabolites arising from glycolysis, TCA cycle and pentose phosphate pathways in themselves can act as intracellular signals that regulate metabolism by allosteric means or promote/ inhibit transcription of various other genes in liver, adipose, or muscle tissue (297). One of these is the recently described carbohydrate response element binding protein (ChREBP) gene whose expression is regulated by an intermediate in the pentose phosphate pathway (297). I am unable to arrive at any inference about the effect of lower dietary glucose from HFD on expression of genes in glucose and fat metabolism without exact feed intake data between LFD and HFD pigs. The starch contents in LFD and HFD were 51.04% and 38.74% respectively. The feed intake for each pig was not available in this study because pigs were group-fed in an open pen ad libitum. Thus, the difference of carbohydrate intake between LFD and HFD pigs is not exactly known. The animal technician did not notice any changes in behavior 135 or an initial rejection of the HFD, and the body weights of pigs on slaughter were not different between LFD and HFD groups. It was possible that LFD pigs still got enough carbohydrate from the diet. Otherwise, the fat in the muscle would be mobilized to produce energy. However, in this study, expression of glucose transport protein (log 2 ratio=0.0623) and genes in fatty acid ?-oxidation was not changed in response to the diet shifting to HFD. In this study, dietary shifting from LFD to HFD did not increase expression of genes in fat oxidation, but I can not conclude that the unchanged expression of genes in fatty acid oxidation was caused by the constant high rate of glucose uptake from HFD without strict feed intake data for each experimental pig. Some more recent newer observations also do not support the glucose-fatty acid cycle theory on the molecular level in explaining the effect of fatty acids on fat oxidation. Saha et al. (319) and Sidossia et al. (325-326) found that the availability of carbohydrate rather than that of fat determines the rate of fat oxidation. Sidossia et al. (325) observed that muscle long-chain acylcarnitine concentration was decreased after infusion of insulin and glucose into human experimental subjects who were constantly infused with long- chain fatty acids. Cameron-Smith (327) found that messenger RNA abundance of FABP, CPT I, and UCP-3 did not differ significantly when rats were fed either a high fat diet or a high carbohydrate diet. Schrauwen-Hinderling et al. (264) found that intramyocellular lipid content in rat skeletal muscle was increased after switching a normal fat diet to a HF diet for 1 week and accompanied by molecular adaptations that favored fat storage in muscle rather than oxidation. They found acetyl-CoA carboxylase 2 (regulator of malonyl-CoA which down regulates CPT-1 activity and thereby controls muscle ?- oxidation) mRNA concentration tended to be increased while hexokinase II, glucose 136 transporter 4, and hormone-sensitive lipase mRNA were unchanged after the HF diet. In my study a tendency of down-regulation of CPT-1 was observed in the muscle after a diet shift from LFD and HFD. In the future studies, assaying the CPT-1 protein concentration and long-chain acylcarnitine concentration may help to determine whether the slightly lower CPT-1 mRNA abundance is related to the dietary starch concentration between LFD and HFD. Ding et al. (48) found that mRNA abundance of CPT-1 tended to be elevated in the muscle of high fat-fed young pigs (P=0.07). This finding would favor the Randle hypothesis. The differences between data from young pigs versus mature rats may be caused by a different developmental phase of animals in the two studies. My study used adult finishing pigs (initial weight was 104~105kg), while young pigs (initial weight was 6.16? 1.01kg) were used in the experiment by Ding et al. (48). Muscle genes involved in the electron transport and oxidative phosphorylation were down regulated, including NADH-ubiquinone oxidoreductase (log 2 ratio= ?0.8985) and NADH dehydrogenase 4 (log 2 ratio= ?1.8858) when the diet was shifted from LFD to HFD in pigs. Sparks (328) et al. observed high fat diet decreased the transcription of six genes involved in oxidative phosphorylation when mice were given high fat diet for 3 weeks including: NADH dehydrogenase 1? subcomplex 3, NADH dehydrogenase 1? subcomplex 5, NADH dehydrogenase flavoprotein1, NADH dehydrogenase Fe-S protein, succinate dehydroganase complex, and solute carrier family 25. In this study, the decreased transcription of oxidative phosphorylation associated genes may be related to the low mRNA abundance of CPT1 and 3-hydroxyacyl-CoA dehydrogenase in the muscle tissue of HFD pigs. In the high-fat-treated animals, expressions of some translation initiation factors and elongation factors in this array 137 profile were also depressed (Table 5); expression of ribosomal protein S4, ?-globin, and non-histone protein, were slightly increased after high fat treatment. Conversely, for different types of collagen genes (collagen alpha 1, 2, VII alpha 1) expression was decreased. Feeding HFD also resulted in a significantly lowered mRNA abundance of myosin light chain kinase and myosin heavy chain in Sus scrofa. While beyond the limits set for this dissertation work, the results on muscle myofibrillar proteins warrant further investigation to explore any putative mechanism whereby dietary fat may modulate muscle protein synthesis, degradation and net accretion in pigs and other animals. CONCLUSION In the present study, the expressions of a set of metabolic genes were compared between LFD and HFD pigs among liver, adipose and muscle tissues. In the liver tissue, HFD down regulated most genes involved in lipid metabolism; in the adipose tissue, expression of genes in TAG synthesis was increased in response to HFD; in the muscle tissue, HFD did not alter expression of most genes involved in carbohydrate and lipid metabolism. Therefore, gene expression changes by HFD are different among tissues, which may be related to physiological functions of different tissues in the pig body. Besides the genes mentioned above, I also found a group of unknown/unidentified genes and other genes in non energy and lipid associated metabolic pathways that were down/up regulated by feeding HFD to pigs. For example, the expression of a group of kinases or phosphatases was changed in liver, adipose and muscle tissue by the shifting from LFD to HFD. The phosphorylation/dephosphorylation cycle is involved in covalent regulation of activity of enzymes in lipid metabolism (i.e., ACC, HSL, 6-PFK) and other 138 data identified a kinase cascade that may be involved in signaling by glucose to transcriptional machinery (294). Results need to be interpreted with prudence because this study determined transcription responses of genes using oligonucleotide microarray without confirming any results by an alternative method, and microarray analysis is a high throughout but low quality technique by nature. Changes in the abundance of mRNA of a given gene may reflect altered rates of gene transcription, mRNA processing, and (or) mRNA stability (329). The principal determinant of the abundance of mRNA may be dependent on developmental stage or may be tissue-dependent (330). Generally, the immediate biological response of cells to changes in the external milieu is regulated (within seconds or minutes) by modification of enzyme activity. In contrast, the adaptation to more prolonged changes depends on the regulation of gene transcription. How expression differences triggered by HFD will affect any final metabolic adaptations in finishing pigs is unclear. Considering that some enzymes in the lipid and carbohydrate metabolism pathways are also controlled by allosteric and covalent regulation, it would be incorrect to make conclusions about the metabolic changes only based on transcriptional responses of genes in biological organisms. Thus, this work was clearly discovery and exploratory in nature. The consequences of consuming a diet of high saturated fat content on cellular activity may involve regulatory effects on gene expression, protein translation, processing, modification, and secretion. Certainly high PUFA containing diets regulate expression of lipogenic genes and affect SREBP processing in rodent liver (101). In this study, a genes of interest list was used to focus my attention on the genes related to 139 hypothesis of researches in this dissertation. To obtain further biological information from the microarray data, one strategy could be to focus on most changed (down-/up- regulated) 10-20 genes. Alternative gene expression techniques can be used to confirm the expression changes of those genes determined by microarray, and then extend the verification of those changes using protein expression and enzyme activity analysis. However it is clear that target genes must be chosen carefully because of the present absence of extensive information on specific sequences for many genes in the porcine genome. For example developing qRT-PCR assays will require designing of sense and antisense primer sequences and specific assay conditions on a gene by gene basis. In summary, this experiment is unique in that, to our knowledge, it is the first to utilize functional genomic techniques to compare gene expression in three tissues in finishing pigs fed either a typical LFD or a tallow-based HFD. A long-range goal of this research is to better understand mechanisms whereby saturated fatty acids regulate expression of genes in lipid metabolism and fat deposition among the porcine tissues. This type of information will enable researchers to develop strategies to modify pork quality in the animal (swine) production in future translational research efforts. Future Research on the Unknown Genes in the Array Platform In this pig micro-array, there are a total of 13,297 spotted 70 mer probes (genes). 7739 probes were designed based on tentative consensus sequences (TCs), each of them containing at least one 3?expresses sequence tag (EST). Since about 58% of spotted probes of the pig array represent unknown or unidentified genes, not surprisingly, expressions of numerous unknown genes were determined to be significantly increased or decreased in finishing pigs when the diet was shifted from LFD to HFD. Therefore, 140 further research is necessary to explore/mine the biological information of the differently expressed unknown genes. Additional data mining of all the unidentified spotted genes that showed significant changes in mRNA abundance may well result in new and unique insights of the effect of high fat consumption on gene expression in pigs, but such a task is beyond the scope of this study. Here a simple process in searching for biological information of unknown genes is proposed; this process was tested for few differently expressed unknown genes. Researchers will likely choose the most differently expressed genes (i.e. a high log 2 ratio) as candidate genes in the following activity after micro-array analysis. The following process is proposed to obtain relevant information about ?unknown? genes. First, the location/identification number on the array from the Qaigen proprietary index for the 70 mer probes (Qaigen file) can be submitted to the TIGR Gene Index Database (SsGI 5.0) (http://www.tigr.org/tigr-scripts/tgi/T_index.cgi?species=pig). From here the consensus sequence (TCs) representing the unknown gene of interest can be obtained. These TCs may include multiple open reading frames (ORF) and/or correspond to multiple SsGI EST. Because limited annotation is provided by the SsGI EST, the TCs may then be aligned against GenBank by running BLASTn. BLASTn may then result in many hits to many genes in different species for the 70 mer probe sequence spotted on the array. Genes with highest BLAST percent identity score with a given 70 mer will then receive further attention. Although this procedure is not completely reliable to reach a final conclusion about the identity of an unknown gene of interest, the information obtained through the above process may be a starting point for future research. An example of searching tentative biological information of one unknown gene is presented in the Appendix J. Also, this process of identifying ?unknown? differentially 141 expressed porcine genes will likely increase our understanding of genome sequence and function of the domestic pig. 142 Table 3. Differential transcription responses in liver to feeding LFD and HFD to pigs. Statistic analysis results presented for liver tissue obtained by SAM. One-class response with 1,000 permutations was used to determine genes whose expression was significantly different from zero (log 2 ratio =log 2 HFD/LFD). At these analysis parameters, the false discovery rate (FDR) for the positive genes was 0.05 (5%); the q value (a measure of significance in terms of the false discovery rate) for all biological replicates were less than 0.01. Different from normal t-test, FDR instead of q value was used to control limits of significant analysis as described in the Chapter 2. In this table, the genes with bold log 2 ratio values are differently expresses genes. Positive value means higher expression in HFD pigs; negative value means lower expression in HFD group. Gene name Log 2 (ratio) Fatty acid oxidation long-chain acyl-CoA dehydrogenase [Sus scrofa] -1.1419 propionyl-CoA carboxylase B [Sus scrofa] -0.6905 enoyl Coenzyme A hydratase short chain 1 mitochondrial {Homo sapiens}, partial (50%) -1.5755 peroxisomal acyl-coenzyme A oxidase {Homo sapiens}, partial (52%) -1.9594 acyl-Coenzyme A dehydrogenase-8 precursor {Homo sapiens}, partial (54%) -1.5876 Similar to 2 4-dienoyl CoA reductase 2 peroxisomal {Homo sapiens}, partial (58%) -1.7430 very-long-chain acyl-CoA dehydrogenase {Homo sapiens}, partial (27%) -3.5427 acyl-Coenzyme A dehydrogenase family member 8 {Homo sapiens}, partial (42%) -2.1410 acyl-CoA oxidase [Sus scrofa] -2.882 long-chain 3-ketoacyl-CoA thiolase [Sus scrofa] -1.7970 novel enoyl coA/acyl coA hydratase/dehydrogenase type protein (isoform 1), complete -0.8277 Fatty synthesis diacylglycerol acyltransferase [Sus scrofa] -1.7742 liver fatty acid binding protein [Sus scrofa] -1.1816 CCAAT/enhancer binding protein alpha [Sus scrofa] -1.7694 cytosolic glycerol-3-phosphate dehydrogenase [Sus scrofa] -2.3805 esterase D [Sus scrofa] -0.6817 HUMAN ATP synthase lipid-binding protein mitochondrial precursor complete -1.2022 acetyl-Coenzyme A acyltransferase {Homo sapiens}, partial (20%) -1.2472 HUMAN Long-chain-fatty-acid--CoA ligase 2 (LACS 2)., partial (24%) -1.5190 HUMAN Sterol regulatory element binding protein-1 (SREBP-1), partial (21%) -1.1527 143 fatty-acid synthase - human, partial (7%) -0.9071 diacylglycerol kinase zeta {Homo sapiens}, partial (28%) -1.5601 HUMAN ATP synthase lipid-binding protein mitochondrial precursor complete -1.6980 ATP lipid-binding protein P1 precursor {Sus scrofa}, complete -1.3098 elongation of very long chain fatty acids-like 1 {Homo sapiens}, complete -2.0498 glucokinase {Homo sapiens}, partial (40%) -1.4927 HUMAN Long-chain-fatty-acid--CoA ligase 3 (Long-chain acyl-CoA synthetase 3) (LACS 3)., partial (11%) -0.2393 fatty acid coenzyme A ligase 5 {Homo sapiens}, partial (14%) -1.4042 fatty acid synthase {Rattus norvegicus}, partial (6%) -0.1057 BOVIN Acetyl-CoA carboxylase 1 (ACC-alpha) partial (7%) -1.9277 C/EBP-induced protein {Homo sapiens}, partial (25%) 0.43178 adipocyte determination and differentiation-dependent factor 1 [Sus scrofa] -0.3364 acetyl-CoA carboxylase [Sus scrofa] -1.5148 CCAAT/enhancer binding protein beta [Sus scrofa] -0.9312 CCAAT/enhancer-binding delta protein {Bos taurus}, partial (54%) -1.9884 Carbohydrate metabolism Fructose-bisphosphate aldolase A [Rabbit], partial (24%) -1.3850 phosphoenolpyruvate carboxykinase 2 {Homo sapiens}, partial (23%) -3.7527 succinyl-CoA synthetase beta-subunit -1.5813 citrate synthase precursor -1.1381 NADPH-specific isocitrate dehydrogenase -0.3766 phosphopyruvate hydratase alpha - human, complete -0.427 pyruvate kinase - rabbit, partial (13%) 0.99343 pyruvate dehydrogenase (lipoamide) [Sus scrofa domestica] -1.8422 glucose-6-phosphatase catalytic subunit [Sus scrofa] -2.9442 UDP glucose pyrophosphorylase [Sus scrofa] -1.6120 ATP-specific succinyl-CoA synthetase beta subunit [Sus scrofa] -1.4984 succinate dehydrogenase {Sus scrofa}, complete 0.3003 Pyruvate ddehydrogenase kinase -4.2257 HMG-CoA lyase -1.557 MOUSE 6-phosphofructokinase liver type (Phosphofructokinase 1) partial (33%) -2.0062 fructokinase {Homo sapiens}, partial (38%) -2.0355 glucose transporter type 2; GLUT-2 [Sus scrofa] -1.8929 Cholesterol metabolism HUMAN C-4 methyl sterol oxidase [Human] partial (43%) -1.3338 steroid membrane binding protein [Sus scrofa] -1.5844 11-beta hydroxysteroid dehydrogenase isoform 1 [Sus scrofa] -2.4192 3-beta-hydroxysteroid dehydrogenase/delta-5-delta-4 isomerase [Sus scrofa] -1.4822 sterol/retinol dehydrogenase {Homo sapiens}, complete -1.0429 3-hydroxymethyl-3-methylglutaryl-Coenzyme A lyase {Homo sapiens}, complete -1.2573 17beta hydroxysteroid dehydrogenase {Homo sapiens}, partial (23%) -1.7631 3-hydroxy-3-methylglutaryl coenzyme A reductase/HMG-CoA reductase [Sus scrofa] -2.1300 steroid 5-alpha-reductase 2 [Sus scrofa] -0.8079 17beta-estradiol dehydrogenase [Sus scrofa] -2.0963 144 7-dehydrocholesterol reductase {Homo sapiens}, complete -3.7507 (or 20beta)-hydroxysteroid dehydrogenase - pig, complete -2.1625 high density lipoprotein receptor SR-BI [Sus scrofa] 0.4785 Similar to high density lipoprotein binding protein {Homo sapiens}, partial (23%) -2.4586 apolipoprotein C-III -2.1489 apolipoprotein E -1.3042 apolipoprotein A-I -1.8754 probable ATP-binding cassette transporter ABC-3 - human, partial (6%) 0.5269 Apolipoprotein A-II precursor (Apo-AII). [Cynomolgus monkey], complete -0.4210 ATP-binding cassette protein M-ABC1 {Homo sapiens}, partial (31%) 0.8241 putative ABC-transporter [Sus scrofa] -0.0700 Electron transport and ATP production NADH dehydrogenase (ubiquinone) 14.5K chain - bovine, complete -0.5583 cytochrome c oxidase subunit I [Sus scrofa] 0.4094 cytochrome b [Sus scrofa] -0.1405 NADH4 [Sus scrofa]NADH5 [Sus scrofa]NADH6 [Sus scrofa] 1.21217 54kDavacuolarH(+)-ATPasesubunit[Susscrofa] -0.1581 sarcoendoplasmicreticulumcalciumATPase[Susscrofa] 2.14926 HUMAN Cytochrome c oxidase polypeptide VIIb mitochondrial precursor [Human] complete 1.19173 Others HUMAN Adenylyl cyclase-associated protein 1 (CAP 1){Homo sapiens}, complete -1.4561 cAMP-dependent protein kinase inhibitor gamma form (PKI-gamma). {Homo sapiens}, complete 1.4151 HUMAN G protein-coupled receptor kinase GRK6 {Homo sapiens}, partial (30%) -1.7287 CREB-binding protein {Homo sapiens}, partial (41%) 0.1304 insulin receptor [Sus scrofa] -0.2312 alpha-1A adrenergic receptor [Sus scrofa] -1.7306 retinoid X receptor beta [Sus scrofa domestica] 0.6841 proteinphosphatase2Aalphasubunit -2.0742 |highdensitylipoproteinreceptorSR-BI[Susscrofa] 0.4785 RABITSerine/threonineproteinphosphatase2A.partial(25%) -1.4554 proteinphosphatase-1delta[Susscrofa] -1.7588 145 Table 4. Differential transcription responses in adipose tissue to feeding LFD and HFD to pigs. Statistic analysis results for adipose tissue were obtained by SAM. One-class response with 1,000 permutations was used to determine genes whose expression was significantly different from zero (log 2 ratio =log 2 HFD/LFD). At these analysis parameters, the false discovery rate (FDR) for the positive genes was 0.05 (5%); the q value (a measure of significance in terms of the false discovery rate) for all biological replicates were less than 0.01. Different from normal t-test, FDR instead of q value was used to control limits of significant analysis as described in the Chapter 2. In this table, the genes with bold log 2 ratio values are differently expresses genes. Positive value means higher expression in HFD pigs; negative value means lower expression in HFD group. Gene Name Log 2 (ratio) Fatty acid oxiaiton Carnitine O-palmitoyltransferase II mitochondrial precursor (CPT II). [Human], partial (34%) 0.3925 long-chain acyl-CoA dehydrogenase [Sus scrofa] 0.5026 short-chain acyl-CoA dehydrogenase [Sus scrofa] 0.2494 mitochondrial 2,4-dienoyl-CoA reductase [Sus scrofa] 0.5058 propionyl-CoA carboxylase B [Sus scrofa] 0.4181 enoyl Coenzyme A hydratase short chain 1 mitochondrial {Homo sapiens}, partial (50%) 0.6780 3-hydroxyacyl-CoA dehydrogenase type II {Bos taurus}, complete 0.0518 peroxisomal acyl-coenzyme A oxidase {Homo sapiens}, partial (52%) 0.5765 acyl-Coenzyme A dehydrogenase-8 precursor {Homo sapiens}, partial (54%) 0.6945 Similar to 2 4-dienoyl CoA reductase 2 peroxisomal {Homo sapiens}, partial (58%) 0.2744 malonyl-CoA decarboxylase {Homo sapiens}, partial (46%) -0.4141 very-long-chain acyl-CoA dehydrogenase {Homo sapiens}, partial (27%) 0.4309 acyl-Coenzyme A dehydrogenase family member 8 {Homo sapiens}, partial (42%) 0.3091 acyl-CoA oxidase [Sus scrofa] 0.6129 long-chain 3-ketoacyl-CoA thiolase [Sus scrofa] 0.2841 novel enoyl coA/acyl coA hydratase/dehydrogenase type protein (isoform 1), complete 0.3607 Fatty acid and TAG synthesis stearyl-CoA desaturase [Sus scrofa] 0.4286 diacylglycerol acyltransferase [Sus scrofa] 0.0450 146 cytosolic glycerol-3-phosphate dehydrogenase [Sus scrofa] 0.9255 esterase D [Sus scrofa] 0.0565 fatty acid-binding protein [Sus scrofa] 1.1244 HUMAN ATP synthase lipid-binding protein mitochondrial precursor complete 0.2422 acetyl-Coenzyme A acyltransferase 2 {Homo sapiens}, partial (20%) 0.1775 HUMAN Long-chain-fatty-acid--CoA ligase 2 partial (24%) 0.7823 pyruvate kinase M2 {Sus scrofa}, complete 0.6373 mitochondrial acetoacetyl-CoA thiolase precursor {Rattus sp.}, partial (40%) 0.5635 HUMAN Sterol regulatory element binding protein-1 (SREBP-1), partial (21%) 0.0242 fatty-acid synthase - human, partial (7%) -0.4467 HUMAN ATP synthase lipid-binding protein mitochondrial precursor complete 0.9169 ATP lipid-binding protein P1 precursor {Sus scrofa}, complete 0.8710 elongation of very long chain fatty acids -like 1 {Homo sapiens}, complete 0.5452 HUMAN Long-chain-fatty-acid--CoA ligase partial (11%) 0.3355 diacylglycerol acyltransferase 2 {Mus musculus}, partial (49%) 0.8850 _BOVIN Acetyl-CoA carboxylase (ACC-alpha partial (7%) 0.0847 C/EBP-induced protein {Homo sapiens}, partial (25%) -0.3895 CCAAT/enhancer binding protein beta [Sus scrofa] 1.3706 succinyl-CoA synthetase alpha subunit [Sus scrofa] 0.1135 CCAAT/enhancer-binding delta protein {Bos taurus}, partial (54%) 0.1274 Arabidopsis thaliana lipid transfer protein 6 0.4245 Carbohydrate metabolism succinyl-CoA synthetase beta-subunit 0.0985 glucose transport protein [Sus scrofa] 0.2785 citrate synthase precursor 0.8103 UDP glucose pyrophosphorylase [Sus scrofa] 0.6706 ATP-specific succinyl-CoA synthetase beta subunit [Sus scrofa] 0.4940 phosphopyruvate hydratase alpha - human, complete 0.6518 Pyruvate kinase M2 isozyme [Rabbit] {Oryctolagus cuniculus}, partial (26%) 0.4117 pyruvate dehydrogenase (lipoamide) [Sus scrofa domestica] 0.6096 HUMAN Succinate dehydrogenase [ubiquinone] complete 0.6024 succinate dehydrogenase {Sus scrofa}, complete 0.6685 Fructose-bisphosphate aldolase A (Muscle-type aldolase). [Rabbit], partial (24%) 0.9142 MOUSE 6-phosphofructokinase liver type (Phosphofructokinase 1) partial (33%) 0.1100 GLUT4 [Sus scrofa] 0.5715 Cholesterol metabolism HUMAN C-4 methyl sterol oxidase [Human] partial (43%) 0.2321 steroidogenic factor-1 SF-1 [Sus scrofa] -0.0602 11-beta hydroxysteroid dehydrogenase isoform 1 [Sus scrofa] -0.0102 sterol regulatory element binding transcription factor 2){Homo sapiens}, partial (18%) 0.3377 sterol/retinol dehydrogenase {Homo sapiens}, complete 0.4294 3-hydroxymethyl-3-methylglutaryl-Coenzyme A lyase {Homo sapiens}, complete 0.2074 17beta hydroxysteroid dehydrogenase {Homo sapiens}, partial (23%) 0.1299 steroid 5-alpha-reductase 2 [Sus scrofa] -0.3321 17beta-estradiol dehydrogenase [Sus scrofa] 0.4718 3alpha(or 20beta)-hydroxysteroiddehydrogenase - pig, complete 0.7596 147 apolipoprotein B -0.4293 apolipoprotein E [Sus scrofa] 0.8726 apolipoprotein A-I [Sus scrofa] -0.1557 ABC-transporter {Gorilla gorilla}, partial (54%) -0.4181 Similar to high density lipoprotein binding protein {Homo sapiens}, partial (23%) 0.6129 putative ABC-transporter [Sus scrofa] -0.2150 Electron transport and ATP production NADH dehydrogenase (ubiquinone) 14.5K chain - bovine, complete 0.5219 NADH4 [Sus scrofa]NADH5 [Sus scrofa]NADH6 [Sus scrofa] 0.5037 H(+)-ATPasesubunit[Susscrofa] 0.5077 Na+./K+ATPasealpha1subunit[Susscrofa] 0.1220 Ca(2+)-transportATPase(class2)[Susscrofa] 0.2191 BOVINVacuolarATPsynthasesubunitE partial(76%) 0.8818 HUMANVacuolarATPsynthasesubunitd)( partial(81%) 0.2563 Others G protein-coupled receptor -0.4972 cAMP-regulated phosphoprotein [Sus scrofa] 0.1242 5'-AMP-activated protein kinase catalytic alpha-2 subunit -0.2688 HUMANCyclic-AMP-dependenttranscriptionfactorATF-4.complete 1.0057 nuclear receptor binding protein {Homo sapiens}, partial (53%) 0.3840 proteinphosphatase2Aalphasubunit 0.6447 actin-relatedprotein3[Susscrofa] 0.3594 leptin[Susscrofa] 0.6220 phospholipaseD2{Homosapiens}.partial(47%) 0.1718 phospholipaseCbeta-3{Rattusnorvegicus}.partial(28%) 0.3438 HUMANSerine/threonineproteinphosphatase2A.partial(59%) 0.3105 HUMANTranscriptioninitiationfactorTFIID31kDasubunit(TAFII- 31Human].partial(41%) 0.3854 transcriptionfactorTFII-I-human.partial(9%) 0.4046 phosphoproteinphosphataseXcatalyticchain-human.partial(48%) 0.2520 HUMANLow-densitylipoproteinreceptor-relatedprotein1precursor(LRP)- 2partial(5%) 0.4505 RATTranscriptioninitiationfactorIIB(TFIIB).[Rat].partial(80%) 0.9901 HUMANTFIIHbasaltranscriptionfactorcomplexp52subunitpartial(44%) 0.1995 RABITSerine/threonineproteinphosphatase2A56kDa partial(25%) 0.9150 BOVINVacuolarATPsynthasesubunitG1.complete 0.9573 RATTranscriptioninitiationfactorIIAgammachaincomplete 0.1436 148 Table 5. Differential transcription responses in skeletal muscle to feeding LFD and HFD to pigs. Statistical analysis results for muscle tissue was obtained by SAM. One-class response with 1,000 permutations was used to determine genes whose expression was significantly different from zero (log 2 ratio =log 2 HFD/LFD). At these analysis parameters, the false discovery rate (FDR) for the positive genes was 0.05 (5%); the q value (a measure of significance in terms of the false discovery rate) for all biological replicates were less than 0.01. Different from normal t-test, FDR instead of q value was used to control limits of significant analysis as described in the Chapter 2. In this table, the genes with bold log 2 ratio values are differently expresses genes. Positive value means higher expression in HFD pigs; negative value means lower expression in HFD group. Gen name Log 2 (ratio) Fatty acid oxidation long-chain acyl-CoA dehydrogenase [Sus scrofa] 0.6594 short-chain acyl-CoA dehydrogenase [Sus scrofa] -0.1393 Propionyl-CoA carboxylase beta chain precursor [Sus scrofa] -0.2702 BOVIN 3-hydroxyacyl-CoA dehydrogenase type II (Type II HADH). [complete] -0.8938 malonyl-CoA decarboxylase {Homo sapiens}, partial (46%) -0.3163 carnitine palmitoyltransferase I {Ovis aries}, partial (32%) -0.7693 very-long-chain acyl-CoA dehydrogenase {Homo sapiens}, partial (27%) 0.4263 novel enoyl coA/acyl coA hydratase/dehydrogenase type protein (isoform 1)), complete -0.1240 Fatty acid and TAG synthesis stearyl-CoA desaturase [Sus scrofa] 0.2820 diacylglycerol acyltransferase [Sus scrofa] -0.0920 CCAAT/enhancer binding protein alpha [Sus scrofa] 0.0413 cytosolic glycerol-3-phosphate dehydrogenase [Sus scrofa] -1.2937 esterase D [Sus scrofa] -0.0981 fatty acid-binding protein [Sus scrofa] 1.2009 acetyl-Coenzyme A acyltransferase 2 {Homo sapiens}, partial (20%) 0.4629 LCFB_HUMAN Long-chain-fatty-acid--CoA ligase 2 partial (24%) 0.2262 mitochondrial acetoacetyl-CoA thiolase precursor {Rattus sp.