PHOSPHORYLATION OF FETUIN-A, A PHYSIOLOGICAL INHIBITOR OF INSULIN ACTION, REGULATED BY INSULIN AND LEPTIN Except where reference is made to the work of others, the work described in this thesis is my own or was done in collaboration with my advisory committee. This thesis does not include proprietary or classified information. __________________________ James B. Papizan Certificate of Approval: ________________________ __________________________ Douglas White Suresh T. Mathews, Chair Associate Professor Assistant Professor Nutrition and Food Science Nutrition and Food Science ________________________ __________________________ Kevin Huggins George T. Flowers Assistant Professor Interim Dean Nutrition and Food Science Graduate School PHOSPHORYLATION OF FETUIN-A, A PHYSIOLOGICAL INHIBITOR OF INSULIN ACTION, REGULATED BY INSULIN AND LEPTIN James B. Papizan A Thesis Submitted to the Graduate Faculty of Auburn University in Partial Fulfillment of the Requirements of the Degree of Master of Science Auburn, Alabama May 10, 2007 iii PHOSPHORYLATION OF FETUIN-A, A PHYSIOLOGICAL INHIBITOR OF INSULIN ACTION, REGULATED BY INSULIN AND LEPTIN James B. Papizan Permission is granted to Auburn University to make copies of this thesis at its discretion, upon request of individuals or institutions at their expense. The author reserves all publication rights. ___________________________ Signature of Author ___________________________ Date of Graduation iv VITA James B. Papizan, son of Bruce and Beverly Papizan, was born in Brooksville, FL on January 19, 1980. Upon graduating from Madison Central High School in Madison, MS, James chose to attend The University of Southern Mississippi on a music scholarship. After studying percussion performance for four years and performing abroad in such places as Paris, France and London, England, he switched his focus to Nutrition. James graduated from Southern Miss in 2005 with a B.S. in Nutrition and Dietetics and with minors in Music Performance and Chemistry. The summer succeeding his graduation, James married the beautiful Ansley Love Nored of Clinton, MS. In August of 2005, James and Ansley moved to Auburn, AL where James enrolled into Auburn University?s Graduate School and pursued a Master of Science Degree in Nutrition in the Department of Nutrition and Food Science under the direction of Dr. Suresh T. Mathews. v THESIS ABSTRACT PHOSPHORYLATION OF FETUIN-A, A PHYSIOLOGICAL INHIBITOR OF INSULIN ACTION, REGULATED BY INSULIN AND LEPTIN James B. Papizan Master of Sciences, May 10 2007 (B.S., University of Southern Mississippi, 2005) 84 Typed Pages Directed by Suresh T. Mathews Fetuin-A, a liver secreted phosphoprotein and rat homologue of human 2-HS- glycoprotein (AHSG), is a physiological inhibitor of insulin action. Fetuin-A-null mice exhibit improved glucose tolerance and resistance to weight gain on a high-fat diet. Recent human data demonstrate a positive correlation of circulating fetuin-A levels with BMI, insulin resistance, and fatty liver. Since phosphorylation is critical for its inhibitory activity, circulating fetuin-A phosphorylation was assayed in animal models of insulin resistance, diabetes, and obesity. Using a phospho-specific ( 312 Ser) fetuin-A antibody, fetuin-A phosphorylation status was assayed in Zucker diabetic fatty (ZDF) rats, ob/ob and db/db mice, streptozotocin-treated rats, and in a rat model of diet-induced obesity (DIO). The leptin receptor-deficient, insulin resistant ZDF rats had significantly higher levels of phosphorylated fetuin-A compared to their lean counterparts. Likewise, the vi leptin deficient, obese ob/ob mice had significantly higher levels of phosphorylated fetuin-A. Consistent with these results, central leptin administration to control and STZ- treated rats, as well as leptin treatment to human hepatoma cell lines, significantly decreased fetuin-A phosphorylation. To further characterize the regulation of fetuin-A phosphorylation, we demonstrate that insulin treatment downregulates phosphorylation in Hep3B and HepG2 cell lines. Similarly, DIO rats that exhibit elevated levels of insulin, albeit not to a significant degree, demonstrate significantly decreased levels of fetuin-A phosphorylation. We demonstrate that insulin and leptin decrease phosphorylation of fetuin-A. Accordingly, in conditions of mild to moderate elevation of insulin (as in the DIO model), fetuin-A phosphorylation is decreased, mitigating the inhibitory effect of fetuin-A on IR and allowing for increased insulin signaling. In conditions of extreme insulin resistance (ZDF and ob/ob models), where insulin is unable to exert its actions on insulin receptors, fetuin-A phosphorylation is increased, potentially exacerbating the diabetic phenotype. These findings suggest that fetuin-A may play a significant role in the regulation of insulin action. vii ACKNOWLEDGEMENTS The author would first like to express his sincere gratitude to his wife, Ansley, for her love, support, and sustenance. He would also like to express his deepest appreciation to his major professor, Dr. Suresh T. Mathews, for his continual guidance, support, and friendship. The author would also like to thank his committee members, Drs. Doug White and Kevin Huggins, for their support and suggestions. A very special appreciation is also extended to Dr. Teayoun Kim for his ceaseless help and support in the laboratory. The author also extends gratitude to his parents, Bruce and Beverly Papizan, as well as his in- laws, Franklin and Beverly Nored, for their endless support, encouragement, and love. viii Style manual of journal used: Diabetes Computer software used: Microsoft Word 2000, Microsoft PowerPoint 2000 ix TABLE OF CONTENTS LIST OF TABLES ..........................................................................................................x LIST OF FIGURES........................................................................................................xi CHAPTER 1. INTRODUCTION.....................................................................................1 CHAPTER 2. REVIEW OF LITERATURE ....................................................................6 Obesity and inflammation.........................................................................6 Diabetes ...................................................................................................7 Insulin signal transduction ........................................................................7 Insulin resistance ....................................................................................11 Negative modulators of insulin action.....................................................12 Fetuin-A.................................................................................................13 Structure .....................................................................................14 IR-TK inhibition .........................................................................19 Gene ...........................................................................................22 Human studies ............................................................................24 Objectives and hypothesis .......................................................................25 CHAPTER 3. PHOSPHORYLATION OF FETUIN-A, A PHYSIOLOGICAL INHIBITOR OF INSULIN ACTION, REGULATED BY INSULIN AND LEPTIN .........................................................................................27 Abstract..................................................................................................27 Introduction............................................................................................29 Materials and methods............................................................................30 Results....................................................................................................33 