}, partial (40%) 0.1597 fatty-acid synthase - human, partial (7%) -0.3833 ATP lipid-binding protein P1 precursor {Sus scrofa}, complete 0.1553 fatty acid coenzyme A ligase 5 {Homo sapiens}, partial (14%) 0.2210 C/EBP-induced protein {Homo sapiens}, partial (25%) 0.1980 149 CCAAT/enhancer binding protein beta [Sus scrofa] -0.8919 HUMAN [Pyruvate dehydrogenase [lipoamide]] kinase isozyme 4 mitochondrial precursor partial (18%) -0.6925 CCAAT/enhancer-binding delta protein {Bos taurus}, partial (54%) -0.7810 Carbohydrate metabolism succinyl-CoA synthetase alpha subunit [Sus scrofa] 0.0739 succinyl-CoA synthetase beta-subunit 0.2177 glucose transport protein [Sus scrofa] 0.0623 citrate synthase precursor 0.2159 pyruvate dehydrogenase (lipoamide) [Sus scrofa domestica] 0.1319 Pyruvate kinase M2 isozyme [Rabbit], partial (26%) -0.2641 malate dehydrogenase precursor 0.0976 ATP-specific succinyl-CoA synthetase beta subunit [Sus scrofa] 0.1448 Fructose-bisphosphate aldolase A (Muscle-type aldolase). [Rabbit], partial (24%) -0.8919 Cholesterol metabolism 3-beta-hydroxysteroid dehydrogenase/delta-5-delta-4 isomerase [Sus scrofa] 0.1527 sterol/retinol dehydrogenase {Homo sapiens}, complete 0.0742 3-hydroxymethyl-3-methylglutaryl-Coenzyme A lyase {Homo sapiens}, complete -0.4520 sterol regulatory element binding protein-2 [Sus scrofa] -0.2470 3-hydroxy-3-methylglutaryl coenzyme A reductase/HMG-CoA reductase [Sus scrofa] -0.3022 17beta-estradiol dehydrogenase [Sus scrofa] 0.2636 3alpha(or 20beta)-hydroxysteroid dehydrogenase - pig, complete 0.1451 apolipoprotein C-III 0.0247 apolipoprotein E [Sus scrofa] -0.7275 apolipoprotein A-I [Sus scrofa] 0.0503 Similar to high density lipoprotein binding protein (vigilin) {Homo sapiens}, partial (23%) -0.3212 Apolipoprotein A-II precursor [Cynomolgus monkey], complete -0.1147 ATP-binding cassette protein M-ABC1 {Homo sapiens}, partial (31%) -0.6341 Protein metabolism Eukaryotic translation initiation factor 3 subunit 7 {Homo sapiens}, partial (38%) -0.8171 beta-globin [Sus scrofa] 0.9614 ribosomal protein S4 [Sus scrofa] 0.4505 40S ribosomal protein S12 [Sus scrofa] 0.6203 smooth muscle myosin light chain kinase [Sus scrofa] 0.1456 HUMAN 40S ribosomal protein S6 (Phosphoprotein NP33). [Rat], complete 0.2182 probable translation initiation factor eIF-2B delta chain ? human partial (61%) 0.1482 ribosomal protein L15 cytosolic [validated] - rat, complete 0.4368 eukaryotic translation initiation factor 3 subunit p42/p44 {Homo sapiens}, complete -1.1871 HUMAN Histone acetyltransferase type B subunit 2 partial (19%) 0.1943 40S ribosomal protein S28. [Rat] {Rattus norvegicus}, complete 0.3755 arginine N-methyltransferase p82 isoform {Cricetulus longicaudatus}, partial (26%) 0.1196 eukaryotic translation initiation factor 4E-like 3 {Homo sapiens}, partial (89%) -0.6320 Similar to argininosuccinate lyase {Mus musculus}, partial (56%) 0.1043 BOVIN Elongation factor Tu mitochondrial precursor. [Bovine] partial (66%) -0.3424 150 151 histone deacetylase 3 {Rattus norvegicus}, partial (50%) -0.4110 translation initiation factor eIF-5A [validated] - human, complete -0.8752 Glutamate dehydrogenase mitochondrial precursor {Mus musculus}, partial (32%) -0.4566 Collagen alpha 1(I) chain precursor. [Dog] partial (5%) -1.0131 Collagen alpha 2(I) chain precursor. [Dog] partial (12%) -1.2133 alanine aminotransferase {Rattus norvegicus}, partial (24%) -0.0594 translation initiation factor eIF-2 alpha chain - rat, partial (62%) -0.3298 collagen VIII alpha 1 [Sus scrofa] -0.9139 myosin light chain kinase -0.9061 myosin heavy chain [Sus scrofa] -0.2212 homologue to EGAD|4603|4479 collagen type VI alpha 1 {Homo sapiens}, (7%) -0.3651 Collagen alpha 1(VI) chain precursor. [Mouse] {Mus musculus}, partial (13%) -0.8547 non-histone protein HMG1 0.2482 glutathione transferase class mu GSTM4 (version 2) - human, complete 0.1365 | mitochondrial branched chain aminotransferase precursor; {Ovis aries}, partial (21%) -0.4397 myosin heavy chain [Sus scrofa] -1.4053 elongation factor 1 alpha {Bos taurus}, partial (24%) -0.3662 Electron transport and ATP synthesis BOVIN NADH-ubiquinone oxidoreductase subunit mitochondrial precursor (37%) -0.8985 cytochrome b [Sus scrofa] -0.3498 NADH4 [Sus scrofa]NADH5 [Sus scrofa]NADH6 [Sus scrofa] -1.8858 Cytochrome c oxidase polypeptide VIIb mitochondrial precursor] {Homo sapiens}, complete -0.4757 Na+/K+ATPasealpha1subunit[Susscrofa] 0.1782 H++K+)-ATPase -0.2512 Others insulin receptor precursor [Sus scrofa] -0.3208 CREB-binding protein {Homo sapiens}, partial (41%) 0.0973 cAMP-regulated phosphoprotein [Sus scrofa] 0.5336 cyclic AMP-responsive element binding protein, delta variant [Sus scrofa] -0.5974 GTP-binding regulatory protein Gs alpha chain partial (54%) 0.4626 leptin[Susscrofa] 0.7110 transmembraneleptinreceptor[Susscrofa] -0.2237 Probablecalcium-transportingATPaseKIAA0703{Homosapiens}.partial(40%) 1.0323 CREB-RP(G13){Homosapiens}.partial(32%) -0.7468 GeneencodinghumansecretedgroupIIIphospholipaseA2{Homosapiens}.partial(38%) -0.6353 cytosolicphospholipaseA2beta;cPLA2beta{Homosapiens}.partial(18%) -0.2098 HUMANTranscriptioninitiationfactorTFIID20/15kDasubunits, partial(68%) 0.2598 TranscriptioninitiationfactorIIB(TFIIB) Rat].partial(80%) 0.0618 calcium-independentphospholipaseA2{Homosapiens}.partial(13%) -0.2182 sarcoendoplasmicreticulumcalciumATPase[Susscrofa] -0.5584 |proteinphosphatase-1delta[Susscrofa] -0.6889 RATTranscriptioninitiationfactorIIAgammachain(TFIIAP12subunit) (TFIIA-12)(TFIIAS).complete 0.2600 proteinphosphatase2Aalphasubunit 0.7904 V. EXPRESSION OF PORCINE GENES RELATED TO FATTY ACID AND CHOLESTEROL METABOLISM IN DIFFERENT PORCINE TISSUES INTRODUCTION Consumption of muscle foods has been typically associated with excess energy and saturated fatty acid intakes. American consumers are cognizant that excessive consumption of high-energy, predominantly saturated fatty acids containing foods may contribute to the onset of obesity, type 2 diabetes, and related cardiovascular maladies. Thus, through genetic selection for more rapidly growing and leaner pigs, through formulation of diets that precisely meet swine nutrient requirements, and through enhanced health care and management practices, over the last 25 years the US pork industry has attempted to produce muscle foods with lower fat content that are more in line with recommendations of the American Heart Association to keep fat calories near 30% of total calorie in the human diet. Unfortunately, this positive change in porcine muscle foods has a down-side in that lean pork lack the traditional taste and juiciness associated with pork. Since the seminal studies of Hammond (331), it has been understood that, in order to achieve fat deposition and the desired juiciness in muscle, subcutaneous and visceral storage lipid depots have to be ?filled? first. Hence during the production cycle, shortly before harvest, pigs have been placed before harvest, on 152 finishing diets to ensure adequate rates of fat gain to promote some intramuscular fat deposition. During industry-wide programs to significantly lower total fat in pork over the last 25 years, finishing programs had been modified and pigs with much lower propensity to deposit fat were utilized. Today the industry is attempting to promulgate a new production strategy that will result in relatively low subcutaneous and visceral fat accumulation coupled with some intramuscular fat deposition. Based on what is known about the biology of fat deposition in storage depots and muscle in pigs, such new strategies will not emerge without a more complete understanding of the temporal and tissue-specific regulation of fat deposition in pigs. As in humans or rodents, fat deposition in the pig is the result of complex interaction between genetic and a range of environmental influences including nutrition (1). Nutritional manipulation such as changing energy sources (fats and carbohydrates) and /or amounts in diets may have a significant effect on both fat accretion and muscle growth (332). The liver is the primary site for de novo fatty acid synthesis in humans and rodents, but adipose tissue in pigs (41) is the principal site of de novo fatty acid synthesis. While the pig has been used as an animal model to study the progression of excess energy intake on fat deposition, obesity and cardiovascular maladies for application to human medicine, many key aspects of lipid metabolism in pig are not exactly identical to human or rodents (13). Thus, to specifically target the pattern of tissue fat deposition in pigs during the finishing phase of production, regulation of porcine lipid metabolism across major tissues (liver, skeletal muscle and adipose tissue) must first be better understood at the molecular level to develop future strategies for specific tissue-targeted fat deposition. 153 Enhancing intramuscular fat accumulation in pigs by increasing dietary fat late during the finishing phase has been previously attempted (333), but has not become a common strategy in the pork industry. In the previous chapter IV described the effect of a sudden shift from a corn-soy low-fat diet to a corn-soy, tallow, corn oil-supplemented high-fat diet on global gene expression in skeletal muscle, liver and adipose tissue in finishing pigs, as evaluated with an oligo/DNA array spotted slide platform. In this chapter, emphasis is placed on expression responses of four targeted genes associated with lipoprotein, triacyglycerol, and cholesterol transport including acyl-CoA cholesterol acyltransferase (ACAT); lethicin-cholesterol acyltransferase (LCAT), ApoB and hepatic lipase (HL). The purpose of this study was to compare the transcription response of these targeted genes after a shift from corn-based high-carbohydrate, low-fat diet (LFD) to a tallow- supplemented high-fat diet (HFD) fed for 2 weeks. The HFD contained a high proportion of saturated fat contributed by the tallow. I propose that an abrupt sudden shift from a typical finishing diet to a diet supplemented with saturated fatty acids in 90-100 kg pigs will result in metabolic adaptations and changes in transcription of genes involved in triacylglycerol and cholesterol trafficking in the animal. While the animal experiment was being conducted, an initial step of this study in the laboratory was to first partially clone a porcine cDNA fragment of LCAT and ACAT. Information provided from these porcine specific sequences along with sequence data for porcine HL and ApoB was then utilized to design primers to determine the expression pattern of these targeted four genes in porcine skeletal muscle, liver, adipose and small intestinal epithelium (gut) and then utilize a semi-quantitative reverse transcriptase-polymerase chain reaction (smqRT- 154 PCR) method to quantify the relative mRNA abundance of ACAT, LCAT, HL and ApoB between pigs fed the low and high fat diets. MATERIALS AND METHODS Animal Feeding Trial Eight adult, crossbred pigs (90 kg) were provided ad libitum access to either a corn and soybean-based, low-fat diet (LFD) (n=4) or a tallow/ corn oil-supplemented high-fat diet (HFD) (n=4) for 14 days. For LFD, 4.3% diet energy was from fat contributed by the corn; while for HFD, 40% dietary energy was contributed by saturated fatty acids from beef tallow plus some additional corn oil. The composition of the diets fed to pigs for the 14 days before slaughter is presented in Table 1. The calculated protein concentration in the experimental diet was 20% and 19.3 %, respectively, for LFD and HFD. Both diets met or exceeded all nutrient requirements for finishing pigs as prescribed by the NRC (199). This experiment was approved by the Auburn University Institutional Animal Care and Use Committee (IACUC #0207-R-2448). The pigs were slaughtered at 14 days, and liver, subcutaneous adipose tissue and skeletal muscle tissues were collected. Pig identification number, diet treatment for each pig, and the day the samples were collected are presented in the Table 2. 155 Table 1. Composition of diets fed to finishing pigs* Ingredient Control (%) High Fat (%) Corn 68.05 51.65 Fat source (Tallow/Sat Fat /corn oil/equiv) 0 13.25 3.25 Soybean Meal 29.00 29.00 Premix Di-Calcium Phosphate 1.00 1.00 Limestone, ground 0.80 0.80 Salt 0.35 0.35 Vitamins & trace mineral mix 0.2 0.2 Additive/fiber 0.5 0.5 Calculated analysis Kcal/gm 4.1 5.2 Total Protein % 20 19.3 Polyunsaturated to sat. fatty acid 0.2 0.2 *Meets all NRC (1998) nutrient requirements for finishing pigs *The formulations are presented on a present as is feed ingredients (not dry metarial corrected). These diets were not formulated to be iso-energetic. Table 2. Identification numbers, assigned dietary treatment, and the date of sample collection for experimental pigs. Pig Length of Treatment (days) Fat Supplemented to diet (%) Date Sample Collected 4901 14 0 (LFD) 11/19/2003 5504 14 0 (LFD) 11/19/2003 5205 14 0 (LFD) 11/19/2003 6002 14 0 (LFD) 11/19/2003 4905 14 16.5 (HFD) 11/19/2003 5207 14 16.5 (HFD) 11/19/2003 5502 14 16.5 (HFD) 11/19/2003 6001 14 16.5 (HFD) 11/19/2003 156 Tissue Collection All the pigs were killed at the Auburn University Meat Laboratory by electrical stunning, followed by exsanguination under USDA/APHIS inspection. Liver, subcutaneous adipose, skeletal muscle and gut tissues were removed immediately snap- frozen in liquid nitrogen as described in the preceding chapter prior to scalding and de- hairing of the swine carcass. Liver samples were removed from the right lobe; tissue samples from the middle layer subcutaneous adipose were removed from the subcutaneous depot near 12 th rib, and skeletal muscle samples were removed from the longissimus muscle between the 10 th and the last ribs. This procedure minimized contamination and product rejection by the federal inspection system and allowed for the further processing of the carcass for eventual human consumption. Analysis of Plasma Triacylglycerol and Cholesterol Concentration Plasma was prepared from blood obtained during slaughter and frozen at -20 ?C until analyzed for total cholesterol and triglyceride concentrations by the Clinical Diagnostic Laboratory at the Auburn University College of Veterinary Medicine. RNA Isolation Total RNA was isolated by using a one step guanidinium-phenol-chloroform extraction procedure (201). One-half gram of frozen adipose tissue from each pig was powdered using a hammer-driven, stainless steel mortar and pestle that was constantly cooled with liquid nitrogen. The tissue was then placed in a 50-ml conical tube containing 10 ml of TriZol reagent (Invitrogen Corporation; Carlsbad, CA), and RNA was isolated according to the instructions provided. 0.5 ?g of total RNA was dissolved in 157 100 ?l of distilled, deionized RNase-free water, and RNA integrity was analyzed on a 1.0 % agarose gel to check the integrity of RNA. Total RNA was quantified using an Ultrospec 3000 UV/ visible spectrophotometer (Amersham Pharmacia Biotech; Piscataway, NJ), and the RNA quality was estimated by observing the smearing of 18S and 28S bands, intensities of the bands and DNA contamination. . Extracted RNA was stored at -80?C in 1?l of RNA Secure/ ?g of RNA (Ambion; Austin, TX). An example of the banding pattern of total RNA is presented in Figure 1. Fig 1. Intact total RNA resolved on a 1.0% agarose gel at 120V for 30 minutes. The bands represented from top to bottom are 28S ribosomal RNA, 18S ribosomal RNA, and transfers/small RNA. 158 Cloning of Porcine LCAT and ACAT Gene Fragments Primer Design Paired sense and anti-sense primers were designed based on known human sequences using the web-based computer software, Primer3 (http://frodo.wi.mit.edu/cgi- bin/primer3/primer3_www.cgi). LCAT and ACAT primers were designed referring to the human LCAT mRNA sequence (Accession No. BC014781.1) and human ACAT mRNA sequence (Accession No. L21934.2) found in GenBank. Primers were used in the following procedure to amplify porcine LCAT and ACAT fragments. The sequence of primers and the expected sizes of amplicons produced by the polymerase chain reaction (PCR) are listed in the Table 3. Table 3. DNA sequence of the primers, annealing temperature used, and number of PCR cycles performed for the semi-quantitative RT-PCR 159 Reverse Transcription-Polymerase Chain Reaction (RT-PCR) One ?g of total RNA from porcine adipose tissue was amplified in the presence of the LCAT or ACAT primer pairs by reverse transcription (RT) reaction and polymerase chain reactions (PCR). These reactions were conducted using the OneStep RT-PCR kit (Qiagen, Inc.) and a PTC-100 programmable thermal cycler (MJ Research, Inc.; Waltham, MA). Within each PCR cycle, the initial denaturation step was at 94 o C for one minute. This was followed by primer annealing step at 59 o C for one minute. Annealing was followed by a one minute extension step at 72 o C. The denaturation, annealing and extension steps were then sequentially repeated 29 more times followed by a final extension step at 72 o C for ten minutes. 20 ?l of RT-PCR reaction product was run on a 1.0% agarose gel to determine if there was an amplicon of the expected length based on a standard 100 bp DNA ladder (Invitrogen Corporation). The expected product lengths derived from sequence data were 453 base pairs (bp), and 815 bp for LCAT and ACAT, respectively. Cloning and Transformation of Porcine Partial Coding Sequences Fresh reverse transcription-polymerase chain reaction products were directly ligated into a pCR-II TOPO vector (Invitrogen) and transfected into TOP10 or TOP10F? E. coli cells (Invitrogen) using the pCR-II TOPO Vector System. In the pCR-II TOPO Vector System there were two selection methods: ampicilin resistance and X-galactosidase (X- gal) activity. After ligation and transformation, 50 ?l and 200 ?l of the transformed bacteria in S.O.C. Media (0.5% Yeast extract, 2.0% tryptone, 10mM NaCl, 2.5mM KCl, 10mM MgCl 2 , 20mM MgSO 4 , 20mM glucose) were plated on LB (Luria-Betani) selective plates containing 200 ?g/ml of ampicillin (Fisher Scientific; Hampton, NH), and 160 53.3 ?g/ml X-gal (Fisher Scientific). For TOP10F? cells, plates also contained 0.13 mM Isopropyl-?-D-thiogalactoside (IPTG) (Fisher Scientific). Plates were incubated at 37?C for 12-16 hours. Positive colonies were selected from the smear plates, streaked on identically prepared LB plates, and incubated 12-15 hours at 37?C. Four colonies positive for the cloned vector containing the insert of interest were then selected from each plate. These colonies were used each to inoculate liquid cultures of 4 ml LB media with 200 ?g/ml ampicillin. These small-scale cultures were incubated on a horizontal shaker at 37?C for 15 hours. Then, plasmid DNA was purified from 1.5 ml of each culture following the microcentrifuge protocol of the Qiaprep Spin Miniprep Kit (Qiagen, Inc.). The pCR II-TOPO cloning vector contains EcoRI restriction sites on either side of the inserted cDNA fragment. Once plasmid DNA was isolated, it was digested by cutting with EcoR1 (Invitrogen Corporation). Sizes of ligated inserts for LCAT and ACAT were verified by a restriction digestion of plasmid DNA with EcoRI restriction enzyme (Invitrogen Corporation) followed by electrophoresis through a 1.0% agarose gel. To produce an ample quantity of cDNA fragments, large-scale cultures of plasmid DNA were obtained by inoculating 200 ml of LB Media containing 200?g/ml ampicillin with 2ml of culture from the colonies of choice in a one-liter culture flask. The cultures were incubated at 37?C for 15 hours with agitation. Plasmid DNA was purified following the Plasmid Purification Maxi Protocol using two Qiagen-tip 500 columns from a Qiafilter Maxi Kit (Qiagen, Inc.). As with the small-scale cultures, restriction digestions were performed and products were run on agarose gels to ensure that purified plasmids contained inserts of appropriate size. Plasmid DNA was prepared for long-term storage by freezing 400?l of culture in 200?l of sterol 100% glycerol at -80?C. 161 Sequencing To confirm RT-PCR cloning and to determine the directionality of the insert, plasmids containing the LCAT and ACAT cDNA fragments were sequenced bi- directionally. Sense and antisense sequences were obtained for the inserts by sequencing with T7 and SP6 primers at the Auburn University Genomics and Sequencing Lab using a modification of the Sanger method (314) with fluorescent dideoxy termination in an automated capillary sequencer (Applied Biosystems; Foster City, CA). The sequence of porcine LCAT and ACAT cDNA fragments were previously unknown. The partial fragment of the porcine LCAT and ACAT mRNA sequence was submitted to Genbank, accepted, and assigned Accession Number AY349156 for LCAT and AY676347 for ACAT. The fragment of pig LCAT is part of the coding region. The translated pig LCAT protein includes 150 amino acids and the protein accession number is AAQ24609 assigned by Genbank. The fragment of ACAT is part of 5? UTR (un- translated region). Sequences obtained from DNA sequencing were submitted to BLASTn (335) to determine the homology of the insert sequences with other sequences in GenBank. The LCAT and ACAT sequences generated here were found to have 92% and 99% homology with the human LCAT and ACAT mRNA sequence, respectively (Fig 5 and Fig 6). The translated partial LCAT protein sequence was submitted to BLASTp (336), and it was found to be 93% homologous with the human LCAT protein (Fig 7). 162 Determining Gene Expression Distribution by RT-PCT Similar RT-PCR procedure as described above was performed to determine the expression pattern of ACAT, LCAT, HL and ApoB in porcine tissues, liver, adipose, muscle and gut ( four tissues). Determining Relative mRNA Abundance by Semi-quantitative PCR A two-step semi-quantitative RT-PCR method was developed to measure gene expression in liver, adipose and gut tissues (337). During the preliminary stages of method development, linearity with respect to RNA and amplicon appearance and PCR cycle number were determined. In the final protocol, cDNA was first synthesized from total RNA using oligo-(dT) 18n primers (Operon, Huntsville, AL) and the Omniscript Reverse Transcription Kit (Qiagen Inc.). In the next step, identical primers with those in the cloning of LCAT and ACAT porcine gene fragments were used for amplification of the cDNA. Primers for apolipoprotein B (ApoB), hepatic lipase (HL) and ? -actin were designed based on the known pig ApoB mRNA sequence (Accession No.L11235.1), pig HL mRNA sequence (Accession No.J03540.1) and pig ?-actin mRNA sequence (AY550069.1). Followig the reverse transcription step, cDNA from different tissues were amplified with ACAT, LCAT, apoB, and HL specific primers using the Taq PCR Core Kit (Qiagen Inc.). The optimal PCR annealing temperatures for each specific gene primer pair and number of PCR cycles to get linear amplification range are presented in Table 3. A housekeeping gene, ?-actin was used as an internal control and normalization gene. During the PCR process for each specific gene, ?-actin primers were added to the same tube when 24 cycles were remaining in each gene?s specified linear amplification range. Finally, the PCR products were electrophoresed on 1.0% agarose gel and base pair sizes 163 of PCR product were determined relative to DNA ladder standards. The gel images were captured by digital still camera (Sony, Tokyo, Japan), and densitometry values were measured with the NIH image J program (http://rsb.info.nih.gov/ij/). RT-PCR values are presented as a ratio of the specified gene?s signal in the selected linear amplification cycle divided by ?-actin signal. Data were analyzed by t-test using SAS software. RESULTS The dietary shift was imposed on the selected pigs without any incremental adaptation. Overall, animal performance for the next 14 d was not affected by the abrupt sudden shift from the LFD to the HFD. Pigs were group-fed in an open pen ad libitum and the animal technician did not notice any changes in behavior or an initial rejection of the HFD. Based on observations only, the HFD was apparently more palatable to the finishing pigs than the control, corn-soy, low-fat diet, and there were no difference in dry matter intake between LFD and HFD pigs. Final bodyweights at slaughter and plasma cholesterol and triacylglycerol concentrations in the experimental animals are presented in Table 4. Table 4. Final body weight of pigs, plasma cholesterol and triacylglycerol on day 14. Each of these physiological parameters is expressed as mean ?standard deviation (SD). Group LFD HFD P-value Final body weight ?SD; kg 105.1 ? 5.4 106.1 ? 2.93 0.34 Plasma cholesterol (mg/100mL) ?SD 91.75? 8.92 101.33? 14.18 0.45 Plasma triacyglycerol (mg/100mL) ?SD 32.75? 6.65 52.33? 19.73 0.11 Both cholesterol and triacylglycerol concentrations were numerically higher in HFD fed pigs but the differences did not approach significance. 164 The tissue distribution pattern of the four genes was first determined by RT-PCR with RNA isolated from liver, adipose, skeletal muscle and gut epithelium. HL (as expected) was expressed only in the liver while apoB was detected in liver, adipose and gut epithelium. LCAT was found in all four tissues, while ACAT was only detected in liver and gut epithelium (Fig 2). ApoB expression was highest in liver, followed by gut and adipose (Fig 2, when normalized to ? actin expression). This ApoB gene expression pattern is similar to that noted in humans and rodents; however, ApoB synthesis, while extremely critical for chylomicron formation (small intestine) and VLDL (liver) synthesis, is not principally regulated at the transcriptional level (338, 339). Based on the present results, the pattern of ApoB mRNA expression in finishing pigs is consistent with previous work (humans, rodents) showing that intestine and liver are primary tissues that export lipids to other tissues utilizing the ApoB protein as a lipoprotein carrier for chylomicrons and VLDL (340). Comparing mRNA abundance/ expression of the four genes in different tissues between LFD and HFD fed pigs (Fig 3), dietary high fat significantly decreased ACAT transcription in porcine liver (P<0.05, Fig 3A). Although mRNA abundance of ACAT was lower in the gut of HFD pigs than LFD pigs from gel image analysis, these results were based on only two pigs (Fig 3C). No changes in mRNA abundance were observed for liver LCAT, ApoB and HL between LFD and HFD fed pigs (Fig 4 A). HFD also did not change the mRNA abundance of LCAT and apoB in the adipose tissue (Fig 4B). 165 Fig 2. Gene distribution pattern in porcine tissues Fig 3. Gel image of relative RT-PCR in porcine tissues A) liver B) adipose C) gut. For A and B, lane 1-4: HCHO diet; lane 5-8: HF diet. For C, lane 1-2:HCHO diet; lane 3-4: HF diet. 166 Fig 4. Relative gene expression in pigs fed HF diet (n=4) relative to the HCHO diet ( n=4), error bars represent standard deviation. Significant difference is represented as * (P<0.05). A) liver, B) adipose A) 0 0.5 1 1.5 2 2.5 ACAT LCAT ApoB HL R e l a t i ve G e n e E x p r es si o n HCHO HF * B) 0 0.05 0.1 0.15 0.2 0.25 0.3 0.35 0.4 0.45 R e l a t i ve G e n e E x p r es si o n LCAT ApoB HCHO HF 167 DISSUSION Feeding diets containing 10% beef tallow to pigs depressed lipogenesis in porcine adipose tissue (46). These findings were based on lipogenic enzyme activity assays in adipose tissues. Since that work, very little new data have accrued on the regulation of lipogenesis, in particular at the gene expression level with respect to high fat/fatty acid intakes in pigs. Further, there are no data on transcription responses of lipid trafficking genes, such as LCAT, ACAT, ApoB and HL to dietary fat intake by pigs. Liver plays an important role in cholesterol trafficking, uptake, excretion and endogenous synthesis in mammals, but such data are primarily from studies with rodents and primates (341). While liver is the primary site of fatty acid synthesis in human and rodents, only small quantities (342) of total de novo fatty acids synthesis occur in the porcine liver, and adipose is the primary tissue for synthesizing fatty acids in pig (343). The small intestinal (gut) epithelium plays an important role in dietary nutrients absorption and metabolism. This is particularly true for uptake of fatty acids arising from intestinal pancreatic lipase activity and consequent transport into the lymph of hydrophobic lipids as lipoproteins synthesized in the gut epithelium from the absorbed fatty acids and monoglycerides (37). In the liver, ACAT catalyzes the esterification of cholesterol with long chain fatty acids, and the derived cholesterol esters are secreted as a component of very low density lipoprotein (VLDL) (344). Similar to the general down-regulated expression pattern of genes in lipid metabolism found in the liver tissue by microarray analysis in Chapter 3, results of semi-quantitative RT-PCR in this study also showed decreased mRNA abundance for ACAT after a diet shift from LFD to HFD in pigs after 14 days. In rodents, 168 high-fat diets are associated with increased synthesis of cholesterol and amounts of blood VLDL and LDL (345) usually necessitating increased ACAT activity. Seo et al. (346) found that dietary fat induced transcription of ACAT in human hepatoma cells. Likewise an increased mRNA abundance of liver ACAT was hypothesized/ anticipated for pigs in this experiment. The lack of agreement between my results and work on primarily rodents may be related to the difference in the species, length of exposure to a high fat diet, and the effects of in vitro vs. in vivo experiments (i.e. cell cultures). Furthermore, because diets containing long-chain fatty acids depressed fatty acid synthesis in pigs (46, 347), it was very possible the HFD diet in this study depressed fatty acid synthesis in pigs. ACAT contributes to cellular cholesterol homeostasis by etherifying free cholesterol and the cholesteroyl ester is deposited in lipid droplets (348). Hence, after the diet shift to the HFD, this putative attenuated fatty acid synthesis, coupled with low dietary cholesterol, might lead to a decreased need for ACAT activity, which may be caused by decreased ACAT mRNA abundance in the liver. In the gut, ACAT plays a role in cholesterol absorption by maintaining a free cholesterol diffusion gradient across the enterocyte surface through the formation of intracellular cholesterol esters (349). The transcription of ACAT also appeared reduced in the gut, but these results are based on only two pigs (low number because of tissue harvesting difficulties beyond the control of the writer of this dissertation). Finally, the oligo array results (see Chapter 3) from liver revealed a basically across the board lowered mRNA abundance of almost all genes involved in lipid and energy metabolism of this set of finishing pigs fed the HFD for 14 days. It is conceivable that a sudden influx of large amount of fatty acids into liver upon the sudden 169 shifting to HFD may have produced a fatty acid overload condition in the liver, which resulted in a general down-regulation of genes in lipid metabolism in the liver to prevent over-accumulation of fat. Based on observation of increased expression of genes in TAG synthesis in the adipose tissue by microarray analysis in the chapter 3, adipose tissue had more potency to absorb and store fatty acids in response to the HFD. This conjecture can not be substantiated at present without fatty acid analysis in plasma and tissues. LCAT catalyzes the initial step in reverse cholesterol transport, the esterification of cell-derived free cholesterol, concomitant with transfer of the esters into the core of high density lipoprotein (HDL) (350). In this study, LCAT was observed highly expressed in liver and adipose tissues, but there were no differences in LCAT mRNA abundance after between LFD and HFD treated pigs. Similar results were reported in mice. Deng et al. (345) found hepatic expression of LCAT was unchanged by high fat-enriched diets in the mice. Hepatic lipase is synthesized and secreted by the hepatocyte. It catalyzes the hydrolysis of the intermediate density protein (IDL) triacylglycerol to produce low density protein (LDL) (351). More recent work has shown that cholesteryl ester transfer protein (CETP) during normal metabolism is responsible for IDL to LDL conversion and the role of HL is to hydrolyze triacylglycerol in HDL (37). CETP is expressed in pigs (AF333037); however, specific roles of HL and CETP in porcine reverse-cholesterol transport is unclear. We did not find any differences in mRNA abundance of HL in pigs fed either LFD to HDL. ApoB is the apoprotein necessary in VLDL and LDL synthesis (37). Two isoforms of apoB, apoB100 and apoB48 have been identified in humans. ApoB100 exclusively is 170 synthesized in human liver, and intestine secretes mainly apoB48 with some apoB 100 (352). ApoB100 and ApoB48 are derived from a single gene, a major mRNA of ~14kb. In the human intestine, a stop codon at 6666 of the apoB messenger RNA terminates the translation and results in the production of a polypeptide known as apoB48 (48% of messenger RNA translated) (353). Therefore, apoB48 is the N-terminal, 48% of full- length apoB100 (354). The porcine apoB gene fragment used here to measure the transcription level in porcine tissues was analyzed to locate its relative position in the human apoB protein. In this process, the porcine mRNA fragment was first translated to obtain the corresponding protein sequence by the TranSeq online software (http://www.ebi.ac.uk/emboss/transeq/). Then the translated porcine apoB protein fragment was aligned with human apoB100 and apoB48 protein sequences (Fig 8). The alignment results showed that the present