Discussion..............................................................................................38 Conclusion .............................................................................................42 BIBLIOGRAPHY .........................................................................................................65 x LIST OF TABLES Table 1. Metabolic indices of high fat fed and chow fed rats..........................................60 xi LIST OF FIGURES Figure 1. Insulin signaling pathway ...............................................................................10 Figure 2. Conserved serine residues on fetuin-A ............................................................17 Figure 3. Protein structure of human AHSG/fetuin-A ...................................................18 Figure 4. Specificity of phospho-fetuin ( 312 Ser) antibody ..............................................44 Figure 5. Body weights of ZDF and lean control rats .....................................................45 Figure 6. Glucose levels in ZDF rats..............................................................................46 Figure 7. Insulin levels in ZDF rats................................................................................47 Figure 8. Phosphorylation of fetuin in insulin resistant, obese ZDF rats (6 weeks) ........................................................................................................48 Figure 9. Phosphorylation of fetuin in insulin resistant, obese ZDF rats (14 weeks).......................................................................................................49 Figure10. Total plasma fetuin levels in insulin resistant, obese ZDF rats (6 weeks) .......................................................................................................50 Figure 11. Total plasma fetuin levels in insulin resistant, obese ZDF rats (14 weeks)....................................................................................................51 Figure 12. Insulin levels in ob/ob and db/db mice ..........................................................52 Figure 13. Plasma leptin levels in ob/ob and db/db mice ................................................53 Figure 14. Phosphorylation of fetuin in obese, diabetic ob/ob and db/db mice..............................................................................................54 xii Figure 15. Total plasma fetuin levels in obese, diabetic ob/ob and db/db mice.............................................................................................55 Figure 16. Glucose levels in STZ-treated rats.................................................................56 Figure 17. Insulin levels in STZ-treated rats...................................................................57 Figure 18. Phosphorylation of fetuin in STZ-treated rats................................................58 Figure 19. Total plasma fetuin levels in STZ-treated rats ...............................................59 Figure 20. Phosphorylation of fetuin in high fat-fed rats ................................................61 Figure 21. Total plasma fetuin levels in high fat-fed rats................................................62 Figure 22. Insulin receptor tyrosine kinase activity in high fat-fed rats...........................63 Figure 23. Regulation of fetuin phosphorylation in Hep3B human hepatoma cells..............................................................................................64 1 Chapter 1: Introduction The incidence of obesity and its associated complications is escalating at an alarming rate worldwide. Obesity predisposes individuals to an increased risk of developing multiple diseases such as cardiovascular disease, arthritis, certain cancers, non-alcoholic fatty liver disease, and diabetes (1-3). The American Obesity Association defines obesity as an individual having a body mass index (BMI) equal to or greater than 30. The BMI is a measure of body fat based on a person?s height and weight, i.e., [weight (kg)/height (m) 2 ]. According to The Alabama Center for Health Statistics, 24.5 percent of adults in Alabama are obese, making Alabama the second heaviest state in the nation. Since obesity is tightly correlated with the prevalence of diabetes, this has caused a relative neologism to creep into medical terminology: Diabesity ? the twin epidemics. Therefore, the cornerstone of much research today is to elucidate the link between obesity and diabetes. Obesity in Western society can partially be attributed to the high-fat diet and sedentary lifestyle. High-fat diet-induced obesity results in an increased secretion of adipocyte-derived nutrients such as free fatty acids (FFA), which have been implicated in the development of insulin-resistance, a hallmark of type 2 diabetes (4). Furthermore, hypertrophic adipocytes secrete less adiponectin, a hormone that has been demonstrated to increase insulin sensitivity (5). Obesity is also regarded as a chronic low-grade 2 inflammatory condition, and a growing amount of evidence has pointed to a link between insulin resistance and obesity-induced inflammation (3; 6). Cytokines associated with inflammation, e.g., tumor necrosis factor-? (TNF-?) and monocyte chemoattractant protein-1 (MCP-1), have been shown to impair insulin action, resulting in diabetes (7-9). Human type 2 diabetes is characterized by two major characteristics: peripheral insulin resistance and impaired insulin secretion by the pancreatic ?-cells (10;11). Normal blood glucose levels are maintained by adaptations of the ?-cells as a compensatory response to insulin resistance. Manifestation of type 2 diabetes results only when the ?-cells? adaptations fail to compensate for insulin resistance. Conversely, human type 1 diabetes is regarded as an autoimmune disease, characterized by the destruction of pancreatic ?- cells, which results in the inability to produce insulin. Type 1 diabetes, or insulin- dependent diabetes mellitus (IDDM), was previously referred to as juvenile diabetes, as the onset of the disease usually occurs during childhood. Type 2 diabetes, or non insulin- dependent diabetes mellitus (NIDDM), was previously referred to as adult-onset diabetes, as the onset of the disease usually occurs during adulthood; however, as the prevalence of obesity in children continues to escalate in Western culture, more and more children are developing type 2 diabetes. Insulin action begins with insulin binding to the insulin receptor (IR), which induces a conformational change thereby phosphorylating tyrosine residues located on the intracellular ?-subunits in a process called autophosphorylation (12). This activates an intrinsic insulin-receptor tyrosine kinase (IR-TK), which phosphorylates the insulin receptor substrate (IRS) proteins, a critical node in insulin signaling (13). The IRS proteins are linked to the other two critical nodes in insulin signaling: the 3 phosphatidylinositol 3-kinase (PI3-K) pathway and the Ras-mitogen-activated protein kinase (MAPK) pathway, which are responsible for the metabolic and mitogenic actions of insulin, respectively (14) (Fig. 1). IRS proteins mediate insulin signaling and, like the IR, are activated by tyrosine phosphorylation and inhibited by serine phosphorylation and protein tyrosine phosphatases (PTPs) (9). While known modulators of insulin action act to sensitize the effects of insulin e.g., adiponectin (5; 15), leptin (16), and thiazolidindiones (TZDs) (17-19), others act to attenuate insulin signaling through mechanisms such as serine phosphorylation and protein tyrosine phosphatase activity. Serine kinases known to down-regulate insulin signaling include c-Jun-N-terminal kinase (JNK), extracellular signal-regulated kinase (ERK), and S6-kinase by phosphorylating serine residues on IRS proteins. Other inhibitors of insulin action include PTP1B, suppressor of cytokine signaling-1 (SOCS1), and plasma-cell-membrane glycoprotein-1 (PC-1), which act at the level of the IR by either sterically blocking the IR/IRS interaction, or by modifying the IR-TK activity (20). Another inhibitor acting at the level of the IR, specifically an IR-TK inhibitor, is fetuin-A, a 63 kD phosphorylated glycoprotein, synthesized and secreted primarily by the liver and belonging to the cystatin family of protease inhibitors (21). Fetuin-A was shown originally to be synthesized and secreted by normal rat hepatocytes as a phosphoprotein called PP 63 (22). Later, PP 63 was shown to be an inhibitor of IR-TK activity only in its phosphorylated form (23). Further investigations have identified bovine fetuin and human ?2-HS-glycoprotein (AHSG) as homologs of rat fetuin-A (24-27). Fetuin-A preferentially interacts with the activated insulin receptor, repressing insulin-induced IR autophosphorylation and subsequent downstream signaling activity in 4 vitro, in intact cells and in vivo (28). Fetuin-A null mice demonstrate improved insulin sensitivity, whole-body glucose utilization, and resistance to weight gain when fed a high fat diet (29). Additionally, these mice are protected against aging-induced obesity and insulin resistance (30). Interestingly, the gene for fetuin-A is localized on chromosome 3q27, a locus that has been shown to be associated with type 2 diabetes and cardiometabolic risk (31). While a polymorphism of the AHSG gene has been shown to be associated with type 2 diabetes, another polymorphism is associated with leanness in Swedish population (32). A growing body of human investigation studies points to a strong association of plasma fetuin-A levels with insulin resistance (33-35), an atherogenic lipid profile (34), and increased BMI and plasma triglyceride levels (36). Plasma concentrations of fetuin-A in humans range from 200-600 ?g/ml, with an average plasma concentration of ~ 300 ?g/ml in healthy adults (37). Fetuin-A exists in both phosphorylated and dephosphorylated forms in human plasma. Pioneering studies by Haglund et al have demonstrated that fetuin-A is phosphorylated on two serine residues, 120 Ser and 312 Ser, and that approximately 20% of circulating fetuin-A is phosphorylated (38). Phosphorylation of fetuin-A has been shown to be critical for its IR tyrosine kinase inhibitory activity (23). While 120 Ser-AHSG has been shown to be constitutively phosphorylated, 312 Ser-AHSG seems to exhibit regulatory covalent modifications, suggesting this site may be critical for its inhibitory activity (38). While previous human studies have investigated the associations of fetuin-A in disease conditions, there are no reports examining its phosphorylation status. Therefore, 5 the goal of this study is to elucidate the phosphorylation status of fetuin-A in animal models of obesity, insulin resistance, and diabetes. 6 Chapter 2: Review of Literature Obesity and inflammation During recent decades, the incidence of obesity and its associated complications, including type 2 diabetes, has risen boundlessly, resulting in obesity-related health concerns associated with increased morbidity and mortality (39; 40). Equally alarming is the concomitant increase in childhood obesity, also resulting in obesity-related health threats, an occurrence that was inscrutable just a few decades ago (41). Adipose tissue was once considered an inert tissue, functioning solely as an energy storage depot. More recently, adipose tissue has been viewed as a dynamic endocrine system, secreting a vast array of adipokines with functions targeting the immune and metabolic systems (42; 43). Adiponectin, leptin, visfatin, resistin, and RBP4 are several adipokines thought to provide an important link or play a role in the development of obesity-induced insulin resistance (2; 3; 44;45). Increasing adiposity is also associated with elevated inflammatory responses, which is characterized by abnormal circulating levels of adipose-derived cytokines (3). Several of these cytokines, such as TNF-? and MCP-1, have been implicated in the development of impaired insulin action, resulting in insulin resistance and diabetes (5; 7; 46). 7 Diabetes The American Diabetes Association defines diabetes as a group of metabolic diseases characterized by hyperglycemia resulting from defects in insulin secretion, insulin action, or both (47). Three types of diabetes exist: type 1, type 2, and gestational diabetes. Type 1 diabetes, or IDDM, develops when the body?s immune system attacks and destroys the pancreatic ?-cells, the cells in which insulin is produced and secreted. In order to survive, individuals with type 1 diabetes must use exogenous insulin as a therapeutic in order to maintain euglycemia. Type 1 diabetes only constitutes 5-10 percent of all diagnosed cases of diabetes in the U.S., while type 2 diabetes, or NIDDM, comprises 90-95 percent of diagnosed cases. Insulin resistance, a complication characterized by the inability of peripheral tissue to respond to circulating insulin, usually preludes type 2 diabetes. As the ?-cells? ability to respond to the body?s demand for insulin attenuates, type 2 diabetes ensues. Gestational diabetes is a form of glucose intolerance that occurs during pregnancy, and women who develop gestational diabetes are at a greater risk for developing type 2 diabetes later in life. Currently there are over 20 million adults in the U.S. with diabetes, and, according to the Alabama Cooperative Extension System, Alabama leads the nation in diabetes with more than 440,000 individuals diagnosed with the disease. This prevalence is costing Alabama more than $3 billion dollars annually. Insulin signal transduction Insulin-mediated events control processes such as glucose uptake, lipid synthesis, protein synthesis, and glycogen deposition. These processes begin with insulin binding to 8 its IR, which is found in virtually all vertebrate tissues; however, the concentration of the receptors varies from tissue to tissue. The IR is a transmembrane, tetrameric protein that consists of two extracellular ?-subunits and two intracellular ?-subunits (48). The extracellular ?-subunits contain the insulin-binding domain and controls the activity of the intracellular, intrinsic tyrosine kinases located on the ?-subunits. Prior to insulin binding, a separation is maintained between the two intracellular tyrosine kinases, inhibiting transphosphorylation. Upon insulin binding to the extracellular ?-subunits, the inhibitory activity is relieved, bringing the intracellular ?-subunits and tyrosine kinases in close enough proximity for transphosphorylation (48). Once autophosphorylated, the IR recruits the IRS proteins, which have both pleckstrin-homology domains (PH domains) and phosphotyrosine binding domains (PTB domains) (49). Six IRS proteins have been identified and have been designated as IRS 1-6 (50; 51). IRS 1 and 2 are found ubiquitously, whereas IRS 3 is expressed in adipocytes and brain, and IRS 4 is limited to embryonic tissues. IRS 5 is found mostly in kidney and liver while IRS 6 is mostly expressed in skeletal muscle (51). The PTB domain of IRS proteins binds directly to the NPXpY-motif of the activated IR. The center and C-terminus of IRS proteins contain multiple potential tyrosine phosphorylation sites that, after phosphorylation by the IR, recruit intracellular molecules containing Src-homology-2 domains (SH2 domains) (49). Two examples of SH2 proteins that bind to phosphorylated IRS proteins are PI3K and Grb2. PI3K mediates the insulin-stimulated metabolic activities and contains a regulatory and a catalytic subunit, which upon activation by IRS proteins, converts phosphatidylinositol bisphosphate (PIP 2 ) to phosphatidylinositol triphosphate (PIP 3 ) (52). PIP 3 then recruits AKT/protein kinase B (AKT/PKB) and 3-phosphoinositide-dependent 9 kinase-1 (PDK-1) to the cell surface where PDK-1 activates AKT/PKB. The GTPase activating protein AKT substrate of 160 kDa (AS160) is one of the AKT/PKB targets responsible for GLUT-4 translocation and glucose uptake (52). Also, AKT/PKB directly phosphorylates GSK3, allowing for insulin-stimulated glycogen deposition. AKT/PKB also phosphorylates forkhead box 01 (FOX01), sequestering it in the cytoplasm, which inhibits gluconeogenesis (52). To coordinate insulin-stimulated mitogenic activities, the SH2 domain of the adaptor molecule, Grb2, binds to IRS proteins. Grb2 then associates with son-of-sevenless (SOS) to activate the Ras-Raf-Mek-MAPK pathway, which relays the message from the membrane to the nucleus (52). 