porcine protein fragment corresponds to a partial amino acid sequence in the C-terminal region of human apoB100. Thus, this particular porcine ApoB cDNA fragment and the resulting primers used here can identify ApoB , but cannot distinguish between ApoB 100 and ApoA 48. In this study, mRNA abundance of apoB was higher in liver than adipose and gut tissues, but mRNA abundance of ApoB in liver, adipose and muscle tissues were not different between LFD and HFD pigs. Previous work has indicated that dietary supply of triacylglycerol alone does not regulate ApoB secretion or plasma concentrations of ApoB (355). A lack of change in the transcription of ApoB in the high fat diet in the rat was previously reported (345). Deng et al. (345) did not detect significance alterations of VLDL apoproteins (apoB, apoE, apoCII, apoCIII) after rats were treated by menhaden oil diets (40% of calories from fat). Furthermore, it has been shown that, in humans, ApoB is mainly 171 regulated at the post-transcriptional level (356). Further research is needed to compare the ApoB protein concentration between LFD and HFD pigs, which may clarify the regulatory mechanisms for ApoB synthesis in the pig. Since this study focused on determination of transcription response of genes, it is impossible here to make any conclusion whether identical regulatory mechanism are applicable in pig as in human or rodents. Lipoproteins transport the majority of cholesterol and triacylglyceride in the circulatory system (357). Further analysis and comparison the lipoprotein profiles of serum and fat content in liver, adipose and muscle tissues between LFD and HFD pigs will likely extend our understanding about the effects of dietary high fat on the expression of genes involved in inter-tissue transport of triacylglycrol and cholesterol. Such data may help explain the observed gene transcription response of these four lipoprotein associated genes upon a shift from LFD to HFD in pigs. CONCLUSIONS This study has established that four genes associated with inter tissue lipid trafficking appear more highly expressed in porcine liver than other tissues studied. These results also showed that a LFD to HFD shift did not change liver LCAT, HL and ApoB gene transcript abundance in pigs, indicating that this 14 day exposure to HFD did not affect the expression of these genes in the liver. Furthermore, no changes were observed in the mRNA abundance of LCAT and ApoB in the adipose tissue between LFD and HFD pigs. This study demonstrated that the transcription response of ACAT differed in various tissues when pigs were shifted from an abundant carbohydrate, low-fat diet to a high-fat 172 diet. Substitution of dietary fat for carbohydrate down-regulated the transcription of ACAT in the porcine liver. To our knowledge, the current study represents the first report of LCAT and ACAT distribution in pigs and on the effect of dietary high fat on the expression of lipid-trafficking genes in pigs. 173 Fig 5. Comparison of LCAT sequences (cds) between pig ( AY349156 ) and human ( BC014781.1 ) by BLAST Score = 632 bits (319), Expect = 2e-178 Identities = 418/451 (92%), Gaps = 0/451 (0%) Strand=Plus/Plus Pig LCAT 1 AGGACCGCTTTATTGATGGCTTCATCTCTCTTGGAGCTCCCTGGGGTGGCTCCACCAAGC 60 |||||||||||||||||||||||||||||||||| ||||||||||||||||||| ||||| Human LCAT 669 AGGACCGCTTTATTGATGGCTTCATCTCTCTTGGGGCTCCCTGGGGTGGCTCCATCAAGC 728 Pig LCAT 61 CCATGCTAGTCTTGGCCTCAGGTGACAACCAGGGCATCCCGATCATGTCCAGCATCAAAC 120 ||||||| |||||||||||||||||||||||||||||||| ||||||||||||||||| | Human LCAT 729 CCATGCTGGTCTTGGCCTCAGGTGACAACCAGGGCATCCCCATCATGTCCAGCATCAAGC 788 Pig LCAT 121 TGAAAGAGGAGCAGCGCATGACAACAACCTCCCCCTGGATGTTTCCCTCCAGCCACGTGT 180 ||||||||||||||||||| || || ||||||||||||||||||||||| || | || Human LCAT 789 TGAAAGAGGAGCAGCGCATAACCACCACCTCCCCCTGGATGTTTCCCTCTCGCATGGCGT 848 Pig LCAT 181 GGCCCGAGGACCATGTGTTCATTTCCACCCCCAGCTTCAACTACACAAGCCATGACTTCC 240 |||| |||||||| |||||||||||||| |||||||||||||||||| ||| |||||||| Human LCAT 849 GGCCTGAGGACCACGTGTTCATTTCCACACCCAGCTTCAACTACACAGGCCGTGACTTCC 908 Pig LCAT 241 AGCGCTTCTTTGCAGACCCGCACTTTGAGGAAGGCTGGTACATGTGGCTACAGTCACGTG 300 | |||||||||||||||| |||||||||||||||||||||||||||||| |||||||||| Human LCAT 909 AACGCTTCTTTGCAGACCTGCACTTTGAGGAAGGCTGGTACATGTGGCTGCAGTCACGTG 968 Pig LCAT 301 ACCTGCTGGCAGGCCTCCCAGCGCCTGGTGTGGAAGTATACTGTCTGTATGGTGTGGGCC 360 |||| |||||||| |||||||| ||||||||||||||||||||||| || || ||||||| Human LCAT 969 ACCTCCTGGCAGGACTCCCAGCACCTGGTGTGGAAGTATACTGTCTTTACGGCGTGGGCC 1028 Pig LCAT 361 TGCCCACACCCCGCACCTACATCTTTGACCACGGCTTCCCCTACACGGACCCTGTGGATG 420 ||||||| |||||||||||||||| ||||||||||||||||||||||||||||||| || Human LCAT 1029 TGCCCACGCCCCGCACCTACATCTACGACCACGGCTTCCCCTACACGGACCCTGTGGGTG 1088 Pig LCAT 421 TGCTCTATGAGGATGGTGATGACACTGTGGC 451 ||||||||||||||||||||||||| ||||| Human LCAT 1089 TGCTCTATGAGGATGGTGATGACACGGTGGC 1119 174 Fig 6. Comparison of ACAT sequences (5?UTR) between pig ( AY676347 ) and human ( L21934.2 ) by BLAST Score = 950 bits (479), Expect = 0.0 Identities = 482/483 (99%), Gaps = 0/483 (0%) Strand=Plus/Plus Pig ACAT 333 CATGGAAAAGTTCTTTACTGGTGATTTCTGAGATTTTAGTTCACCCCTTATCCTGAGCAG 392 |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| Human ACAT 636 CATGGAAAAGTTCTTTACTGGTGATTTCTGAGATTTTAGTTCACCCCTTATCCTGAGCAG 695 Pig ACAT 393 TGTACACTGTTCCCAATATGTAGCCTTTTATCCCTCACCCCCTCTAAGTTCAAGAAGACT 452 |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| Human ACAT 696 TGTACACTGTTCCCAATATGTAGCCTTTTATCCCTCACCCCCTCTAAGTTCAAGAAGACT 755 Pig ACAT 453 ATGGTCCTGCAGAAAGCTTTATATGTAATTAACATATCTTTATCTTTATCTTTATAGGCA 512 |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| Human ACAT 756 ATGGTCCTGCAGAAAGCTTTATATGTAATTAACATATCTTTATCTTTATCTTTATAGGCA 815 Pig ACAT 513 GTAGACTCATCTTTTGAAACAGATTCCATTAAGAGTGAATGTGTACCCTCCCTCTAGCCT 572 |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| Human ACAT 816 GTAGACTCATCTTTTGAAACAGATTCCATTAAGAGTGAATGTGTACCCTCCCTCTAGCCT 875 Pig ACAT 573 TTATTATTACTGTTTTTGCTATTACATGTGTTAGTGTATGTGAATTTAATGCTTAAAAAT 632 |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| Human ACAT 876 TTATTATTACTGTTTTTGCTATTACATGTGTTAGTGTATGTGAATTTAATGCTTAAAAAT 935 Pig ACAT 633 GTATCCCATTGGCTACTATGGCAAAAGGTTGACTCATAAGAGTTTAGCACGGGTTAAGAT 692 |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| Human ACAT 936 GTATCCCATTGGCTACTATGGCAAAAGGTTGACTCATAAGAGTTTAGCACGGGTTAAGAT 995 Pig ACAT 693 CTGAAAGTTTTCCCCCAGCCTCTTATCACTGGCGCAGACTTCACAATTCATGGAAGCCAC 752 |||||||||||| ||||||||||||||||||||||||||||||||||||||||||||||| Human ACAT 996 CTGAAAGTTTTCTCCCAGCCTCTTATCACTGGCGCAGACTTCACAATTCATGGAAGCCAC 1055 Pig ACAT 753 CAGTGAGATGACATTGCCTCAGGCAGTTACTATTTTTATATTCTATAACTCGAGGAGCTC 812 |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| Human ACAT 1056 CAGTGAGATGACATTGCCTCAGGCAGTTACTATTTTTATATTCTATAACTCGAGGAGCTC 1115 Pig ACAT 813 AGG 815 ||| Human ACAT 1116 AGG 1118 175 Fig 7. Comparison of LCAT protein sequences between pig (AAQ24609.1) and human ( AAA59500.0) by BLAST Score = 302 bits (774), Expect = 2e-80 Identities = 140/150 (93%), Positives = 142/150 (94%), Gaps = 0/150 (0%) Query 212 DRFIDGFISLGAPWGGSIKPMLVLASGDNQGIPIMSSIKLKEEQRITTTSPWMFPSRMAW 271 DRFIDGFISLGAPWGGS KPMLVLASGDNQGIPIMSSIKLKEEQR+TTTSPWMFPS W Sbjct 1 DRFIDGFISLGAPWGGSTKPMLVLASGDNQGIPIMSSIKLKEEQRMTTTSPWMFPSSHVW 60 Query 272 PEDHVFISTPSFNYTGRDFQRFFADLHFEEGWYMWLQSRDLLAGLPAPGVEVYCLYGVGL 331 PEDHVFISTPSFNYT DFQRFFAD HFEEGWYMWLQSRDLLAGLPAPGVEVYCLYGVGL Sbjct 61 PEDHVFISTPSFNYTSHDFQRFFADPHFEEGWYMWLQSRDLLAGLPAPGVEVYCLYGVGL 120 Query 332 PTPRTYIYDHGFPYTDPVGVLYEDGDDTVA 361 PTPRTYI+DHGFPYTDPV VLYEDGDDTVA Sbjct 121 PTPRTYIFDHGFPYTDPVDVLYEDGDDTVA 150 176 Fig 8. Multiple sequence alignment of human apoB 100 protein (NP_000375.1), apoB48 (AAA51741.1) and translated fragment of pig apoB mRNA by CLUSTAL W (1.82) Sequence 1 Human APoB100 precursor amino acids (aa) sequence: gi|4502153|ref|NP_000375.1 Note: In the apoB100 precursor, apoB 100 begins from 1670, the previous sequences contain regions for signal peptide, multiple disulfide bonds, glycosylation regions, variant regions and unknown function domains. (Knott,T.J, 1986) Sequence 2 Human ApoB 48 amino acid sequence: gi|178732|gb|AAA51741.1 (Hardman,D.A.,1987) Sequence 3 Translated pig amino acid sequence based on the sequenced PCR amplicons in this study: translated sequence gi|4502153|ref|NP_000375.1| MDPPRPALLALLALPALLLLLLAGARAEEEMLENVSLVCPKDATRFKHLR 50 gi|178732|gb|AAA51741.1| -------------------------------------------------- translated -------------------------------------------------- ??????? gi|4502153|ref|NP_000375.1| GISTSATTNLKCSLLVLENELNAELGLSGASMKLTTNGRFREHNAKFSLD 1700 gi|178732|gb|AAA51741.1| --------------------LNAELGLSGASMKLTTNGRFREHNAKFSLD 30 translated -------------------------------------------------- gi|4502153|ref|NP_000375.1| GKAALTELSLGSAYQAMILGVDSKNIFNFKVSQEGLKLSNDMMGSYAEMK 1750 gi|178732|gb|AAA51741.1| GKAALTELSLGSAYQAMILGVDSKNIFNFKVSQEGLKLSNDMMGSYAEMK 80 translated -------------------------------------------------- gi|4502153|ref|NP_000375.1| FDHTNSLNIAGLSLDFSSKLDNIYSSDKFYKQTVNLQLQPYSLVTTLNSD 1800 gi|178732|gb|AAA51741.1| FDHTNSLNIAGLSLDFSSKLDNIYSSDKFYKQTVNLQLQPYSLVTTLNSD 130 translated -------------------------------------------------- gi|4502153|ref|NP_000375.1| LKYNALDLTNNGKLRLEPLKLHVAGNLKGAYQNNEIKHIYAISSAALSAS 1850 gi|178732|gb|AAA51741.1| LKYNALDLTNNGKLRLEPLKLHVAGNLKGAYQNNEIKHIYAISSAALSAS 180 translated -------------------------------------------------- gi|4502153|ref|NP_000375.1| YKADTVAKVQGVEFSHRLNTDIAGLASAIDMSTNYNSDSLHFSNVFRSVM 1900 gi|178732|gb|AAA51741.1| YKADTVAKVQGVEFSHRLNTDIAGLASAIDMSTNYNSDSLHFSNVFRSVM 230 translated -------------------------------------------------- gi|4502153|ref|NP_000375.1| APFTMTIDAHTNGNGKLALWGEHTGQLYSKFLLKAEPLAFTFSHDYKGST 1950 gi|178732|gb|AAA51741.1| APFTMTIDAHTNGNGKLALWGEHTGQLYSKFLLKAEPLAFTFSHDYKGST 280 translated -------------------------------------------------- gi|4502153|ref|NP_000375.1| SHHLVSRKSISAALEHKVSALLTPAEQTGTWKLKTQFNNNEYSQDLDAYN 2000 gi|178732|gb|AAA51741.1| SHHLVSRKSISAALEHKVSALLTPAEQTGTWKLKTQFNNNEYSQDLDAYN 330 translated -------------------------------------------------- gi|4502153|ref|NP_000375.1| TKDKIGVELTGRTLADLTLLDSPIKVPLLLSEPINIIDALEMRDAVEKPQ 2050 gi|178732|gb|AAA51741.1| TKDKIGVELTGRTLADLTLLDSPIKVPLLLSEPINIIDALEMRDAVEKPQ 380 translated -------------------------------------------------- gi|4502153|ref|NP_000375.1| EFTIVAFVKYDKNQDVHSINLPFFETLQEYFERNRQTIIVVVENVQRNLK 2100 gi|178732|gb|AAA51741.1| EFTIVAFVKYDKNQDVHSINLPFFETLQEYFERNRQTIIVVLENVQRNLK 430 translated -------------------------------------------------- gi|4502153|ref|NP_000375.1| HINIDQFVRKYRAALGKLPQQANDYLNSFNWERQVSHAKEKLTALTKKYR 2150 gi|178732|gb|AAA51741.1| HINIDQFVRKYRAALGKLPQQANDYLNSFNWERQVSHAKEKLTALTKKYR 480 translated -------------------------------------------------- gi|4502153|ref|NP_000375.1| ITENDIQIALDDAKINFNEKLSQLQTYMIQFDQYIKDSYDLHDLKIAIAN 2200 gi|178732|gb|AAA51741.1| ITENDIQIALDDAKINFNEKLSQLQTYMIQFDQYIKDSYDLHDLKIAIAN 530 translated -------------------------------------------------- gi|4502153|ref|NP_000375.1| IIDEIIEKLKSLDEHYHIRVNLVKTIHDLHLFIENIDFNKSGSSTASWIQ 2250 177 gi|178732|gb|AAA51741.1| IIDEIIEKLKSLDEHYHIRVNLVKTIHDLHLFIENIDFNKSGSSTASWIQ 580 translated -------------------------------------------------- gi|4502153|ref|NP_000375.1| NVDTKYQIRIQIQEKLQQLKRHIQNIDIQHLAGKLKQHIEAIDVRVLLDQ 2300 gi|178732|gb|AAA51741.1| NVDTKYQIRIQIQEKLQQLKRHIQNIDIQHLAGKLKQHIEAIDVRVLLDQ 630 translated -------------------------------------------------- gi|4502153|ref|NP_000375.1| LGTTISFERINDVLEHVKHFVINLIGDFEVAEKINAFRAKVHELIERYEV 2350 gi|178732|gb|AAA51741.1| LGTTISFERINDVLEHVKHFVINPYWDFEVAEKINAFRAKVHELIERYEV 680 translated -------------------------------------------------- gi|4502153|ref|NP_000375.1| DQQIQVLMDKLVELTHQYKLKETIQKLSNVLQQVKIKDYFEKLVGFIDDA 2400 gi|178732|gb|AAA51741.1| DQHIQVLMDKLVELAHQYKLKETIQKLSNVLQQVKIKDYFEKLVGFID-- 728 translated -------------------------------------------------- gi|4502153|ref|NP_000375.1| VKKLNELSFKTFIEDVNKFLDMLIKKLKSFDYHQFVDETNDKIREVTQRL 2450 gi|178732|gb|AAA51741.1| -------------------------------------------------- translated -------------------------------------------------- gi|4502153|ref|NP_000375.1| NGEIQALELPQKAEALKLFLEETKATVAVYLESLQDTKITLIINWLQEAL 2500 gi|178732|gb|AAA51741.1| -------------------------------------------------- translated -------------------------------------------------- gi|4502153|ref|NP_000375.1| SSASLAHMKAKFRETLEDTRDRMYQMDIQQELQRYLSLVGQVYSTLVTYI 2550 gi|178732|gb|AAA51741.1| -------------------------------------------------- translated -------------------------------------------------- gi|4502153|ref|NP_000375.1| SDWWTLAAKNLTDFAEQYSIQDWAKRMKALVEQGFTVPEIKTILGTMPAF 2600 gi|178732|gb|AAA51741.1| -------------------------------------------------- translated -------------------------------------------------- gi|4502153|ref|NP_000375.1| EVSLQALQKATFQTPDFIVPLTDLRIPSVQINFKDLKNIKIPSRFSTPEF 2650 gi|178732|gb|AAA51741.1| -------------------------------------------------- translated -------------------------------------------------- gi|4502153|ref|NP_000375.1| TILNTFHIPSFTIDFVEMKVKIIRTIDQMQNSELQWPVPDIYLRDLKVED 2700 gi|178732|gb|AAA51741.1| -------------------------------------------------- translated -------------------------------------------------- gi|4502153|ref|NP_000375.1| IPLARITLPDFRLPEIAIPEFIIPTLNLNDFQVPDLHIPEFQLPHISHTI 2750 gi|178732|gb|AAA51741.1| -------------------------------------------------- translated -------------------------------------------------- gi|4502153|ref|NP_000375.1| EVPTFGKLYSILKIQSPLFTLDANADIGNGTTSANEAGIAASITAKGESK 2800 gi|178732|gb|AAA51741.1| -------------------------------------------------- translated -------------------------------------------------- gi|4502153|ref|NP_000375.1| LEVLNFDFQANAQLSNPKINPLALKESVKFSSKYLRTEHGSEMLFFGNAI 2850 gi|178732|gb|AAA51741.1| -------------------------------------------------- translated -------------------------------------------------- gi|4502153|ref|NP_000375.1| EGKSNTVASLHTEKNTLELSNGVIVKINNQLTLDSNTKYFHKLNIPKLDF 2900 gi|178732|gb|AAA51741.1| -------------------------------------------------- translated -------------------------------------------------- gi|4502153|ref|NP_000375.1| SSQADLRNEIKTLLKAGHIAWTSSGKGSWKWACPRFSDEGTHESQISFTI 2950 gi|178732|gb|AAA51741.1| -------------------------------------------------- translated -------------------------------------------------- gi|4502153|ref|NP_000375.1| EGPLTSFGLSNKINSKHLRVNQNLVYESGSLNFSKLEIQSQVDSQHVGHS 3000 gi|178732|gb|AAA51741.1| -------------------------------------------------- translated -------------------------------------------------- gi|4502153|ref|NP_000375.1| VLTAKGMALFGEGKAEFTGRHDAHLNGKVIGTLKNSLFFSAQPFEITAST 3050 gi|178732|gb|AAA51741.1| -------------------------------------------------- translated -------------------------------------------------- gi|4502153|ref|NP_000375.1| NNEGNLKVRFPLRLTGKIDFLNNYALFLSPSAQQASWQVSARFNQYKYNQ 3100 gi|178732|gb|AAA51741.1| -------------------------------------------------- translated -------------------------------------------------- gi|4502153|ref|NP_000375.1| NFSAGNNENIMEAHVGINGEANLDFLNIPLTIPEMRLPYTIITTPPLKDF 3150 gi|178732|gb|AAA51741.1| -------------------------------------------------- translated -------------------------------------------------- gi|4502153|ref|NP_000375.1| SLWEKTGLKEFLKTTKQSFDLSVKAQYKKNKHRHSITNPLAVLCEFISQS 3200 gi|178732|gb|AAA51741.1| -------------------------------------------------- translated -------------------------------------------------- 178 179 gi|4502153|ref|NP_000375.1| IKSFDRHFEKNRNNALDFVTKSYNETKIKFDKYKAEKSHDELPRTFQIPG 3250 gi|178732|gb|AAA51741.1| -------------------------------------------------- translated INSFNRHFETVRDKALDFFTESYNEIKITFDKYKVEKPLDQQPRTFQIPG gi|4502153|ref|NP_000375.1| YTVPVVNVEVSPFTIEMSAFGYVFPKAVSMPSFSILGSDVRVPSYTLILP 3300 gi|178732|gb|AAA51741.1| -------------------------------------------------- translated YTIPVINIDVSPFTVKMETFGYVIPKEISTPNITILGSGISVPSYTLGLQ gi|4502153|ref|NP_000375.1| SLELPVLHVPRNLKLSLPHFKELCTISHIFIPAMGNITYDFSFKSSVITL 3350 gi|178732|gb|AAA51741.1| -------------------------------------------------- translated FLELPALDVPRNLQISLPEL------------------------------ ??????????????????????. gi|4502153|ref|NP_000375.1| YMKLAPGELTIIL 4563 gi|178732|gb|AAA51741.1| ------------- translated ------------- VI. CONCLUSION AND PERSPECTIVE Genetic selection, and the development and application of repartitioning agents to shift nutrients from fat deposition to protein deposition in the production of leaner animals, are research areas of major economic importance to the livestock producer in providing food for improved nutrition and health of consumers. Nutritional manipulation of energy to protein ratio and feed energy intake during the production cycle is an additional option to reduce fatness without compromising efficiency of growth. The theory of nutrient partitioning, as originally proposed by Sir John Hammond, for the maintenance and growth of individual tissues occurs in a hierarchial manner to those tissues essential for survival of the species. In growing animals, muscle and adipose tissues (edible meat) develop relative to genetic potential, but are of low priority with respect to nutrient utilization for maintenance requirements (358). Nutrient partitioning to optimize efficiency of meat production can be gained 1) through improvement of genetic composition by selection or gene alteration, and 2) hormonal and neuroendocrine strategies to alter appetite, reduce stress and favor muscle accretion rather than adipose tissue deposition in a healthy environment for animal well-being (359). From a genomics perspective, nutritients are dietary signals that are detected by the cellular sensor systems that influence gene expression, protein translation, and metabolite synthesis (360). Genomic tools can be used in two different but complementary strategies 180 in molecular nutrition research. One is the traditional hypothesis-driven approach: specific genes and proteins, the expression of which is influenced by nutrients, and identified using genomic tools (361). The other strategy is largely theoretical at this stage, is the ?systems biology? approach where gene, protein and metabolite signatures that are associated with specific nutrients, or nutritional regimes, are catalogued, and might provide ?early warning? molecular biomarkers for nutrient-induced changes to homeostasis (362). In this dissertation, the first strategy was used but expanded to a discovery micorarray approach, to get detailed molecular data on whole genome responses in muscle, liver and adipose tissues in pigs administered RAC or fed high fat diets. On the basis of my experiments, it seems prudent to conclude that 1) These studies showed that micro-arrays are able to detect transcriptional changes resulting from feeding 60 ppm ractopamine and an 16 % increase in dietary fat. 2) Ractopamine up regulated the expression of genes PPARa and CPTII, and decreased expression of genes encoding enzymes in fatty acid synthesis and electron transport in the adipose tissue in pigs fed the beta adrenergic agonist for 28 days. 3) After the diet was shifted from LFD to HFD, the mRNA level of genes in fatty acid synthesis, FABP and fatty acid oxidation were decreased in the liver; expression of FABP and genes in the TAG synthesis were increased in the adipose tissue; transcription of CPT1 and 3-hydroxyacyl-CoA dehydrogenase and some genes in the oxidative phosphorylation were up regulated in muscle tissue. Comparing effects of RAC and high fat on the gene expression in pigs, RAC is a ?- adrenergic agonist added to the diet as a pure compound in small doses, but RAC acts 181 with high affinity and selectivity for a limited number of biological targets. Most nutrients (carbohydrate, protein and fat) are softer dietary signals, and their net effect must be considered in the context of chronic exposure (363). An animal has to process a large number of different nutrients and other diet components, but nutrients can reach high concentrations without becoming toxic. Each nutrient can also bind to numerous targets with different affinities and specificities. In this study, the oligo-array analysis detected wider and stronger transcription response in RAC-treated pigs than those noted in pigs after the diet was shifted from LFD to HFD. In RAC study, transcription of 1,299 genes was altered while around 847 genes/ESTs in pig tissues switched to a HFD showed significant differences in expression. One of the major challenges that remain from the work is how to interpret differently expressed genes/transcripts, such as a group of phosphatases in the muscle of high-fat treated pigs that were not part of the gene filter assignment corresponding to the hypothesis. Future data mining, even of results generated here, may be important to fully exploit my micro-array result. The type of follow-ups may range from bioinformatic analysis of presently non analyzed raw data in the arrays, application of real-time PCR analysis of specific genes, promoter function and structure analyses to designing new experiments to further explore gene expression changes from in pigs exposed to RAC or other dietary combinations. Perhaps more importantly, my study showed that there are significant numbers of uncharacterized transcripts in this micro-array platform. Using bioinformaticsl tools and molecular methods, it is possible to discover new genes, which may provide opportunities 182 for characterization of encoded proteins of unknown function and their interaction in the process of metabolism pathways. When I first joined the Bergen?s laboratory, research activities focused on developing gene-specific cDNA probes by cloning new sequences/DNA fragments of genes rate limiting in porcine lipid metabolism and measuring or determining mRNA abundance of various transcripts in porcine adipose tissue using Northern blotting. Obviously, these methods were laborious, and the number of characterized genes available for such studies are limiting. My study was the first to comprehensively characterize expression responses in the porcine transcriptome to ractopamine and dietary high fat. If the high-fat study were to be repeated, I would first like to increase the dietary fat content; I would further increase the number of animals per group and measure the plasma metabolone and complete plasma lipoproteins profile for all treatments. In addition, I would confirm the many of differently expressed genes in high fat micro-array analysis with real-time PCR. This would require a lot of preliminary work to obtain appropriate primes for each of the genes, and would take extensive laboratory work to set up many qRT-PCR assays and would be quite costly. I would also like to expand the dietary lipid profile to include substantial amounts cholesterol and study its effect on the transcription of genes in lipoprotein and cholesterol metabolism pathways. Furthermore, I would add another experimental group with even higher fat content and thus lower dietary carbohydrate content to explore the expression of genes that may cross-talk in fatty acid and glucose oxidation. Additionally, I might recommend a study applying Western blot or enzyme activity analysis to determine if changes happened on the proteins encoded by genes with changed expression in transcription level. For those 183 genes expressing opposite changes of mRNA level in different tissues (e.g transcription of FABP was decreased in the liver but increased in adipose and muscle in response to high fat diet) if confirmed by qRT-PCR, there is a need to characterize the potential differences in the promoter regions of these gene isoforms in various tissues. Such a characterization may help to explain gene expression response differences observed in this study between tissues that use fatty acids, store fatty acids and in the adipose tissue lipogenic response to treatments. Data presented in this dissertation provide the first description of the transcriptome response of lipid metabolism genes in the finishing pigs treated by ractopamine or high fat. In the absence of complete gene information in domestic pig, an oligo-array platform was used to determine the transcription profile of fatty acid metabolism in the pig. Data from Chapter 2 confirmed that short-term effects of ractopamine on decreasing fatty acid synthesis and increasing fatty acid ?-oxidation at transcription level. Results in Chapter 3 presented evidence that transcription responses to dietary fat were specific in different tissues. This work provided a basis to understand the relationship between dietary fat and gene expression among tissues of pigs. Micro-array from my experience is a useful tool to discover genes that may be important as targets in the further research. However, we should be aware that simply accumulating micro-array datasets alone can not lead to important insights. 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Integrity of the total RNA was evaluated by gel electrophoresis of ~1 ?g RNA on an 1% agarose 0.5? tris(hydroxymethyl)aminomethane (Tris)-borate-EDTA buffer (TBE) gel containing ethidium bromide, at 120V for 30 minutes using 1? TBE as the running buffer. Images of the gels were taken under ultraviolet (UV) light using Polaroid instant film number 55 to generate printed image of the gel. All RNAs were resoled and imaged using same electrophoresis and imaging methods. Fig 1-10 were images of RNA captured during the period of microarray analysis, and Fig 11-13 were images of RNA captures on April of 2006 after receiving comments and suggestion from the committees. Fig 1. Images of RNAs developed on April 4, 2005. RNAs were isolated from liver tissue in sudden dietary shift study (Chapter 3). The lanes from left to right are: DNA marker, #5207, #5502, #6001. Fig 2. Images of RNAs developed on May 23, 2005. RNAs were isolated from liver tissue in sudden dietary shift study (Chapter 3). The lanes from left to right are: #4901, #6002, #5504, #4905. 210 Fig 3. Images of DNase-treated RNAs developed on May 24, 2005. RNAs were isolated from liver tissue in sudden dietary shift study (Chapter 3). The lanes from left to right are: #6002, #5504, #4905, control pool RNA. Fig 4. Images of RNAs developed on June 18, 2005. RNAs were isolated from liver tissue in sudden dietary shift study (Chapter 3). The lanes from left to right are: #4901, #6002, #5504, #5205, #4905. Fig 5. Images of RNAs developed on July 13, 2005. RNAs were isolated from liver tissue in sudden dietary shift study (Chapter 3). The lanes from left to right are: #4901, #6002, #5504, #5205, #5207, #4905, #5502, #6001. 211 Fig 6. Images of RNAs developed on July 21, 2005. RNAs were isolated from adipose tissue in sudden dietary shift study (Chapter 3). The lanes from left to right are: #4901, #6002, #5504, #5205, #5207, #6001, #5502, #4905. Fig 7. Images of RNAs developed on July 26, 2005. RNAs were isolated from adipose tissue in sudden dietary shift study (Chapter 3). The lanes from left to right are: #4901, #5205, #5504, #6002. 212 Fig 8. Images of RNAs developed on August 2, 2005. RNAs were isolated from adipose tissue in sudden dietary shift study (Chapter 3). The lanes from left to right are: #5502, #5207, #4905, #6001, #6002, #5205, #5504, #4901. Fig 9. Images of RNAs developed on Sempter 13, 2005. RNAs were isolated from muscle tissue in sudden dietary shift study (Chapter 3). The lanes from left to right are: #4901, #5205, #5504, #6002, #4905, #5207, #5502, #6001. Fig 10. Images of RNAs developed on Sempter 13, 2005. RNAs were isolated from adipose tissue in Paylean study (Chapter 2). The lanes from left to right are: #787, #784, #779, #796, #807, #826. 213 Fig 11. Images of RNAs developed on April 13, 2006. RNAs were isolated from adipose tissue in Paylean study (Chapter 2). The lanes from left to right are: #784, #796, #779, #826, #807. Fig 12. Images of RNAs developed on April 14, 2006. RNAs were isolated from liver tissue in sudden diet shift study (Chapter 3). The lanes from left to right are: #4901, #5207, #4905, #6002. 214 Fig 13. Images of RNAs developed on April 14, 2006. RNAs were isolated from muscle tissue in sudden diet shift study (Chapter 3). The lanes from left to right are: #5502, #5207, #4905, #6001, #4901, #5205,#6002,#5504. 215 Appendix B. Microarray Analysis Protocol References: Amino Allyl Labeling of RNA for microarrays protocol. Functional Genomics Lab, W. M. Keck Center for Functional and Comparative Genomics, University of Illinois at Urbana-Champaign. AMINOALLYL LABELING OF RNA FOR MICROARRAYS 1. PURPOSE This protocol describes the labeling of eukaryotic RNA with aminoallyl labeled nucleotides via first strand cDNA synthesis followed by a coupling of the aminoallyl groups to either Cyanine 3 or 5 (Cy 3/Cy5) fluorescent molecules. 2. SCOPE This procedural format is utilized by Human Colon Cancer and Mouse microarray projects under the supervision of within the Eukaryotic Genomics Dept. TIGR 3. MATERIALS 3.1 5-(3-aminoallyl)-2?deoxyuridine-5?-triphosphate (AA-dUTP) (Sigma; Cat # A0410) 3.2 100 mM dNTP Set PCR grade 3.3 Random Hexamer primers (3mg/mL) or polydT 18-22 oligo 3.4 SuperScript III RT (200U/uL) (Invitrogen Cat # 18080-044) 3.5 Cy Dye Post Labelling reactive dye pack (Amersham-GE RPN5661) or Invitrogen- Molecular Probes Alexa Fluor Dye Decapack A32755 3.7 QIAquick PCR Purification Kit (Qiagen; Cat # 28106) 3.8 RNeasy? Mini Kit (Qiagen; Cat # 74106) 4. REAGENT PREPARATION 4.1 Phosphate Buffers 4.1.1 Prepare 2 solutions: 1M K2HPO4 and 1M KH2PO4 4.1.2 To make a 1M Phosphate buffer (KPO4, pH 8.5-8.7) combine: 1M K2HPO4??..9.5 mL 1M KH2PO4??..0.5 mL 4.1.3 For 100 mL Phosphate wash buffer (5 mM KPO4, pH 8.0, 80% EtOH) mix: 1 M KPO4 pH 8.5?. 0.5 mL MilliQ water???... 15.25 mL 95% ethanol???... 84.25 mL Note: Wash buffer will be slightly cloudy. 4.1.4 Phosphate elution buffer is made by diluting 1 M KPO4, pH 8.5 to 4 mM with MilliQ water. 4.2 Aminoallyl dUTP 4.2.1 For a final concentration of 100mM add 19.1 ?L of 0.1 M KPO4 buffer (pH 7.5) to a stock vial containing 1 mg of aa-dUTP. Gently vortex to mix and transfer the aa-dUTP solution into a new microfuge tube. Store at ?20 o C. 216 4.2.2 Measure the concentration of the aa-dUTP solution by diluting an aliquot 1:5000 in 0.1 M KPO4 (pH 7.5) and measuring the OD289. (Stock concentration in mM = OD289 x 704) 4.3 Labeling Mix (50X) with 2:3 aa-dUTP: dTTP ratio 4.3.1 Mix the following reagents: Final concentration dATP (100 mM)???5?L?... (25 mM) dCTP (100 mM)???5?L?... (25 mM) dGTP (100 mM)???5?L?... (25 mM) dTTP (100 mM)???3?L??(15 mM) aa-dUTP (100 mM)?...2?L??(10 mM) Total: 20?L 4.3.1 Store unused solution at ?20 o C. 4.4 Sodium Carbonate Buffer (Na2CO3): 1M, pH 9.0 4.4.1 Dissolve 2.7 gram sodium carbonate in 20 ml and adjust pH to 9.0 with 12N HCl. Fill to 25 ml. 4.4.2 To make a 0.1 M solution for the dye coupling reaction dilute 1:10 with water. Note: Carbonate buffer changes composition over time; make it fresh each day when use. 4.5 Cy-dye esters 4.5.1 Cy3-ester and Cy5-ester are provided as a dried product in 5 tubes. Resuspend a tube of dye ester in 45 uL of DMSO before use. 4.5.2 Wrap all reaction tubes with foil and keep covered as much as possible in order to prevent photobleaching of the dyes. Note: Dye esters must either be used immediately or aliquoted and stored at ?80 o C. Any introduced water to the dye esters will result in a lower coupling efficiency due to the hydrolysis of the dye esters. Since DMSO is hygroscopic (absorbs water from the atmosphere) store it well sealed in desiccant. 5. PROCEDURE 5.1 Aminoallyl Labeling 5.1.1 To 10 ug (10-20 ug) of total RNA (or 2 ug poly(A+) RNA) which has been DNase I-treated and Qiagen RNeasy purified, add 2 uL Random Hexamer primers (3mg/mL) (We use 3 ug of dT 18 primer for total RNA), spiking controls (if desired), and bring the final volume up to 18.5 L with RNase-free or DEPC treated water. 5.1.2 Mix well and incubate at 70 o C for 10 minutes. 5.1.3 Place on ice for 30 seconds, centrifuge. 5.1.4 Add: 5X First Strand buffer????.. 6 uL 0.1 M DTT????????.. 3 uL 50X aminoallyl-dNTP mix??.. 0.6 uL SuperScript III RT (200U/uL)?.. 2 uL 217 (We also add 0.5 ul or 20U RNAse inhibitor) 5.1.5 Mix and incubate at 46 o C for 3 hours to overnight. 5.1.6 To hydrolyze RNA, add: 1 M NaOH 10 uL 0.5 M EDTA 10 uL mix and incubate at 65 o C for 15 minutes. 5.1.7 Add 10 uL of 1 M HCl to neutralize pH. Add 10 ul sodium acetate 3M. 5.2 Reaction Purification I: Removal of unincorporated aa-dUTP and free amines (Qiagen PCR Purification Kit) Note: This purification protocol is modified from the Qiagen QIAquick PCR purification kit protocol. The phosphate wash and elution buffers (prepared in 4.1.3 & 4.1.4) are substituted for the Qiagen supplied buffers because the Qiagen buffers contain free amines which compete with the Cy dye coupling reaction. 5.2.1 Mix cDNA reaction with 300 uL (5X reaction volume) buffer PB (Qiagen supplied) and transfer to QIAquick column. 5.2.2 Place the column in a 2 ml collection tube (Qiagen supplied) and centrifuge at ~13,000 rpm (10,000g) for 1 minute. Empty collection tube. 5.2.3 To wash, add 750 uL phosphate wash buffer to the column and centrifuge at ~13,000 rpm (10,000g) for 1 minute. 5.2.4 Empty the collection tube and repeat the wash and centrifugation step (5.2.3). 5.2.5 Empty the collection tube and centrifuge column an additional 1 minute at maximum speed. 5.2.6 Transfer column to a new 1.5 mL microfuge tube and carefully add 30 uL phosphate elution buffer (see 4.1.4) to the center of the column membrane. 5.2.7 Incubate for 1 minute at room temperature. 5.2.8 Elute by centrifugation at ~13,000 rpm(10,000g) for 1 minute. 5.2.9 Elute a second time into the same tube by repeating steps 5.2.6- 5.2.8. The final elution volume should be ~60 uL. 5.2.10 Dry sample in a speed vac. (about 1 hr.) 5.3 Coupling aa-cDNA to Cy Dye Ester. 5.3.1 Resuspend single pack of dye in 9.0 ul 0.1M sodium carbonate. Transfer dye to sample and mix. Note: To prevent photobleaching of the Cy dyes wrap all reaction tubes in foil and keep them sequestered from light as much as possible. 5.3.3 Incubate the reaction for 1 hour in the dark at room temperature. 5.4 Reaction Purification II: Removal of uncoupled dye (Qiagen PCR Purification Kit) 5.4.1 To the reaction add 35 uL 3M NaOAc pH 5.2. 5.4.2 Add 250 uL (5X reaction volume) Buffer PB (Qiagen supplied). 5.4.3 Place a QIAquick spin column in a 2 mL collection tube (Qiagen supplied), apply the sample to the column, and centrifuge at 10,000 g for 1 minute. Empty collection tube. 218 5.4.4 To wash, add 0.75 mL Buffer PE (Qiagen supplied) to the column and centrifuge at ~13,000 rpm (10,000g) for 1 minute. Note: Make sure Buffer PE has added ethanol before using (see label for correct volume). 5.4.5 Empty collection tube and centrifuge column for an additional 1 minute at maximum speed. 5.4.6 Place column in a clean 1.5 mL microfuge tube and carefully add 40 uL Buffer EB (Qiagen supplied) to the center of the column membrane. 5.4.7 Incubate for 1 minute at room temperature. 5.4.8 Elute by centrifugation at ~13,000 rpm (10,000g) for 1 minute. 5.4.9 Elute a second time into the same tube by repeating steps 5.4.6- 5.4.8. The final elution volume should be ~80 uL. Note: This protocol is modified from the Qiagen QIAquick Spin 5.5 Analysis of Labeling Reaction 5.5.1 Use an 80 uL Beckman quartz MicroCuvette to analyze the entire undiluted sample in a spectrophotometer. 5.5.2 Wash the cuvette with water and blow dry with compressed air duster. 5.5.3 Pipette sample into cuvette and place cuvette in spectrophotometer. 5.5.4 For each sample measure absorbance at 260 nm and either 550 nm for Cy3 or 650 nm for Cy5, as appropriate. 5.5.5 Pipette sample from cuvette back into the original sample tube. 5.5.6 For each sample calculate the total picomoles of cDNA synthesized using: pmol nucleotides = [OD260 * volume (uL) * 37 ng/uL * 1000 pg/ng] 324.5 pg/pmol Note: 1 OD260 = 37 ng/uL for cDNA; 324.5 pg/pmol average molecular weight of a dNTP) 5.5.7 For each sample calculate the total picomoles of dye incorporation (Cy3 or Cy5 accordingly) using: pmol Cy3 = OD550 * volume (uL) /0.15 pmol Cy5 = OD650 * volume (uL) /0.25 nucleotides/dye ratio = pmol cDNA/pmol Cy dye Note: >30 pmol of dye incorporation per sample and a ratio of less than 50 nucleotides/dye molecules is optimal for hybridizations. 5.5.9 Dry the Cy3/Cy5 probes to completion in a speed vac. Hybridization (Corning GAPS II Aminosilane coated slides) Prehybridization 1. Preheat prehybridization solution in 42C water bath. Place slides in Coplin jar with the array towards the bottom of the jar. Fill with prehyb solution until array is covered. Allow to incubate in 42C water bath for 45 min. shaking occasionally. 2. Wash in millipore water 5 times and then isopropanol. Spin dry. (Spin in 50 ml tube with kimwipe stuffed in the bottom in a swing bucket rotor. Place array side up) 219 3. Hybridize immediately (or as soon as possible). TIGR Prehyb buffer 5 X SSC 25 ml 20 X SSC 0.1% SDS 1 ml 10% SDS 1% BSA 1 g BSA (Sigma A-9647) fill to 100 ml. Alternative Prehyb Buffer: 250 ml. (This is a hybridization buffer that we traditionally used on membranes but works well on Aminosilane coated slides as well) 20% Formamide 50 ml Formamide 5X Denhardts 25 ml 50X Denhards 6X SSC 83 ml 20X SSC 0.1% SDS 2.5 ml 10% SDS 25 ug/ml tRNA 0.625 ml 10 mg/ml tRNA H 2 O 88.5 ml 250 ml Hybridization 1. For a 48 pin slide (22 x 60mm Lifterslip coverslip) add 20 ug COT-1, 20 ug PolyA to one probe and fill to 40ul with sterile water. Transfer solution to second probe and mix well with pipettor. 2. Probe is heated at 95 o C for 2-3 min. 3. Position slide in corning hyb chamber with Lifterslip covering array area. 4. Add 40 ul of 2X hybridization buffer preheated to 42 C and apply to slide. 2X Hybridization buffer: 50% Formamide 5 ml 10X SSC 5 ml 20X SSC 0.2% SDS 200 ul 10% SDS 5. Slide is placed in Corning Hybridization Chamber with 2 small pieces of tissue paper saturated with water on either end of the slide, and hybridized 16-20 hours (overnight) or longer at 42C. (It has been shown that 2 or 3 days gives even better results.) Wash 1. First wash- 1X SSC, 0.2% SDS at 42C, coverslip is removed by agitation 5 min. 2. Second wash- 0.1%X SSC, 0.2% SDS at room temperature, agitation 5 min. 3. Third wash- 0.1X SSC, agitation 5 min. Repeat step 3 in wash 3 again. 4. An extra wash may be needed if high background is visible after scanning. 5. Spin dry immediately. Any droplet that dries on the surface of the slide will leave background haze. Wash 1: 50 ml 20XSSC, 20 ml 10% SDS, fill to 1 liter Wash 2: 5 ml 20XSSC, 20 ml 10% SDS, fill to 1 liter Wash 3: 5 ml 20XSSC, fill to 1 liter 220 Appendix C. Images of fluorescent dye Cy-5 labeled cDNA probe for slides hybridization in microarray analysis Fig 1-15. Cy5-labeled cDNA probe was resolved on a 2% agarose gel at 130V for 45 minutes, the gel was imaged on Typhoon 9410 (Amersham Pharmacia Biotech) at PMT 600 using emission filter for Cy5 (laser 633nm). All the probes were resoled and imaged using same electrophoresis and imaging condition. Fig 1-3 were images of probes used in Paylean study (Chapter 2) and Fig 4-15 were images of probes used in sudden dietary shift study (Chapter 3). Fig 1. Image of Cy5 labeled cDNA probe developed from RNA preparation of adipose tissue (pig number: #784) in Paylean study (Chapter 2). The probe was prepared on November 3, 2005. 221 Fig 2. Image of Cy5 labeled cDNA probe developed from RNA preparation of adipose tissue (pig number: #796) in Paylean study (Chapter 2). The probe was prepared on November 8, 2005. Fig 3. Image of Cy5 labeled cDNA probe developed from control pool RNA preparation of adipose tissue in Paylean study (Chapter 2). The probe was prepared on November 17, 2005. 222 Fig 4. Image of Cy5 labeled cDNA probe developed from RNA preparation of liver tissue (pig number: #6001) in sudden dietary shift study (Chapter 3). The probe was prepared on July 11, 2005. Fig 5. Image of Cy5 labeled cDNA probe developed from control pool RNA preparation of liver tissue in sudden dietary shift study (Chapter 3). The probe was prepared on July 18, 2005. 223 Fig 6. Image of Cy5 labeled cDNA probe developed from control pool RNA preparation of liver tissue in sudden dietary shift study (Chapter 3). The probe was prepared on July 24, 2005. Fig 7. Image of Cy5 labeled cDNA probe developed from RNA preparation of liver tissue (pig number: #5207) in sudden dietary shift study (Chapter 3). The probe was prepared on July 27, 2005. 224 Fig 8. Image of Cy5 labeled cDNA probe developed from control pool RNA preparation of adipose tissue in sudden dietary shift study (Chapter 3).The probe was prepared on August 1, 2005. Fig 9. Image of Cy5 labeled cDNA probe developed from RNA preparation of adipose tissue (pig number: #6001) in sudden dietary shift study (Chapter 3). The probe was prepared on August 5, 2005. 225 Fig 10. Image of Cy5 labeled cDNA probe developed from control pool RNA preparation of adipose tissue in sudden dietary shift study (Chapter 3). The probe was prepared on September 9, 2005. Fig 11. Image of Cy5 labeled cDNA probe developed from RNA preparation of adipose tissue (pig number: #5502) in sudden dietary shift study (Chapter 3). The probe was prepared on Octorber10, 2005. 226 Fig 12. Image of Cy5 labeled cDNA probe developed from RNA preparation of muscle tissue (pig number: #4905) in sudden dietary shift study (Chapter 3). The probe was prepared on September 19, 2005. Fig 13. Image of Cy5 labeled cDNA probe developed from control pool RNA preparation of muscle tissue in sudden dietary shift study (Chapter 3). The probe was prepared on September 26, 2005. 227 Fig 14. Image of Cy5 labeled cDNA probe developed from control pool RNA preparation of muscle tissue in sudden dietary shift study (Chapter 3). The probe was prepared on September 30, 2005. Fig 15. Image of Cy5 labeled cDNA probe developed from RNA preparation of muscle tissue (pig number: #5207) in sudden dietary shift study (Chapter 3). The probe was prepared on October 3, 2005. 228 Appendix D. Pig Genome Oligo Set and Pig Genome Oligo Extension Set 1.0 Reference: QIAGEN Array-Ready Oligo Sets TM for the Pig Genome and the Pig Genome Oligo Extension Set, Version 1.0. Gene sequence source and selection All probes are designed from The Institute of Genome Research (TIGR) Gene Index Database SsGI Release5.0, released on Octable 1, 2002. (http://www.tigr.org/tdb/tgi/ssgi/). SsGI Release 5.0 contains a total of 49,201 unique sequences including 17,354 tentative consensus sequences (TCs), singleton 494 expressed transcripts (ETs), and 31.353 singleton ESTs. ETs represent mature transcripts. Table1. Gene sequence source of Pig Genome Oligo Set and Extension Set Version 1.0 TIGR Pig SsGI Release 5.0 Database Number of sequences in SsGI Release 5.0 Pig Genome Oligo Set Version 1.0 Pig Genome Oligo Extension Set Version 1.0 TCs 17,354 10,313 2632 Singleton ETs 494 352 0 Singleton ESTS 31,353 0 0 Total 49,201 10,665 2632 Pig Genome Oligo Set sequence selection All TCs and singleton ETs were aligned using BLAST versus known gene transcripts for human, mouse, and pig. These TCs and singleton ETs were aligned to 27,628 known human gene trancripts from ENSEMBL, 28,097 known mouse gene trancripts, and 1897 known pig gene transcripts from the Pig UniGene Build #10 (http://www.ncbi.nlm.nih.gov). Both the human and mouse ENSEMBL database are from January 2003 and were obtained from http://www.ensembl.org. All TCs and singleton ETs with a >75% identity over at least 100 bases to a known human, mouse, or pig gene transcript and yielding a designed probe <=70 crosshybridization identity is included in this set. Pig Genome Oligo Extension Set sequence selection All component ESTs used to make the TCs were obtained from GenBank at http://www.ncbi.nlm.nih.gov. All ESTs with the keyword 3?, denoting a 3 primer EST, were marked. A total of 7739 TCs were found at least one 3? EST. The TCs that contain at least one 3? EST and are not present in the Pig Genome Oligo Set are included in the Pig Genome Oligo Extension Set. 229 Table 2. Oligo sequence selection Pig Genome Oligo Set and Extension Set Version 1.0 Pig Genome Oligo Set Version 1.0 Pig Genome Oligo Extension Set Version 1.0 Number of oligos designed from a TC with at least one 3? EST 5005 2632 Number of oligos designed from a sequence with a hit to a known human, mouse, or pig gene transcript 10,665 172 Numer of oligos that have a <=70% crosshybridization identity to another sequence 10,665 2538 Total 10,665 2632 Sequence orientation TIGR obtains and predicts orientation for all the tentative consensus sequences and singletons based on various techniques including alignments to known proteins and poly A trimming. After SsGI 5.0 was released, TIGR later updated orientation of 7218 of the sequences in this database. A total of 628 of these are TCs and the rest are singleton ESTs. Probes for these 628 TCs that appear in the sets are therefore designed in the updated orientation. In the gene list, a column indicates the orientation of the probe to the original TC sequence. All probes are designed in the sense strand as given by TIGR. 230 Table 3. Probe design and selection rules for Pig Genome Oligo Set and Extension Set 1.0 Oligo selection criteria Criteria values Number of oligos in the Pig Genome Oligo Set Version 1.0 satisfying these criteria Number of oligos in the Pig Genome Extension Set Version 1.0 satisfying these criteria Length 70mer Melting temperature 78 o C ? 5 o C Location from 3? end <=1000 Poly(N) tract length <8 Stem length in protein hairpin <9 Cross-hybridization identity to all other sequences <=70% Contiguous base match to any other sequence <=20 10,607 2418 Total number of oligos not satisfying one or more of the above criteria 58 214 Length 50mer 27* 10 ? Location from 3? end >1000 31* 6 ? Contiguous base match to any other sequence >20 0* 176 ? Cross-hybridization to all other sequences >70% 0* 94 ? Total 10,665 2632 *Out of 58 probes ? Out of 214 probes Quality check of probe design specification Once the final oligo set has been selected to represent a gene, each oligo undergoes design specifications quality control where we use an independent method to confirm that 231 all oligos have met the specified design specifications. The table below summarizes data from Qiagen quality check for probe design specifications for all probes. Table 4. Qiagen quality check for probe design specifications Probe design specification Expected value Verified range Number of oligos pig genome oligo set version 1.0 Number of oligos pig genome extension set version 1.0 Melting temperature ( o C) 78 o C ? 5 o C 73.6-82.9 10,665 2632 Cross- hybridization identity (%) <=70 31-70 10,665 2538 Cross- hybridization identity (%) >70 71-100 0 94 The following graphs and illustrations show the distribution of all 10,665 oligos representing the Pig Genome Oligo Set Version 1.0 followed by the 2632 oligos from the Pig Genome Oligo Extension Set for the melting temperature, GC content, location from 3? end, and longest stem length, and cross-hybridization identity. Fig1. Melting temperature-Pig genome oligo set 232 Fig2. GC content-Pig genome oligo set Fig3. Location from 3? end-Pig genome oligo set 233 Fig4. Longest hairpin stem length-Pig genome oligo set Fig5. Cross-hybridization identity-Pig genome oligo set 234 Fig6. Melting temperature-Pig genome oligo set Fig7. GC content-pig genome oligo extension set 235 Fig8. Location from 3? end-pig genome oligo extension set Fig9. Longest hairpin stem length-Pig genome oligo extension set 236 Fig10. Cross-hybridization identity-pig genome oligo extension set 237 Appendix E. Microarray images of Ractopamine experiment (Chapter 2) Fig 1. Ratio image of hybridization of adipose tissue RNAs from control pool RNA labeled by Cy5 and RNA from pig #779 labeled by Cy3. The image was obtained when scanning channels 532nm and 635 nm together by AXON 4000B laser scanner, and shown as ratio imaging (635/532). Detail scanning method was described in Materials and Methods in Chapter 2. 238 Fig 2. Ratio image of hybridization of adipose tissue RNAs from control pool RNA labeled by Cy3 and RNA from pig #796 labeled by Cy5. The image was obtained when scanning channels 532nm and 635 nm together by AXON 4000B laser scanner, and shown as ratio imaging (635/532). Detail scanning method was described in Materials and Methods in Chapter 2. 239 Fig 3. Ratio image of hybridization of adipose tissue RNAs from control pool RNA labeled by Cy3 and RNA from pig #784 labeled by Cy5. The image was obtained when scanning channels 532nm and 635 nm together by AXON 4000B laser scanner, and shown as ratio imaging (635/532). Detail scanning method was described in Materials and Methods in Chapter 2. 240 Appendix F. M-A plots of pro- and post- LOWESS normalization for Ractopamine experiment (Chapter 2) Fig 1. Median pixel intensities from adipose tissue of #779 (T) Cy3 labeled RNA and control pool (C) Cy5 labeled RNA hybridized to pig array presented as scatter plots of M= log 2 (T/C) vs. A= log 2 (T*C)/2. (A) before (pro-) normalization, (B) after (post-) LOWESS normalization (A) (B) Post-LOWESS-normalization 241 Fig 2. Median pixel intensities from adipose tissue of #796 (T) Cy5 labeled RNA and control pool (C) Cy3 labeled RNA hybridized to pig array presented as scatter plots of M= log 2 (T/C) vs. A= log 2 (T*C)/2. (A) before (pro-) normalization, (B) after (post-) LOWESS normalization (A) (B) 242 Fig 3. Median pixel intensities from adipose tissue of #784 (T) Cy5 labeled RNA and control pool (C) Cy3 labeled RNA hybridized to pig array presented as scatter plots of M= log 2 (T/C) vs. A= log 2 (T*C)/2. (A) before (pro-) normalization, (B) after (post-) LOWESS normalization (A) (B) 243 Appendix G. Microarray images of diet shifting experiment (Chapter 3) Fig 1. Ratio image of hybridization of liver tissue RNAs from control pool RNA labeled by Cy5 and RNA from pig #4905 labeled by Cy3. The image was obtained when scanning channels 532nm and 635 nm together by AXON 4000B laser scanner, and shown as ratio imaging (635/532). Detail scanning method was described in Materials and Methods in Chapter 2. 244 Fig 2. Ratio image of hybridization of liver tissue RNAs from control pool RNA labeled by Cy3 and RNA from pig #5207 labeled by Cy5. The image was obtained when scanning channels 532nm and 635 nm together by AXON 4000B laser scanner, and shown as ratio imaging (635/532). Detail scanning method was described in Materials and Methods in Chapter 2. 245 Fig 3. Ratio image of hybridization of liver tissue RNAs from control pool RNA labeled by Cy5 and RNA from pig #5502 labeled by Cy3. The image was obtained when scanning channels 532nm and 635 nm together by AXON 4000B laser scanner, and shown as ratio imaging (635/532). Detail scanning method was described in Materials and Methods in Chapter 2. 246 Fig 4. Ratio image of hybridization of liver tissue RNAs from control pool RNA labeled by Cy3 and RNA from pig #6001 labeled by Cy5. The image was obtained when scanning channels 532nm and 635 nm together by AXON 4000B laser scanner, and shown as ratio imaging (635/532). Detail scanning method was described in Materials and Methods in Chapter 2. 247 Fig 5. Ratio image of hybridization of adipose tissue RNAs from control pool RNA labeled by Cy5 and RNA from pig #4905 labeled by Cy3. The image was obtained when scanning channels 532nm and 635 nm together by AXON 4000B laser scanner, and shown as ratio imaging (635/532). Detail scanning method was described in Materials and Methods in Chapter 2. 248 Fig 6. Ratio image of hybridization of adipsoe tissue RNAs from control pool RNA labeled by Cy5 and RNA from pig #5207 labeled by Cy3. The image was obtained when scanning channels 532nm and 635 nm together by AXON 4000B laser scanner, and shown as ratio imaging (635/532). Detail scanning method was described in Materials and Methods in Chapter 2. 249 Fig 7. Ratio image of hybridization of adipsoe tissue RNAs from control pool RNA labeled by Cy3 and RNA from pig #5502 labeled by Cy5. The image was obtained when scanning channels 532nm and 635 nm together by AXON 4000B laser scanner, and shown as ratio imaging (635/532). Detail scanning method was described in Materials and Methods in Chapter 2. 250 Fig 8. Ratio image of hybridization of adipose tissue RNAs from control pool RNA labeled by Cy3 and RNA from pig #6001 labeled by Cy5. The image was obtained when scanning channels 532nm and 635 nm together by AXON 4000B laser scanner, and shown as ratio imaging (635/532). Detail scanning method was described in Materials and Methods in Chapter 2. 251 Fig 9. Ratio image of hybridization of muscle tissue RNAs from control pool RNA labeled by Cy3 and RNA from pig #5207 labeled by Cy5. The image was obtained when scanning channels 532nm and 635 nm together by AXON 4000B laser scanner, and shown as ratio imaging (635/532). Detail scanning method was described in Materials and Methods in Chapter 2. 252 Fig 10. Ratio image of hybridization of muscle tissue RNAs from control pool RNA labeled by Cy5 and RNA from pig #5502 labeled by Cy3. The image was obtained when scanning channels 532nm and 635 nm together by AXON 4000B laser scanner, and shown as ratio imaging (635/532). Detail scanning method was described in Materials and Methods in Chapter 2. 253 Fig 11. Ratio image of hybridization of muscle tissue RNAs from control pool RNA labeled by Cy3 and RNA from pig #4905 labeled by Cy5. The image was obtained when scanning channels 532nm and 635 nm together by AXON 4000B laser scanner, and shown as ratio imaging (635/532). Detail scanning method was described in Materials and Methods in Chapter 2. 254 Fig 12. Ratio image of hybridization of muscle tissue RNAs from control pool RNA labeled by Cy5 and RNA from pig #6001 labeled by Cy3. The image was obtained when scanning channels 532nm and 635 nm together by AXON 4000B laser scanner, and shown as ratio imaging (635/532). Detail scanning method was described in Materials and Methods in Chapter 2. 255 Appendix H. M-A plots of pro- and post- LOWESS normalization for diet shifting experiment (Chapter 3) Fig 1. Median pixel intensities from liver tissue of #4905 (T) Cy3 labeled RNA and control pool (C) Cy5 labeled RNA hybridized to pig array presented as scatter plots of M=log 2 (T/C) vs. A= log 2 (T*C)/2. (A) before (pro-) normalization, (B) after (post-) LOWESS normalization (A) (B) 256 Fig 2 Median pixel intensities from liver tissue of #5207 (T) Cy5 labeled RNA and control pool (C) Cy3 labeled RNA hybridized to pig array presented as scatter plots of M= log 2 (T/C) vs. A= log 2 (T*C)/2. (A) before (pro-) normalization, (B) after (post-) LOWESS normalization (A) (B) 257 Fig 3 Median pixel intensities from liver tissue of #5502 (T) Cy3 labeled RNA and control pool (C) Cy5 labeled RNA hybridized to pig array presented as scatter plots of M= log 2 (T/C) vs. A= log 2 (T*C)/2. (A) before (pro-) normalization, (B) after (post-) LOWESS normalization (A) (B) 258 Fig 4. Median pixel intensities from liver tissue of #6001 (T) Cy5 labeled RNA and control pool (C) Cy3 labeled RNA hybridized to pig array presented as scatter plots of M= log 2 (T/C) vs. A= log 2 (T*C)/2. (A) before (pro-) normalization, (B) after (post-) LOWESS normalization (A) (B) 259 Fig 5 Median pixel intensities from adipose tissue of #4905 (T) Cy3 labeled RNA and control pool (C) Cy5 labeled RNA hybridized to pig array presented as scatter plots of M= log 2 (T/C) vs. A= log 2 (T*C)/2. (A) before (pro-) normalization, (B) after (post-) LOWESS normalization (A) (B) 260 Fig 6. Median pixel intensities from adipose tissue of #5207 (T) Cy3 labeled RNA and control pool (C) Cy5 labeled RNA hybridized to pig array presented as scatter plots of M= log 2 (T/C) vs. A= log 2 (T*C)/2. (A) before (pro-) normalization, (B) after (post-) LOWESS normalization (A) (B) 261 Fig 7. Median pixel intensities from adipose tissue of #5502 (T) Cy5 labeled RNA and control pool (C) Cy3 labeled RNA hybridized to pig array presented as scatter plots of M= log 2 (T/C) vs. A= log 2 (T*C)/2. (A) before (pro-) normalization, (B) after (post-) LOWESS normalization (A) (B) 262 Fig 8. Median pixel intensities from adipose tissue of #6001 (T) Cy5 labeled RNA and control pool (C) Cy3 labeled RNA hybridized to pig array presented as scatter plots of M= log 2 (T/C) vs. A= log 2 (T*C)/2. (A) before (pro-) normalization, (B) after (post-) LOWESS normalization (A) (B) 263 Fig9. Median pixel intensities from muscle tissue of #4905 (T) Cy5 labeled RNA and control pool (C) Cy3 labeled RNA hybridized to pig array presented as scatter plots of M= log 2 (T/C) vs. A= log 2 (T*C)/2. (A) before (pro-) normalization, (B) after (post-) LOWESS normalization (A) (B) 264 Fig 10. Median pixel intensities from muscle tissue of #5207 (T) Cy5 labeled RNA and control pool (C) Cy3 labeled RNA hybridized to pig array presented as scatter plots of M= log 2 (T/C) vs. A= log 2 (T*C)/2. (A) before (pro-) normalization, (B) after (post-) LOWESS normalization (A) (B) 265 Fig 11 Median pixel intensities from muscle tissue of #5502 (T) Cy3 labeled RNA and control pool (C) Cy5 labeled RNA hybridized to pig array presented as scatter plots of M= log 2 (T/C) vs. A= log 2 (T*C)/2. (A) before (pro-) normalization, (B) after (post-) LOWESS normalization (A) (B) 266 Fig 12 Median pixel intensities from muscle tissue of #6001 (T) Cy3 labeled RNA and control pool (C) Cy5 labeled RNA hybridized to pig array presented as scatter plots of M= log 2 (T/C) vs. A= log 2 (T*C)/2. (A) before (pro-) normalization, (B) after (post-) LOWESS normalization (A) (B) 267 Appendix I. List of transcripts up/down regulated (top 200 highest/lowest Log 2 ratio value) by dietary ractopamine supplement or dietary shifting (including Log 2 ratio of each microarray analysis for biological replications) Table 1. Transcripts highly up/down regulated determined by oligo-array in the adipose tissue by dietary supplement of Paylean T (60ppm) (Chapter 2). For each transcript, log 2 ratio=log 2 (Paylean T treated/ no Paylean T treated).The positive value means higher mRNA abundance in Paylean T treated pigs; negative value means lower mRNA abundance in Paylean T treated pigs Gene name Log 2 (R1/ G1) Log 2 (G2/ R2) Log 2 (G3/ R3) Average NADH/NADPHthyroidoxidasep138-tox[Susscrofa] 1.7701 2.7176 3.7438 2.7438 unknown 2.0000 1.0875 4.5437 2.5437 unknown 0.0825 2.5236 4.3030 2.3030 unknown 1.7814 1.5236 3.1525 2.1525 unknown 2.2095 0.8845 3.0470 2.0470 homologuetoGP2828068gbAAB99978.1BRCA1- associatedRINGdomainprotein{Homosapiens}.partial(15%) 1.1623 0.8826 4.0225 2.0225 surfactantproteinB[Susscrofa] 1.2224 1.8074 3.0149 2.0149 unknown 1.6699 1.3219 2.9959 1.9959 unknown 0.8365 1.0875 3.9620 1.9620 similartoGP15594005embCAC69823.p373c6.1(novelC2H2typezincfi ngerprotein){Homosapiens}.partial(19%) 1.6699 1.2479 2.9589 1.9589 25-hydroxyvitaminD31alpha-hydroxylase[Susscrofa] 1.6521 1.2630 2.9576 1.9576 unknown 1.3847 1.5146 2.9496 1.9496 homologuetoGP4235275gbAAD13152.1talin{Homosapiens}.partial(5 %) 1.3890 1.4406 2.9148 1.9148 similartoSPP51690ARSE_HUMANArylsulfataseEprecursor(EC3.1.6.- )(ASE).[Human]{Homosapiens}.partial(38%) 1.2044 0.6245 3.9144 1.9144 unknown 1.8875 0.9349 2.9112 1.9112 butyrophilin[Susscrofa] 1.8845 0.9260 2.9053 1.9053 GP2224539dbjBAA20759.1KIAA0299{Homosapiens}.partial(9%) 1.1375 1.6699 2.9037 1.9037 porcineInterleukin2[Susscrofa]interleukin- 2[Susscrofa]interleukin2precursor[Susscrofa] 1.3049 1.7948 2.5998 1.8998 unknown 1.6245 1.8699 2.1972 1.8972 unknown 1.6699 1.3155 2.6927 1.8927 pituitarytranscriptionfactor1beta[Susscrofa] 1.2505 1.8236 2.5871 1.8871 VLA-2[Susscrofa] 0.4150 2.0219 3.1685 1.8685 unknown 1.3536 1.3785 2.8661 1.8661 unknown 1.6130 1.3155 2.6642 1.8642 unknown 1.3626 1.4388 2.7507 1.8507 unknown 1.4919 1.7016 2.3467 1.8467 unknown 1.8182 0.6745 3.0463 1.8463 unknown 1.2016 1.8739 2.4378 1.8378 unknown 1.8231 1.2074 2.4152 1.8152 pot.ORF1(aa1-44)[Susscrofa]pot.ORF2(aa1- 19)[Susscrofa]mAChR(aa1-460)[Susscrofa] 2.0000 0.9215 2.5107 1.8107 h2-calponin[Susscrofa] 1.7801 1.3219 2.3010 1.8010 estradiolreceptorbeta[Susscrofa]estrogenreceptorbeta[Susscrofa]estr ogenreceptorbeta[Susscrofa] 1.1605 1.9150 2.2878 1.7878 268 matrixmetalloproteinase3precursor[Susscrofa] 1.1876 1.5656 2.5766 1.7766 similartoGP1684843gbAAB48302.1pinin{Bostaurus}.partial(19%) 1.0593 1.9686 2.2639 1.7639 unknown 1.0431 1.6507 2.5469 1.7469 unknown 0.9069 1.5850 2.7459 1.7459 Na+/H+exchangerisoform4[Susscrofa] 0.5525 1.4386 3.2456 1.7456 weaklysimilartoPIRI38488I38488trophinin-human.partial(37%) 1.4493 1.3297 2.4395 1.7395 homologuetoGP1054873gbAAA80977.1alpha- 2IXcollagen{Homosapiens}.partial(31%) 1.2663 1.2876 2.6270 1.7270 homologuetoGP6331022dbjBAA86578.1KIAA1264protein{Homosapi ens}.partial(9%) 1.3074 0.9375 2.9225 1.7225 unknown 0.8406 2.0000 2.3203 1.7203 homologuetoGP16444660gbAAL16407.1muscleatrophyF- boxprotein{Homosapiens}.partial(80%) 1.2630 1.4592 2.4111 1.7111 similartoSPO15375MOT6_HUMANMonocarboxylatetransporter6(MC T6)(MCT5).[Human]{Homosapiens}.partial(62%) 1.5677 1.2509 2.3093 1.7093 interleukin12receptorbeta2chain[Susscrofa]interleukin- 12receptorbeta2[Susscrofa] 1.5224 1.0931 2.5078 1.7078 weaklysimilartoGP15082550gbAAH12183.1Unknown(proteinforMGC: 20470){Homosapiens}.partial(34%) 1.3049 1.1069 2.7059 1.7059 unknown 1.1864 1.7224 2.2044 1.7044 unknown 1.2756 1.2325 2.6040 1.7040 unknown 1.9370 0.6699 2.5034 1.7034 homologuetoGP11693028gbAAG38938.1calcineurin- bindingproteincalsarcin-1{Homosapiens}.partial(49%) 1.5850 0.7140 2.7995 1.6995 DNA-directedRNApolymeraseIIpolypeptideB;POLR2B[Susscrofa] 1.3283 1.3677 2.3980 1.6980 unknown 1.0641 1.0219 2.9930 1.6930 unknown 1.5025 0.8814 2.6919 1.6919 unknown 0.8785 2.0000 2.1893 1.6893 myoglobin 1.4996 0.8745 2.6870 1.6870 unknown 1.5850 1.2802 2.1826 1.6826 steroidogenicfactor-1SF-1[Susscrofa] 1.5110 1.1541 2.3826 1.6826 weaklysimilartoSPQ14690RRP5_HUMANRRP5proteinhomolog(Frag ment).