10 INTRACELLULAR, INSULIN-DEPENDENT SIGNALING PATHWAYS FIG. 1. Metabolic and mitogenic responses to insulin. In the presence of insulin, the IR becomes phosphorylated, and IRS proteins are recruited to the IR to mediate the PI3-K and MAPK pathways. Fetuin-A is shown here inhibiting the IR-TK activity, thus repressing insulin-mediated events. 11 Insulin resistance Insulin resistance is characterized by an attenuated ability of peripheral tissue to respond to circulating insulin. While the underlying cellular mechanisms of insulin resistance have not been comprehensively characterized, multiple factors have been implicated in its manifestation. The first link between obesity-induced inflammation and insulin resistance was reported by Hotamisligil and co-workers (7). Their findings illustrated an increase in TNF-? mRNA in adipose tissue of different rodent models of diabetes and obesity, and that neutralization of TNF-? in these animals significantly increased peripheral glucose uptake in response to insulin. Corresponding human studies showed increased TNF-? expression in adipose tissue of obese individuals, with decreased expression following weight loss (53). As a mechanism to explain inflammation-induced insulin resistance, TNF-? has been shown to activate JNK and I?K kinase ? (IKK?), which have both been demonstrated to phosphorylate serine residues on IRS-1, attenuating insulin action (54-56). Insulin-dependent tyrosine phosphorylation of IRS proteins is a critical step in insulin action, as these proteins connect IR activation to essential downstream kinase events leading to glucose uptake and gene expression. In the event where IRS proteins are absent in the liver of rodents, a systemic insulin resistant, glucose intolerant phenotype results, confirming the indispensability of these proteins (57). Further, in animal models of diet-induced obesity (DIO), JNK activation and IRS serine phosphorylation has been shown to be upregulated (58; 59). Drugs that act to sensitize insulin action have been shown to increase IRS-1 tyrosine phosphorylation (60), and endogenous insulin sensitizers, such as adiponectin, have been demonstrated to 12 reduce serine phosphorylation, and concomitantly increase tyrosine phosphorylation of IRS-1, increasing insulin-dependent downstream cellular events (61). These findings illustrate the importance of IR activation and its subsequent communication with post- receptor signaling molecules. Negative modulators of insulin action A small number of insulin resistance cases have demonstrated mutations of the IR gene, suggesting insulin resistance occurs at the level of the receptor. Conversely, in the vast majority of patients with insulin resistance, the IR gene has been found to be normal, indicating a post-receptor phenomenon (62). However, several molecules have recently been identified that act at the level of the receptor, attenuating insulin action. PTP-1B acts as a negative regulator of insulin action by dephosphorylating tyrosine residues on the IR. Furthermore, PTP-1B expression in muscle and adipose tissue is increased in obese humans and rodent models, and overexpression of PTP-1B results in severe insulin resistance and decreased glucose uptake (63; 64). Other proteins, such as the suppressors of cytokine signaling (SOCS-1 and -3), mitigate insulin signaling by binding to essential kinase domains of the IR, thereby inhibiting the IR/IRS interaction (65; 66). This is of particular interest since SOCS-1 and -3 have been found to be increased in different rodent models of obesity and insulin resistance (67), and knockdown of these proteins demonstrate lower glucose levels and increased insulin sensitivity (68). Another protein responsible for contributing to insulin resistance by interacting with the IR is plasma-cell- membrane glycoprotein-1 (PC-1). PC-1 is an ectoenzyme present in the cell membrane that exhibits pyrophosphatase and phosphodiesterase activities (69). Maddux and co- 13 workers have demonstrated that PC-1 directly interacts with the ?-subunit of the IR, and overexpression of PC-1 is sufficient to induce insulin resistance and hyperglycemia (70; 71). Further, PC-1 has been shown to be elevated in insulin-resistant tissues and fibroblasts (72; 73). PC-1 does not block insulin binding to the IR, rather it abates the IR?s insulin-induced conformational change that leads to IR-TK activity and autophosphorylation (70; 74). Fetuin-A: Introduction Fetuin-A (also known as ?2-HS-glycoprotein, AHSG), a 63 kD phosphorylated glycoprotein, synthesized and secreted primarily by the liver and belonging to the cystatin family of protease inhibitors, is a natural inhibitor of IR-TK activity (23). Fetuin- A preferentially interacts with the activated insulin receptor, repressing insulin-induced IR autophosphorylation and subsequent downstream signaling activity in vitro, in intact cells, and in vivo (28). Mice lacking fetuin-A demonstrate improved insulin sensitivity, whole-body glucose utilization, and resistance to weight gain when fed a high fat diet (29). Additionally, these mice are protected against aging-induced obesity and insulin resistance (30). Interestingly, the gene for fetuin-A is localized on chromosome 3q27, a locus that has been shown to be associated with type 2 diabetes and cardiometabolic risk (31). While a polymorphism of the AHSG gene has been shown to be associated with type 2 diabetes, another polymorphism is associated with leanness in Swedish population (32). A growing body of human investigation studies point to a strong association of plasma fetuin-A levels with insulin resistance (33-35), an atherogenic lipid profile (34), and increased BMI and plasma triglyceride levels (36). Recently, a second member of the 14 fetuin family has been described, which designated the previously mentioned fetuin (AHSG, fetuin, pp63) as fetuin-A (75). Fetuin-B demonstrates domain homology, conservation of cysteine resisdues, and chromosomal assignments with fetuin-A; however, fetuin-A and ?B exhibit significant differences in amino acid sequence, protein structure, and gene expression, suggesting discrete functions for the proteins (75). Structure Human ?2-HS-glycoprotein (AHSG), named after the discoverers Heremans, Schmid, and B?rgi (76; 77), is the species homologue of fetuin-A, first reported by Pederson as the most abundant protein in fetal bovine serum (78). Fetuin-A belongs to the class of cysteine protease inhibitors of the cystatin superfamily (79), which encompasses proteins that exhibit several cystatin-like domains. Fetuin-A is composed of two chains, a large A-chain and a smaller B-chain, that are linked by a short connecting peptide and an interchain disulfide bridge. It has been shown that the two chains are encoded by a single mRNA transcript (80), and the connecting peptide is partially or completely cleaved before the protein is released into circulation (81). Characterization of the A-chain revealed that it consists of 282 amino acid residues, while exhibiting 29% ?- helix, 24% ?