[Human]{Homosapiens}.partial(13%) 1.4964 1.0601 2.4783 1.6783 unknown 1.3081 1.0484 2.6782 1.6782 unknown 1.3923 0.9635 2.6779 1.6779 unknown 0.7536 2.0000 2.2768 1.6768 voltage-dependentKchannel[Susscrofa] 1.0000 3.3505 0.6752 1.6752 unknown 2.5443 1.7975 0.6709 1.6709 unknown 2.4044 1.9260 0.6652 1.6652 homologuetoGP14017779dbjBAB47410.MEGF11protein(KIAA1781){ Homosapiens}.partial(25%) 1.8679 2.0546 1.0612 1.6612 unknown 1.7162 2.6049 0.6605 1.6605 unknown 1.2388 2.6809 1.0599 1.6599 unknown 1.3985 0.8981 2.6483 1.6483 homologuetoGP17384624embCAC81020.kainatereceptorsubunit{Ho mosapiens}.partial(22%) 1.5146 1.1814 2.2480 1.6480 homologuetoGP13276649embCAB66508.hypotheticalprotein{Homos apiens}.partial(32%) 0.8625 1.4021 2.6323 1.6323 unknown 1.5146 2.1500 1.2323 1.6323 unknown 1.0000 1.7563 2.1282 1.6282 similartoSPQ9Y4L1OXRP_HUMAN150kDaoxygen- regulatedproteinprecursor(Orp150).[Human]{Homosapiens}.partial(17 %) 1.1979 1.3506 2.3243 1.6243 enamelinprecursor[Susscrofa] 1.3479 0.8931 2.6205 1.6205 269 homologuetoGP11138034dbjBAB17758.KIAA1173protein{Homosapi ens}.partial(31%) 1.3440 2.3931 1.1185 1.6185 sarcoendoplasmicreticulumcalciumATPase[Susscrofa] 1.2701 0.9668 2.6185 1.6185 homologuetoGP15559571gbAAH14146.1Unknown(proteinforMGC:2 0704){Homosapiens}.partial(57%) 0.9345 2.0000 1.9172 1.6172 unknown 0.7370 1.3854 2.7112 1.6112 myosinlightchain[Susscrofa]fastmyosinlightchain1F[Susscrofa] 1.3785 0.8413 2.6099 1.6099 weaklysimilartoGP7671629embCAB89275.2bA145L22.2(novelKRAB boxcontainingC2H2typezincfingerprotein){Homosapiens}.partial(27%) 1.3344 1.0835 2.4090 1.6090 similartoPIRA45771A457712-5A-dependentRNAase- human.partial(23%) 1.1454 1.4653 2.2054 1.6054 similartoGP8574032embCAB94769.1b24o18.4(proteaseserine16(thy mus)){Homosapiens}.partial(26%) 1.3668 0.8433 2.6050 1.6050 unknown 1.1926 1.3150 2.3038 1.6038 unknown 1.2439 0.9635 2.6037 1.6037 alpha-lactalbumin 0.6031 2.0000 2.2015 1.6015 unknown 0.9220 1.0768 2.7994 1.5994 homologuetoGP1665809dbjBAA13401.1SimilartoC.eleganshypotheti cal37.7kDprotein{Homosapiens}.partial(21%) 1.6130 0.5850 2.5990 1.5990 unknown 1.2288 0.9658 2.5973 1.5973 unknown 1.3561 1.0365 2.3963 1.5963 GP13905302gbAAH06949.1SimilartoATPaseclassIItype9A{Musmusc ulus}.partial(19%) 1.4150 0.7776 2.5963 1.5963 similartoGP13279167gbAAH04300.1Similartovillin- like{Homosapiens}.partial(18%) 0.7427 0.7004 3.3357 1.5929 similartoGP10438776dbjBAB15338.unnamedproteinproduct{Homosa piens}.partial(22%) 1.2630 1.9228 1.5929 1.5929 unknown 1.0153 1.7699 1.9926 1.5926 unknown 1.1864 0.9936 2.5900 1.5900 opticin[Susscrofa] 1.4330 1.2254 2.1092 1.5892 unknown 1.8657 0.7105 2.1881 1.5881 unknown 1.2563 0.9175 2.5869 1.5869 L-gulono-gamma-lactoneoxidase[Susscrofa] 1.0923 1.3809 2.2866 1.5866 unknown 1.6101 0.9607 2.1854 1.5854 similartoGP12840537dbjBAB24873.putative{Musmusculus}.partial(80 %) 0.7313 1.9359 2.0836 1.5836 unknown 1.1283 1.0381 2.5832 1.5832 interleukin-13[Susscrofa]interleukin-13[Susscrofa] 1.7162 0.6406 2.3784 1.5784 GP1145789gbAAA97870.1neuroligin2{Rattusnorvegicus}.partial(21% ) 1.1844 1.2696 2.2770 1.5770 unknown 1.3520 2.0000 1.3760 1.5760 unknown 1.0919 1.0566 2.5743 1.5743 weaklysimilartoGP8896138gbAAF81254.1pregnancy- associatedglycoprotein4{Susscrofa}.partial(25%) 0.8375 2.0000 1.8688 1.5688 unknown 1.0395 0.2969 3.3682 1.5682 unknown 0.8667 0.9685 2.8676 1.5676 albumin[Susscrofa] 1.3312 2.0000 1.3656 1.5656 apolipoproteinB 0.9069 0.8224 2.9646 1.5646 unknown 0.7271 2.0000 1.9636 1.5636 ART5protein[Susscrofa] 1.0105 1.7166 1.9635 1.5635 unknown 1.2479 0.8745 2.5612 1.5612 homologuetoGP1373169gbAAC50520.1autosomaldominantpolycysti ckidneydiseasetypeII{Homosapiens}.partial(19%) 1.1319 1.2875 2.2597 1.5597 unknown 1.2323 1.4854 1.9588 1.5588 270 unknown 1.3692 1.0454 2.2573 1.5573 unknown 1.2345 0.8745 2.5545 1.5545 homologuetoGP3114828embCAA06754.1JM5{Homosapiens}.partial( 31%) 1.4044 1.0004 2.2524 1.5524 similartoGP13324451gbAAK18752.1putativeGprotein- coupledreceptorGPCR1precursor{Homosapiens}.partial(64%) 1.5475 0.8525 2.2500 1.5500 unknown 0.9386 1.3575 2.3481 1.5481 weaklysimilartoGP12859945dbjBAB31821.putative{Musmusculus}.pa rtial(56%) 1.3458 1.2485 2.0471 1.5471 unknown 1.3785 1.0127 2.2456 1.5456 unknown 0.9831 1.6054 2.0442 1.5442 unknown 1.2410 1.3413 2.0412 1.5412 Tcellreceptorbeta-chain[Susscrofa]Tcellreceptorbeta- chain[Susscrofa] 1.1970 1.0826 2.3398 1.5398 unknown 1.3720 0.7063 2.5391 1.5391 unknown 1.0000 1.2753 2.3376 1.5376 unknown 1.1699 1.1931 2.2315 1.5315 homologuetoGP9651081dbjBAB03553.1hypotheticalprotein{Macacaf ascicularis}.partial(58%) 1.1979 1.3551 2.0265 1.5265 transmembraneleptinreceptor[Susscrofa] 1.1069 1.1411 2.3240 1.5240 similartoGP13183568gbAAK15262.1GTRGEO22{Musmusculus}.parti al(60%) 0.6903 2.0565 1.8234 1.5234 homologuetoGP3003021gbAAC08996.1acetylcholinesteraseglycoph ospholipid-anchoredformprecursor{Feliscatus}.partial(19%) 1.3505 0.9939 2.2222 1.5222 unknown 0.7444 2.0000 1.8222 1.5222 erythropoietinreceptor[Susscrofa] 0.9280 1.4150 2.2215 1.5215 unknown 1.0661 1.2758 2.2209 1.5209 unknown 1.5339 2.0000 1.0170 1.5170 voltage-dependentpotassiumchannel[Susscrofa]potassiumvoltage- gatedchannel[Susscrofa] 1.6479 1.7843 1.1161 1.5161 weaklysimilartoGP16550968dbjBAB71080.unnamedproteinproduct{H omosapiens}.partial(46%) 0.7776 1.8538 1.9157 1.5157 unknown 0.9260 1.4043 2.2152 1.5152 unknown 1.2928 1.2370 2.0149 1.5149 GP9501803dbjBAB03308.1potassiumchannelinteractingprotein1{Ratt usnorvegicus}.partial(37%) 1.1699 1.9587 1.4143 1.5143 unknown 1.3104 2.0144 1.2124 1.5124 unknown 1.6571 1.6656 1.2114 1.5114 homologuetoPIRT00390T00390KIAA0614protein- human(fragment).partial(7%) 1.0304 0.9912 2.5108 1.5108 unknown 1.5546 1.8634 1.1090 1.5090 homologuetoGP7637906gbAAF65253.1Ralguaninenucleotideexchan gefactorRalGPS1A{Homosapiens}.partial(55%) 1.0785 1.6374 1.8080 1.5080 similartoGP13249297gbAAK16734.1bicarbonatetransporter- relatedproteinBTR1{Homosapiens}.partial(14%) 0.9456 1.2704 2.3080 1.5080 similartoGP12833761dbjBAB22654.putative{Musmusculus}.complete 1.0614 1.1511 2.3062 1.5062 unknown 0.8260 1.5844 2.1052 1.5052 SPQ9NP90RB9L_HUMANRas-relatedproteinRab-9L(RAB9- likeprotein).[Human]{Homosapiens}.partial(46%) 0.9220 1.3875 2.2047 1.5047 unknown 1.0850 1.4245 2.0047 1.5047 similartoGP16923707gbAAL31549.1glutathionetransferaseT1- 1{Homosapiens}.complete 1.2996 1.0090 2.2043 1.5043 homologuetoGP7022449dbjBAA91602.1unnamedproteinproduct{Ho mosapiens}.partial(86%) 1.0614 0.9449 2.5031 1.5031 homologuetoSPP48443RXRG_HUMANRetinoicacidreceptorRXR- gamma.[Human]{Homosapiens}.partial(30%) 1.2479 1.2549 2.0014 1.5014 271 unknown 1.2395 1.9615 1.3005 1.5005 growthdifferentiationfactor9B[Susscrofa] 1.0375 1.0635 2.4005 1.5005 unknown 0.8921 1.3060 2.2991 1.4991 unknown 1.3329 1.1615 1.9972 1.4972 unknown 1.5505 1.6405 1.2955 1.4955 unknown 0.9098 1.3797 2.1948 1.4948 unknown 1.0211 1.2674 2.1942 1.4942 unknown 1.2854 1.3004 1.8929 1.4929 unknown 1.0271 1.0578 2.3925 1.4925 apolipoproteinC-III 1.2713 1.4078 1.7896 1.4896 somatostatin 1.4594 1.8194 1.1894 1.4894 weaklysimilartoSPQ05588UPAR_BOVINUrokinaseplasminogenactiv atorsurfacereceptorprecursor(U- PAR)(CD87).[Bovine]{Bostaurus}.partial(46%) 0.8047 1.1699 2.4873 1.4873 brain-derivedneurotrophicfactorprecursor(AA- 18to234)[Susscrofa]brain-derivedneurotrophicfactor[Susscrofa] 1.1769 1.2975 1.9872 1.4872 unknown 1.5333 1.8382 1.0858 1.4858 homologuetoGP12833806dbjBAB22670.putative{Musmusculus}.parti al(43%) 1.0261 1.4406 1.9833 1.4833 homologuetoGP17226390gbAAL37760.1ventricularmyosinlightchain 2{Canisfamiliaris}.partial(85%) 1.3388 1.8262 1.2825 1.4825 unknown 1.5288 1.6262 1.2925 1.4825 transforminggrowthfactor-betatypeIIIreceptor 0.8962 1.0677 2.4820 1.4820 homologuetoGP14669471gbAAK71934.1lysyloxidase- relatedproteinC{Homosapiens}.partial(14%) 1.4245 1.2377 1.7811 1.4811 homologuetoGP12803161gbAAH02384.1methionine- tRNAsynthetase{Homosapiens}.partial(12%) 1.0297 1.2316 2.1807 1.4807 unknown 0.7063 2.5538 1.1800 1.4800 unknown 1.3480 2.1085 0.9783 1.4783 unknown 1.5219 1.6301 1.2760 1.4760 unknown 0.9749 1.3765 2.0757 1.4757 similartoGP12654233gbAAH00936.1Similartohypotheticalproteinclon e1-2{Homosapiens}.partial(30%) 1.5146 1.0361 1.8753 1.4753 weaklysimilartoGP15020653embCAC44536.hypotheticalprotein{Hom osapiens}.partial(14%) 1.2801 1.2699 1.8750 1.4750 unknown 1.0000 1.3475 2.0738 1.4738 Tcellreceptorbeta-chain[Susscrofa]TCRbetachainV-beta- T13Vregion[Susscrofa] 1.4058 1.8408 1.1733 1.4733 unknown 1.6395 1.9069 0.8732 1.4732 unknown 0.9773 1.2678 2.1725 1.4725 unknown 1.6147 1.5219 1.2683 1.4683 unknown 1.1321 1.6045 1.6683 1.4683 unknown 1.0780 1.3580 1.9680 1.4680 similartoGP5911433gbAAD55791.1putativephosphate/phosphoenolp yruvatetranslocator{Rattusnorvegicus}.partial(33%) 1.7232 1.5090 1.1661 1.4661 homologuetoSPO88413TUL3_MOUSETubbyrelatedprotein3(Tubby- likeprotein3).[Mouse]{Musmusculus}.partial(44%) 1.2630 1.0674 2.0652 1.4652 homologuetoGP7020121dbjBAA91002.1unnamedproteinproduct{Ho mosapiens}.partial(58%) 1.1623 1.2672 1.9647 1.4647 unknown 1.3699 1.7574 1.2637 1.4637 unknown 1.3410 1.2850 1.7630 1.4630 unknown 1.4515 1.6730 1.2622 1.4622 homologuetoGP12846941dbjBAB27371.putative{Musmusculus}.parti al(84%) 1.1561 1.2682 1.9622 1.4622 272 273 interleukin- 7[Susscrofa]interleukin7precursor[Susscrofa]interleukin7[Susscrofa] 1.1882 1.7332 1.4607 1.4607 unknown 1.6743 1.7451 0.9597 1.4597 similartoGP177107gbAAA51533.1arachidonate12- lipoxygenase{Homosapiens}.partial(13%) 1.2451 1.8724 1.2587 1.4587 similartoGP12833402dbjBAB22510.putative{Musmusculus}.complete 0.9723 1.2438 2.1581 1.4581 homologuetoGP1239957gbAAB17015.1estrogenreceptor- relatedprotein{Homosapiens}.partial(17%) -1.3255 -2.0660 -2.5208 -1.9708 unknown -1.5035 -2.4689 -1.9412 -1.9712 unknown -1.4224 -2.3212 -2.1868 -1.9768 unknown -1.5392 -2.1456 -2.2474 -1.9774 unknown -1.7202 -2.5352 -1.6777 -1.9777 homologuetoSPP05386RLA1_HUMAN60SacidicribosomalproteinP1.[ Human]{Homosapiens}.complete -1.5211 -2.7257 -1.6884 -1.9784 homologuetoGP15559423gbAAH14079.1Unknown(proteinforMGC:2 0582){Homosapiens}.partial(48%) -1.5067 -2.5428 -1.8898 -1.9798 weaklysimilartoGP2072963gbAAC51270.1p40{Homosapiens}.partial( 35%) -1.5593 -2.6037 -1.7815 -1.9815 unknown -1.7952 -2.5115 -1.6433 -1.9833 SPP43331SMD3_HUMANSmallnuclearribonucleoproteinSmD3(snR NPcoreproteinD3)(Sm-D3).[Human]{Homosapiens}.complete -1.3167 -2.5626 -2.0746 -1.9846 SPP02383RS26_HUMAN40SribosomalproteinS26.[Rat]{Rattusnorve gicus}.complete -1.3618 -2.2981 -2.2949 -1.9849 unknown -1.5287 -2.0630 -2.3659 -1.9859 unknown -1.6356 -2.6808 -1.6482 -1.9882 unknown -1.3952 -2.8515 -1.7184 -1.9884 similartoSPO18778PAHX_BOVINPhytanoyl- CoAdioxygenaseperoxisomalprecursor(EC1.14.11.18)(Phytanoyl- CoAalpha-hydroxylase).partial(34%) -1.6731 -2.1330 -2.1731 -1.9931 homologuetoSPP48201AT93_HUMANATPsynthaselipid- bindingproteinmitochondrialprecursor(EC3.6.1.34).complete -1.5888 -2.0404 -2.3546 -1.9946 unknown -1.1893 -2.3631 -2.4362 -1.9962 unknown -1.4708 -2.1326 -2.3867 -1.9967 unknown -1.6850 -2.1302 -2.1776 -1.9976 unknown -1.4604 -2.6504 -1.8904 -2.0004 unknown -1.4105 -2.4621 -2.1313 -2.0013 GP13542790gbAAH05598.1Similartodendriticcellprotein{Musmuscul us}.partial(54%) -1.2386 -2.4611 -2.3149 -2.0049 homologuetoSPQ9Z2U0PSA7_MOUSEProteasomesubunitalphatype 7(EC3.4.25.1)(ProteasomesubunitRC6- 1).[Mouse]{Musmusculus}.complete -1.7126 -2.7463 -1.5694 -2.0094 unknown -1.8745 -2.0309 -2.1327 -2.0127 similartoGP13569612gbAAK31162.1ubiquitinA- 52residueribosomalproteinfusionproduct1{Homosapiens}.partial(73%) -1.7062 -2.5392 -1.7927 -2.0127 homologuetoGP12847259dbjBAB27498.putative{Musmusculus}.parti al(51%) -1.2436 -2.4694 -2.3365 -2.0165 homologuetoGP13274518gbAAK17960.1complement- c1qtumornecrosisfactor-relatedprotein{Homosapiens}.partial(78%) -1.4663 -2.6573 -1.9268 -2.0168 unknown -1.7457 -2.0390 -2.2674 -2.0174 GP12654655gbAAH01165.1N-ethylmaleimide- sensitivefactorattachmentproteinalpha{Homosapiens}.partial(56%) -1.4053 -2.7307 -1.9180 -2.0180 unknown -1.6773 -2.1301 -2.2487 -2.0187 unknown -0.9711 -3.1709 -1.9210 -2.0210 homologuetoGP13325337gbAAH04480.1Unknown(proteinforMGC:1 0520){Homosapiens}.partial(38%) -0.9636 -2.4803 -2.6219 -2.0219 GP15929961gbAAH15405.1ribosomalproteinS5{Homosapiens}.comp lete -1.6558 -2.4389 -1.9729 -2.0225 rig-analogDNA-bindingprotein[Susscrofa] -1.5339 -2.1577 -2.3858 -2.0258 unknown -1.3786 -2.8659 -1.8373 unknown -1.6446 -2.0443 -2.3995 -2.0295 unknown -1.5881 -2.3114 -2.1898 -2.0298 -1.0037 -2.9479 -2.1408 -2.0308 unknown -2.8649 -1.7227 -2.0327 GP15824485gbAAL09365.1DiGeorgesyndrome- relatedproteinFKSG5{Homosapiens}.complete -0.9937 -2.2349 -2.0349 unknown -1.6038 -2.1364 -2.0273 PIRJC2329JC2329translationinitiationfactoreIF-2betachain- rabbit.partial(36%) -1.5104 -2.8760 -2.3651 -2.0351 cystatinB[Susscrofa] -1.4337 -2.7633 -1.9185 -2.0385 similartoPIRS11021S1102124-dienoyl- CoAreductase(NADPH)(EC1.3.1.34)-rat.partial(20%) -1.4304 -2.4676 -2.2190 -2.0390 homologuetoGP15080078gbAAH11819.1DEAD/H(Asp-Glu-Ala- Asp/His)boxpolypeptide3{Homosapiens}.partial(19%) -1.6332 -2.5422 -1.9527 -2.0427 unknown -1.6715 -2.2190 -2.2452 -2.0452 unknown -1.6011 -2.9412 -1.5962 -2.0462 SPP01252THYA_BOVINProthymosinalpha.[Bovine]{Bostaurus}.com plete -1.4431 -2.5628 -2.1380 -2.0480 homologuetoSPP37980IPYR_BOVINInorganicpyrophosphatase(EC3 .6.1.1)(Pyrophosphatephospho- hydrolase)(PPase).[Bovine].partial(49%) -1.6521 -2.6740 -1.8180 -2.0480 unknown -1.7909 -2.6572 -1.6991 -2.0491 unknown -1.6012 -2.7828 -1.7720 -2.0520 homologuetoGP3986482gbAAC84044.1translationinitiationfactoreIF3 p40subunit;eIF3p40{Homosapiens}.partial(44%) -1.6177 -2.3145 -2.2361 -2.0561 unknown -1.6634 -2.6468 -1.8851 -2.0651 unknown -1.3015 -2.3801 -2.5158 -2.0658 unknown -1.3151 -2.2811 -2.6081 -2.0681 unknown -1.2508 -2.3485 -2.6096 -2.0696 unknown -1.5582 -2.2420 -2.4101 -2.0701 ]NADH2[Susscrofa]NADHdehydrogenasesubunit2[Susscrofa]NADHd ehydrogenasesubunit2[Susscrofa] -1.5597 -2.8514 -1.8005 -2.0705 homologuetoSPQ02375NUYM_BOVINNADH- ubiquinoneoxidoreductase18kDasubunitmitochondrialprecursor(EC1. 6.5.3)(EC1.6.99.3).complete -1.6727 -2.7615 -1.7821 -2.0721 unknown -1.6269 -2.8750 -1.7260 -2.0760 similartoGP14290496gbAAH09016.1Similartocomplementcomponent 1qsubcomponentcpolypeptide{Homosapiens}.complete -1.4862 -2.3459 -2.3961 -2.0761 unknown -1.2846 -3.1695 -1.7771 -2.0771 similartoSPP28268TBA_EUPVATubulinalphachain.{Euplotesvannus} .partial(39%) -1.2640 -2.6705 -2.3123 -2.0823 unknown -1.7512 -3.4136 -1.0824 -2.0824 PIRS42409S42409proteintranslocationcomplexSec61betachainendo plasmicreticulum-dog.complete -1.9453 -2.0631 -2.2442 -2.0842 PIRS08228S08228ribosomalproteinS2cytosolic- human(fragment).partial(94%) -1.5355 -2.7670 -1.9562 -2.0862 unknown -1.3967 -2.4920 -2.3793 -2.0893 unknown -1.6358 -2.6898 -1.9528 -2.0928 homologuetoGP4929553gbAAD34037.1CGI- 41protein{Homosapiens}.partial(44%) -1.6054 -2.3822 -2.2938 -2.0938 cytochromeb5[Susscrofa] -1.9852 -2.6529 -1.6440 -2.0940 homologuetoPIRB54211B54211H+- transportingATPsynthase(EC3.6.1.34)chaing-bovine.complete -1.7981 -2.1604 -2.3242 -2.0942 unknown -1.6053 -2.7134 -1.9643 -2.0943 unknown -1.7935 -2.6881 -1.8058 -2.0958 similartoGP13093775embCAC29495.hypotheticalprotein{Homosapie -1.3951 -2.2853 -2.6502 -2.1102 274 ns}.partial(61%) unknown -1.6818 -2.3407 -2.3263 -2.1163 unknown -1.5866 -2.4693 -2.2979 -2.1179 unknown -1.0593 -3.3267 -1.9680 -2.1180 preprocathepsinH[Susscrofa] -1.3166 -2.9811 -2.0588 -2.1188 unknown -1.7721 -2.3517 -2.2569 -2.1269 unknown -1.7421 -2.8345 -1.8083 -2.1283 homologuetoSPP27952RS2_RAT40SribosomalproteinS2.[Rat]{Rattu snorvegicus}.partial(91%) -1.2222 -2.7869 -2.3795 -2.1295 unknown -1.6887 -2.7542 -1.9515 -2.1315 unknown -1.7285 -2.4573 -2.2279 -2.1379 tropomyosin4[Susscrofa] -1.5159 -2.8729 -2.0294 -2.1394 unknown -1.4850 -2.6167 -2.3209 -2.1409 cytolytictriggermoleculeG7CD16A.c[Susscrofa] -1.8050 -2.8470 -1.7860 -2.1460 unknown -1.6393 -2.4566 -2.3479 -2.1479 decorin[Susscrofa] -1.6619 -2.8898 -1.9009 -2.1509 PIRA22632UQHUR7ubiquitin/ribosomalproteinS27acytosolic[validate d]-human.complete -1.7489 -2.4233 -2.2811 -2.1511 40SribosomalproteinS12[Susscrofa] -1.9041 -2.7892 -1.7617 -2.1517 unknown -1.1594 -3.4360 -1.8777 -2.1577 apolipoproteinRprecursor[Susscrofa]apolipoproteinRprecursor[Susscr ofa] -1.9525 -2.6341 -1.8883 -2.1583 homologuetoPIRD53737D53737phosphatecarrierproteinprecursormit ochodrialspliceformB-bovine.partial(69%) -1.7823 -2.3560 -2.3391 -2.1591 unknown -1.5135 -2.3774 -2.5904 -2.1604 homologuetoGP5106998gbAAD39918.1HSPC040protein{Homosapie ns}.complete -1.6951 -2.8689 -1.9220 -2.1620 similartoSPP14622COXR_BOVINCytochromecoxidasepolypeptideVII I-livermitochondrialprecursor(EC1.9.3.1)(IX).[Bovine].complete -1.7873 -2.7675 -1.9324 -2.1624 unknown -0.3628 -3.5660 -2.5644 -2.1644 unknown -1.8910 -3.0859 -1.5175 -2.1648 unknown -1.6164 -2.1783 -2.7074 -2.1674 homologuetoPIRS11696A29170phosphopyruvatehydratase(EC4.2.1. 11)alpha-human.complete -1.8712 -2.9757 -1.6584 -2.1684 hyaluronansynthase2[Susscrofa] -1.6703 -2.4731 -2.3717 -2.1717 unknown -1.6929 -3.1517 -1.6723 -2.1723 SPP12947RL31_HUMAN60SribosomalproteinL31.[Pig]{Susscrofa}.c omplete -1.5726 -2.9856 -1.9641 -2.1741 similartoSPP42929HS27_CANFAHeatshock27kDaprotein(HSP27).[D og]{Canisfamiliaris}.complete -1.4851 -2.7896 -2.2724 -2.1824 unknown -2.0000 -2.6352 -1.9126 -2.1826 SPP18621RL17_HUMAN60SribosomalproteinL17(L23).[Human]{Ho mosapiens}.complete -1.7138 -2.4646 -2.3742 -2.1842 SPP38663RL24_HUMAN60SribosomalproteinL24(L30).[Bovine]{Bost aurus}.complete -1.9013 -2.7468 -1.9191 -2.1891 similartoGP3126984gbAAC16021.1CAG- isl7{Homosapiens}.complete -1.6517 -2.3731 -2.5524 -2.1924 SPQ9C005DP30_HUMANDpy-30- likeprotein.[Human]{Homosapiens}.complete -1.7751 -2.4289 -2.4020 -2.2020 PIRG01229G01229cappingproteinalpha- human(fragment).partial(25%) -1.4773 -2.2999 -2.8336 -2.2036 homologuetoGP12843076dbjBAB25849.putative{Musmusculus}.parti al(85%) -1.3065 -2.9674 -2.3469 -2.2069 PIRS55913S55913ribosomalproteinL21cytosolic-human.complete -1.8416 -2.7528 -2.0272 -2.2072 unknown -1.5838 -2.3854 -2.6596 -2.2096 275 unknown -1.8738 -2.8696 -1.8917 -2.2117 unknown -1.3306 -3.5409 -1.7757 -2.2157 homologuetoSPQ95140RLA0_BOVIN60SacidicribosomalproteinP0(L 10E)(Fragment).[Bovine]{Bostaurus}.complete -2.0490 -2.9203 -1.6896 -2.2196 SPP05209TBA1_CRIGRTubulinalpha- 1chain.[Chinesehamster]{Cricetulusgriseus}.partial(42%) -1.6812 -2.1309 -2.8511 -2.2211 GP17932958dbjBAB79470.ribosomalproteinL34{Homosapiens}.com plete -1.8143 -2.3622 -2.4932 -2.2232 cytoplasmiclight-chaindynein[Susscrofa] -1.8473 -2.7941 -2.0357 -2.2257 unknown -1.8339 -2.1644 -2.6891 -2.2291 similartoGP16197488dbjBAB69947.legumain{Bostaurus}.complete -2.0236 -2.3476 -2.3206 -2.2306 homologuetoPIRS18294EFHU2translationelongationfactoreEF-2- human.partial(19%) -1.6235 -2.1689 -2.9012 -2.2312 unknown -1.5823 -2.5354 -2.5839 -2.2339 homologuetoPIRI51803I51803TAXREB107-human.partial(88%) -2.0782 -2.8705 -1.7693 -2.2393 weaklysimilartoGP2648023embCAB09994.1cICF0811.6(chromosom e6openreadingframe11(BING4)){Homosapiens}.partial(49%) -1.7698 -2.1737 -2.7817 -2.2417 unknown -1.0183 -3.7430 -1.9756 -2.2456 PIRS38962S38962serpin-pig.partial(64%) -1.5418 -3.2145 -1.9882 -2.2482 unknown -1.8697 -2.4605 -2.4351 -2.2551 similartoGP14250636gbAAH08782.1nuclearfactorofkappalightpolype ptidegeneenhancerinB-cellsinhibitor-like2{Homosapiens}.partial(24%) -1.7898 -2.2719 -2.7059 -2.2559 unknown -1.0286 -3.8500 -1.8993 -2.2593 weaklysimilartoGP11345388gbAAG34681.1lysosomalthiolreductasep recursor{Musmusculus}.complete -1.7908 -2.3406 -2.6507 -2.2607 homologuetoSPP13272UCRI_BOVINUbiquinol- cytochromeCreductaseiron- sulfursubunitmitochondrialprecursor(EC1.10.2.2).partial(49%) -2.1490 -2.6899 -1.9995 -2.2795 similartoGP15779050gbAAH14597.1SimilartoRIKENcDNA1700052K 15gene{Homosapiens}.partial(28%) -1.5267 -3.4388 -1.8828 -2.2828 homologuetoPIRA26437UQHUBpolyubiquitin3-human.partial(85%) -1.8709 -2.1356 -2.8432 -2.2832 unknown -1.3980 -2.6921 -2.7800 -2.2900 PIRS49326S49326nascentpolypeptide- associatedcomplexalphachain-human.complete -1.8686 -2.9801 -2.0344 -2.2944 GP14719845gbAAD20460.3ribosomalproteinL11{Homosapiens}.com plete -1.7992 -2.9785 -2.1188 -2.2988 PIRS34755S3475514-3-3protein(clone1054)-human.complete -1.5856 -3.6472 -1.7014 -2.3114 unknown -1.7802 -2.9802 -2.1852 -2.3152 GP7688693gbAAF67487.130kDaprotein{Homosapiens}.partial(63%) -1.8751 -2.3442 -2.7547 -2.3247 unknown -2.0421 -3.0284 -1.9352 -2.3352 homologuetoGP15186717dbjBAB62888.TdTbindingprotein{Homosap iens}.partial(50%) -1.0900 -3.6706 -2.2453 -2.3353 unknown -2.0587 -3.0932 -1.8560 -2.3360 homologuetoSPO88322NID2_MOUSENidogen-2precursor(NID- 2)(Entactin-2).[Mouse]{Musmusculus}.partial(9%) -2.0179 -2.3137 -2.7058 -2.3458 GP6624731embCAB63859.1putativenonclassicalMHCclassIantigen{ Susscrofa}.complete -1.7254 -2.8693 -2.4474 -2.3474 dihydrolipoamideacetyltransferase[Susscrofa] -1.9272 -2.0611 -3.0592 -2.3492 unknown -2.2722 -2.7953 -1.9938 -2.3538 unknown -2.0615 -2.4976 -2.5046 -2.3546 PIRJC4662JC4662ribosomalproteinS3acytosolic-human.complete -2.0549 -3.2136 -1.8542 -2.3742 SPP23131RL23_HUMAN60SribosomalproteinL23(L17).[Pig]{Susscro fa}.complete -2.1121 -2.3681 -2.6451 -2.3751 unknown -1.4334 -3.6848 -2.0341 -2.3841 malatedehydrogenasedecarboxylase(NADP+)[Susscrofa] -1.8353 -2.4937 -2.8395 -2.3895 276 277 unknown -2.2063 -2.3490 -2.6527 -2.4027 homologuetoSPQ9DBS5KLC3_MOUSEProbablekinesinlightchain3(K LC3).[Mouse]{Musmusculus}.partial(37%) -1.5627 -3.9234 -1.7981 -2.4281 similartoGP12654605gbAAH01138.1SimilartohexosaminidaseA(alph apolypeptide){Homosapiens}.partial(40%) -1.8925 -3.2687 -2.1456 -2.4356 Igkappachain -1.5681 -3.6811 -2.0846 -2.4446 homologuetoGP12804601gbAAH01722.1CGI- 99protein{Homosapiens}.complete -2.2066 -2.3516 -2.7891 -2.4491 fibrillin-1precursor[Susscrofa] -2.2117 -3.2714 -1.8716 -2.4516 unknown -1.9590 -2.4565 -2.9577 -2.4577 homologuetoGP14286220gbAAH08906.1enoylCoenzymeAhydratase shortchain1mitochondrial{Homosapiens}.partial(50%) -1.7922 -3.6168 -2.0295 -2.4795 unknown -1.4482 -3.6817 -2.3550 -2.4950 similartoGP29539embCAA28407.1precursorofC1r(AA- 17to688){Homosapiens}.partial(10%) -1.8083 -3.6475 -2.0529 -2.5029 unknown -2.0807 -2.3916 -3.0462 -2.5062 unknown -1.7158 -3.8660 -1.9809 -2.5209 homologuetoSPQ9UL46PSE2_HUMANProteasomeactivatorcomplex subunit2(Proteasomeactivator28- betasubunit)(PA28beta)(PA28b).complete -2.0141 -2.9879 -2.6210 -2.5410 similartoGP558908gbAAA67727.1reversetranscriptase{Musmusculus }.partial(10%) -1.8824 -2.6948 -3.0836 -2.5536 unknown -1.6446 -3.2375 -2.8010 -2.5610 similartoGP12833323dbjBAB22481.putative{Musmusculus}.complete -2.1386 -2.4848 -3.0617 -2.5617 homologuetoSPP17665COXO_MOUSECytochromecoxidasepolypep tideVIIcmitochondrialprecursor(EC1.9.3.1).[Mouse]{Musmusculus}.co mplete -2.2571 -2.3134 -3.1702 -2.5802 S100Cprotein[Susscrofa]S100C[Susscrofa] -1.8383 -2.8750 -3.0317 -2.5817 similartoGP14279576gbAAK58638.1interferon-inducedprotein1- 8U{Bostaurus}.partial(89%) -2.1011 -3.5838 -2.1074 -2.5974 MHCclassIIDR-alpha -2.2021 -3.6807 -1.9214 -2.6014 homologuetoSPO95445APOM_HUMANApolipoproteinM(ApoM)(G3a )(HSPC336).[Human]{Homosapiens}.complete -1.5496 -3.3457 -2.9127 -2.6027 Ca2+ATPaseoffasttwitch1skeletalmusclesarcoplasmicreticulum[Suss crofa] -2.5535 -2.0683 -3.2109 -2.6109 unknown -2.4346 -2.6497 -2.7522 -2.6122 homologuetoGP10717134gbAAG22029.1carbonicanhydraseIII{Mus musculus}.complete -1.5791 -2.6975 -3.7133 -2.6633 unknown -1.8741 -3.9888 -2.1965 -2.6865 unknown -2.1522 -3.1928 -2.8075 -2.7175 unknown -2.3915 -3.8634 -1.9125 -2.7225 homologuetoPIRT14797T14797hypotheticalproteinDKFZp564B167.1 -human.complete -2.0901 -2.6139 -3.4770 -2.7270 unknown -2.7707 -2.3274 -3.1041 -2.7341 homologuetoGP12835239dbjBAB23198.putative{Musmusculus}.parti al(24%) -2.6937 -3.6766 -1.8652 -2.7452 homologuetoGP13879314gbAAH06632.1Unknown(proteinforIMAGE: 3481996){Musmusculus}.partial(23%) -2.6148 -3.8678 -1.7563 -2.7463 stearyl-CoAdesaturase[Susscrofa] -2.3930 -3.6949 -2.2040 -2.7640 MHCclassIantigen[Susscrofa]MHCclassIantigen[Susscrofa] -2.1832 -2.9851 -3.1691 -2.7791 similartoGP18139943gbAAL60202.1X- boxbindingproteinprocessedisoform{Musmusculus}.partial(47%) -3.9642 -2.8622 -1.5532 -2.7932 homologuetoPIRA40119SNHUINinsulysin(EC3.4.24.56)[validated]- human.partial(16%) -1.8098 -3.1682 -3.7640 -2.9140 SLA-DR1beta1domain[Susscrofa] -2.4055 -4.2679 -2.1817 -2.9517 immunoglobulinlambda-chainimmunoglobulinlambdachain[Susscrofa] -1.4962 -4.8754 -2.5708 -2.9808 similartoSPQ28022MGP2_BOVINMicrofibril- associatedglycoprotein2precursor(MAGP- -2.7404 -2.6714 -3.5309 -2.9809 Table 2. Transcripts highly up/down regulated determined by oligo-array in the liver tissue by the dietary shifting from LFD to HFD (Chapter 3). For each transcript, log 2 ratio=log 2 (HFD/LFD).The positive value means higher mRNA abundance in HFD pigs; negative value means lower mRNA abundance in HFD pigs 2)(MP25).[Bovine]{Bostaurus}.partial(85%) SLA-DQbeta1domain[Susscrofa]SLA-DQbeta1domain[Susscrofa] -2.4513 -3.6160 -2.9586 -3.0086 putativeolfactoryreceptor-likeprotein[Susscrofa] -2.7296 -3.6798 -2.6497 -3.0197 unknown -2.9464 -3.5569 -2.8466 -3.1166 beta2-microglobulinbeta-2-microglobulinprotein[Susscrofa] -2.3536 -3.0963 -3.9849 -3.1449 similartoGP443671gbAAB59537.1complementcomponentC4A{Homo sapiens}.partial(6%) -2.3474 -3.6868 -3.5271 -3.1871 unknown -3.1231 -3.9207 -2.9819 -3.3419 unknown -3.3282 -3.8706 -3.7944 -3.6644 salivarylipocalin[Susscrofa] -4.9285 -5.0744 -6.1415 -5.3815 Gene Name Log 2 (R1/ G1) Log 2 (R2/ G2) Log 2 (G3/ R3) Log 2 (G4/ R4) Average unknown 3.2084 5.8379 6.8249 2.2214 4.5232 unknown 3.1699 5.3576 5.2436 3.2839 4.2637 unknown 3.8387 4.1352 1.8144 6.1594 3.9869 homologue to GP|6331022|dbj|BAA86578.1 KIAA1264 protein {Homo sapiens}, partial (9%) 3.5748 4.2365 1.6245 6.1868 3.9057 unknown 3.6472 4.1085 2.1203 5.6355 3.8779 unknown 3.3400 4.2245 2.9696 4.5949 3.7823 unknown 2.4150 5.0255 3.8188 3.6218 3.7203 unknown 2.7975 4.4897 3.6209 3.6663 3.6436 unknown 3.7197 3.5560 2.0196 5.2562 3.6379 homologue to SP|Q62813|LAMP_RAT Limbic system- associated membrane protein precursor (LSAMP). [Rat] {Rattus norvegicus}, partial (37%) 3.4072 3.8480 1.3269 5.9283 3.6276 unknown 3.6483 3.5826 2.2303 5.0005 3.6154 endothelin receptor subtype A, ETA receptor [swine, lung, Peptide, 427 aa] 3.8000 3.3808 4.6049 2.5759 3.5904 unknown 3.3385 3.7318 4.5850 2.4853 3.5351 unknown 3.6537 3.2745 2.6731 4.2551 3.4641 unknown 4.1906 2.7205 1.2668 5.6442 3.4555 unknown 4.3155 2.5104 1.9701 4.8558 3.4129 unknown 3.7761 2.9871 1.6781 5.0851 3.3816 homologue to PIR|A38096|A38096 perlecan precursor - human, partial (4%) 3.2693 3.4650 0.2382 6.4961 3.3671 unknown 3.4842 3.2364 1.4994 5.2212 3.3603 unknown 3.4941 3.2042 1.2763 5.4220 3.3491 unknown 3.9152 2.6759 0.3551 6.2360 3.2956 unknown 3.4692 3.0506 1.0000 5.5199 3.2599 unknown 3.0211 3.4849 0.1389 6.3671 3.2530 homologue to GP|8100510|gb|AAF72335.1| Y-box protein ZONAB-A {Canis familiaris}, partial (26%) 3.6153 2.8407 1.4085 5.0475 3.2280 unknown 3.4936 2.9617 0.9155 5.5398 3.2277 unknown 3.3663 3.0184 0.7119 5.6728 3.1924 unknown 3.1608 3.1933 1.8569 4.4972 3.1770 unknown 3.4429 2.9005 0.4171 5.9263 3.1717 278 unknown 3.2224 3.1085 0.0995 6.2314 3.1655 unknown 3.1375 3.0205 0.8553 5.3027 3.0790 unknown 2.7987 3.2835 -0.2774 6.3596 3.0411 unknown 3.7445 2.2722 0.6138 5.4029 3.0083 unknown 3.2920 2.7225 0.7551 5.2594 3.0072 unknown 3.0433 2.9053 -0.1585 6.1071 2.9743 unknown 3.0255 2.8698 1.3956 4.4997 2.9477 unknown 2.6135 3.2345 1.1024 4.7456 2.9240 unknown 3.7442 1.9929 0.2360 5.5011 2.8685 unknown 3.4948 2.2332 -0.3699 6.0979 2.8640 unknown 3.5236 2.2029 1.1255 4.6010 2.8633 unknown 3.2730 2.4485 0.5372 5.1843 2.8607 unknown 2.8560 2.8138 0.0970 5.5727 2.8349 homologue to GP|12860213|dbj|BAB31879. putative {Mus musculus}, partial (65%) 3.2386 2.4046 2.0386 3.6046 2.8216 unknown 3.1651 2.4525 0.4985 5.1191 2.8088 unknown 2.7389 2.8402 1.1497 4.4294 2.7896 unknown 3.1797 2.3863 0.7029 4.8631 2.7830 homologue to GP|16306782|gb|AAH01585.1 ligatin {Homo sapiens}, partial (30%) 3.2854 2.1753 0.3930 5.0677 2.7304 unknown 3.5327 1.9031 1.0112 4.4246 2.7179 unknown 2.8309 2.5900 0.6651 4.7558 2.7105 unknown 3.2957 2.0979 0.7092 4.6844 2.6968 unknown 2.6189 2.6909 0.5450 4.7648 2.6549 unknown 2.9417 2.3635 2.0959 3.2093 2.6526 unknown 2.8770 2.3959 0.9773 4.2956 2.6365 unknown 2.8284 2.3890 1.0568 4.1606 2.6087 unknown 3.1009 2.0894 1.5783 3.6120 2.5951 unknown 3.2536 1.9160 1.3813 3.7882 2.5848 unknown 2.3612 2.7803 1.4150 3.7264 2.5707 homologue to GP|1389694|gb|AAB02905.1| FX-induced thymoma transcript {Mus musculus}, partial (43%) 3.9854 1.1375 1.0173 4.1056 2.5614 unknown 2.1615 2.9329 0.8646 4.2298 2.5472 unknown 2.7225 2.3154 1.2402 3.7976 2.5189 unknown 2.4245 2.6122 1.3740 3.6627 2.5183 unknown 3.2056 1.8282 1.7370 3.2969 2.5169 unknown 3.0334 1.9728 1.6781 3.3282 2.5031 unknown 3.3278 1.6632 1.4477 3.5434 2.4955 unknown 1.8948 3.0875 1.0904 3.8919 2.4911 unknown 2.2161 2.7646 0.2730 4.7076 2.4903 unknown 2.0199 2.9584 1.5590 3.4194 2.4892 unknown 2.6677 2.3029 2.1525 2.8181 2.4853 unknown 2.6705 2.2876 1.9955 2.9626 2.4791 homologue to GP|12803695|gb|AAH02683.1 neuritin {Homo sapiens}, partial (88%) 1.3312 3.6245 2.0810 2.8747 2.4778 unknown 1.7843 3.1699 -0.3440 5.2982 2.4771 unknown 2.9190 2.0293 1.8604 3.0879 2.4742 unknown 3.3817 1.5361 1.0012 3.9166 2.4589 unknown 3.1775 1.7056 1.6960 3.1870 2.4415 279 280 unknown 1.7708 3.1085 0.1255 4.7538 2.4397 calcineurin catalytic subunit delta isoform [Sus scrofa] 3.1528 1.6861 0.4393 4.3996 2.4195 unknown 2.3376 2.4935 1.7952 3.0359 2.4156 unknown 2.6845 2.1113 1.4594 3.3363 2.3979 homologue to GP|12655231|gb|AAH01474.1 Unknown (protein for IMAGE:3138844) {Homo sapiens}, partial (13%) 3.2283 1.5375 1.8211 2.9447 2.3829 similar to GP|7329074|gb|AAF59902.1| collagen type V alpha 3 chain {Homo sapiens}, partial (4%) 2.9936 1.7667 0.6586 4.1018 2.3802 unknown 3.3124 1.4374 0.6249 4.1249 2.3749 unknown 2.6695 2.0461 1.0364 3.6792 2.3578 unknown 3.3397 1.3735 0.1239 4.5893 2.3566 unknown 1.8981 2.8142 2.6951 2.0172 2.3562 unknown 2.7446 1.9542 0.9637 3.7351 2.3494 unknown 1.7472 2.9307 1.7081 2.9699 2.3390 similar to GP|6467994|gb|AAF13271.1| CBL-B {Rattus norvegicus}, partial (49%) 1.2350 3.3923 1.7675 2.8599 2.3137 unknown 2.5797 2.0381 0.2469 4.3709 2.3089 unknown 2.3428 2.2521 1.0984 3.4965 2.2975 unknown 2.2184 2.3717 1.4406 3.1495 2.2951 unknown 2.9404 1.6384 1.0189 3.5600 2.2894 unknown 1.8398 2.7235 0.9704 3.5929 2.2817 unknown 2.4530 2.1037 1.7821 2.7746 2.2783 unknown 2.7959 1.7608 2.3453 2.2114 2.2783 unknown 1.8346 2.7199 1.1484 3.4061 2.2772 unknown 1.8931 2.6521 1.5715 2.9736 2.2726 unknown 3.0190 1.5070 1.1754 3.3505 2.2630 unknown 2.7588 1.7442 0.7272 3.7758 2.2515 unknown 2.4561 2.0388 1.4222 3.0727 2.2474 unknown 3.7521 0.7182 0.9272 3.5431 2.2352 unknown 2.1893 2.2801 -0.2073 4.6768 2.2347 unknown 3.1849 1.2551 2.2166 2.2234 2.2200 unknown 2.2455 2.1926 0.9133 3.5249 2.2191 unknown 3.1085 1.3293 0.8850 3.5529 2.2189 unknown 2.9165 1.5045 1.0636 3.3574 2.2105 similar to SP|Q16790|CAH9_HUMAN Carbonic anhydrase IX precursor (EC 4.2.1.1) (Carbonate dehydratase IX) (CA- IX) (CAIX), partial (56%) 3.2088 1.2099 1.1262 3.2924 2.2093 unknown 2.1788 2.2168 0.6384 3.7572 2.1978 unknown 2.9747 1.4099 0.8497 3.5349 2.1923 unknown 2.8219 1.5552 1.2687 3.1084 2.1886 unknown 2.3440 2.0258 0.2009 4.1689 2.1849 homologue to GP|16550722|dbj|BAB71035. unnamed protein product {Homo sapiens}, partial (34%) 3.1864 1.1725 0.8881 3.4708 2.1794 unknown 3.3337 0.9907 0.9069 3.4175 2.1622 unknown 2.6551 1.6473 0.9334 3.3690 2.1512 sarcoendoplasmic reticulum calcium ATPase [Sus scrofa] 1.8835 2.4150 0.6394 3.6591 2.1493 unknown 2.6280 1.6666 1.0191 3.2755 2.1473 unknown 2.4631 1.8257 0.8564 3.4323 2.1444 weakly similar to SP|O95661|RHOI_HUMAN Rho-related GTP-binding protein RhoI. [Human] {Homo sapiens}, partial 2.7349 1.5431 0.4034 3.8747 2.1390 (79%) homologue to SP|P13213|SPRC_BOVIN SPARC precursor (Secreted protein acidic and rich in cysteine) (Osteonectin) (ON), partial (50%) 1.0000 3.2538 1.3491 2.9046 2.1269 unknown 2.0718 2.1809 0.9310 3.3218 2.1264 unknown 2.9075 1.3405 0.7768 3.4713 2.1240 unknown 2.4868 1.7517 1.1553 3.0832 2.1193 unknown 2.4874 1.7304 0.2208 3.9970 2.1089 unknown 2.7442 1.4481 1.0935 3.0987 2.0961 unknown 2.8931 1.2987 1.1279 3.0638 2.0959 homologue to GP|6634025|dbj|BAA20833.2 KIAA0379 protein {Homo sapiens}, partial (21%) 3.7127 0.4780 1.1354 3.0553 2.0954 unknown 2.7142 1.4496 1.1066 3.0573 2.0819 unknown 2.1240 2.0092 1.3692 2.7640 2.0666 unknown 2.3410 1.7625 0.8480 3.2555 2.0518 unknown 2.2479 1.8532 1.2778 2.8233 2.0505 unknown 3.2888 0.8074 1.7147 2.3814 2.0481 unknown 3.0361 1.0521 0.4594 3.6288 2.0441 unknown 2.8103 1.2587 1.2337 2.8354 2.0345 immunoglobulin heavy chain [Sus scrofa] 2.6229 1.4395 0.5956 3.4669 2.0312 unknown 1.3219 2.7291 0.4263 3.6248 2.0255 unknown 1.9475 2.0623 0.9622 3.0476 2.0049 unknown 2.6689 1.3012 1.2787 2.6914 1.9850 unknown 2.6963 1.2704 0.7049 3.2618 1.9834 unknown 3.0306 0.9018 0.7012 3.2312 1.9662 unknown 3.2224 0.7078 0.2559 3.6743 1.9651 unknown 2.2801 1.6101 0.6250 3.2652 1.9451 unknown 3.2388 0.6439 -2.9579 6.8406 1.9413 unknown 1.9087 1.9260 2.0000 1.8347 1.9174 unknown 2.5078 1.3219 1.1069 2.7228 1.9149 unknown 1.7955 2.0202 1.3962 2.4195 1.9079 unknown 2.6660 1.1410 1.1708 2.6361 1.9035 homologue to GP|11041489|dbj|BAB17282. hypothetical protein {Macaca fascicularis}, partial (58%) 1.5361 2.2679 0.5257 3.2783 1.9020 unknown 2.6393 1.1634 0.0137 3.7891 1.9014 unknown 2.1520 1.6464 0.7144 3.0839 1.8992 unknown 2.5142 1.2812 2.2082 1.5872 1.8977 unknown 2.5632 1.2317 1.3877 2.4072 1.8975 GP|15489242|gb|AAH13725.1 Unknown (protein for IMAGE:3859726) {Homo sapiens}, partial (52%) 2.8415 0.9438 2.5321 1.2532 1.8927 unknown 1.7718 2.0000 0.2990 3.4729 1.8859 homologue to GP|12060855|gb|AAG48269.1 serologically defined breast cancer antigen NY-BR-96 {Homo sapiens}, partial (24%) 3.2987 0.4581 0.9329 2.8240 1.8784 unknown 2.7618 0.9903 2.1451 1.6070 1.8761 unknown 1.6848 2.0611 1.7817 1.9642 1.8730 unknown 2.1988 1.5429 1.6004 2.1413 1.8708 unknown 2.8319 0.8981 2.0166 1.7134 1.8650 unknown 3.1155 0.6114 2.0129 1.7140 1.8635 unknown 1.5146 2.2016 1.8305 1.8857 1.8581 281 unknown 2.6893 1.0194 1.6362 2.0725 1.8543 galanin-like peptide precursor [Sus scrofa] 1.2400 2.4628 1.8631 1.8397 1.8514 unknown 2.2216 1.4809 0.6701 3.0324 1.8512 homologue to SP|P47240|PX8A_CANFA Paired box protein PAX-8 isoform 8A. [Dog] {Canis familiaris}, partial (26%) 3.2793 0.4072 1.3265 2.3601 1.8433 unknown 2.7340 0.9471 0.3857 3.2954 1.8405 unknown 2.6387 1.0421 0.8596 2.8211 1.8404 CD11b 2.8352 0.8455 1.7236 1.9571 1.8403 unknown 2.0310 1.6208 0.2970 3.3549 1.8259 unknown 1.7521 1.8838 1.0753 2.5606 1.8179 homologue to PIR|JC5952|JC5952 mitogen-activated protein kinase-activated protein kinase (EC 2.7.-.-) 5 - mouse, partial (29%) 2.3896 1.2395 0.0056 3.6234 1.8145 unknown 2.5816 1.0459 1.1433 2.4843 1.8138 unknown 2.7937 0.8237 1.7326 1.8849 1.8087 unknown 2.4984 1.1161 1.2642 2.3502 1.8072 unknown 2.4010 1.2025 1.3396 2.2639 1.8017 homologue to GP|11907601|gb|AAG41237.1 protein kinase HIPK2 {Mus musculus}, partial (7%) 3.3509 0.2515 1.5417 2.0608 1.8012 unknown 2.5699 1.0086 1.8729 1.7055 1.7892 unknown 3.1859 0.3812 0.5923 2.9748 1.7836 homologue to GP|12654557|gb|AAH01113.1 U3 snoRNP- associated 55-kDa protein {Homo sapiens}, partial (21%) 3.2040 0.3463 2.1657 1.3846 1.7752 unknown 1.5626 1.9758 1.4499 2.0884 1.7692 homologue to GP|4337109|gb|AAD18085.1| BAT3 {Homo sapiens}, partial (12%) 2.9355 0.6020 1.2655 2.2720 1.7687 unknown 1.4199 2.0969 0.2876 3.2292 1.7584 unknown 1.5850 1.9220 1.2964 2.2106 1.7535 unknown 2.1399 1.3646 0.7585 2.7460 1.7523 unknown 2.1532 1.3365 0.7885 2.7013 1.7449 homologue to GP|7959345|dbj|BAA96063.1 KIAA1539 protein {Homo sapiens}, partial (29%) 2.9048 0.5798 2.0690 1.4155 1.7423 unknown 2.4466 1.0344 2.4075 1.0735 1.7405 unknown 2.8655 0.5905 0.4663 2.9897 1.7280 precursor protein (partial) (AA -24 to 392) [Sus scrofa] 2.3906 1.0638 0.1680 3.2864 1.7272 unknown 2.0000 1.4415 2.1320 1.3095 1.7208 GP|14272235|emb|CAC39629. bA183L8.1 (lipoma HMGIC fusion partner) {Homo sapiens}, partial (53%) 2.8645 0.5749 1.7287 1.7107 1.7197 unknown 2.5006 0.9260 3.0224 0.4042 1.7133 unknown 3.3879 0.0208 3.0219 0.3868 1.7043 unknown 2.4442 0.9635 0.8904 2.5173 1.7038 unknown 3.1370 0.2641 0.5067 2.8943 1.7005 unknown 1.8593 1.5380 0.2473 3.1499 1.6986 unknown 3.2763 0.0966 1.7975 1.5754 1.6864 unknown 1.8142 1.5568 1.0314 2.3397 1.6855 unknown 1.5525 1.8125 0.8178 2.5472 1.6825 unknown 2.4386 0.9199 1.1497 2.2088 1.6793 unknown 2.2736 1.0728 1.5230 1.8234 1.6732 homologue to GP|17511743|gb|AAH18727.1 Unknown (protein for MGC:3183) {Homo sapiens}, partial (15%) 1.7776 1.5686 1.3553 1.9909 1.6731 unknown 2.1940 1.1352 0.6594 2.6698 1.6646 282 unknown 1.7943 1.5255 1.0163 2.3034 1.6599 unknown 1.7792 1.5138 1.7830 1.5100 1.6465 similar to SP|O15228|DAPT_HUMAN Dihydroxyacetone phosphate acyltransferase (EC 2.3.1.42) (DHAP-AT) (DAP- AT), partial (27%) 3.2244 0.0611 1.5638 1.7217 1.6428 unknown 1.3847 1.8937 0.6821 2.5963 1.6392 unknown 1.8931 1.3626 1.2279 2.0278 1.6278 -2.5705 unknown -2.9319 -2.2091 -1.7044 -3.4366 -2.5750 fibrinogen A-alpha-chain [Sus scrofa] -0.3862 -4.7639 -1.2507 -3.8994 -2.5797 unknown -1.9651 -3.1942 -1.4173 -3.7420 -2.5882 unknown -3.9596 -1.2169 -2.4301 -2.7464 -2.5984 unknown -2.5494 -2.6474 -2.4874 -2.7094 -2.6008 PIR|S38962|S38962 serpin - pig, partial (64%) -0.7701 -4.4314 -1.7929 -3.4086 -2.6018 homologue to SP|P15586|GL6S_HUMAN N- acetylglucosamine-6-sulfatase precursor (EC 3.1.6.14) (G6S) (Glucosamine-6-sulfatase). [Human], partial (34%) -3.1503 -2.0533 -1.8919 -3.3117 -2.6080 similar to GP|7582395|gb|AAF64308.1| class mu glutathione S-transferase {Bos taurus}, partial (55%) -3.4719 -1.7440 -2.1642 -3.0517 -2.6083 unknown -3.3982 -1.8184 -1.9872 -3.2294 -2.6097 unknown -3.5059 -1.7135 -1.4397 -3.7797 -2.6116 homologue to SP|P35227|ME18_HUMAN DNA-binding protein Mel-18 (Zinc finger protein 144). [Human] {Homo sapiens}, partial (61%) -2.9457 -2.2776 -1.3583 -3.8649 -2.6182 unknown -2.6234 -2.6129 -1.3358 -3.9005 -2.6242 similar to GP|14602473|gb|AAH09742.1 ladinin 1 {Homo sapiens}, partial (21%) -2.4974 -2.7510 -3.3424 -1.9060 -2.6272 unknown -3.0718 -2.1826 -1.2948 -3.9595 -2.6273 homologue to GP|1730288|gb|AAC50934.1| acetolactate synthase homolog {Homo sapiens}, partial (61%) -4.3899 -0.8648 -1.6598 -3.5948 -2.6294 unknown -2.5369 -2.7218 -1.9194 -3.3394 similar to GP|1311661|gb|AAC50471.1| hepatocyte growth factor-like protein {Homo sapiens}, partial (36%) -3.9958 -1.2715 -3.1755 -2.0919 -2.6337 similar to GP|16552719|dbj|BAB71368. unnamed protein product {Homo sapiens}, partial (80%) -2.7323 -2.5387 -1.7370 -3.5341 -2.6355 similar to GP|3687387|emb|CAA69957.1 ranbp3 {Homo sapiens}, partial (26%) -1.7630 -3.5110 -2.2692 -3.0047 -2.6370 GP|14495652|gb|AAH09434.1 Unknown (protein for MGC:15765) {Homo sapiens}, partial (4%) -2.5294 -2.7617 -1.8910 -3.4000 -2.6455 unknown -2.5449 -2.7530 -1.4220 -3.8759 -2.6489 unknown -3.4073 -1.9010 -1.5578 -3.7505 -2.6542 unknown -2.8305 -2.4812 -0.7994 -4.5123 -2.6558 similar to SP|P06681|CO2_HUMAN Complement C2 precursor (EC 3.4.21.43) (C3/C5 convertase). [Human] {Homo sapiens}, partial (26%) -2.7072 -2.6071 -1.5634 -3.7509 -2.6572 SP|P29053|TF2B_RAT Transcription initiation factor IIB (TFIIB) (RNA polymerase II alpha initiation factor). [Rat], partial (80%) -2.0740 -3.2479 -2.4249 -2.8971 -2.6610 homologue to SP|P53007|TXTP_HUMAN Tricarboxylate transport protein mitochondrial precursor (Citrate transport protein) (CTP), partial (86%) -2.5373 -2.8010 -1.9130 -3.4253 -2.6692 unknown -3.2091 -2.1434 -2.5603 -2.7922 -2.6763 similar to GP|12802994|gb|AAH01099.1 Unknown (protein for IMAGE:3510317) {Homo sapiens}, partial (71%) -3.6818 -1.7067 -3.1313 -2.2572 -2.6943 SP|Q00380|A2S1_MOUSE Clathrin coat assembly protein AP17 (Clathrin coat associated protein AP17), partial (59%) -2.3172 -3.0753 -2.2709 -3.1216 -2.6963 unknown -2.6465 -2.7612 -2.3873 -3.0204 -2.7039 283 similar to GP|12852439|dbj|BAB29412. putative {Mus musculus}, partial (18%) -3.7987 -1.6090 -1.8268 -3.5809 -2.7039 weakly similar to GP|16878257|gb|AAH17327.1 Unknown (protein for MGC:29726) {Homo sapiens}, partial (23%) -1.5740 -3.8448 -2.7423 -2.6766 -2.7094 unknown -3.3015 -2.1316 -2.4386 -2.9945 -2.7165 homologue to GP|8515870|gb|AAF76218.1| bridging integrator-3 {Homo sapiens}, partial (67%) -3.4191 -2.0150 -2.3873 -3.0467 -2.7170 similar to SP|P21195|PDI_RABIT Protein disulfide isomerase precursor (PDI) (EC 5.3.4.1) (Prolyl 4- hydroxylase beta subunit), partial (46%) -3.0709 -2.3691 -1.7676 -3.6724 -2.7200 similar to GP|15080110|gb|AAH11831.1 Unknown (protein for MGC:20496) {Homo sapiens}, partial (72%) -2.5744 -2.8669 -2.6535 -2.7878 -2.7206 endothelin-converting enzyme 1 [Sus scrofa] -2.7067 -2.7380 -2.4240 -3.0206 -2.7223 homologue to GP|12653059|gb|AAH00294.1 Unknown (protein for IMAGE:2819455) {Homo sapiens}, complete -1.5438 -3.8887 -2.3752 -3.0821 -2.7224 homologue to GP|13279068|gb|AAH04266.1 Unknown (protein for IMAGE:3613103) {Homo sapiens}, partial (45%) -2.5481 -2.9040 -2.5180 -2.9341 -2.7261 homologue to GP|14290607|gb|AAH09084.1 Similar to selenium binding protein 1 {Homo sapiens}, partial (30%) -3.1525 -2.3121 -3.3243 -2.1402 -2.7323 vascular endothelial growth factor -2.9024 -2.5685 -2.1981 -3.2729 -2.7355 homologue to SP|O60907|TBL1_HUMAN Transducin beta- like 1 protein. [Human] {Homo sapiens}, partial (47%) -2.6931 -2.8027 -2.1375 -3.3583 -2.7479 similar to GP|2645879|gb|AAB87523.1| molybdenum cofactor biosynthesis protein A {Homo sapiens}, partial (46%) -1.9311 -3.5757 -2.2697 -3.2370 -2.7534 similar to GP|2224569|dbj|BAA20773.1 KIAA0314 {Homo sapiens}, partial (22%) -3.0078 -2.5025 -2.4854 -3.0249 -2.7552 cytochorme P450 2A19 [Sus scrofa] -3.3379 -2.1767 -2.2105 -3.3041 -2.7573 homologue to GP|11275667|gb|AAG33699.1 oxidized-LDL responsive gene 2 {Homo sapiens}, partial (36%) -2.9915 -2.5240 -1.1441 -4.3714 -2.7577 similar to GP|16516591|emb|CAD10242. unnamed protein product {Homo sapiens}, partial (56%) -3.4348 -2.0865 -0.6715 -4.8498 -2.7607 homologue to SP|P02469|LMB1_MOUSE Laminin beta-1 chain precursor (Laminin B1 chain). [Mouse] {Mus musculus}, partial (15%) 0.0931 -5.6147 -3.6221 -1.8995 -2.7608 metallothionein isoform [Sus scrofa]metallothionein isoform [Sus scrofa]metallothionein isoform [Sus scrofa]metallothionein isoform [Sus scrofa]metallothionein isoform [Sus scrofa]metallothionein isoform [Sus scrofa]metallothionein isoform [S -4.1415 -1.3899 -2.7291 -2.8023 -2.7657 homologue to GP|14336773|gb|AAK61300.1 annexin A2 like - ? : selenoprotein X {Homo sapiens}, complete -3.2463 -2.2891 -2.1356 -3.3998 -2.7677 homologue to GP|1840045|gb|AAB47236.1| transporter protein {Homo sapiens}, partial (27%) -3.1409 -2.3955 -1.0699 -4.4665 -2.7682 unknown -2.3780 -3.1621 -1.2742 -4.2658 -2.7700 gag protein [Sus scrofa]pol protein [Sus scrofa]env protein [Sus scrofa]gag-pol precursor [Sus scrofa domestica]protease [Sus scrofa]protease [Sus scrofa]protease [Sus scrofa]protease [Sus scrofa]protease [Sus scrofa]polyprotein [Sus sc -0.9403 -4.6098 -1.6868 -3.8633 -2.7750 homologue to GP|14043628|gb|AAH07788.1 Similar to eukaryotic translation initiation factor 4 gamma 1 {Homo sapiens}, partial (35%) -2.9371 -2.6162 -1.6998 -3.8536 -2.7767 unknown -3.0365 -2.5271 -4.4150 -1.1485 -2.7818 homologue to GP|12804417|gb|AAH01611.1 Similar to bromodomain-containing 7 {Homo sapiens}, partial (32%) -2.6049 -2.9830 -1.3419 -4.2459 -2.7939 complement component C1s [Sus scrofa] -2.7853 -2.8118 -1.3415 -4.2556 -2.7986 GP|567053|gb|AAA56751.1|| beta 5 tubulin {Xenopus laevis}, partial (22%) -4.6795 -0.9783 -2.5973 -3.0605 -2.8289 unknown -2.7875 -2.8739 -0.8875 -4.7739 -2.8307 unknown -2.9519 -2.7134 -0.8495 -4.8158 -2.8326 284 homologue to GP|12832716|dbj|BAB22226. putative {Mus musculus}, partial (30%) -3.8374 -1.8291 -1.2025 -4.4639 -2.8332 similar to SP|P58058|PPNK_MOUSE Putative inorganic polyphosphate/ATP-NAD kinase (EC 2.7.1.23) (Poly(P)/ATP NAD kinase). [Mouse], partial (55%) -2.8913 -2.7767 -1.3164 -4.3516 -2.8340 mature porcine factor I -2.4647 -3.2060 -2.1344 -3.5363 -2.8353 unknown -3.0822 -2.5984 -2.9882 -2.6924 -2.8403 similar to GP|13477137|gb|AAH05025.1 Similar to metalloprotease 1 (pitrilysin family) {Homo sapiens}, partial (20%) -3.2389 -2.4490 -1.9511 -3.7369 -2.8440 matricin -2.0796 -3.6400 -3.0965 -2.6230 -2.8598 polypyrimidine tract-binding protein [Sus scrofa] -3.1578 -2.5674 -2.2410 -3.4842 -2.8626 similar to GP|3882165|dbj|BAA34442.1 KIAA0722 protein {Homo sapiens}, partial (9%) -2.6613 -3.0902 -2.6416 -3.1099 -2.8758 acyl-CoA oxidase [Sus scrofa] -3.1852 -2.5802 -1.9946 -3.7708 -2.8827 similar to GP|17512170|gb|AAH19069.1 Unknown (protein for MGC:29596) {Homo sapiens}, partial (29%) -2.7710 -2.9982 -1.6339 -4.1354 -2.8846 GP|12803403|gb|AAH02524.1 KIAA0064 gene product {Homo sapiens}, partial (81%) -2.9709 -2.8004 -3.4968 -2.2744 -2.8856 similar to GP|14602654|gb|AAH09849.1 Unknown (protein for MGC:15400) {Homo sapiens}, partial (56%) -3.4883 -2.2898 -2.3806 -3.3975 -2.8891 similar to SP|Q9XT77|SL56_RABIT Sodium-dependent multivitamin transporter (Na(+)-dependent multivitamin transporter). [Rabbit], partial (17%) -1.7521 -4.0279 -1.7143 -4.0657 -2.8900 unknown -3.0350 -2.7714 -1.4303 -4.3761 -2.9032 unknown -3.8885 -1.9257 -1.8609 -3.9533 -2.9071 homologue to GP|15929704|gb|AAH15276.1 inter-alpha trypsin inhibitor heavy chain 3 {Mus musculus}, partial (21%) -3.3755 -2.4548 -1.5124 -4.3179 -2.9151 similar to SP|O43615|IM44_HUMAN Import inner membrane translocase subunit TIM44 mitochondrial precursor. [Human] {Homo sapiens}, partial (38%) -0.4780 -5.3707 -3.2487 -2.6000 -2.9244 similar to GP|37996|emb|CAA46158.1|| Xeroderma Pigmentosum Group C Complementing factor {Homo sapiens}, partial (23%) -2.3902 -3.4725 -1.9368 -3.9259 -2.9314 SP|P42891|ECE1_BOVIN Endothelin-converting enzyme 1 (EC 3.4.24.71) (ECE-1). [Bovine] {Bos taurus}, partial (14%) -3.7493 -2.1140 -1.6683 -4.1950 -2.9317 vascular endothelial growth factor [Sus scrofa] -2.9890 -2.8953 -2.1244 -3.7598 -2.9421 glucose-6-phosphatase catalytic subunit [Sus scrofa] -3.1862 -2.7024 -0.8747 -5.0138 -2.9443 unknown -3.2909 -2.6204 -1.9983 -3.9130 -2.9557 similar to SP|P15169|CBPN_HUMAN Carboxypeptidase N catalytic chain precursor (EC 3.4.17.3) (Arginine carboxypeptidase) (Kininase 1), partial (32%) -2.7057 -3.2089 -1.3984 -4.5161 -2.9573 homologue to GP|3800742|gb|AAC68839.1| RGC-32 {Rattus norvegicus}, partial (90%) -2.7942 -3.1340 -2.1017 -3.8265 -2.9641 similar to GP|12658433|gb|AAK01138.1 interferon regulatory factor 1 {Ovis aries}, partial (61%) -2.4690 -3.4594 -2.5584 -3.3701 -2.9642 similar to SP|Q92484|AS3A_HUMAN Acid sphingomyelinase-like phosphodiesterase 3a (EC 3.1.4.-) (ASM-like phosphodiesterase 3a), partial (49%) -2.8748 -3.0666 -1.7651 -4.1763 -2.9707 similar to GP|12803319|gb|AAH02477.1 Unknown (protein for MGC:3090) {Homo sapiens}, partial (7%) -2.8516 -3.1035 -0.5678 -5.3872 -2.9775 gal beta-1,3 GalNAc alpha-2,3 sialyltransferase -0.1526 -5.8159 -1.6941 -4.2745 -2.9843 similar to GP|7644350|gb|AAF65550.1| golgi matrix protein GM130 {Homo sapiens}, partial (9%) -3.5180 -2.4665 -0.6118 -5.3727 -2.9922 GP|12654655|gb|AAH01165.1 N-ethylmaleimide-sensitive factor attachment protein alpha {Homo sapiens}, partial (56%) -3.3293 -2.6581 -0.4894 -5.4980 -2.9937 homologue to GP|2285790|dbj|BAA21659.1 p47 {Rattus norvegicus}, partial (76%) -2.7822 -3.2072 -1.7340 -4.2554 -2.9947 285 homologue to GP|15341753|gb|AAH12406.1 Unknown (protein for MGC:7221) {Mus musculus}, partial (41%) -3.0480 -2.9512 -0.5729 -5.4263 -2.9996 homologue to GP|2624718|pdb|1RGP| Gtpase-Activation Domain From Rhogap, partial (88%) -2.7109 -3.2955 -0.9057 -5.1007 -3.0032 similar to GP|10441934|gb|AAG17244.1 unknown {Homo sapiens}, partial (29%) -3.4183 -2.5897 -4.0602 -1.9478 -3.0040 homologue to GP|13559033|emb|CAC36008. bA11M20.3.1 (novel protein similar to Pleurodeles waltlii RAP55 protein isoform 1) {Homo sapiens}, partial (48%) -2.3330 -3.6818 -2.3814 -3.6334 -3.0074 homologue to GP|13785926|gb|AAK39520.1 BTB domain protein {Homo sapiens}, partial (34%) -2.8387 -3.2072 -0.4954 -5.5505 -3.0229 homologue to SP|P22234|PUR6_HUMAN Multifunctional protein ADE2, partial (54%) -2.5758 -3.4720 -0.8815 -5.1663 -3.0239 unknown -3.3773 -2.7031 -1.3742 -4.7063 -3.0402 similar to GP|3372677|gb|AAC29066.1| tumorous imaginal discs protein Tid56 homolog {Homo sapiens}, partial (35%) -2.8217 -3.2617 -0.7370 -5.3464 -3.0417 similar to GP|15214665|gb|AAH12461.1 Similar to RIKEN cDNA 2310061O04 gene {Homo sapiens}, partial (63%) -0.5450 -5.5546 -1.3717 -4.7278 -3.0498 similar to PIR|A31870|A31870 amine oxidase (flavin- containing) (EC 1.4.3.4) B - rat, partial (32%) -3.3733 -2.7538 -2.2373 -3.8898 -3.0635 homologue to GP|10440073|dbj|BAB15639. unnamed protein product {Homo sapiens}, partial (29%) -2.4378 -3.7188 -1.5054 -4.6513 -3.0783 similar to PIR|T12469|T12469 hypothetical protein DKFZp564C1940.1 - human (fragment), partial (93%) -4.1196 -2.0395 -1.3194 -4.8396 -3.0795 unknown -4.0080 -2.1583 -1.6560 -4.5103 -3.0832 homologue to GP|14486422|gb|AAK61367.1 retina ubiquilin {Bos taurus}, partial (38%) -2.2398 -3.9542 -0.9016 -5.2924 -3.0970 homologue to SP|Q07954|LRP1_HUMAN Low-density lipoprotein receptor-related protein 1 precursor (LRP) (Alpha-2-macroglobulin receptor), partial (5%) -3.7630 -2.4454 -0.5451 -5.6633 -3.1042 homologue to GP|12653075|gb|AAH00303.1 phosphoglycerate dehydrogenase {Homo sapiens}, partial (31%) -4.0861 -2.1504 -1.1034 -5.1331 -3.1182 homologue to GP|10717134|gb|AAG22029.1 carbonic anhydrase III {Mus musculus}, complete -3.2913 -2.9463 -0.9393 -5.2983 -3.1188 similar to GP|18089178|gb|AAH20844.1 Unknown (protein for MGC:23971) {Homo sapiens}, partial (84%) -1.9437 -4.3143 -2.3109 -3.9471 -3.1290 MHC class I antigen [Sus scrofa]MHC PD1 major transplantation antigenMHC PD1a major transplantation antigenMHC class I antigen heavy chain [Sus scrofa] -2.0010 -4.2753 -4.8067 -1.4696 -3.1382 similar to GP|13477197|gb|AAH05060.1 Similar to quinolinate phosphoribosyltransferase, partial (58%) -3.9014 -2.4673 -1.9480 -4.4207 -3.1843 homologue to GP|8925838|gb|AAF81636.1| acidic alpha- glucosidase {Bos taurus}, partial (23%) -3.7638 -2.6496 -0.6636 -5.7498 -3.2067 similar to GP|12652817|gb|AAH00161.1 secretory carrier membrane protein 3 {Homo sapiens}, partial (35%) -3.3108 -3.2254 -1.2670 -5.2692 -3.2681 similar to GP|15209782|emb|CAC51180. unnamed protein product {Homo sapiens}, complete -3.7078 -2.8719 -2.4695 -4.1103 -3.2899 similar to SP|P98153|IDD_HUMAN Integral membrane protein DGCR2/IDD precursor. [Human] {Homo sapiens}, partial (94%) -3.0263 -3.5850 -2.3824 -4.2288 -3.3056 homologue to GP|4590328|gb|AAD26531.1| valyl-tRNA synthetase {Mus musculus}, partial (16%) -2.9069 -3.7188 -1.0547 -5.5710 -3.3129 similar to EGAD|88612|96537 lysophosphatidic {Homo sapiens}, partial (37%) -4.2724 -2.4102 -0.8945 -5.7881 -3.3413 similar to GP|12858330|dbj|BAB31278. putative {Mus musculus}, partial (37%) -3.5317 -3.2333 -3.3609 -3.4040 -3.3825 unknown -3.1455 -3.6727 -1.8301 -4.9881 -3.4091 unknown -3.4700 -3.3916 -2.1400 -4.7216 -3.4308 GP|12654775|gb|AAH01230.1 Similar to CGI-78 protein {Homo sapiens}, partial (53%) -4.0867 -2.8314 -1.9796 -4.9386 -3.4591 sialyltransferase [Sus scrofa] -1.8864 -5.0490 -1.6261 -5.3094 -3.4677 homologue to PIR|I39174|I39174 seven trans-membrane domain protein AD3LP/AD5 - human, partial (61%) -3.8841 -3.0952 -0.9941 -5.9852 -3.4896 286 similar to SP|P17177|CP27_RABIT Cytochrome P450 27 mitochondrial precursor (EC 1.14.-.-) (Cytochrome P- 450C27/25), partial (34%) -4.2203 -2.7629 -0.2183 -6.7650 -3.4916 similar to SP|P35292|RB17_MOUSE Ras-related protein Rab-17. [Mouse] {Mus musculus}, partial (68%) -4.1898 -2.8138 -2.3385 -4.6652 -3.5018 similar to GP|3273228|dbj|BAA29057.1 very-long-chain acyl-CoA dehydrogenase {Homo sapiens}, partial (27%) -3.6789 -3.4065 -1.6499 -5.4356 -3.5427 homologue to GP|6319138|gb|AAF07179.1| ALG-2 interacting protein 1 {Rattus norvegicus}, partial (38%) -3.3612 -3.7694 -2.7249 -4.4057 -3.5653 homologue to EGAD|136325|145398 neutral and basic amino acid transporter protein {Sus scrofa}, complete -2.3267 -4.8142 -4.7970 -2.3439 -3.5705 similar to GP|4826565|emb|CAB42884.1 cathepsin F {Mus musculus}, partial (63%) -2.8979 -4.2576 -1.3684 -5.7871 -3.5777 homologue to PIR|G01236|G01236 enhancer of split m9/m10 (groucho protein) - human, partial (92%) -2.2910 -4.8899 -1.8802 -5.3007 -3.5904 similar to SP|P23588|IF4B_HUMAN Eukaryotic translation initiation factor 4B (eIF-4B). [Human] {Homo sapiens}, partial (33%) -3.5004 -3.7213 -3.1055 -4.1162 -3.6108 GP|14550508|gb|AAH09504.1 Similar to CG8974 gene product {Homo sapiens}, partial (66%) -2.9194 -4.3607 -1.6157 -5.6645 -3.6401 immunoglobulin kappa light chain VJ region [Sus scrofa]immunoglobulin kappa light chain VJ region [Sus scrofa] -3.5288 -3.7567 -2.8242 -4.4614 -3.6428 acid-labile subunit [Sus scrofa] -3.7897 -3.5591 -2.7039 -4.6450 -3.6744 similar to GP|12652617|gb|AAH00054.1 7- dehydrocholesterol reductase {Homo sapiens}, complete -3.7207 -3.7808 -1.5133 -5.9882 -3.7508 similar to GP|12655193|gb|AAH01454.1 phosphoenolpyruvate carboxykinase 2 (mitochondrial) {Homo sapiens}, partial (23%) -5.8303 -1.6752 -5.0303 -2.4753 -3.7528 weakly similar to GP|881921|gb|AAC50161.1|| interferon- inducible peptide precursor {Homo sapiens}, partial (95%) -3.9620 -3.6104 -4.0468 -3.5256 -3.7862 unknown -4.2590 -3.3177 -2.1305 -5.4462 -3.7884 similar to GP|5596693|emb|CAB51405.1 hypothetical protein {Homo sapiens}, partial (43%) -3.0494 -4.5427 -1.0710 -6.5211 -3.7961 similar to GP|12654625|gb|AAH01149.1 Similar to KIAA0266 gene product {Homo sapiens}, partial (21%) -3.5990 -4.0224 -3.4185 -4.2029 -3.8107 homologue to SP|P34897|GLYM_HUMAN Serine hydroxymethyltransferase mitochondrial precursor (EC 2.1.2.1) (Serine methylase), partial (31%) -4.0647 -3.6516 -4.3602 -3.3561 -3.8581 homologue to GP|6330847|dbj|BAA86562.1 KIAA1248 protein {Homo sapiens}, partial (50%) -4.6183 -3.1515 -3.1856 -4.5843 -3.8849 homologue to SP|Q98TR3|RNT1_FUGRU Putative regulator of nonsense transcripts 1. [Japanese pufferfish Takifugu rubripes], partial (14%) -2.5366 -5.2854 -4.1544 -3.6676 -3.9110 similar to GP|17511927|gb|AAH18918.1 Unknown (protein for MGC:12603) {Homo sapiens}, complete -5.5236 -2.4537 -3.5483 -4.4290 -3.9886 homologue to GP|7022137|dbj|BAA91499.1 unnamed protein product {Homo sapiens}, partial (48%) -2.2416 -5.7495 -5.4242 -2.5670 -3.9956 GP|11514162|pdb|1FKN|A Chain A Structure Of Beta- Secretase Complexed With Inhibitor, partial (66%) -1.4101 -6.5925 -2.2818 -5.7208 -4.0013 homologue to GP|12857441|dbj|BAB31012. putative {Mus musculus}, partial (36%) -4.2044 -3.8563 -4.4581 -3.6027 -4.0304 homologue to SP|Q9NSE2|CISH_HUMAN Cytokine- inducible SH2-containing protein (Suppressor of cytokine signaling) (SOCS) (CIS1) (G18)., partial (54%) -2.1398 -6.1579 -3.2977 -5.0000 -4.1488 similar to SP|P28800|A2AP_BOVIN Alpha-2-antiplasmin precursor (Alpha-2-plasmin inhibitor) (Alpha-2-PI) (Alpha-2- AP). [Bovine], partial (50%) -4.7630 -3.5694 -3.2089 -5.1235 -4.1662 SP|O55096|DPP3_RAT Dipeptidyl-peptidase III (EC 3.4.14.4) (DPP III) (Dipeptidyl aminopeptidase III), partial (21%) -3.8515 -4.4998 -3.1655 -5.1859 -4.1757 homologue to GP|13543295|gb|AAH05811.1 pyruvate dehydrogenase kinase isoenzyme 2 {Homo sapiens}, partial (53%) -4.9206 -3.5308 -3.8883 -4.5631 -4.2257 similar to GP|12003293|gb|AAG43523.1 organic anion -5.0924 -3.4217 -3.0395 -5.4746 -4.2571 287 transporter 2 {Homo sapiens}, partial (28%) similar to PIR|I56095|C4HU complement C4A precursor [validated] - human, partial (9%) -5.7787 -2.8958 -4.8162 -3.8584 -4.3373 similar to PIR|A56619|A56619 female sterile homeotic (fsh) homolog RING3 - human, partial (27%) -4.4403 -4.2919 -3.6348 -5.0974 -4.3661 homologue to GP|4938304|emb|CAA07619.2 lysine- ketoglutarate reductase /saccharopine dehydrogenase {Homo sapiens}, partial (15%) -0.8021 -7.9530 -4.4003 -4.3548 -4.3775 similar to GP|467528|dbj|BAA01185.1| alanine aminotransferase {Rattus norvegicus}, partial (24%) -4.5250 -4.2790 -3.2394 -5.5645 -4.4020 similar to GP|3327162|dbj|BAA31649.1 KIAA0674 protein {Homo sapiens}, partial (14%) -3.6082 -5.2854 -3.7888 -5.1048 -4.4468 similar to SP|P30519|HO2_HUMAN Heme oxygenase 2 (EC 1.14.99.3) (HO-2). [Human] {Homo sapiens}, complete -4.7502 -4.2364 -5.0230 -3.9636 -4.4933 similar to GP|14789873|gb|AAH10812.1 Unknown (protein for IMAGE:4211034) {Mus musculus}, partial (50%) -4.8527 -4.5506 -4.3326 -5.0706 -4.7016 similar to SP|O75891|FTDH_HUMAN 10- formyltetrahydrofolate dehydrogenase (EC 1.5.1.6) (10- FTHFDH). [Human] {Homo sapiens}, partial (18%) -5.0176 -4.4480 6.2603 -15.7258 -4.7328 similar to GP|14603281|gb|AAH10100.1 Unknown (protein for MGC:19693) {Homo sapiens}, partial (22%) -3.7384 -5.8159 -5.6939 -3.8604 -4.7771 similar to GP|17389278|gb|AAH17692.1 Similar to quiescin Q6 {Homo sapiens}, partial (30%) -4.4499 -5.4378 -3.6599 -6.2277 -4.9438 alpha-2,6-sialyltransferase [Sus scrofa] -4.8471 -5.6660 -3.7079 -6.8052 -5.2565 Table 3. Transcripts highly up/down regulated determined by oligo-array in the adipose tissue by the dietary shifting from LFD to HFD (Chapter 3). For each transcript, log tio=log e means higher mRNA abundance in HFD pigs; negative value means lower mRNA abundance in HFD pigs Gene name Log 1/ G1) Log 2/ G2) Log 3/ R3) Average 2 ra 2 (HFD/LFD).The positive valu 2 (R 2 (R 2 (G Log 2 (G4/ R4) similartoGP13810568dbjBAB43955.Toll- likereceptor5{Homosapiens}.partial(19%) 3.5025 4.4811 4.3977 3.5859 3.9918 DNA- directedRNApolymeraseIIpolypeptideB;POLR2B[Susscr ofa] 2.1876 5.3576 2.2878 5.2574 3.7726 unknown 2.1769 5.3129 1.8452 5.6445 3.7449 alpha-1-antichymotrypsin3[Susscrofa] 2.8413 5.3672 2.4207 3.9059 3.6338 homologuetoGP12805589gbAAH02274.1Unknown(prot einforMGC:7676){Musmusculus}.complete 1.4150 6.3750 1.7276 4.0624 3.3950 AMP- activatedproteinkinasegammasubunit[Susscrofa]AMPKg ammasubunit[Susscrofa] 2.8875 3.3219 2.1494 4.0601 3.1047 unknown 3.9069 2.0544 1.1019 4.8595 2.9807 unknown 0.6280 5.3219 0.5070 5.4430 2.9750 similartoGP7981270embCAB91983.1hypotheticalprotein {Homosapiens}.partial(56%) 0.9434 4.9189 2.2912 3.5710 2.9311 homologuetoGP3327126dbjBAA31631.1KIAA0656protei n{Homosapiens}.partial(22%) 2.0000 3.8413 2.3383 3.5030 2.9207 unknown 0.2137 6.0444 1.2617 4.1415 2.9153 unknown 0.5305 5.2095 0.1959 5.5440 2.8700 homologuetoGP2828149gbAAC00006.1cyclophilin- 33A{Homosapiens}.partial(72%) 0.5850 5.1293 0.1507 5.5636 2.8571 weaklysimilartoGP13623713gbAAH06486.1Unknown(pr oteinforMGC:771){Homosapiens}.partial(61%) 1.9069 3.6724 1.6980 3.8813 2.7897 unknown 0.5146 5.0444 2.0780 3.4810 2.7795 similartoGP17389636gbAAH17843.1cholesterol25- 1.1127 5.4263 0.9189 3.6201 2.7695 288 289 maantigenP15){Homosapiens}.partial(88%) intermediate-conductancecalcium- activatedpotassiumchannel[Susscrofa] 0.6705 3.5361 1.6613 2.5452 2.1033 unknown 1.4910 2.7004 1.4443 2.7471 2.0957 homologuetoPIRJC5392JC5392zincfingerproteinKF- 2.4594 1.7253 1.8519 2.3328 2.0924 hydroxylase{Homosapiens}.partial(60%) similartoGP12803737gbAAH02705.1chromosome22ope nreadingframe3{Homosapiens}.partial(84%) 1.3692 4.1155 1.1699 4.3148 2.7424 unknown 0.2801 5.1699 0.4005 5.0495 2.7250 similartoGP183997gbAAA58640.1heregulin- beta2{Homosapiens}.partial(17%) 2.1699 3.2095 2.2473 3.1320 2.6897 unknown 0.8480 4.4919 1.1234 4.2165 2.6699 unknown 0.7370 4.5850 1.1629 4.1590 2.6610 unknown 10.2041 5.4919 1.6479 -6.7683 2.6439 unknown 0.0875 5.1293 0.5580 4.6588 2.6084 unknown 0.2801 4.8413 1.3866 3.7348 2.5607 unknown 0.4657 4.5850 0.7590 4.2916 2.5253 unknown 0.5771 4.4150 0.6558 4.3362 2.4960 unknown 0.2479 4.6439 1.6923 3.1995 2.4459 unknown 1.0182 3.8413 0.7628 4.0967 2.4298 similartoGP10435722dbjBAB14652.unnamedproteinpro duct{Homosapiens}.partial(81%) 1.9296 2.9069 2.3923 2.4442 2.4183 unknown 2.0419 2.7655 0.8766 3.9308 2.4037 similartoGP10438452dbjBAB15247.unnamedproteinpro duct{Homosapiens}.partial(42%) 1.7645 3.0356 2.3079 2.4923 2.4001 unknown 0.4475 5.2095 1.2462 2.6209 2.3810 similartoGP1770426embCAA66469.1G- proteincoupledreceptorkinase{Homosapiens}.partial(75 %) 3.1699 1.5850 0.7977 3.9572 2.3774 GP13938376gbAAH07321.1Unknown(proteinforMGC:13 46){Homosapiens}.complete 0.4639 4.2801 0.8417 3.9023 2.3720 similartoGP1732422gbAAB51326.1C3f{Homosapiens}.p artial(20%) 0.3626 4.3576 0.4500 4.2701 2.3601 unknown 0.1159 4.4919 1.2613 3.3465 2.3039 unknown 2.4594 2.0809 1.8729 2.6674 2.2702 unknown 0.5850 3.9542 2.3862 2.1530 2.2696 unknown 1.2458 3.2801 4.0608 0.4651 2.2629 unknown 1.0704 4.5850 0.0907 3.2831 2.2573 weaklysimilartoGP16306780gbAAH01584.1Unknown(pr oteinforIMAGE:3461401){Homosapiens}.partial(54%) 1.0378 3.4594 1.9316 2.5656 2.2486 CCAAT/enhancerbindingproteinbeta[Susscrofa] 1.3301 3.1375 0.8598 3.6078 2.2338 homologuetoSPQ15329E2F5_HUMANTranscriptionfact orE2F5(E2F-5).[Human]{Homosapiens}.partial(40%) 1.5384 2.9175 2.1716 2.2843 2.2280 SPP29053TF2B_RATTranscriptioninitiationfactorIIB(TFII B)(RNApolymeraseIIalphainitiationfactor).[Rat].partial(80 %) 0.8074 3.6439 0.1683 4.2829 2.2256 similartoGP13591537embCAC36352.dJ1033H22.2(brea stcanceranti- estrogenresistance3){Homosapiens}.partial(50%) 1.4150 2.9798 0.4386 3.9563 2.1974 unknown 0.6134 3.7814 2.3299 2.0648 2.1974 homologuetoSPO14493CLD4_HUMANClaudin- 4(Clostridiumperfringensenterotoxinreceptor)(CPE- receptor)(CPE-R).