-pleated sheet, and 26% reverse turns (82). Four glycosylation sites were also identified; two N-glycosylation sites were observed on 138 Asn and 158 Asn, while two O-glycans were identified on 238 Thr and 252 Thr. The B-chain also has been characterized, consisting of 27 amino acid residues and a trisaccharide consisting of sialic acid, galactose, and N-acetylgalactosamine O-glycosidically linked to 328 Ser (83). The 40 amino acid connecting peptide joins the A-chain at 282 Leu and the B-chain at 323 Thr. 15 Some controversy exists as to whether the connecting peptide is posttransitionally cleaved by limited proteolysis (83; 84), or if the A-chain retains 39 of the 40 amino acids of the connecting peptide, resulting in a single-chain circulating protein (81; 85). Fetuin-A exists in both phosphorylated and dephosphorylated forms in human plasma. Haglund et al, have demonstrated that fetuin-A is phosphorylated on two serine residues, 120 Ser and 312 Ser, two of the phosphorylation sites suggested by Jahnen-Dechent et al. (85), and that approximately 20% of circulating fetuin-A is phosphorylated (38). Phosphorylation of fetuin-A has been shown to be critical for its IR tyrosine kinase inhibitory activity (23). While 120 Ser has been shown to be constitutively phosphorylated, 312 Ser seems to exhibit regulatory covalent modifications, suggesting this site may be critical for its inhibitory activity (38). Furthermore, these two phosphorylation sites are conserved in human, mouse, rat, sheep, pig, and cow (82; 83; 86; 23; 87; 25; 88) (Fig. 2.). The disulfide loops present in fetuin-A and its similarity with cystatins has been elucidated by Kellerman et al. (81). They report the presence of 12 half-cysteine residues on the molecule, and that 11 of them are present on the large A-chain while only one resides on the smaller B-chain, forming six disulfide bridges. The first and last half- cysteine residues engage to form a disulfide loop, connecting the A- and B-chains at 14 Cys and 140 Cys. The other five disulfide loops are found on the A-chain, which exhibit a similar disulfide loop arrangement found in those of the cystatin family, hence fetuin-A?s classification in the cystatin superfamily. From the amino- to the carboxyl-terminus, fetuin-A consists of three domains. Domains 1 and 2 (D1-2), located on the A-chain, are 16 cystatin-like domains, whereas domain 3 (D3) is of a hydrophobic nature, composed of a proline-rich region and the connecting peptide (81) (Fig. 3). In an effort to determine the conserved regions of fetuin-A, the cDNA?s encoding human (82; 83), rat (23; 86), mouse (87), cow (25), sheep, and pig (88) fetuin have been cloned and sequenced, and comparisons between these species reveals strong similarity (89). Dziegielewska and Brown have shown that human fetuin-A shares 64, 65, 64, 56, and 59% sequence homology with sheep, pig, cow, rat, and mouse fetuin, respectively. Of particular interest is the conservation of the Asn-linked N-glycans on the A-chain, and 120 Ser and 312 Ser of the A-chain and connecting peptide, respectively, which, as mentioned previously, have been reported to be phosphorylated (38; 85). The conservation of these posttranslationally modified sites may serve as sites of regulation of fetuin-A?s functions. 17 CONSERVED SERINE RESIDUES ON FETUIN-A FIG. 2. Conserved serine residues. Fetuin is phosphorylated on 120 Ser and 312 Ser, and the sequence alignment of rat, mouse, and human are shown. Conserved 120 Ser and 312 Ser residues are indicated by arrows. The underlined sequence denotes the peptide from which phospho- 312 Ser-fetuin-A antibody was generated. 18 PROTEIN STRUCTURE OF HUMAN AHSG/FETUIN-A FIG. 3. Fetuin Protein structure (81; 85). The A-chain, comprising the signal peptide (SP), domains A and B, a proline rich segment (Pro) and 39 of the 40 amino acids of the connecting peptide (CP), is linked to the B-chain, or light chain, by a single disulfide bond. The conserved N- and O-linked glycosylation and phosphorylation sites are represented by N, O, and P, respectively. 19 IR-TK inhibition Fetuin-A was first identified as a 63-kD phosphoprotein (PP 63 ), secreted from isolated rat hepatocytes (22). The same group later characterized PP 63 as a natural inhibitor of the IR-TK, and showed that only the phosphorylated protein was active as an IR-TK inhibitor (23). Subsequently, a search for related protein sequences revealed that human AHSG and bovine fetuin shared 56 and 60% amino acid sequence identity, respectively, to that of rat PP 63 , indicating its classification as a mammalian fetuin (24). Srinivas et al. (90) demonstrated that AHSG is specific for the tyrosine kinase activity of the IR, inhibiting insulin-induced autophosphorylation and tyrosine kinase activity in vitro and in vivo. This group further demonstrated that AHSG inhibits insulin-stimulated IRS-1 tyrosine phosphorylation and its subsequent association with PI3-K. Inhibition of insulin-mediated mitogenesis in rat hepatoma cells with ASHG was also demonstrated. To assess whether AHSG competes with insulin for binding sites on the ?-subunit of the insulin receptor, Srinivas et al, performed competitive binding studies using 125 I-labeled insulin in the presence or absence of AHSG. AHSG was found not to alter specific insulin binding to the IR (90). Further characterization of AHSG?s inhibitory effects has been elucidated. Srinivas et al. (91), using recombinant AHSG, illustrated that insulin- stimulated GRB2 association with IRS-1 was blunted. Furthermore, the insulin- dependent GRB2/Sos complex, which docks to IRS-1, was found to be inhibited by more than 75% in the presence of AHSG. AHSG also inhibited the insulin-dependent tyrosine phosphorylation of Shc and the Ras-GTP formation. Consequently, phosphorylation of the downstream targets Raf and MEK were also inhibited in the presence of ASHG. However, AHSG failed to blunt the metabolic effects of insulin, i.e., glucose uptake and 20 amino acid transport (91). Similar reports of the inhibitory activity of human recombinant AHSG have been illustrated (92). Kalabay et al. expressed ASHG in insect cells using the baculovirus system and showed that the purified protein had the connecting peptide intact. The connecting peptide contains the 312 Ser residue reported to be phosphorylated, and phosphorylation has been shown to be critical for AHSG?s inhibitory effects. The paradox lies in the concept that AHSG inhibited PI3-K (90), but was without effect on glucose uptake (91). PI3-K has been shown to be critical in insulin-induced glucose uptake (93) and has been well characterized since then (20). One explanation was that AHSG?s inhibition of PI3-K was not complete and, therefore, remaining activity of PI3-K may have been sufficient to mediate glucose uptake. Another contributing factor could have been the degree of activity, i.e., phosphorylation of AHSG. AHSG?s effects on insulin-stimulated glucose uptake are currently under investigation and preliminary studies by Kim et al. demonstrate that phosphorylated AHSG inhibits insulin-stimulated glucose uptake in skeletal muscle cells and adipocytes (94). The homology of fetuins, human AHSG, and PP 63 have been reported, and similar inhibitory effects of PP 63 and human AHSG have been demonstrated (23; 90-92). Subsequently, bovine fetuin, like its homologs AHSG and PP 63 , was reported to act as a negative regulator of insulin action by inhibiting the IR-TK activity (95). Bovine fetuin completely blocks insulin-stimulated autophosphorylation in vitro and significantly blunted IR-TK activity in HIRcB cells. Additionally, bovine fetuin completely abrogated insulin-stimulated DNA synthesis in rat H-35 hepatoma cells. These studies demonstrate a common IR-TK inhibitory function for the fetuin homologs; however, the studies were carried out in cells overexpressing human IR?s, not in legitimate targets of insulin such as 21 adipocytes. Chen et al. showed, in a physiological perspective, that human AHSG, indeed, inhibits insulin-dependent mitogenic activity in rat adipose cells but not glucose uptake (96). Mathews et al. have demonstrated the time-course and specificity of inhibition, its interaction with IR, and described a probable physiological role for AHSG (28). They demonstrated that incubation of 1.8 ?M AHSG in HIRcB cells is sufficient to inhibit insulin-induced IR autophosphorylation by over 80%. This inhibitory effect was abated to half in 60 minutes and had no effect on insulin-like growth factor-1-induced cognate receptor (IGF-IR) autophosphorylation. To further characterize the specificity of AHSG, Mathews et al. treated HIRcB cell lysates, in the presence or absence of insulin, with increasing concentrations of AHSG. Immunoprecipitation with anti-AHSG antibody demonstrated that AHSG preferentially interacts with the activated insulin receptor; only in the highest concentration (4.0 ?M) did AHSG interact with the IR in basal conditions, while concentrations as low as 0.9 ?M AHSG interacted with the activated IR. This investigation also illustrated that AHSG interacts with ?