[Human].complete 1.8480 2.4831 1.7989 2.5322 2.1655 unknown 0.9260 3.3576 0.0674 4.2162 2.1418 unknown 0.3219 4.5850 1.0972 2.5220 2.1315 unknown 0.5361 3.7004 0.1660 4.0705 2.1182 similartoGP13751639embCAC37285.C367G8.1(melano 1.0473 3.1832 2.2684 1.9621 2.1153 290 unknown 0.3959 3.2479 1.6987 1.9452 1.8219 similartoPIRT08771T08771hypotheticalproteinDKFZp58 6L151.1-human(fragment).partial(35%) 0.9260 2.7162 0.8845 2.7577 1.8211 homologuetoSPQ9ULW2FZ10_HUMANFrizzled10precu rsor(Frizzled-10)(Fz- 0.7687 2.8413 -2.4780 6.0880 1.8050 1precursor-human.partial(42%) unknown 1.5824 4.3458 0.5652 1.7534 2.0617 unknown -0.5619 4.6439 3.0972 0.9847 2.0410 cyclin- dependentkinaseinhibitor.p16[Susscrofa]cyclindependan tkinaseinhibitor[Susscrofa]cyclindependantkinaseinhibito r[Susscrofa] 2.0000 2.0780 1.1405 2.9375 2.0390 unknown 0.6101 3.4594 0.3660 3.7035 2.0347 unknown 0.1155 4.1699 0.2370 3.5865 2.0272 weaklysimilartoGP17315166gbAAH14156.1Unknown(pr oteinforMGC:20834){Homosapiens}.partial(62%) -0.0893 4.1247 2.3231 1.7123 2.0177 homologuetoGP11527783dbjBAB18652.ubiquitin- conjyugatingenzymeE2{Homosapiens}.partial(59%) 2.0477 1.9676 1.4617 2.5535 2.0076 unknown 0.5703 3.4429 0.2630 3.7502 2.0066 unknown 1.0000 3.0000 1.0658 2.9342 2.0000 unknown 0.3720 3.5607 0.9977 2.9350 1.9663 unknown 0.9411 2.9819 2.4517 1.4713 1.9615 similartoSPP30519HO2_HUMANHemeoxygenase2(EC1 .14.99.3)(HO-2).[Human]{Homosapiens}.complete 2.0324 1.8745 0.5017 3.4052 1.9534 similartoGP17736920gbAAL41029.1asparaginase- likespermautoantigen{Rattusnorvegicus}.partial(42%) 0.3626 3.5443 1.1050 2.8019 1.9534 similartoGP12654233gbAAH00936.1Similartohypothetic alproteinclone1-2{Homosapiens}.partial(86%) 1.3847 2.5146 1.2104 2.6889 1.9496 unknown 1.9386 1.9434 0.8573 3.0248 1.9410 unknown 1.7549 2.0926 0.6642 3.1833 1.9238 homologuetoGP1054835embCAA63313.1ICA105{Rattu snorvegicus}.partial(14%) 1.0375 2.8074 0.8604 2.9844 1.9224 homologuetoGP496887embCAA56071.1betatubulin{Ho mosapiens}.partial(56%) -0.0931 3.9307 0.7457 3.0919 1.9188 homologuetoGP207008gbAAA42158.1smallnuclearribon ucleoparticle- associatedprotein{Rattusnorvegicus}.partial(56%) 1.8074 2.0000 2.2515 1.5558 1.9037 unknown 0.3175 3.4854 1.9916 1.8114 1.9015 unknown 1.7004 2.0995 1.5294 2.2705 1.9000 similartoSPP20132SDHL_HUMANL- serinedehydratase(EC4.2.1.13)(L- serinedeaminase).[Human]{Homosapiens}.partial(37%) 0.6139 3.1699 1.0203 2.7635 1.8919 homologuetoGP12231878gbAAG49297.1copineI{Homo sapiens}.partial(83%) 0.8074 2.9386 1.2746 2.4713 1.8730 GP12746394gbAAK07475.1CUG-BPandETR- 3likefactor4{Homosapiens}.partial(21%) 1.0661 3.8074 0.6868 1.9222 1.8706 similartoGP16878298gbAAH17344.1Similartohypothetic alproteinFLJ23469{Homosapiens}.complete 1.5850 2.1520 2.3281 1.4089 1.8685 homologuetoGP4521249dbjBAA76297.1DNAhelicase{M usmusculus}.partial(31%) 0.5850 3.1375 1.4788 2.2436 1.8612 unknown 2.2928 1.4288 1.4797 2.2420 1.8608 unknown 0.4374 3.2801 1.6828 2.0347 1.8588 unknown 0.7370 2.9798 1.0740 2.6428 1.8584 unknown 0.4361 3.2801 1.1716 2.5446 1.8581 homologuetoGP6729336dbjBAA89782.1seventransmem branedomainorphanreceptor{Homosapiens}.partial(58%) 0.3626 3.3219 0.2288 3.4557 1.8422 similartoGP13277562gbAAH03690.1SimilartoRIKENcD NA8430408O15gene{Homosapiens}.partial(35%) 0.7574 2.9260 0.1191 3.5644 1.8417 unknown 2.3388 1.3093 1.5445 2.1036 1.8241 291 otein{Homosapiens}.complete unknown 0.5305 2.6699 1.2873 1.9130 1.6002 similartoSPO75031HBP2_HUMANHeatshockfactor2bind ingprotein.[Human]{Homosapiens}.partial(73%) 1.3969 1.5850 2.0354 1.3589 1.5940 10)(hFz10)(FzE7).[Human]{Homosapiens}.partial(27%) unknown 0.6374 2.9635 1.9820 1.6189 1.8005 unknown 0.1612 3.4263 1.4363 2.1511 1.7937 GP13436092gbAAH04869.1Unknown(proteinforIMAGE: 3834272){Homosapiens}.partial(47%) 0.0000 3.5850 1.6680 1.9170 1.7925 unknown 0.6521 2.9260 0.3565 3.2216 1.7890 unknown 0.5206 4.0875 1.0540 1.4718 1.7834 unknown 1.5146 2.0211 1.9980 1.5376 1.7678 unknown 0.5721 2.9594 0.9686 2.5629 1.7657 homologuetoGP15930203gbAAH15534.1Unknown(prot einforMGC:9474){Homosapiens}.partial(50%) 0.2895 3.8074 1.7359 1.2030 1.7589 unknown 0.5850 2.9307 0.7922 2.7235 1.7578 unknown 0.1420 2.3692 1.1546 3.3567 1.7556 similartoGP16198523gbAAH15943.1Unknown(proteinfor MGC:9325){Homosapiens}.partial(84%) 0.7279 2.7655 1.4666 2.0269 1.7467 homologuetoGP603953dbjBAA07893.1Thisgeneisnovel. {Homosapiens}.partial(18%) 2.5361 0.9438 1.4416 2.0383 1.7399 unknown 0.6215 2.8524 1.9243 1.5496 1.7370 interleukin-15[Susscrofa] 1.7162 1.7432 1.5878 1.8716 1.7297 unknown 1.1575 2.2928 0.5792 2.8712 1.7252 unknown 0.4854 2.9635 1.3326 2.1163 1.7245 similartoGP14714476gbAAH10364.1mitochondrialriboso malproteinS18A{Homosapiens}.complete 1.3994 3.8220 0.6874 0.9365 1.7113 similartoPIRA42912A429123alpha(or20beta)- hydroxysteroiddehydrogenase(EC1.1.1.53)-pig.complete 0.5305 2.8679 0.7199 2.6785 1.6992 unknown 0.3858 2.9635 2.4069 0.9424 1.6746 unknown 0.5261 2.8202 1.0921 2.2541 1.6731 homologuetoGP11141704gbAAG32038.1SIR2L2{Musm usculus}.partial(50%) 0.4330 2.9069 0.0931 3.2467 1.6699 unknown 1.5850 1.7549 0.5679 2.7720 1.6699 unknown 0.6630 2.6738 1.8005 1.5362 1.6684 similartoGP12406680embCAC24973.unnamedproteinpr oduct{Homosapiens}.partial(65%) 0.8737 2.4429 2.4001 0.9166 1.6583 homologuetoGP13938463gbAAH07375.1Similartotumor differentiallyexpressed1{Homosapiens}.partial(56%) 1.5208 1.7935 0.2196 3.0948 1.6572 unknown 0.7004 2.6114 1.3337 1.9782 1.6559 homologuetoSPP48443RXRG_HUMANRetinoicacidrece ptorRXR-gamma.[Human]{Homosapiens}.partial(41%) 0.1699 3.1293 1.9361 1.3631 1.6496 GP16041715gbAAH15733.1Unknown(proteinforMGC:22 977){Homosapiens}.partial(31%) -0.2988 3.5850 0.1415 3.1446 1.6431 unknown 2.1155 1.1699 1.1678 2.1176 1.6427 unknown 0.7814 1.4975 2.3803 1.8986 1.6394 homologuetoGP15919176gbAAL10712.1buddinguninhib itedbybenzimidazoles1beta{Homosapiens}.partial(16%) 0.5097 2.7655 1.1021 2.1731 1.6376 unknown 0.6101 2.6605 1.9502 1.3204 1.6353 similartoGP1469205dbjBAA09490.1TheKIAA0141genep roductisnovel.{Homosapiens}.partial(36%) 1.0589 3.3219 0.2847 1.8606 1.6315 similartoGP12652921gbAAH00217.1Similartohypothetic alprotein{Homosapiens}.complete 1.4094 1.8398 0.9104 2.3389 1.6246 unknown 0.4150 2.8301 1.0835 2.1616 1.6226 unknown 1.2451 1.4594 1.6848 2.0393 1.6072 homologuetoGP12053249embCAB66806.hypotheticalpr 0.3785 2.8301 0.8257 2.3828 1.6043 292 myosinregulatorylightchainventricularisoform[Susscrofa] myosinlightchain2[Susscrofa] 1.4935 1.4279 1.7475 1.4624 1.5328 unknown 1.3112 1.7506 0.5146 2.5473 1.5309 unknown 1.1699 1.8894 1.7649 1.2945 1.5297 homologuetoGP12655091gbAAH01396.1AD- 003protein{Homosapiens}.partial(62%) 1.3281 3.5110 0.7245 0.8021 1.5914 similartoGP13177635gbAAK14906.1phospholipaseCbet a-3{Rattusnorvegicus}.partial(28%) 0.1069 3.0661 0.7427 2.4303 1.5865 unknown 0.5850 2.5850 0.3799 2.7900 1.5850 SPP52433RPB7_HUMANDNA- directedRNApolymeraseII19kDapolypeptide(EC2.7.7.6)( RPB7).[Rat]{Rattusnorvegicus}.complete 1.6323 1.5311 1.5334 1.6299 1.5817 unknown 2.1699 0.9922 1.1210 2.0411 1.5811 homologuetoSPP55931ETFD_PIGElectrontransferflavop rotein- ubiquinoneoxidoreductasemitochondrialprecursor(EC1.5 .5.1).partial(22%) 1.8931 1.2666 1.8083 1.3514 1.5799 weaklysimilartoGP6841340gbAAF29023.1HSPC051{Ho mosapiens}.partial(86%) 1.6049 1.5493 2.7632 0.3910 1.5771 homologuetoGP13625170gbAAK34944.1NDR3{Homosa piens}.partial(67%) 0.6374 2.5166 1.5406 1.6134 1.5770 unknown 1.0995 2.0531 1.8455 1.3072 1.5763 unknown 0.0000 3.1468 1.0106 2.1363 1.5734 unknown 1.6571 1.4780 1.6234 1.5117 1.5676 homologuetoGP10441970gbAAG17262.1unknown{Hom osapiens}.partial(20%) 1.0000 2.1346 1.6650 1.4697 1.5673 homologuetoGP17223689gbAAK77940.1F- boxproteinFBG3{Homosapiens}.partial(74%) 0.6414 2.4919 1.8489 1.2843 1.5666 similartoGP12653567gbAAH00557.1phosphatidylethano lamineN-methyltransferase{Homosapiens}.partial(86%) 0.1031 3.0297 0.3037 2.8292 1.5664 homologuetoGP17224454gbAAL36982.1nanos{Homosa piens}.partial(78%) 0.6668 2.4594 2.3667 0.7595 1.5631 unknown 1.1491 1.2730 2.2821 1.5434 1.5619 GP12652557gbAAH00018.1Similartoactinrelatedprotein 2/3complexsubunit5(16kD){Homosapiens}.complete 0.7885 2.3219 1.8734 1.2371 1.5552 unknown 0.6630 2.4475 0.5995 2.5110 1.5552 unknown 1.4330 1.6738 1.4599 1.6468 1.5534 unknown 0.5117 2.5850 0.5406 2.5561 1.5483 unknown 1.3219 1.7726 1.4010 1.6935 1.5473 GP13540389gbAAK29448.1ceramideglucosyltransferas e{Cricetulusgriseus}.partial(25%) 1.5850 1.5070 0.0625 3.0294 1.5460 unknown 1.1979 1.8931 1.4527 1.6383 1.5455 unknown 0.9386 2.1520 1.1944 1.8962 1.5453 homologuetoGP1523871embCAA65861.1cofactorA{Bos taurus}.complete 0.7965 2.2912 1.8723 1.2154 1.5438 unknown 1.5850 1.5025 0.7262 2.3612 1.5437 unknown 0.1394 2.9475 1.5171 1.5699 1.5435 SPP38384S61G_HUMANProteintransportproteinSEC61 gammasubunit.[Dog]{Canisfamiliaris}.complete 1.7419 1.3426 1.4014 1.6830 1.5422 similartoPIRA53028A53028isopentenyl- diphosphateDelta-isomerase(EC5.3.3.2)homolog- human.complete 1.2861 1.7979 0.8402 2.2437 1.5420 homologuetoSPQ9NPD3RR41_HUMANExosomecompl exexonucleaseRRP41(EC3.1.13.- )(RibosomalRNAprocessingprotein41).[Human].partial(7 7%) 1.5850 1.4919 1.0111 2.0658 1.5384 similartoGP3789868gbAAC67525.1signaltransducerand activatoroftranscription6{Homosapiens}.partial(30%) 0.9329 2.1375 0.7578 2.3126 1.5352 GP12803667gbAAH02669.1nuclearreceptorsubfamily2g roupFmember6{Homosapiens}.partial(50%) 0.6477 2.4215 1.3731 1.6961 1.5346 293 orylasemuscleform(EC2.4.1.1)(Myophosphorylase).[She ep]{Ovisaries}.partial(26%) similartoGP16359275gbAAH16100.1Unknown(proteinfor MGC:27672){Musmusculus}.partial(35%) -1.7631 -0.8277 -1.6362 -0.9546 -1.2954 similartoPIRB34087B34087hypotheticalprotein(L1H3'reg -1.1279 -2.7225 -0.3859 -0.9529 -1.2973 homologuetoGP14250686gbAAH08809.1Similartoangio- associatedmigratorycellprotein{Homosapiens}.partial(80 %) 0.1806 2.8745 1.3219 1.7331 1.5275 voltage-dependentanionchannel2[Susscrofa] 1.5471 1.4932 2.9776 0.0627 1.5201 unknown 1.2274 1.2605 1.0060 2.5723 1.5166 homologuetoSPQ00013EM55_HUMAN55kDaerythrocyt emembraneprotein(P55).[Human]{Homosapiens}.partial( 23%) 0.6183 2.4126 2.5056 0.5252 1.5154 unknown -0.1013 3.1265 1.8059 1.2193 1.5126 similartoSPQ9Y3Q8TIZ2_HUMANTSC22- relatedinducibleleucinezipperprotein2(Tsc-22- likeproteinTHG-1).[Human]{Homosapiens}.partial(35%) 0.8688 2.1468 0.8278 2.1878 1.5078 unknown 0.0352 2.9798 1.5395 1.4755 1.5075 unknown 1.3219 1.6859 0.7203 2.2875 1.5039 unknown 0.7935 2.2115 1.5328 1.4723 1.5025 unknown 1.3591 1.6408 1.4545 1.5454 1.4999 similartoGP17512436gbAAH19177.1Similartoalveolarsof tpartsarcomachromosomeregioncandidate1{Musmuscul us}.partial(44%) 0.3870 2.6088 1.1641 1.8318 1.4979 homologuetoGP13374079embCAC34475.TAFII140prote in{Homosapiens}.partial(20%) 1.3576 1.6374 1.7812 1.2138 1.4975 similartoGP16740625gbAAH16196.1Unknown(proteinfor MGC:27580){Musmusculus}.partial(56%) -0.0544 3.0484 1.9482 1.0457 1.4970 unknown 0.3440 2.6394 2.4658 0.5175 1.4917 similartoGP12839167dbjBAB24454.putative{Musmuscul us}.partial(58%) 0.3049 2.6781 0.9845 1.9984 1.4915 similartoGP9930614gbAAG02116.1steroidreceptorRNA activatorisoform3{Homosapiens}.partial(73%) 1.1155 1.8667 1.0208 1.9615 1.4911 unknown 1.0614 2.9069 0.7549 1.2134 1.4841 unknown 0.5850 2.3785 1.4542 1.5093 1.4817 similartoGP13097537gbAAH03494.1Unknown(proteinfor MGC:6943){Musmusculus}.partial(75%) 0.8886 2.0671 1.5022 1.4535 1.4778 homologuetoGP493132gbAAC41688.1creatinetransport er{Homosapiens}.partial(27%) -1.8373 -0.6744 -0.4606 -2.0511 -1.2558 tissueinhibitorofmetalloproteinase-3[Susscrofa] -0.3219 -2.1979 -1.2856 -1.2343 -1.2599 unknown -1.0916 -1.4384 -1.2830 -1.2470 -1.2650 hyaluronidase -1.0845 -1.4475 -0.6053 -1.9267 -1.2660 unknown -1.1473 -1.3923 -1.3851 -1.1545 -1.2698 weaklysimilartoPIRS68191S68191triadin- human.partial(19%) -1.5424 -1.0909 -2.0107 -0.4530 -1.2742 unknown -1.6812 -0.8780 -0.0328 -2.5264 -1.2796 homologuetoGP5821375dbjBAA83793.1MTH1b(p22)MT H1c(p21)MTH1d(p18){Homosapiens}.partial(72%) -1.2482 -1.3152 -0.1880 -2.3754 -1.2817 5- HT1Dreceptor[Susscrofa]serotoninreceptor1D[Susscrofa ] -0.6674 -1.2310 -1.8894 -1.3393 -1.2818 homologuetoSPQ13887KLF5_HUMANKrueppel- likefactor5(Intestinal-enrichedkrueppel- likefactor)(Colonkrueppel-likefactor).partial(52%) -0.7453 -1.8210 -1.5146 -1.0517 -1.2831 weaklysimilartoSPP23606TGLK_RATProtein- glutaminegamma- glutamyltransferaseK(EC2.3.2.13)(TransglutaminaseK)( TGaseK)(TGK).partial(20%) -0.1587 -2.7402 -1.3186 -0.9456 -1.2908 unknown -0.7400 -1.6595 -1.2614 -1.5101 -1.2928 homologuetoSPO18751PHS2_SHEEPGlycogenphosph -0.6206 -1.9683 -1.4211 -1.1678 -1.2944 294 unknown -1.0356 -1.8379 -2.3991 -0.3319 -1.4012 unknown -1.3395 -1.4671 -2.3992 -0.4074 -1.4033 homologuetoSPP23588IF4B_HUMANEukaryotictranslati oninitiationfactor4B(eIF- -0.1444 -2.6651 -0.8293 -1.9802 -1.4048 ion)-human.partial(11%) aminopeptidaseN.[Susscrofa]aminopeptidaseN[Susscrof a]aminopeptidase[Susscrofa] -1.1806 -1.4150 -0.6975 -1.8981 -1.2978 unknown -0.2065 -2.3923 -1.4766 -1.1222 -1.2994 unknown -0.4019 -2.1987 -1.1022 -1.4984 -1.3003 beta-tropomyosin[Susscrofa]beta- tropomyosin[Susscrofa] -0.4656 -3.0692 -1.5866 -0.0858 -1.3018 unknown -1.1609 -1.4435 -1.4080 -1.1964 -1.3022 similartoGP11385352gbAAG34759.1aminoacidtransport erSLC3A1{Canisfamiliaris}.partial(20%) -0.7590 -1.3646 -1.0701 -2.0175 -1.3028 unknown -0.1312 -2.4780 -0.9236 -1.6857 -1.3046 insulin- likegrowthfactorbindingprotein2[Susscrofa]IGFbindingpro tein-2 -1.0701 -1.5425 -1.8340 -0.7787 -1.3063 similartoGP14424509gbAAH09274.1Unknown(proteinfor MGC:10681){Homosapiens}.partial(53%) -1.6039 -1.0224 -0.7960 -1.8303 -1.3131 unknown -0.3049 -2.3219 -0.1608 -2.4660 -1.3134 unknown -1.6660 -0.9621 -1.6950 -0.9331 -1.3140 unknown 0.0000 -2.6521 -1.9486 -0.7035 -1.3260 homologuetoGP13177775gbAAH03656.1minichromoso memaintenancedeficient(S.cerevisiae)5(celldivisioncycle 46){Homosapiens}.partial(48%) -1.3588 -1.2971 -1.2035 -1.4524 -1.3279 unknown -0.8352 -1.8257 -0.8234 -1.8375 -1.3305 homologuetoGP1430783embCAA65075.1X- linkedmentalretardationcandidategene{Homosapiens}.pa rtial(11%) -1.5850 -1.2479 -1.6325 -0.8605 -1.3315 similartoPIRT12462T12462hypotheticalproteinDKFZp56 4I122.1-human(fragment).partial(97%) -0.6125 -2.0506 -1.6442 -1.0190 -1.3316 homologuetoSPO46392CA21_CANFACollagenalpha2(I) chainprecursor.[Dog]{Canisfamiliaris}.partial(12%) -1.5459 -1.1493 -0.7951 -1.9000 -1.3476 heparin-bindingepidermalgrowthfactor- likegrowthfactor[Susscrofa]Heparin- bindingepidemialgrowthfactor[Susscrofa] -0.0791 -2.7753 -1.0372 -1.5009 -1.3481 unknown -1.5472 -1.1492 -0.9429 -1.7535 -1.3482 unknown -0.4044 -1.1043 -2.0044 -1.8867 -1.3500 similartoGP15862322embCAC88591.unnamedproteinpr oduct{Homosapiens}.partial(52%) -0.4971 -2.2130 -1.2213 -1.4888 -1.3550 similartoGP14714906gbAAH10609.1hypotheticalprotein FLJ22167{Homosapiens}.partial(37%) -1.1844 -1.8972 -1.1108 -1.2331 -1.3564 homologuetoSPP27706EF12_MOUSEElongationfactor1 -alpha2(EF-1-alpha-2)(Elongationfactor1A-2)(eEF1A- 2)(StatinS1).[Rat].partial(15%) 0.1830 -2.8970 -1.6056 -1.1084 -1.3570 unknown -1.7004 -2.4263 -0.5992 -0.7258 -1.3629 unknown -1.4737 -1.2575 -0.4418 -2.2894 -1.3656 homologuetoGP10438696dbjBAB15314.unnamedprotei nproduct{Homosapiens}.partial(30%) -1.3612 -1.3735 -0.2825 -2.4521 -1.3673 similartoGP14160858gbAAK07671.1ADP- ribosepyrosphosphataseNUDT9{Homosapiens}.partial(2 8%) -0.8684 -1.8707 -1.5075 -1.2316 -1.3695 ART5protein[Susscrofa] -1.6114 -2.0176 -1.8153 -0.0479 -1.3731 unknown -0.9733 -1.7776 -1.1133 -1.6376 -1.3755 similartoPIRT18522T18522tubulin-foldingcofactorD- bovine.partial(15%) -0.2266 -2.5361 -0.1139 -2.6487 -1.3813 titin[Susscrofa] -0.8091 -1.5959 -1.8917 -1.2769 -1.3934 unknown -0.0544 -2.7327 -1.1455 -1.6417 -1.3936 295 unknown -2.8074 -1.9658 -0.2471 -1.2967 -1.5792 unknown -0.6521 -2.5081 -1.4372 -1.7229 -1.5801 similartoGP3327808gbAAC39879.1latenttransforminggr owthfactor- -1.3036 -1.8581 -1.5202 -1.6415 -1.5809 4B).[Human]{Homosapiens}.partial(27%) homologuetoGP15862466embCAC88632.unnamedprot einproduct{Homosapiens}.partial(79%) -0.3609 -2.4594 -1.3070 -1.5134 -1.4102 Igkappachain -0.7583 -2.0641 -0.2524 -2.5700 -1.4112 similartoGP10438524dbjBAB15267.unnamedproteinpro duct{Homosapiens}.partial(82%) -0.3752 -2.4478 -0.2870 -2.5360 -1.4115 homologuetoGP18027794gbAAL55858.1unknown{Hom osapiens}.partial(11%) -1.6697 -1.1623 -1.1283 -1.7037 -1.4160 unknown -1.4150 -1.2479 -1.3421 -1.6607 -1.4164 unknown -0.7555 -2.0921 -0.8444 -2.0032 -1.4238 homologuetoSPQ9P2W9STXH_HUMANSyntaxin18.[Hu man]{Homosapiens}.partial(28%) -1.1575 -1.0156 -0.5696 -2.9733 -1.4290 homologuetoGP1800225gbAAC50950.1JAK3{Homosapi ens}.partial(6%) -0.6501 -2.2121 -1.1329 -1.7293 -1.4311 unknown -1.6677 -1.1983 -0.4986 -2.3674 -1.4330 GP15929669gbAAH15264.1sialyltransferase5{Musmusc ulus}.partial(42%) -0.0458 -2.8346 -0.4919 -2.3885 -1.4402 similartoSPP10074HKR3_HUMANKrueppel- relatedzincfingerprotein3(HKR3protein).[Human]{Homos apiens}.partial(33%) -1.6630 -1.5443 -1.4721 -1.0833 -1.4407 unknown -1.1405 -1.0444 -2.3305 -1.2925 -1.4520 weaklysimilartoGP3218467embCAA07090.1putativepho sphatase{Gallusgallus}.partial(66%) -0.1715 -2.7370 -0.6228 -2.2856 -1.4542 cartilageaggregatingproteoglycan[Susscrofa]aggrecanC S2domain[Susscrofa] -1.3219 -1.2479 -2.4056 -0.8765 -1.4630 readingframe[Susscrofa] -2.2668 -1.2224 -1.6481 -0.7739 -1.4778 similartoGP2564320dbjBAA22955.1KIAA0286{Homosap iens}.partial(26%) -0.2392 -1.1964 -1.5967 -2.8821 -1.4786 homologuetoSPQ92176CO1A_BOVINCoronin- likeproteinp57(Coronin1A).[Bovine]{Bostaurus}.partial(27 %) -0.6420 -2.3219 -1.3894 -1.5745 -1.4819 homologuetoPIRA28442TPRBCStroponinCfastskeletalm uscle-rabbit.complete -0.2808 -1.2504 -2.0127 -2.3952 -1.4848 unknown -1.4561 -1.4263 -1.8284 -1.2296 -1.4851 similartoGP6683128dbjBAA20800.2KIAA0342protein{Ho mosapiens}.partial(13%) -1.4721 -1.4854 -0.8215 -2.2477 -1.5067 similartoGP6179932gbAAF05716.1tektin{Canisfamiliaris }.partial(32%) -0.0618 -1.9696 -2.3955 -1.6359 -1.5157 unknown -1.1444 -1.1876 -1.4240 -2.3305 -1.5216 GP14250798gbAAH08869.1hypotheticalproteinF17127_ 1{Homosapiens}.partial(41%) -1.0193 -2.0324 -0.4529 -2.5988 -1.5259 triadin[Susscrofa] -0.4448 -1.5110 -2.3896 -1.7870 -1.5331 unknown -2.0056 -1.0744 -1.4050 -1.6526 -1.5344 mineralocorticoidreceptor[Susscrofa] -0.5753 -2.5025 -1.4373 -1.6405 -1.5389 unknown -0.8470 -2.2327 -1.9130 -1.1666 -1.5398 similartoGP12854557dbjBAB30070.putative{Musmuscul us}.partial(83%) -1.4150 -1.5025 -1.9980 -1.2594 -1.5437 similartoGP8896138gbAAF81254.1pregnancy- associatedglycoprotein4{Susscrofa}.partial(34%) -0.9386 -2.1619 -0.7975 -2.3030 -1.5502 similartoGP2351683gbAAB68608.1nucleolarfibrillarcent erprotein{Homosapiens}.partial(31%) -0.8301 -1.9542 -1.6761 -1.7879 -1.5621 ubiquitousTPRmotifproteinubiquitousTPRmotifproteinUT Y[Susscrofa]ubiquitousTPRmotifproteinUTX[Susscrofa] -0.4321 -1.5677 -1.3440 -2.9274 -1.5678 Gprotein-coupledreceptor -1.0815 -2.2200 -1.6981 -1.2773 -1.5693 homologuetoGP10437164dbjBAB15001.unnamedprotei nproduct{Homosapiens}.partial(16%) -1.4930 -1.6550 -0.7757 -2.3723 -1.5740 296 weaklysimilartoGP1196432gbAAA88037.1unknownprote in{Homosapiens}.partial(10%) -0.3479 -2.2530 -1.2484 -3.3525 -1.8005 dipeptidaseprecursor[Susscrofa]dipeptidase[Susscrofa] -1.3103 -1.9260 -1.5431 -2.4518 -1.8078 similartoSPQ9Y2B2PIGL_HUMANN-acetylglucosaminyl- phosphatidylinositolde-N-acetylase(EC3.5.1.- -1.0000 -2.6630 -1.5932 -2.0698 -1.8315 betabindingprotein4S{Homosapiens}.partial(13%) similartoSPQ9H9D4PRDH_HUMANPR- domainzincfingerprotein17.[Human]{Homosapiens}.parti al(18%) -0.5519 -2.6280 -1.3073 -1.8726 -1.5900 unknown -0.6406 -2.5465 -1.7507 -1.4364 -1.5935 unknown -1.8074 -1.0297 -1.1294 -2.4783 -1.6112 unknown -1.6439 -1.8704 -0.8019 -2.1369 -1.6133 homologuetoGP13184897embCAC33267.unnamedprot einproduct{Homosapiens}.partial(18%) -1.9594 -1.2288 -1.9952 -1.3555 -1.6347 unknown -1.3219 -1.6049 -0.2884 -3.3507 -1.6415 homologuetoGP4325215gbAAD17301.1single- strandselectivemonofunctionaluracilDNAglycosylase{Ho mosapiens}.partial(77%) -0.6868 -1.9790 -1.9578 -1.9607 -1.6461 skeletalmusclespecificcalpain;Ca2+- dependentcysteineprotinase[Susscrofa]skeletalmuscle- specificcalpaincalpainlargepolypeptideL3[ -0.4019 -2.8938 -0.8864 -2.4093 -1.6478 smallproline-richprotein -1.4739 -2.7866 -2.3012 -0.0636 -1.6563 homologuetoGP5689525dbjBAA83046.1KIAA1094protei n{Homosapiens}.partial(42%) -0.9285 -2.3923 -2.5461 -0.7747 -1.6604 typeIIcollagenalpha1[Susscrofa] -1.6665 -1.6557 -0.1148 -3.2075 -1.6611 similartoSPQ9Z262CLD6_MOUSEClaudin- 6.[Mouse]{Musmusculus}.complete -0.2854 -3.6245 -0.9223 -1.8460 -1.6695 homologuetoGP11139720gbAAG31814.1polyadenylatio nproteinCSTF64{Musmusculus}.partial(17%) -1.1756 -2.1743 -0.7256 -2.6242 -1.6749 similartoGP16552606dbjBAB71352.unnamedproteinpro duct{Homosapiens}.partial(35%) -0.1363 -3.2224 -0.9856 -2.3731 -1.6794 similartoGP12840019dbjBAB24733.putative{Musmuscul us}.partial(52%) -1.3833 -1.7549 -0.7992 -2.8057 -1.6858 unknown -0.6699 -2.7090 -1.2224 -2.1564 -1.6894 homologuetoGP17900927embCAD19357.unnamedprot einproduct{Homosapiens}.partial(9%) -0.6292 -2.0128 -2.0693 -2.0560 -1.6918 homologuetoGP9651109dbjBAB03567.1TTYH1{Macaca fascicularis}.partial(30%) -0.4109 -3.0000 -1.9461 -1.4648 -1.7054 unknown -0.4748 -2.9386 -1.4043 -2.0091 -1.7067 apoptosisinhibitorsurvivin[Susscrofa] -1.3458 -2.1043 -1.1935 -2.2566 -1.7251 homologuetoSPP29562IF41_RABITEukaryoticinitiationf actor4A-I(eIF-4A-I)(eIF4A- I)(Fragment).[Rabbit]{Oryctolaguscuniculus}.partial(18%) -1.9175 -1.3750 -2.1098 -1.5126 -1.7288 similartoGP12407338gbAAG53461.1proteinO- mannosyltransferase1{Rattusnorvegicus}.partial(27%) -1.3476 -2.1184 -1.8292 -1.6369 -1.7330 unknown -0.1150 -3.3646 -2.5025 -0.9771 -1.7398 similartoSPP28667MRP_MOUSEMARCKS- relatedprotein(MAC- MARCKS)(BrainproteinF52).[Mouse]{Musmusculus}.part ial(78%) -1.7935 -1.7391 -2.3743 -1.1582 -1.7663 alpha2A-adrenergicreceptor(PORA2AR)alpha- 2Aadrenergicreceptor[Susscrofa] -0.7899 -2.7608 -2.4065 -1.1442 -1.7754 unknown -0.9055 -2.6462 -1.4869 -2.0647 -1.7758 homologuetoGP5081463gbAAD39394.1bigMAPkinase1 a{Musmusculus}.partial(14%) -1.4637 -2.0952 -0.6167 -2.9421 -1.7794 unknown -0.9069 -2.6650 -0.6193 -2.9526 -1.7859 unknown -0.7776 -2.8074 -0.8195 -2.7655 -1.7925 typeIcollagenalpha1[Susscrofa] -0.4978 -3.0999 -2.1502 -1.4474 -1.7988 homologuetoGP12804735gbAAH01799.1Unknown(prot einforIMAGE:3354600){Homosapiens}.partial(35%) -1.4657 -2.0661 -1.9750 -1.6941 -1.8002 297 preproacrosin[Susscrofa]acrosinprecursor(EC3.4.21.10) -2.1069 -2.6439 -1.3536 -2.9695 -2.2685 homologuetoEGAD2653127372discs- largehomologg3{Homosapiens}.partial(27%) -0.5761 -3.9696 -1.3654 -3.1803 -2.2729 gagprotein[Susscrofa]polprotein[ -2.5850 -3.9765 -1.2091 -1.3524 -2.2808 ).partial(34%) similartoGP6841522gbAAF29114.1HSPC150{Homosapi ens}.partial(40%) -1.1545 -2.8413 -1.3650 -2.0128 -1.8434 unknown -1.6167 -2.0969 -0.8418 -2.8718 -1.8568 homologuetoGP6329074dbjBAA86388.1UDP-GlcNAc:a- 13-D-mannosideb-14-N- acetylglucosaminyltransferaseIV{Homosapiens}.partial(3 4%) -2.3923 -2.1085 -2.0335 -0.8981 -1.8581 weaklysimilartoGP37996embCAA46158.1XerodermaPig mentosumGroupCComplementingfactor{Homosapiens}. partial(18%) -1.0314 -1.7597 -1.4426 -3.2230 -1.8642 homologuetoSPP24140GPT_CRIGRUDP-N- acetylglucosamine--dolichyl-phosphateN- acetylglucosaminephosphotransferase(EC2.7.8.15).parti al(42%) -1.6200 -2.1257 -1.6356 -2.1101 -1.8729 unknown -0.7014 -3.0495 -1.5733 -2.1776 -1.8755 unknown -1.8114 -2.5859 -1.0075 -2.1442 -1.8873 similartoGP9367763embCAB97494.1zincfingerproteinC ezanne{Homosapiens}.partial(25%) -1.7608 -1.5443 -1.0695 -3.1923 -1.8918 unknown -0.9042 -2.9292 -0.8336 -2.9999 -1.9167 similartoGP18089247gbAAH20966.1Unknown(proteinfor MGC:9127){Homosapiens}.partial(19%) -0.3923 -3.2395 -1.7530 -2.3095 -1.9236 homologuetoGP3721838dbjBAA33714.1NIK{Homosapie ns}.partial(29%) -0.6575 -3.2197 -1.1283 -2.7488 -1.9386 unknown -2.3626 -1.2479 -1.7912 -2.3690 -1.9427 unknown -1.0913 -3.9819 -1.1915 -1.5164 -1.9453 homologuetoGP12804537gbAAH01679.1Unknown(prot einforMGC:2722){Homosapiens}.partial(49%) -0.3771 -3.5850 -1.5429 -2.4192 -1.9810 unknown -1.1520 -2.1155 -1.5504 -3.1091 -1.9817 Ca2+ATPaseoffasttwitch1skeletalmusclesarcoplasmicret iculum[Susscrofa] -0.3460 -3.3113 -2.1326 -2.1407 -1.9826 unknown -1.6854 -3.2873 -0.9303 -2.0424 -1.9863 unknown -1.1225 -2.8580 -1.3578 -2.6227 -1.9902 homologuetoGP434775dbjBAA04946.1KIAA0014{Homo sapiens}.partial(36%) -1.4530 -2.5406 -1.9845 -2.0091 -1.9968 homologuetoGP6330597dbjBAA86534.1KIAA1220protei n{Homosapiens}.partial(24%) -1.4448 -2.4485 -1.6566 -2.4575 -2.0018 matrixmetalloproteinase -1.0265 -2.0000 -1.8395 -3.1869 -2.0132 similartoGP1684843gbAAB48302.1pinin{Bostaurus}.part ial(19%) -1.0506 -3.0875 -2.0151 -1.9205 -2.0184 weaklysimilartoPIRG01880G01880fatty- acidsynthase(EC2.3.1.85)(version2)-human.partial(7%) -1.4838 -2.5914 -1.4604 -2.6148 -2.0376 melanocortintype4receptor[Susscrofa]melanocortin4rece ptor[Susscrofa]melanocortin-4receptorMC4R[Susscrofa] -1.7241 -2.4739 -1.0626 -3.1354 -2.0990 unknown -0.4868 -3.7188 -1.3956 -2.8100 -2.1028 similartoGP15823629dbjBAB69011.ALS2CR4{Homosap iens}.partial(21%) -0.5096 -3.7391 -2.0698 -2.1789 -2.1243 unknown -1.3155 -2.5831 -1.5394 -3.0972 -2.1338 homologuetoGP12856351dbjBAB30641.putative{Musmu sculus}.complete -2.0400 -1.2449 -2.2090 -3.0759 -2.1425 unknown -2.3169 -2.6245 -1.7190 -1.9550 -2.1538 similartoSPQ92636FAN_HUMANProteinFAN(Factorass ociatedwithN-SMaseactivation).partial(22%) -1.6280 -2.9542 -1.7468 -2.3233 -2.1631 similartoGP1669621dbjBAA13700.1latexin{Musmusculu s}.partial(36%) -1.3049 -4.7549 -1.4522 -1.3882 -2.2250 unknown -1.5443 -3.0444 -1.8908 -2.5207 -2.2500 similartoSPO43246CTR4_HUMANCationicaminoacidtra nsporter-4(CAT- 4)(CAT4).[Human]{Homosapiens}.partial(62%) -2.4815 -2.0875 -0.8999 -3.6691 -2.2845 homologuetoGP12314190embCAC16281.dJ445H2.2(no velprotein){Homosapiens}.partial(30%) -1.6101 -2.1898 -1.7970 -3.5627 -2.2899 unknown -1.5077 -4.0931 -1.7061 -1.8947 -2.3004 homologuetoSPP50461CSR3_HUMANLIMdomainprotei ncardiac(MuscleLIMprotein)(Cysteine- richprotein3)(CRP3).[Human].partial(76%) -1.6940 -2.9260 -2.8826 -1.7374 -2.3100 similartoSPP35605COPP_BOVINCoatomerbeta'subunit( Beta'-coatprotein)(Beta'- COP)(p102).[Bovine]{Bostaurus}.partial(8%) -2.0000 -2.7004 -0.9733 -3.7272 -2.3502 unknown -1.8074 -3.5236 -2.5192 -1.5823 -2.3581 homologuetoSPP15407FRA1_HUMANFOS- relatedantigen1.[Human]{Homosapiens}.partial(31%) -1.7127 -3.4919 -1.8177 -2.5360 -2.3896 unknown -1.3985 -4.3837 -1.2012 -2.5811 -2.3911 unknown -1.4381 -3.2533 -2.6152 -2.3238 -2.4076 similartoPIRA44128A44128(N-acetylneuraminyl)- galactosylglucosylceramideN- acetylgalactosaminyltransferase(EC2.4.1.92)- .partial(46%) -2.3219 -2.5648 -1.9772 -2.9095 -2.4434 homologuetoGP8515870gbAAF76218.1bridgingintegrat or-3{Homosapiens}.partial(67%) -2.3665 -2.5317 -3.4693 -1.4289 -2.4491 homologuetoPIRS04090S04090myosinheavychain3skel etalmuscleembryonic-human.partial(4%) -2.2738 -3.6684 -1.2549 -2.6873 -2.4711 unknown -2.0238 -3.9674 -1.3740 -2.6173 -2.4956 fattyacidsynthase[Susscrofa] -1.7643 -4.3325 -2.4084 -1.6885 -2.5484 unknown -2.5305 -5.8074 -0.8235 -1.3923 -2.6384 weaklysimilartoGP7671629embCAB89275.2bA145L22.2 (novelKRABboxcontainingC2H2typezincfingerprotein){H omosapiens}.partial(27%) -2.4721 -3.0000 -2.8841 -2.6997 -2.7640 titin[Susscrofa] -3.0307 -2.9505 -3.5721 -2.4091 -2.9906 unknown -4.3141 -2.6147 -1.9807 -3.6917 -3.1503 unknown -4.0643 -2.2368 -3.5633 -2.7378 -3.1506 unknown -3.0729 -2.4896 -3.1690 -4.1018 -3.2083 unknow -1.8826 -5.4094 -1.9647 -3.7968 -3.2634 Table 4. Transcripts highly up/down regulated determined by oligo-array in the muscle tissue by the dietary shifting from LFD to HFD (Chapter 3). For each transcript, log tio=log e means higher mRNA abundance in HFD pigs; negative value means lower mRNA abundance in HFD pigs Gene name Log 1/ G1) Log 2/ R2) Log 3/ G3) Avrage 2 ra 2 (HFD/LFD).The positive valu 2 (R 2 (G 2 (R Log 2 (G4/ R4) unknown 2.6663 1.8616 2.7270 1.8010 2.2640 ribosomalproteinL19[Susscrofa ] 2.5662 1.8995 2.0796 2.3861 2.2328 homologuetoPIR|S71405|S71405helix-loop- helixproteinID3longspliceform-human.partial(65%) 1.4332 2.9359 3.0173 1.3518 2.1845 similartoSP|O00212|RHOD_HUMANRho-relatedGTP- bindingproteinRhoD(Rho- relatedproteinHP1)(RhoHP1).[Human]{Homosapiens}. partial(91%) 3.2661 0.9472 2.1781 2.0352 2.1067 homologuetoSP|P55931|ETFD_PIGElectrontransferfl avoprotein- ubiquinoneoxidoreductasemitochondrialprecursor(EC 1.5.5.1).partial(22%) 2.1843 1.9564 2.5663 1.5744 2.0704 homologuetoSP|P22392|NDKB_HUMANNucleosidedi phosphatekinaseB(EC2.7.4.6)(NDKB)(NDPkinaseB)(n m23-H2).complete 2.7958 1.2525 2.3009 1.7475 2.0242 298 299 homologuetoGP|3282771|gb|AAC33845.1|actin- bindingproteinhomologABP- 278{Homosapiens}.partial(9%) 2.1387 1.0000 1.0682 2.0706 1.5694 unknown 1.9225 1.1907 0.3723 2.7408 1.5566 unknown 2.7031 1.2880 2.9856 1.0056 1.9956 unknown 2.8269 1.1632 1.8722 2.1178 1.9950 PIR|A48045|A48045ribosomalproteinS27cytosolic- human.complete 2.6471 1.8754 1.7109 1.6117 1.9613 homologuetoGP|12833968|dbj|BAB22732.putative{M usmusculus}.partial(23%) 2.1672 1.7244 2.2182 1.6733 1.9458 homologuetoGP|5531805|gb|AAD44477.1|16.7Kdprot ein{Homosapiens}.complete 3.2370 0.6302 2.0132 1.8540 1.9336 unknown 2.3772 1.4493 1.6647 2.1618 1.9132 medium-chainacyl-CoAdehydrogenase 2.6677 1.0346 1.2100 2.4923 1.8511 homologuetoSP|P82664|RT10_HUMANMitochondrial 28SribosomalproteinS10(MRP- S10)(MSTP040).[Human]{Homosapiens}.partial(30%) 2.2430 1.4406 2.0695 1.6140 1.8418 unknown 3.2359 0.4258 2.0773 1.5843 1.8308 unknown 2.5573 1.0780 2.5619 1.0735 1.8177 90-kDaheatshockprotein[Susscrofa] 2.4052 1.0389 1.8896 1.5545 1.7220 homologuetoSP|P07471|COXD_BOVINCytochromec oxidasepolypeptideVIa- heartmitochondrialprecursor(COXVIAH).partial(75%) 2.3281 1.1100 1.7416 1.6965 1.7190 homologuetoGP|2547076|dbj|BAA22860.1A+U- richelementRNAbindingfactor{Homosapiens}.partial(3 3%) 2.5399 0.8919 1.4815 1.9502 1.7159 unknown 1.9664 1.4185 1.0646 2.3203 1.6924 unknown 2.6746 0.6738 1.3466 2.0018 1.6742 similartoGP|12407437|gb|AAG53507.1tripartitemotifpr oteinTRIM16{Musmusculus}.partial(66%) 2.3643 0.9590 1.3274 1.9960 1.6617 homologuetoSP|P29350|PTN6_HUMANProtein- tyrosinephosphatasenon- receptortype6(EC3.1.3.48)(Protein- tyrosinephosphatase1C).partial(63%) 2.2657 1.0541 1.7946 1.5252 1.6599 homologuetoGP|12843392|dbj|BAB25965.putative{M usmusculus}.complete 0.9076 2.4103 1.3579 1.9600 1.6589 unknown 1.5768 1.7370 2.0641 1.2497 1.6569 similartoGP|12053165|emb|CAB66762.hypotheticalpr otein{Homosapiens}.partial(19%) 1.5670 1.7404 1.9786 1.3287 1.6537 weaklysimilartoGP|14336751|gb|AAK61280.1unknow n{Homosapiens}.partial(27%) 2.2877 1.0000 1.6845 1.6032 1.6438 unknown 2.3820 0.8963 1.1022 2.1761 1.6391 beta2-microglobulinbeta-2- microglobulinprotein[Susscrofa] 2.2755 0.9919 1.0502 2.2171 1.6337 unknown 2.5115 0.7498 1.2974 1.9639 1.6306 unknown 2.3909 0.8425 1.2039 2.0294 1.6167 similartoSP|Q13283|G3BP_HUMANRas-GTPase- activatingproteinbindingprotein1(GAPSH3- domainbindingprotein1)(G3BP-).