-subunits of the IR, and insulin- dependent conformational change of the ?-subunits was not required for AHSG?s inhibitory actions. To demonstrate this, HIRc B cells were treated with trypsin, which results in proteolysis of the IR ?-subunit at 576 Arg- 577 Arg, making the IR-TK activity constitutively active (97). To confirm the insulin-binding site was removed by trypsin, 125 I-labled insulin was unable to bind to IRs from trypsin-treated cells, while bound to IRs from control cells. AHSG (0.1 ?M) completely inhibited both insulin-stimulated and trypsin-activated IR-TK activity. To corroborate these findings in vivo, 2 ?M/120 ?g bolus AHSG was injected into rats at the portal vein in the presence or absence of insulin. 22 ASHG inhibited insulin-stimulated IR autophosphorylation and IRS-1 tyrosine phosphorylation in intact rat liver and hind-limb muscle, further characterizing and adding physiological relevance to AHSG?s inhibitory activity (28). The human AHSG gene is localized on chromosome 3q27, a recently established type 2 diabetes susceptibility locus (98). Additionally, Kissebah et al. have demonstrated a quantitative trait locus on chromosome 3q27 strongly linked to the metabolic syndrome (31) Mice null for the Ahsg gene are fertile and exhibit no gross abnormalities with the exception of ectopic microcalcifications in a minority of females breeders (99). Mathews et al. explored the role of fetuin in insulin action in fetuin-deficient mice (29). Fetuin knockout (KO) mice demonstrated enhanced basal and insulin-stimulated IR autophosphorylation and increased MAPK and Akt phosphorylation. The KO mice also exhibited increased glucose tolerance and insulin sensitivity, and when challenged with a high-fat diet, remained insulin sensitive, exhibited decreased fat mass, and were resistant to weight gain. Additionally, the fetuin KO mice demonstrated protection from obesity and insulin resistance associated with aging (30). These findings illustrate a role for fetuin in regulating insulin sensitivity, weight gain, and fat accumulation and demonstrate a novel therapeutic target for combating the deleterious symptoms of type 2 diabetes, obesity, and insulin resistance. AHSG gene The cDNA encoding AHSG has been cloned and chromosomally mapped to 3q27 (80). Osawa et al. (100) have described the structure and organization of the AHSG gene by comparing human AHSG cDNA sequence (80), to the genetic sequence of rat PP 63 23 (101). A total of 8,748 bp were delineated from PCR fragments, and sequence analysis revealed the presence of seven exons and six introns, which spanned 8.2 kb. The ATG start codon was found on the first exon, while the TAG stop codon was found on the seventh exon at nucleotide positions 7787 and 7789, relative to the ATG start codon. Further analysis and comparisons indicated that the positions of the intron interruptions corresponded exactly with that of rat PP 63 (fetuin) gene as described by Falquerho et al (101). These findings, along with conserved protein sequences, demonstrate the identity of human AHSG and rat fetuin. Osawa et al. further demonstrate the presence of the CCAAT enhancer binding protein (C/EBP)-binding sequence (TTATGCAAT) and hepatocyte nuclear factor (HNF)-binding sequence (TGTTTGC) on the promoter. The C/EBP family of transcription factors is involved in metabolism and differentiation of cells, especially in liver and adipose tissue (102), while HNF is involved in the transcription of various liver specific genes (103). Furthermore, C/EBP and hepatocyte nuclear factor 3? (HNF-3?) have been demonstrated to mediate dexamethasone-induced upregulation of the AHSG gene (104). Treatment with dexamethasone to primary rat hepatocytes increased C/EBP protein levels and transcription of AHSG. Accordingly, it was demonstrated that C/EBP and HNF-3? bind to the steroid response unit (SRU), which is highly conserved in rat, mouse, and human within the promoter, resulting in enhanced transcription of AHSG. Dexamethasone has been well characterized as an insulin antagonist, opposing the effects of insulin (105). The study by Woltje et al. suggests that dexamethasone-mediated upregulation of AHSG via C/EBP and HNF-3?, may contribute to dexamethasone-induced insulin resistance. In addition, regulation of the AHSG gene in response to high fat (HF) and low fat (LF) diets has been reported in 24 rats (106). Osborne-Mendel (OM) and S5B/PI rats differ in their susceptibility to obesity when challenged with a HF diet. S5B/PI rats remain lean, while OM rats exhibit a much higher propensity to weight gain. Further analyses demonstrate that the S5B/PI rats exhibit an impaired expression of AHSG, further implicating AHSG in the involvement of insulin resistance and fat accumulation. Human Studies Polymorphisms of human AHSG have been reported (107; 108). One study reports differences in six nucleotide arrangements at amino acid positions: 84,107, 172, 230, 238, and 252 of AHSG cDNA obtained by RT-PCR (107). Positions 107, 230, and 238 give rise to amino acid substitutions, while 84, 172, and 252 do not. Interestingly, amino acid positions 230 Thr and 238 Thr harbor two of the O-linked glycosylates (See Fig. 2). A single nucleotide polymorphism (SNP) in the 5? region has been identified and associated with insulin-mediated inhibition of lipolysis and stimulation of lipogenesis, as well as two additional SNPs being associated with circulating cholesterol (108), providing more evidence for AHSG/fetuin-A?s role in fat accumulation. Further, in a Swedish population, substitutions at Thr230Met and Thr238Ser, two of the variations described by Osawa et al. (107), are associated with decreased AHSG protein levels, which correspond to an increased propensity for leanness among men (32). These results are in concert with findings from the Ahsg KO mice, which report resistance to weight gain in Ahsg null mice when fed a high fat diet (see reference 29). Other investigations report AHSG SNPs associated with type 2 diabetes in French Caucasians and increased adipocyte ?2-adrenoceptor function (109; 110), further demonstrating AHSG/fetuin-A?s role in regulating body mass and adiposity. 25 Several reports demonstrate decreased levels of AHSG are associated with leanness and resistance to weight gain. Other studies reciprocate these findings by showing increased AHSG levels positively correlate with body mass and insulin resistance. Gestational diabetes is defined as carbohydrate intolerance that is first recognized during pregnancy. Kalabay et al., show that gestational diabetes patients have significantly higher levels of fetuin-A than do healthy pregnant women and non-pregnant controls (35). Data from the Heart and Soul Study indicate that fetuin-A levels are positively associated with the metabolic syndrome as well as an atherogenic lipid profile (34). Mori et al. have compared fetuin-A levels between patients with type 2 diabetes and non-diabetic controls (36). There were no significant differences in fetuin-A levels between non-diabetic and type 2 diabetic groups; however, fetuin-A levels were highly associated with HOMA, BMI, and triglyceride levels in the non-diabetic group. Additionally, fetuin-A levels were shown to be associated with insulin resistance and a fatty liver in individuals without diabetes (33). These human reports imply a significant role of fetuin-A in regulating body mass, fat accumulation, and insulin resistance. However, these studies have only examined total fetuin-A levels. Since phosphorylation of fetuin-A has been has been shown to be a critical regulator of its IR-TK inhibitory activity, assessment of fetuin-A phosphorylation in conditions of diabetes, obesity, and insulin resistance can significantly contribute to our understanding of the role of AHSG in these conditions. Objectives and hypotheses The objective of the present study was to elucidate the phosphorylation status of fetuin-A in animal models of diabetes, obesity, and insulin resistance. Fetuin-A is a 26 physiological inhibitor of the IR-TK, and phosphorylation has been shown to be critical for its inhibitory activity. Human studies demonstrate that fetuin-A is positively correlated with BMI, insulin resistance, fatty liver, and an atherogenic blood profile; however, no studies have examined fetuin-A phosphorylation in obese, diabetic, or insulin resistant conditions. The 120 Ser and 312 Ser phosphorylation sites on fetuin-A are conserved in human, mouse, rat, pig, sheep, and cow, which suggests a regulatory role at these sites. The majority of the phosphorylation has been shown to occur at 312 Ser. Accordingly, we have generated an anti-phospho- 312 Ser-fetuin-A antibody in order to assay fetuin-A phosphorylation in animal models of obesity, diabetes, and insulin resistance. The animal models in the present study are well characterized and widely used in scientific investigations. We hypothesize that in conditions of extreme insulin resistance or frank diabetes animals will exhibit increased phospho-fetuin-A levels. Additionally, in conditions of slightly elevated diabetic parameters, i.e., insulin, leptin, and glucose levels, animals will exhibit decreased phospho-fetuin-A levels as a compensatory mechanism to allow for increased insulin action. 27 CHAPTER 3: PHOSPHORYLATION OF FETUIN-A, A PHYSIOLOGICAL INHIBITOR OF INSULIN ACTION, REGULATED BY INSULIN AND LEPTIN ABSTRACT Fetuin-A, a liver secreted phosphoprotein and rat homologue of human 2-HS- glycoprotein (AHSG), is a physiological inhibitor of insulin action. Fetuin-A-null mice exhibit improved glucose tolerance and resistance to weight gain on a high-fat diet. Recent human data demonstrate a positive correlation of circulating fetuin-A levels with BMI, insulin resistance, and fatty liver. Since phosphorylation is critical for its inhibitory activity, circulating fetuin-A phosphorylation was assayed in animal models of insulin resistance, diabetes, and obesity. Using a phospho-specific ( 312 Ser) fetuin-A antibody, fetuin-A phosphorylation status was assayed in Zucker diabetic fatty (ZDF) rats, ob/ob and db/db mice, streptozotocin-treated rats, and in a rat model of diet-induced obesity (DIO). The leptin receptor-deficient, insulin resistant ZDF rats had significantly higher levels of phosphorylated fetuin-A compared to their lean counterparts. Likewise, the leptin-deficient, obese ob/ob mice had significantly higher levels of phosphorylated fetuin-A. Consistent with these results, central leptin administration to control and STZ- treated rats, as well as leptin treatment to human hepatoma cell lines, significantly decreased fetuin-A phosphorylation. To further characterize the regulation of fetuin-A phosphorylation, we demonstrate that insulin treatment downregulates phosphorylation in 28 Hep3B and HepG2 cell lines. Similarly, DIO rats that exhibit elevated levels of insulin, albeit not to a significant degree, demonstrate significantly decreased levels of fetuin-A phosphorylation. We demonstrate that insulin and leptin decrease phosphorylation of fetuin-A. Accordingly, in conditions of mild to moderate elevation of insulin (as in the DIO model), fetuin-A phosphorylation is decreased, mitigating the inhibitory effect of fetuin-A on IR and allowing for increased insulin signaling. In conditions of extreme insulin resistance (ZDF and ob/ob models), where insulin is unable to exert its actions on insulin receptors, fetuin-A phosphorylation is increased, potentially exacerbating the diabetic phenotype. These findings suggest that fetuin-A may play a significant role in the regulation of insulin action. 29 INTRODUCTION Insulin resistance is characterized by an attenuated ability of peripheral tissue to respond to circulating insulin. Individuals with insulin resistance have a higher propensity for developing type 2 diabetes. Of the reported 20 million people who suffer from diabetes in the U.S., 90-95% is affected by type 2 diabetes; ergo, enhancing insulin action in this population is of paramount importance. Though the underlying cellular mechanisms leading to insulin resistance have not been clearly delineated; several physiological regulators of insulin action, including free fatty acids (4), TNF? (7), leptin (111), adiponectin (112), resistin (113), and RBP4 (45), have been implicated in modulating insulin sensitivity. Some of these molecules have been demonstrated to inhibit insulin action at the level of the insulin receptor (IR), thus decreasing downstream, insulin-dependent signaling events. Fetuin-A (also known as ?2-HS-glycoprotein, AHSG), a 63 kD phosphorylated glycoprotein, synthesized and secreted primarily by the liver and belonging to the cystatin family of protease inhibitors, is a natural inhibitor of IR tyrosine kinase activity (21). Fetuin-A preferentially interacts with the activated insulin receptor, repressing insulin-induced IR autophosphorylation and subsequent downstream signaling activity in vitro, in intact cells and in vivo (28). Mice lacking fetuin-A demonstrate improved insulin sensitivity, whole-body glucose utilization, and resistance to weight gain when fed a high fat diet (29). Additionally, these mice are protected against aging-induced obesity and insulin resistance (30). Interestingly, the gene for fetuin-A is localized on chromosome 30 3q27, a locus that has been shown to be associated with type 2 diabetes and cardiometabolic risk (31). While a polymorphism of the AHSG gene has been shown to be associated with type 2 diabetes, another polymorphism is associated with leanness in Swedish population (32). Increased plasma fetuin-A levels have been reported in insulin resistance in humans. Further, a growing body of human investigation studies point to a strong association of plasma fetuin-A levels with insulin resistance (33-35), an atherogenic lipid profile (34), increased BMI and plasma triglyceride levels (36). Plasma concentrations of fetuin-A in humans range from 200-600 ?g/ml, with an average plasma concentration of ~ 300 ?g/ml in healthy adults (37). Fetuin-A exists in both phosphorylated and dephosphorylated forms in human plasma, and studies by Haglund et al, have demonstrated that fetuin-A is phosphorylated on two serine residues, 120 Ser and 312 Ser, and that approximately 20% of circulating fetuin-A is phosphorylated (38). Phosphorylation of fetuin-A has been shown to be critical for its IR tyrosine kinase inhibitory activity (22). While 120 Ser has been shown to be constitutively phosphorylated, 312 Ser seems to exhibit regulatory covalent modifications, suggesting this site may be critical for its inhibitory activity (38). While previous human studies have investigated the associations of fetuin-A in disease conditions, there are no reports examining its phosphorylation status. Therefore, the aim of this study is to elucidate the phosphorylation status of fetuin-A in obesity, insulin resistance, and diabetes. 