[Human].partial(54%) 2.4975 0.7291 2.2798 0.9468 1.6133 homologuetoGP|15072481|gb|AAK71328.1LOH1CR1 2{Homosapiens}.partial(83%) 1.7751 1.4429 0.7618 2.4562 1.6090 heatshockprotein70[Susscrofa]heatshockprotein70.hs p70 2.3166 0.8931 1.3994 1.8103 1.6048 similartoGP|6573163|gb|AAF17574.1|ubiquitinspecific processingprotease{Rattusnorvegicus}.partial(43%) 1.6300 1.5729 2.1155 1.0874 1.6014 unknown 2.7362 0.4594 1.4070 1.7886 1.5978 homologuetoGP|286011|dbj|BAA02792.1|KIAA0002{ Homosapiens}.partial(20%) 2.4962 0.6969 0.5958 2.5974 1.5966 homologuetoGP|12804349|gb|AAH03035.1Unknown( proteinforMGC:4355){Homosapiens}.partial(38%) 1.1237 2.0156 1.7370 1.4024 1.5697 300 relatedproteinRab-7.[Dog]{Canisfamiliaris}.complete similartoEGAD|3524|3493collagentypeVIalpha3{Hom osapiens}.partial(18%) 1.5267 1.3499 2.2718 0.6048 1.4383 unknown 2.0756 0.7907 1.8549 1.0113 1.4331 similartoSP|O75185|ATC4_HUMANProbablecalcium- 1.6494 1.2115 1.3714 1.4896 1.4305 unknown 2.6843 0.4208 1.2242 1.8809 1.5526 GP|12654423|gb|AAH01037.1ribosomalproteinL35a{ Homosapiens}.complete 1.8096 1.2918 1.3009 1.8005 1.5507 similartoGP|6624920|emb|CAB63941.1DMBT1prototy pe{Homosapiens}.partial(10%) 1.8891 1.2033 0.0929 2.9996 1.5462 homologuetoPIR|I38191|I38191nucleicacidbindingprot ein-human(fragment).partial(96%) 2.6905 0.3612 1.4150 1.6367 1.5258 unknown 1.6820 1.3692 0.9086 2.1427 1.5256 unknown 2.2230 0.8274 1.1103 1.9401 1.5252 unknown 1.7776 1.2706 1.3862 1.6621 1.5241 homologuetoGP|4929617|gb|AAD34069.1|CGI- 74protein{Homosapiens}.partial(78%) 2.4470 0.5850 1.0969 1.9351 1.5160 homologuetoGP|11138955|gb|AAG31556.115kDasele noprotein{Homosapiens}.complete 2.3346 0.6878 1.2224 1.7999 1.5112 similartoSP|P32019|I5P2_HUMANTypeIIinositol-145- trisphosphate5- phosphataseprecursor(EC3.1.3.56)(5PTASE)(Fragme nt)..partial(22%) 1.7489 1.2701 1.7225 1.2965 1.5095 homologuetoSP|P00819|ACYM_PIGAcylphosphatase muscletypeisozyme(EC3.6.1.7)(Acylphosphatephosph ohydrolase).[Pig].complete 2.1315 0.8823 2.0952 0.9186 1.5069 homologuetoGP|13940506|gb|AAK50397.1GDP- fucosetransporter{Homosapiens}.partial(92%) 0.9860 2.0247 1.7232 1.2875 1.5053 homologuetoSP|P70698|PYRG_MOUSECTPsynthas e(EC6.3.4.2)(UTP-- ammonialigase)(CTPsynthetase).[Mouse]{Musmuscul us}.partial(20%) 1.3408 1.6684 1.6542 1.3550 1.5046 unknown 0.4548 2.5538 1.6807 1.3279 1.5043 unknown 2.3942 0.5726 1.6158 1.3510 1.4834 homologuetoGP|10439498|dbj|BAB15508.unnamedpr oteinproduct{Homosapiens}.partial(75%) 1.6067 1.3579 1.8057 1.1589 1.4823 unknown 1.1184 1.8407 2.0458 0.9133 1.4795 ATPsynthasegammasubunit1[Susscrofa] 2.6754 0.2828 1.3895 1.5687 1.4791 unknown 1.9361 1.0199 1.0354 1.9206 1.4780 unknown 3.2049 0.2580 1.2885 1.1424 1.4735 unknown 1.4671 1.4739 1.1137 1.8274 1.4705 SP|P23821|RS7_HUMAN40SribosomalproteinS7(S8). [Rat]{Rattusnorvegicus}.complete 2.5080 0.4241 1.3388 1.5933 1.4660 similartoGP|12845654|dbj|BAB26840.putative{Musmu sculus}.complete 2.5002 0.4288 1.2323 1.6967 1.4645 unknown 2.7203 0.2027 1.0555 1.8675 1.4615 unknown 2.9003 1.0117 0.9831 0.9290 1.4560 SP|P33552|CKS2_HUMANCyclin- dependentkinasesregulatorysubunit2(CKS- 2).[Human]{Homosapiens}.complete 2.1028 0.8074 1.3108 1.5994 1.4551 homologuetoGP|15524116|emb|CAC69312.unnamed proteinproduct{Homosapiens}.partial(25%) 2.2926 0.6117 1.3845 1.5198 1.4522 unknown 2.3703 0.5305 2.0721 0.8287 1.4504 homologuetoGP|7321168|emb|CAB82246.1dJ860F19 .3(metallocarboxypeptidaseCPX- 1){Homosapiens}.partial(54%) 1.8884 1.0092 1.3462 1.5515 1.4488 homologuetoEGAD|45512|479773-hydroxyisobutyryl- coenzymeAhydrolase{Homosapiens}.partial(49%) 1.3072 1.5850 1.9569 0.9352 1.4461 unknown 2.3914 0.4975 2.2890 0.5999 1.4444 homologuetoSP|P18067|RAB7_CANFARas- 2.3868 0.4902 1.0643 1.8127 1.4385 301 C:28311){Musmusculus}.partial(45%) similartoGP|2935442|gb|AAC78563.1|ribonucleaseH1 {Homosapiens}.partial(46%) 2.0729 0.5590 1.1057 1.5262 1.3159 unknown 1.9571 0.6658 1.3375 1.2855 1.3115 transportingATPaseKIAA0703(EC3.6.3.8).[Human]{H omosapiens}.partial(40%) homologuetoGP|15012167|gb|AAH10991.1hypothetic alproteinFLJ21977{Homosapiens}.partial(56%) 0.5637 2.2946 1.2655 1.5929 1.4292 arachidonate12- lipoxygenase(EC1.13.11.31)arachidonate12- lipoxygenase[Susscrofa] 0.1354 2.7178 1.2572 1.5960 1.4266 SP|P00829|ATPB_BOVINATPsynthasebetachainmito chondrialprecursor(EC3.6.3.14).[Bovine]{Bostaurus}.p artial(48%) 2.7907 0.0501 1.8585 0.9824 1.4204 dihydropyrimidinedehydrogenase 1.5025 1.3219 1.4996 1.3249 1.4122 homologuetoGP|14250712|gb|AAH08825.1DEAD/H(A sp-Glu-Ala- Asp/His)boxpolypeptide16{Homosapiens}.partial(19%) 1.6095 1.2100 1.2455 1.5740 1.4097 unknown 2.2605 0.5539 1.1926 1.6218 1.4072 unknown 1.4516 1.3567 1.7567 1.0516 1.4041 similartoSP|P27213|PTPS_RAT6- pyruvoyltetrahydrobiopterinsynthaseprecursor(EC4.6. 1.10)(PTPS)(PTPsynthase).[Rat].partial(93%) 2.1546 0.6521 0.4586 2.3480 1.4033 similartoGP|9864062|gb|AAG01291.1|MOG1isoformB {Homosapiens}.complete 1.9074 0.8974 0.2604 2.5444 1.4024 GP|13172662|gb|AAK14178.1ubiquitin- like5protein{Homosapiens}.complete 2.4691 0.3334 1.8402 0.9623 1.4012 unknown 1.9894 0.8045 1.0641 1.7298 1.3970 unknown 2.0977 1.3053 1.0635 1.1183 1.3962 PIR|S05014|R5RT37ribosomalproteinL37acytosolic[v alidated]-rat.complete 1.3136 1.4772 1.5563 1.2346 1.3954 unknown 1.5677 1.2168 0.0943 2.6902 1.3922 metallothionein-III[Susscrofa] 0.6833 2.0959 1.1777 1.6015 1.3896 unknown 1.0596 1.7137 1.7797 0.9936 1.3867 homologuetoPIR|S60062|S60062hevinprecursor- human.partial(21%) 1.3229 1.4301 1.2122 1.5409 1.3765 neuron-derivedorphanreceptor- 1beta[Susscrofa]neuron-derivedorphanreceptor- 1alfa[Susscrofa] 1.5236 1.2224 1.8679 0.8781 1.3730 similartoGP|13279278|gb|AAH04341.1SimilartoRIKE NcDNA5730568A12gene{Homosapiens}.partial(32%) 0.3940 2.3472 1.1676 1.5736 1.3706 unknown 1.4285 1.3093 1.4060 1.3318 1.3689 unknown 0.4231 2.3108 0.7894 1.9444 1.3669 homologuetoGP|13177724|gb|AAH03639.1Unknown( proteinforIMAGE:3346359){Homosapiens}.partial(95% ) 0.7410 1.9894 0.3800 2.3503 1.3652 c- Fosprotein[Susscrofa]earlyimmediategeneexpression[ Susscrofa] 1.9784 0.7498 1.3758 1.3524 1.3641 unknown 1.8426 0.8711 0.1729 2.5407 1.3568 unknown 1.7774 0.9319 1.7339 0.9754 1.3546 long-chainacyl-CoAdehydrogenase[Susscrofa] 1.8144 0.8713 0.0491 2.6366 1.3429 unknown 2.1063 0.5778 0.4578 2.2263 1.3420 unknown 2.2496 0.4316 1.6481 1.0331 1.3406 unknown 1.2729 1.3883 1.6426 1.0187 1.3306 SP|Q28151|OZF_BOVINZincfingerproteinOZF.[Bovin e]{Bostaurus}.partial(19%) 1.8324 0.8220 0.8306 1.8238 1.3272 proteinphosphatase2Aalphasubunit 2.0760 0.5702 1.3930 1.2532 1.3231 GP|18043683|gb|AAH20043.1Unknown(proteinforMG -0.7370 3.3798 1.3699 1.2729 1.3214 302 human.partial(88%) homologuetoGP|18088472|gb|AAH20773.1Unknown( proteinforMGC:22685){Homosapiens}.partial(48%) 1.6374 0.8433 0.1293 2.3514 1.2404 unknown 2.2440 0.2333 1.1017 1.3756 1.2387 unknown 1.1707 1.4365 1.4429 1.1642 1.3036 unknown 0.9254 1.6738 1.4552 1.1439 1.2996 homologuetoGP|10439230|dbj|BAB15467.unnamedpr oteinproduct{Homosapiens}.partial(32%) 2.5688 0.0159 1.3252 1.2596 1.2924 unknown 1.3164 1.2645 0.5811 1.9998 1.2905 SP|P41276|ARL1_RATADP-ribosylationfactor- likeprotein1.[Rat]{Rattusnorvegicus}.partial(56%) 2.2327 0.3479 0.2990 2.2817 1.2903 similartoGP|13325194|gb|AAH04415.1hypotheticalpro teinFLJ13154{Homosapiens}.partial(87%) 1.5787 1.0000 1.5003 1.0784 1.2894 GP|12834526|dbj|BAB22945.putative{Musmusculus}. complete 0.9494 1.6268 1.6663 0.9100 1.2881 fattyacid-bindingprotein[Susscrofa]heartfattyacid- bindingprotein[Susscrofa] 1.2023 1.3732 0.7592 1.8162 1.2877 similartoGP|12382294|gb|AAG53094.13- methylcrotonyl- CoAcarboxylasebetasubunit{Homosapiens}.partial(19 %) 1.3809 1.1915 0.5000 2.0724 1.2862 homologuetoGP|14603084|gb|AAH10013.1Similartop utativeDNAbindingprotein{Homosapiens}.partial(29%) 0.9125 1.6592 1.3677 1.2040 1.2859 similartoGP|12654491|gb|AAH01075.1mitochondrialu ncouplingprotein1{Homosapiens}.partial(43%) 1.4829 1.0875 1.5850 0.9854 1.2852 similartoPIR|T12474|T12474hypotheticalproteinDKFZ p564K2062.1-human(fragment).complete 0.5521 2.0078 1.2082 1.3517 1.2800 similartoGP|15530220|gb|AAH13889.1Unknown(prote inforMGC:11192){Homosapiens}.partial(23%) 0.5300 2.0297 0.5236 2.0362 1.2799 unknown 1.3581 1.1993 0.2893 2.2682 1.2787 similartoGP|14035880|emb|CAC38536.unnamedprote inproduct{Homosapiens}.partial(47%) 1.6339 0.9228 0.3008 2.2560 1.2784 unknown 2.6826 0.1309 0.7580 1.5319 1.2758 homologuetoGP|10432840|dbj|BAB13857.unnamedpr oteinproduct{Homosapiens}.partial(30%) 0.6946 1.8516 1.0625 1.4837 1.2731 similartoSP|Q02380|NISM_BOVINNADH- ubiquinoneoxidoreductaseSGDHsubunitmitochondrial precursor(EC1.6.5.3)(EC1.6.99.3).complete 2.3257 0.2153 1.4924 1.0486 1.2705 unknown 2.2955 0.2439 1.2008 1.3385 1.2697 beta-globin[Susscrofa] 0.1162 2.6487 1.1197 1.1803 1.2662 similartoGP|12751447|gb|AAK07659.1minorhistocom patibilityantigenprecursor{Musmusculus}.complete 1.2390 1.2876 0.1715 2.3551 1.2633 unknown 1.2619 1.2640 0.3714 2.1546 1.2630 heatshockprotein70.2[Susscrofa] 1.7897 0.7357 1.1511 1.3744 1.2627 unknown 1.2571 1.2563 0.1712 2.3423 1.2567 homologuetoGP|12314036|emb|CAC10469.dJ383J4. 1(AKelchmotif- containingprotein){Homosapiens}.partial(22%) 1.0866 1.4263 1.5850 0.9279 1.2564 unknown 1.5971 0.9143 0.0693 2.4421 1.2557 MADH4protein[Susscrofa]Smad4 0.8724 1.6386 0.6130 1.8981 1.2555 unknown 0.9942 1.5164 0.4824 2.0283 1.2553 unknown 1.5237 0.9851 1.1896 1.3192 1.2544 homologuetoGP|3043720|dbj|BAA25524.1KIAA0598p rotein{Homosapiens}.partial(54%) 1.7586 0.7323 0.7822 1.7087 1.2454 PIR|S11393|R5HU32ribosomalproteinL32- human.complete 2.5469 -0.0613 0.9744 1.5112 1.2428 weaklysimilartoGP|15489209|gb|AAH13712.1Unknow n(proteinforIMAGE:3155889){Musmusculus}.partial(55 %) 1.1490 1.3350 0.7834 1.7006 1.2420 homologuetoPIR|I51803|I51803TAXREB107- 2.8150 0.3325 0.9458 0.8719 1.2413 303 teinFLJ22637{Homosapiens}.partial(24%) homologuetoEGAD|31767|32837T1/ST2receptor- bindingprotein{Homosapiens}.complete -2.1198 -1.9110 -1.8583 -0.5284 -1.6044 homologuetoGP|10438296|dbj|BAB15220.unnamedpr oteinproduct{Homosapiens}.partial(40%) -2.1553 -1.0540 -0.4403 -2.7689 -1.6046 homologuetoGP|4406524|gb|AAD20016.1|tip- associatedproteinTAP{Homosapiens}.partial(45%) 0.8539 1.6215 1.3491 1.1262 1.2377 unknown 1.0213 1.4525 0.3000 2.1738 1.2369 homologuetoGP|6435831|gb|AAC25580.2|bithoraxoid -likeprotein{Rattusnorvegicus}.complete 2.0257 0.4475 1.0931 1.3801 1.2366 unknown 2.6684 0.2015 0.9179 1.1460 1.2335 unknown 0.5983 1.8667 0.9582 1.5068 1.2325 SP|Q93068|SM33_HUMANUbiquitin- likeproteinSMT3Cprecursor).complete 1.3495 1.1155 0.7182 1.7467 1.2325 unknown 1.1724 1.2801 0.9044 1.5481 1.2262 homologuetoGP|13097153|gb|AAH03350.1SimilartoG proteinpathwaysuppressor1{Musmusculus}.partial 0.9748 1.4698 0.6818 1.7628 1.2223 unknown 0.5297 1.9069 1.4643 0.9723 1.2183 unknown 0.3920 2.0403 0.5755 1.8568 1.2161 unknown 1.9154 0.4969 0.4799 1.9324 1.2062 preproSPAI-2[Susscrofa]SPAI-2[Susscrofa]SPAI- 2[Susscrofa] 1.2722 1.1375 0.1349 2.2747 1.2048 homologuetoSP|P47197|AKT2_RATRAC- betaserine/threonineproteinkinase(EC2.7.1.-)(RAC- PK-beta)(ProteinkinaseAkt-2).partial(25%) 1.6449 0.7593 0.3118 2.0924 1.2021 homologuetoGP|183227|gb|AAB59563.1||glucokinase {Homosapiens}.partial(40%) 2.1928 0.1908 1.5149 0.8688 1.1918 similartoGP|14035956|emb|CAC38574.unnamedprote inproduct{Homosapiens}.partial(46%) 1.3219 1.0589 1.0627 1.3181 1.1904 adipocytefattyacidbindingprotein[Susscrofa] 0.8330 2.2115 1.2792 0.4333 1.1893 unknown 0.7876 1.5850 0.4172 1.9553 1.1863 CD40ligand[Susscrofa]CD40L[Susscrofa] 1.3978 0.9676 0.5100 1.8553 1.1827 unknown 1.6732 0.6840 1.2577 1.0995 1.1786 unknown 1.6815 0.6741 0.0602 2.2953 1.1778 GP|4689156|gb|AAD27787.1|unrprotein{Homosapien s}.partial(36%) 0.6693 1.6781 0.8310 1.5164 1.1737 unknown 2.3238 1.0225 0.7713 0.5749 1.1731 unknown 0.7853 1.5449 0.1569 2.1733 1.1651 unknown 1.7321 0.5963 1.3281 1.0003 1.1642 homologuetoSP|Q9Y2X7|GIT1_HUMANARFGTPase- activatingproteinGIT1(Gprotein- coupledreceptorkinase- interactor1).[Human].partial(53%) -2.7726 -1.4150 -0.8260 -1.3617 -1.5938 homologuetoGP|12804745|gb|AAH01808.1nucleoside diphosphatekinasetype6(inhibitorofp53- inducedapoptosis-alpha){Homosapiens}.partial(38%) -1.5220 -2.6684 -0.5773 -1.6130 -1.5952 similartoSP|P30519|HO2_HUMANHemeoxygenase2( EC1.14.99.3)(HO- 2).[Human]{Homosapiens}.complete -1.5397 -1.3458 -0.5220 -2.9805 -1.5970 GP|6463679|dbj|BAA86954.1Fzr1{Homosapiens}.parti al(47%) -2.6521 -1.4557 -1.1293 -1.1558 -1.5982 homologuetoEGAD|45385|47850envoplakin{Homosa piens}.partial(10%) -2.3692 -1.1709 -0.3997 -2.4567 -1.5992 GP|4679028|gb|AAD27002.1|HSPC021{Homosapiens }.partial(44%) -1.1474 -1.0525 -2.0121 -2.1878 -1.5999 homologuetoGP|14043628|gb|AAH07788.1Similartoe ukaryotictranslationinitiationfactor4gamma1{Homosapi ens}.partial(35%) -1.2900 -1.0866 -1.0529 -2.9773 -1.6017 unknown -1.5031 -1.2980 -1.3427 -2.2664 -1.6025 similartoGP|16306850|gb|AAH06548.1hypotheticalpro -1.9806 -1.7751 -0.5968 -2.0585 -1.6028 304 or1-human(fragment).partial(23%) homologuetoGP|12654907|gb|AAH01299.1putativetra nsmembraneprotein;homologofyeastGolgimembranep roteinYif1p.partial(52%) -1.5115 -1.1155 -1.6293 -2.5357 -1.6980 homologuetoSP|P78537|GC5L_HUMANGCN5- likeprotein1(RT14protein).[Human]{Homosapiens}.co mplete -2.5699 -1.3581 -1.3458 -1.1498 -1.6059 collagenVIIIalpha1[Susscrofa] -2.3390 -2.1193 -0.8836 -1.0975 -1.6099 homologuetoEGAD|140573|149904pyruvatekinaseM2 {Susscrofa}.complete -1.7650 -1.5392 -1.3851 -1.7624 -1.6129 unknown -1.5353 -1.3046 -0.7719 -2.8496 -1.6154 homologuetoSP|O43826|G6PU_HUMANGlucose6- phosphatetranslocase(Glucose5- phosphatetransporter)(PRO0685).[Human]{Homosapi ens}.complete -2.1187 -0.8789 -1.6042 -1.8778 -1.6199 similartoGP|11177148|gb|AAG32154.1mitoribosomalp roteinL12{Homosapiens}.complete -2.3219 -1.0677 -0.4519 -2.6668 -1.6271 similartoSP|Q95108|THI2_BOVINThioredoxinmitocho ndrialprecursor(MT- TRX).[Bovine]{Bostaurus}.complete -2.1898 -0.9307 -1.4588 -1.9388 -1.6295 unknown -1.9208 -1.6610 -2.4915 -0.4462 -1.6299 homologuetoGP|17224454|gb|AAL36982.1nanos{Ho mosapiens}.partial(78%) -2.6033 -1.3429 -0.4977 -2.0770 -1.6302 unknown -2.0806 -1.1973 -1.5159 -1.7620 -1.6390 homologuetoPIR|JE0086|JE0086SH3- domainbindingproteinSab-human.partial(42%) -2.9511 -1.3272 -0.5907 -1.6875 -1.6391 homologuetoGP|3449362|gb|AAC32546.1|slowskelet almuscletroponinT{Musmusculus}.partial(51%) -2.8819 -1.4012 -0.4495 -1.8336 -1.6415 similartoGP|10438452|dbj|BAB15247.unnamedprotein product{Homosapiens}.partial(42%) -2.2132 -1.0723 -0.4027 -2.8828 -1.6427 unknown -1.6746 -1.6267 -2.5215 -0.7797 -1.6506 homologuetoEGAD|3033|3004collagentypeIValpha2{ Homosapiens}.partial(35%) -2.0463 -1.2630 -1.0000 -2.3093 -1.6547 unknown -2.8791 -1.4327 -1.1672 -1.1447 -1.6559 homologuetoGP|11611554|dbj|BAB18991.hypothetica lprotein{Macacafascicularis}.partial(43%) -2.3750 -1.0612 -1.9903 -1.2012 -1.6569 PIR|B31486|FIHUAtranslationinitiationfactoreIF- 5A[validated]-human.complete -2.2427 -1.0737 -1.2508 -2.0656 -1.6582 unknown -2.2033 -1.1155 -1.9866 -1.3322 -1.6594 unknown -2.6630 -1.3440 -1.2157 -1.4154 -1.6595 similartoGP|14336708|gb|AAK61240.1similartoAK001 902{Homosapiens}.partial(53%) -1.3399 -2.0180 -1.6666 -1.6192 -1.6609 unknown -2.2625 -1.0635 -1.0891 -2.2368 -1.6630 similartoSP|P51449|RORG_HUMANNuclearreceptor ROR-gamma(NuclearreceptorRZR- gamma).[Human]{Homosapiens}.partial(16%) -2.1420 -1.1846 -0.6746 -2.6520 -1.6633 similartoGP|11559826|gb|AAG38105.1hepatopoietinp rotein{Homosapiens}.complete -2.2946 -1.0447 -1.5646 -1.7747 -1.6696 unknown -2.0238 -1.3219 -1.0468 -2.2990 -1.6729 similartoGP|12652989|gb|AAH00255.1Unknown(prote inforMGC:2495){Homosapiens}.complete -1.8981 -1.4499 -1.2785 -2.0695 -1.6740 homologuetoGP|12653265|gb|AAH00401.1splicingfac tor3bsubunit2145kD{Homosapiens}.partial(18%) -2.4813 -0.8707 -0.8989 -2.4531 -1.6760 calcium-sensingreceptor[Susscrofa] -2.5988 -1.2414 -1.5850 -1.2895 -1.6787 homologuetoGP|5442038|gb|AAD43218.1|stromalcell- derivedreceptor-1alpha{Homosapiens}.partial(81%) -2.1806 -1.1846 -0.0196 -3.3456 -1.6826 unknown -1.9069 -1.4710 -1.5909 -1.7870 -1.6889 unknown -2.2161 -1.8369 -1.2994 -1.4060 -1.6896 unknown -1.7405 -1.6423 -2.0118 -1.3710 -1.6914 similartoPIR|S27962|S27962modulatorrecognitionfact -1.7175 -2.3296 -1.3385 -1.3902 -1.6940 305 similartoGP|10047355|dbj|BAB13465.KIAA1639protei n{Homosapiens}.partial(9%) -2.1463 -2.4669 -0.5530 -2.0602 -1.8066 similartoGP|2463531|dbj|BAA22541.1Fln29{Homosap iens}.partial(24%) -2.9009 -1.7126 -1.2648 -1.3487 -1.8067 homologuetoGP|16041122|dbj|BAB69728.hypothetica lprotein{Macacafascicularis}.partial(32%) -2.0517 -1.3502 -0.9926 -2.4093 -1.7010 GP|2791680|gb|AAC26843.1|26SproteasomeATPase subunit{Homosapiens}.partial(39%) -2.7682 -1.3604 -2.1242 -0.5628 -1.7039 homologuetoSP|P79334|PHS2_BOVINGlycogenphos phorylasemuscleform(EC2.4.1.1)(Myophosphorylase). [Bovine]{Bostaurus}.partial(22%) -2.1562 -1.2521 -1.9779 -1.4304 -1.7041 homologuetoGP|3327044|dbj|BAA31590.1KIAA0615p rotein{Homosapiens}.partial(15%) -1.9740 -1.5657 -2.0643 -1.2125 -1.7041 similartoGP|2914185|pdb|1FSU|4- Sulfatase(Human).partial(52%) -2.1024 -1.3072 -1.6674 -1.7422 -1.7048 caspase-3[Susscrofa] -2.4594 -1.0418 -1.0339 -2.3000 -1.7088 weaklysimilartoGP|463552|gb|AAA16956.1||AF- 1{Homosapiens}.partial(69%) -3.1346 -1.2930 -0.9364 -1.4913 -1.7138 homologuetoSP|P53620|COPG_BOVINCoatomergam masubunit(Gamma-coatprotein)(Gamma- COP).[Bovine]{Bostaurus}.partial(10%) -2.7312 -0.7004 -1.1085 -2.3231 -1.7158 unknown -2.1515 -1.2968 -1.9574 -1.4909 -1.7241 unknown -2.3999 -2.0566 -0.5850 -1.8716 -1.7283 GP|551606|gb|AAA67650.1||RNApolymeraseIIelongat ionfactorSIIIp15subunit{Homosapiens}.complete -2.7395 -1.2735 -1.2925 -1.6266 -1.7330 homologuetoGP|833776|emb|CAA32002.1|adrenodox inreductase(338AA){Bostaurus}.partial(65%) -2.0830 -1.3851 -0.8235 -2.6446 -1.7340 unknown -2.9449 -0.5305 -1.0209 -2.4545 -1.7377 GP|2102696|gb|AAC51317.1|karyopherinbeta3{Homo sapiens}.partial(21%) -3.1315 -0.3491 -1.0870 -2.3935 -1.7403 homologuetoSP|Q92785|REQU_HUMANZinc- fingerproteinubi- d4(Requiem)(Apoptosisresponsezincfingerprotein).[H uman].partial(43%) -1.9849 -1.5004 -1.2366 -2.2470 -1.7422 homologuetoGP|14715064|gb|AAH10696.1proprotein convertasesubtilisin/kexintype7{Homosapiens}.partial( 79%) -1.5363 -1.0480 -2.5700 -1.8224 -1.7442 heparin-bindingepidermalgrowthfactor- likegrowthfactor[Susscrofa]Heparin- bindingepidemialgrowthfactor[Susscrofa] -2.3337 -1.1649 -2.0852 -1.4134 -1.7493 similartoGP|12858123|dbj|BAB31206.putative{Musmu sculus}.partial(20%) -2.0875 -1.5850 -1.7590 -1.5736 -1.7513 weaklysimilartoGP|12839600|dbj|BAB24608.putative{ Musmusculus}.partial(33%) -1.2226 -2.2801 -1.0713 -2.4313 -1.7513 similartoSP|Q13588|GRAP_HUMANGRB2- relatedadaptorprotein.[Human]{Homosapiens}.comple te -1.9228 -1.4154 -0.8603 -2.8164 -1.7537 homologuetoGP|12845499|dbj|BAB26774.putative{M usmusculus}.partial(43%) -1.6491 -1.1350 -1.5081 -2.7361 -1.7571 fourandahalfLIMdomains1protein.isoformCskeletalmu scleLIMprotein[Susscrofa] -3.1099 -0.4158 -1.2156 -2.3101 -1.7628 similartoGP|17512422|gb|AAH19171.1SimilartoRIKE NcDNA2310010G13gene{Musmusculus}.partial(53%) -2.6622 -1.1098 -1.8069 -1.5259 -1.7762 TATAboxbindingprotein(TBP)associatedfactor[Susscr ofa] -2.5952 -0.9870 -1.5511 -2.0310 -1.7911 unknown -2.7726 -1.1844 -1.4001 -1.8192 -1.7941 homologuetoGP|13785926|gb|AAK39520.1BTBdomai nprotein{Homosapiens}.partial(34%) -2.5496 -2.0422 -1.4031 -1.1887 -1.7959 homologuetoSP|P17844|DDX5_HUMANProbableRN A-dependenthelicasep68(DEAD- boxproteinp68)(DEAD- boxprotein5).[Human].partial(13%) -2.1076 -1.5096 -0.9890 -2.5897 -1.7990 unknown -2.4851 -1.1166 -0.6742 -2.9275 -1.8009 306 unknown -1.7509 -2.2377 -1.8106 -2.1780 -1.9943 homologuetoSP|P18754|RCC1_HUMANRegulatorofc hromosomecondensation(Cellcycleregulatoryprotein).[ Human]{Homosapiens}.partial(41%) -2.8213 -1.1727 -1.0113 -2.9827 -1.9970 5-hydroxytryptaminereceptor2c[Susscrofa] -2.0000 -1.8695 -2.5247 -1.6058 -2.0000 homologuetoSP|P00883|ALFA_RABITFructose- bisphosphatealdolaseA(EC4.1.2.13)(Muscle- typealdolase).[Rabbit].partial(24%) -2.7379 -0.8856 -1.6784 -1.9451 -1.8117 unknown -1.3324 -1.2916 -2.2985 -2.3255 -1.8120 unknown -2.9696 -1.3410 -1.4671 -1.4794 -1.8143 similartoGP|1694954|dbj|BAA13745.1Neuroblastoma{ Homosapiens}.partial(87%) -2.2033 -1.4339 -1.3896 -2.2476 -1.8186 olfactoryreceptor[Susscrofa] -2.4946 -1.1468 -1.5600 -2.0814 -1.8207 unknown -2.5546 -1.1255 -1.2355 -2.4446 -1.8401 similartoSP|Q04857|CA16_MOUSECollagenalpha1(V I)chainprecursor.[Mouse]{Musmusculus}.partial(13%) -2.6743 -1.9783 -1.4598 -1.2796 -1.8480 sodium/hydrogenexchangerisoform3[Susscrofa] -2.6994 -1.0066 -1.0916 -2.6145 -1.8530 unknown -2.7407 -1.0148 -1.2252 -2.4711 -1.8630 unknown -1.6553 -2.0774 -1.5302 -2.2025 -1.8663 similartoGP|13182763|gb|AAK14927.1CDA03{Homos apiens}.partial(60%) -2.7885 -0.9477 -1.6599 -2.0763 -1.8681 similartoSP|P52758|UK14_HUMAN14.5kDatranslatio nalinhibitorprotein(p14.5)(UK114antigenhomolog).[Hu man]{Homosapiens}.partial(75%) -2.9069 -1.1220 -2.0000 -1.5409 -1.8925 similartoSP|P58058|PPNK_MOUSEPutativeinorganic polyphosphate/ATP- NADkinase(EC2.7.1.23)(Poly(P)/ATPNADkinase).[Mo use].partial(55%) -3.6781 -1.1198 -1.5510 -1.2469 -1.8990 homologuetoSP|P42208|SEP2_MOUSESeptin2(NED D5protein).[Mouse]{Musmusculus}.partial(54%) -3.0444 -1.2303 -1.0457 -2.3078 -1.9070 unknown -2.8413 -1.0234 -2.2303 -1.5409 -1.9090 PIR|JC4949|JC4949ADP-ribosylationfactor5- mouse.partial(97%) -2.2896 -1.4610 -0.8480 -3.0586 -1.9143 unknown -2.0100 -2.1740 -1.8639 -1.6240 -1.9180 similartoGP|16605472|emb|CAC82744.acyl- malonylcondensingenzyme{Homosapiens}.partial(53 %) -2.1448 -2.3080 -1.8665 -1.3543 -1.9184 long-chainenoyl-CoAhydratase:3-hydroxyacyl- CoAdehydrogenaseprecursor[Susscrofa]gastrin- bindingprotein -2.3969 -1.5484 -2.0291 -1.7224 -1.9242 homologuetoGP|11527783|dbj|BAB18652.ubiquitin- conjyugatingenzymeE2{Homosapiens}.partial(59%) -2.6821 -1.8237 -0.9750 -2.2360 -1.9292 weaklysimilartoGP|12848483|dbj|BAB27972.putative{ Musmusculus}.partial(78%) -2.5169 -1.3473 -0.7494 -3.1148 -1.9321 homologuetoSP|Q9XSJ7|CA11_CANFACollagenalph a1(I)chainprecursor.[Dog]{Canisfamiliaris}.partial(5%) -2.1026 -2.2174 -1.7998 -1.6505 -1.9426 homologuetoGP|7020143|dbj|BAA91010.1unnamedpr oteinproduct{Homosapiens}.partial(37%) -2.4444 -1.5591 -1.3021 -2.4650 -1.9426 homologuetoGP|17390440|gb|AAH18197.1Similartoh ypotheticalprotein{Musmusculus}.partial(23%) -3.5341 -0.3704 -1.3070 -2.5976 -1.9523 homologuetoGP|12832202|dbj|BAB22006.putative{M usmusculus}.partial(85%) -2.5105 -1.6051 -1.7652 -1.9302 -1.9527 similartoSP|O94833|ACFX_HUMANTrabeculin- beta(Fragment).[Human]{Homosapiens}.partial(11%) -1.5742 -2.3443 -1.1998 -2.7187 -1.9593 similartoGP|14250483|gb|AAH08682.1actin- relatedprotein3-beta{Homosapiens}.partial(45%) -1.9189 -2.0000 -1.3399 -2.5790 -1.9594 unknown -2.2730 -2.3479 -1.7799 -1.4494 -1.9625 GP|12654649|gb|AAH01162.1signalrecognitionparticl ereceptor('dockingprotein'){Homosapiens}.partial(22% ) -2.1403 -1.8025 -1.5555 -2.3872 -1.9714 similartoGP|7020512|dbj|BAA91159.1unnamedprotein product{Homosapiens}.partial(22%) -2.9079 -1.0503 -1.6773 -2.2809 -1.9791 307 homologuetoGP|11141704|gb|AAG32038.1SIR2L2{M usmusculus}.partial(50%) -2.7549 -2.1993 -1.6599 -2.4970 -2.2778 Ca2+ATPaseoffasttwitch1skeletalmusclesarcoplasmic reticulum[Susscrofa] -2.7532 -2.1902 -1.4926 -2.6902 -2.2815 eag-relatedgene[Susscrofa] -2.1964 -1.1844 -1.6554 -2.9878 -2.0060 similartoGP|12804145|gb|AAH02927.1HSCARGprotei n{Homosapiens}.partial(53%) -2.1699 -2.1579 -1.3226 -2.3735 -2.0060 homologuetoPIR|A26711|A26711translationinitiationfa ctoreIF-2alphachain-rat.partial(29%) -1.5572 -1.5396 -2.2249 -2.7135 -2.0088 similartoGP|15430292|gb|AAK95951.1musclealpha- kinase{Homosapiens}.partial(5%) -2.1085 -2.0825 -1.0641 -2.7970 -2.0130 homologuetoGP|13506235|gb|AAG24463.1ST7protei nform3splicevariantb{Musmusculus}.partial(57%) -2.7814 -1.7270 -1.7370 -1.8634 -2.0272 similartoPIR|T08778|T08778hypotheticalproteinDKFZ p586I1520.1-human(fragment).partial(12%) -2.6245 -1.5682 -1.4444 -2.4755 -2.0281 homologuetoGP|12654907|gb|AAH01299.1putativetra nsmembraneprotein;homologofyeastGolgimembranep roteinYif1p.partial(52%) -2.2446 -1.1672 -1.8507 -2.8922 -2.0387 unknown -1.9668 -1.8889 -2.3930 -1.9071 -2.0390 similartoGP|13477137|gb|AAH05025.1Similartometall oprotease1(pitrilysinfamily){Homosapiens}.partial(20% ) -1.6163 -1.5280 -2.4213 -2.6109 -2.0441 GP|10280562|gb|AAG15419.1eukaryotictranslationinit iationfactor3subunitp42/p44{Homosapiens}.complete -2.3399 -2.2479 -1.4929 -2.1032 -2.0460 homologuetoGP|179830|gb|AAA35636.1||caldesmon{ Homosapiens}.partial(33%) -1.7310 -1.3901 -2.3898 -2.7313 -2.0605 homologuetoGP|13430408|gb|AAK25826.1BTBD2pro tein{Homosapiens}.partial(37%) -1.6196 -1.5295 -2.4433 -2.7058 -2.0745 unknown -2.0297 -2.1283 -1.6229 -2.5351 -2.0790 homologuetoSP|O46392|CA21_CANFACollagenalpha 2(I)chainprecursor.[Dog]{Canisfamiliaris}.partial(12%) -2.0798 -2.0937 -1.3513 -2.8222 -2.0867 unknown -2.2883 -2.0988 -1.4884 -2.5034 -2.0947 homologuetoPIR|T47177|T47177hypotheticalproteinD KFZp762H157.1-human(fragment).partial(43%) -2.0224 -1.7997 -2.4150 -2.2082 -2.1113 weaklysimilartoGP|2981631|dbj|BAA25253.1ORF2{C anisfamiliaris}.partial(26%) -2.3063 -1.9231 -0.4364 -3.7930 -2.1147 PIR|T50638|T50638synapticglycoproteinSC2[importe d]-human.complete -2.2967 -2.0640 -1.0425 -3.0621 -2.1163 similartoPIR|B34087|B34087hypotheticalprotein(L1H3 'region)-human.partial(11%) -2.4030 -1.8340 -1.3133 -2.9237 -2.1185 homologuetoGP|4071321|gb|AAC98673.1|Y- boxproteinMSY2{Musmusculus}.partial(47%) -3.1729 -1.0745 -1.6032 -2.6442 -2.1237 GP|567053|gb|AAA56751.1||beta5tubulin{Xenopuslae vis}.partial(22%) -2.3294 -2.0780 -1.0995 -2.9958 -2.1257 homologuetoSP|O95716|RB3D_HUMANRas- relatedproteinRab- 3D.[Human]{Homosapiens}.partial(83%) -2.0790 -2.1785 -1.9131 -2.3443 -2.1287 SP|Q14449|GRBE_HUMANGrowthfactorreceptor- boundprotein14(GRB14adapterprotein).[Human]{Hom osapiens}.partial(30%) -1.9707 -2.3041 -1.7285 -2.5463 -2.1374 fibroblastgrowthfactor9[Susscrofa] -2.5850 -2.2224 -1.8301 -2.0877 -2.1813 homologuetoPIR|JC5252|JC5252mitogen- activatedproteinkinase(EC2.7.1.-)p38gamma- human.partial(61%) -2.1665 -2.2204 -1.4342 -2.9528 -2.1935 homologuetoGP|1000746|gb|AAC50214.1|Pro- a2(XI){Homosapiens}.partial(12%) -2.9387 -1.4789 -2.3429 -2.1591 -2.2299 gagprotein[Susscrofa]polprotein[Susscrofa] -2.2384 -2.2261 -1.9760 -2.4885 -2.2322 similartoPIR|A55050|A55050enigma- human.partial(32%) -2.5618 -1.9211 -2.2940 -2.1889 -2.2414 homologuetoSP|Q92830|GCL2_HUMANGeneralcontr olofaminoacidsynthesisprotein5-like2(EC2.3.1.- ).partial(26%) -3.5041 -0.9938 -2.8034 -1.6945 -2.2489 unknown -3.4321 -1.1070 -1.1882 -3.3508 -2.2695 homologuetoGP|5821375|dbj|BAA83793.1MTH1b(p2 2)MTH1c(p21)MTH1d(p18){Homosapiens}.partial(72 %) -2.7915 -1.2266 -2.0855 -3.0263 -2.2825 homologuetoGP|7959283|dbj|BAA96035.1KIAA1511p rotein{Homosapiens}.partial(10%) -2.6147 -2.0107 -2.2268 -2.3558 -2.3020 similartoGP|14272678|emb|CAC39777.unnamedprote inproduct{Homosapiens}.partial(28%) -2.8541 -1.7843 -2.0418 -2.5966 -2.3192 unknown -2.3374 -1.6894 -2.2811 -2.9880 -2.3240 homologuetoSP|P24140|GPT_CRIGRUDP-N- acetylglucosamine--dolichyl-phosphateN- acetylglucosaminephosphotransferase(EC2.7.8.15).p artial(42%) -2.1207 -2.4725 -2.4833 -2.2199 -2.3241 cytosolicglycerol-3- phosphatedehydrogenase[Susscrofa] -2.2630 -2.4019 -2.5453 -2.1197 -2.3325 PIR|A24156|BCBOIAS-100proteinalphachain- bovine.partial(85%) -2.1458 -2.5299 -1.7774 -2.8984 -2.3379 homologuetoSP|O18751|PHS2_SHEEPGlycogenpho sphorylasemuscleform(EC2.4.1.1)(Myophosphorylase ).[Sheep]{Ovisaries}.partial(26%) -2.7762 -1.9125 -2.3896 -2.2990 -2.3443 unknown -2.9069 -1.7984 -2.4330 -2.2724 -2.3527 similartoGP|13540827|gb|AAF15294.2LRP16{Homos apiens}.complete -2.8912 -2.1606 -1.1350 -3.2743 -2.3653 homologuetoGP|2145060|gb|AAB58413.1|TTF- Iinteractingpeptide20;TIP20;TranscriptionTermination FactorIInteractingPeptide20.partial(33%) -3.2029 -2.3954 -1.1179 -2.8988 -2.4038 homologuetoGP|493132|gb|AAC41688.1||creatinetran sporter{Homosapiens}.partial(27%) -2.9203 -3.0588 -1.4981 -2.2460 -2.4308 similartoGP|13397120|emb|CAC34689.unnamedprote inproduct{Homosapiens}.partial(6%) -3.5064 -2.4663 -1.8694 -2.1033 -2.4863 unknown -3.0284 -2.0310 -2.6483 -2.2872 -2.4987 unknown -3.2184 -1.8331 -1.4501 -3.6014 -2.5258 homologuetoSP|Q15113|PCO1_HUMANProcollagen C- proteinaseenhancerproteinprecursor(PCPE).partial(41 %) -1.6724 -2.5757 -3.2398 -2.7055 -2.5484 homologuetoGP|3413926|dbj|BAA32327.1KIAA0483p rotein{Homosapiens}.partial(54%) -1.9069 -2.3515 -2.8365 -3.4219 -2.6292 KCNE4[Susscrofa] -3.1793 -3.1080 -1.6995 -2.5878 -2.6436 similartoGP|6330358|dbj|BAA86507.1KIAA1193protei n{Homosapiens}.partial(68%) -2.3576 -4.0080 -3.5387 -0.7949 -2.6748 homologuetoGP|9931474|gb|AAG02184.1|RNAbindin gproteinMCG10{Homosapiens}.partial(33%) -2.7857 -3.4236 -1.8986 -2.6163 -2.6811 homologuetoSP|Q14324|MYPF_HUMANMyosin- bindingproteinCfast-type(FastMyBP-C)(C- proteinskeletalmusclefast- isoform).[Human].partial(11%) -3.1693 -2.5337 -2.1753 -3.3928 -2.8178 GP|10438718|dbj|BAB15321.unnamedproteinproduct{ Homosapiens}.partial(92%) -3.4079 -3.1286 -3.1198 -2.9023 -3.1396 immunoglobulinmuheavychainconstantregion[swine.s pleen.PeptidePartial.403aa]immunoglobulinmuchainc h4andsecretedomainsofswineIgM[Susscrofa] -4.8284 -2.5642 -2.3685 -3.0241 -3.1963 SP|P06366|RS14_HUMAN40SribosomalproteinS14(P roteinPRO2640).[Chinesehamster]{Cricetulusgriseus}. complete -2.9971 -2.6899 -3.7448 -3.9422 -3.3435 typeIIcollagenalpha1[Susscrofa] -4.7687 -2.9978 -2.7636 -3.0029 -3.3833 308 Appendix J. An example of searching tentative biological information for one unknown transcript (gene) In the result of adipose tissue, transcription of an unknown gene was significantly increased with the average normalized log e GenePix Array List (GAL) file, the oligo ID of the unknown gene was SS0009090. Then searching the pig array Axon Text File (ATF) and the probe sequence was CGCGTCCGGTCTCGGATAAAAGTCCTGGATTTTCCATTGGTTTTCATAATGGG TGTTTATATAAAACTAC . Then, the sequence was submitted to TIGR Gene Index Database (SsGI 5.0) (http://www.tigr.org/tigr-scripts/tgi/T_index.cgi?species=pig). It returned the gene ID of TC60740. Then, the sequence information of TC60740 was recruited from SsGI, and the information was presented in the Fig 1. 2 ratio of 2.98. Tracking th Results showed four open reading frames were included by the sequence; each of them corresponds to an expression sequence tag (EST). The accession numbers of the four ESTs were CF179926, AW785390, BF711816 and BM659677 respectively. Since the ESTs provided limited biological information about the unknown gene, the tentative consensus sequence of the unknown gene was aligned against GenBank by running BLASTn (Stephen, F. et al, 1997 ). BLASTn returned 128 hits (Fig 2). Further research may be designed to characterize the unknown gene based on the result with highest BLAST percent identity score and the knowledge in the pig biology. 309 Fig 1: Recruiting information of TC60740 by searching TIGR Porcine (Sus scrofa) Gene Index (SsGI). (http://www.tigr.org/tigr-scripts/tgi/T_index.cgi?species=pig) 310 311 Fig 2. Result of aligning tentative consensus sequence of the unknown gene against GenBank by BLASTn