31 MATERIALS AND METHODS Animals: Animal use and euthanasia protocols were reviewed and approved by Auburn University?s Institutional Animal Care and Use Committee. Five-week-old male Zucker diabetic fatty (fa/fa) and lean Zucker (fa/-) rats were purchased from Charles River Laboratories (Indianapolis, IN). Rats were housed on a 12-hour light/dark cycle and fed a standard chow diet ad libitum with free access to water. Blood samples were collected from the saphenous vein bimonthly. At the time of sacrifice, a section of the liver was rapidly removed and stored in RNA later for gene expression analysis. Male Wistar rats were purchased from Charles River Laboratories and were treated with or without streptozotocin (30 mg/kg intravenously via the tail vein) and/or central leptin (5 ?g ICV/day/10 days), resulting in four groups: Control, STZ-treated, leptin-treated, and STZ-leptin-treated. Pooled plasma samples from ob/ob and db/db animals were obtained from Jackson Laboratories (Bar Harbor, ME). For the high-fat fed rodent model, we obtained frozen liver tissues and plasma samples from our collaborator, Dr. Catherine Jen, Professor and Head, Department of Nutrition and Food Science at Wayne State University, MI. For this model, Wistar rats were fed a 40% high-fat diet or standard rat chow for 6 weeks. Antibodies: Rabbit polyclonal phospho-specific anti- 321 Ser-Fetuin-A antibody was generated using the following peptide: HTFMGVVSLGSPS(PO 4 )GEVSHPR. Antibody- containing serum was affinity purified using the above peptide (Affinity BioReagents, Golden, CO). Pan-fetuin-A antibody was purchased from INCSTAR (Stillwater, MN). 32 Metabolic studies: Blood glucose levels were determined bimonthly using an Accu- Chek glucometer (Roche Diagnostics). Insulin and leptin levels were measured by ELISA using rat insulin or leptin as a standard (Linco Research Inc.). Cell culture: Hep-3B and Hep-G2 cells were cultured in improved modified Eagle?s medium (IMEM) supplemented with 10% fetal bovine serum. Cells were grown to confluence then starved overnight in a serum-free 0.1% BSA IMEM. The cells were then washed with PBS, treated for 24 hours with insulin (0.01 and 1.0 ?M) (Roche Diagnostics) or leptin (1.0 and 100 nM) (R&D Systems, Minneapolis, MN), and maintained in serum-free media. Cells were then washed and treated again with insulin or leptin. Media was collected 6 hours later for SDS-PAGE and Western analysis. SDS-PAGE and Western analysis: Plasma from whole blood samples was diluted in sterile saline to 1:100 and was quantified using the Bradford protein assay. Normalized samples were boiled in SDS-buffer and separated on 4-20% SDS-PAGE, transferred to nitrocellulose membrane, and developed with antibodies against human fetuin-A (INCSTAR) and 312 Ser-Fetuin-A (Affinity BioReagents). Media samples from cell culture were similarly assayed. Quantitative RT-PCR analysis: Total RNA was isolated from rat liver tissue using an RNeasy kit (QIAGEN). cDNA was prepared from 1 ?g of RNA using the iQ TM SYBR Green Supermix (BioRad). Quantitative real-time PCR was performed on an iCycler real- time PCR detection system (BioRad), using the following primers: Fetuin forward 5?ACGTGGTCCACACTGTCAAA?3, reverse 5?CGCAGCTATCACAAACTCCA?3 (Invitrogen). Expression level were standardized to ?-actin by calculating ?Ct (?Ct = Gene Ct - ?-actin Ct). Gene expression was determined using the ??Ct method (114). 33 Partial purification of IR autophosphorylation and IR-TK activity: IR?s were partially purified from whole liver tissue of high-fat fed and chow-fed rats using wheat germ agglutinin (WGA)-agarose columns and eluted with 0.3 mol/l N-acetylglucosamine. Total protein was normalized to 1 ?g/ml using the Bradford protein assay. IR autophosphorylation was carried in the presence or absence of insulin using [?- 32 P]ATP in combination with 5 mmol MnCl 2 , 50 ?mol/l ATP, 50 mmol/l HEPES, pH 7.6, and 0.1% Triton X-100. IR-TK activity was assayed by quantitation of phosphorylation on exogenous poly(Glu 80 Tyr 20 ) (115). Statistical analyses: Data are presented as means ? SE. Statistical significance was determined using a one-way ANOVA and unpaired t tests where appropriate. Significance was declared for P values < 0.05. (GraphPad Instat). RESULTS: Phospho-specific anti- 312 Ser fetuin antibody: Phospho-specific antibody was tested for its ability to detect fetuin 312 Ser- phosphorylation. Fetuin was purified from HepG2 cell- media on a Jacalin column and was dephosphorylated by addition of calf intestinal alkaline phosphatase (CIAP) (Promega) in 10 mM Tris HCl, pH 8.0 for 2 h at 37? C. Samples, with and without treatment of CIAP, were assayed for phosphorylation using Western blotting. We show that the phospho-specific anti- 312 Ser fetuin antibody detects only phosphorylated fetuin, while the pan-fetuin antibody detects both phosphorylated and dephosphorylated fetuin (Fig. 4). Phospho-fetuin and total fetuin levels in rodent models of diabetes and obesity: Zucker Diabetic Fatty Rats (ZDF): 34 Male ZDF rats, having a defect in the long-form of the leptin receptor (OB-R), exhibit significantly higher body weights at ages 6 weeks through 12 weeks compared to lean controls; however, by week 14 there was no significant difference in body weights between the two groups (Fig. 5). Although at 6 weeks there was no apparent difference in blood glucose levels, levels began to escalate at 7 weeks and plateau at 10 weeks in the ZDF rats (Fig. 6). Conversely, plasma insulin levels were significantly elevated at 6 weeks, indicating insulin resistance, and began to decline at 9 weeks, suggesting ? cell failure (Fig. 7). Calculation of the homeostasis model assessment of insulin resistance (HOMA-IR) indicates that the ZDF rats were significantly more insulin resistant than their lean controls at week 6 (84.54 ? 25.4 vs. 3.84 ?0.9) (p = 0.01) and week 8 (247.56 ? 11.06 vs. 3.53 ? 1.12) (p < 0.001). After week 8, the ZDF rats began to exhibit ? cell failure indicated by declining insulin levels, suggesting a shift from insulin resistance to overt diabetes. This decline in circulating insulin would alter the HOMA calculation; therefore, insulin resistance data was not calculated for 9 through 14 weeks of age. Since previous reports have only assayed total circulating levels of fetuin in diabetic and insulin resistant conditions, it was of interest to ascertain the phosphorylation of fetuin-A in the ZDF rat, a genetic model of insulin resistance and progressive ?-cell failure, leading to diabetes. Using the phospho-fetuin anti- 312 Ser antibody, we found plasma phospho-fetuin levels to be significantly increased in 6-week old ZDF rats compared to lean controls (Fig. 8). However, by 14 weeks there were no appreciable differences in plasma phospho-fetuin between the groups (Fig. 9). Similarly, there were no significant differences in total plasma fetuin levels at 6 or 14 weeks (Fig. 10, 11), despite an escalating trend in total plasma fetuin in the ZDF rats by 14 weeks (Fig. 11). Since 35 phospho-fetuin levels in the 6-8 weeks old, insulin and leptin resistant ZDF rat were significantly elevated, it was of interest to examine phosphorylation status of fetuin in other animal models of obesity and diabetes. Obese ob/ob and diabetic db/db mouse models: The ob/ob and db/db mice have mutations in the leptin gene and the long-form of the leptin receptor (ObR), respectively (98; 116). Pooled plasma samples from 8 week old ob/ob mice and ob/+ littermate controls and db/db mice and db/+ littermate controls were purchased form Jackson Laboratories, ME. Both of these mice exhibit hyperinsulinemia (Fig. 12). The db/db mouse is hyperleptinemic while the ob/ob is devoid of circulating leptin due to mutations in the long form of the leptin receptor and the leptin gene, respectively (Fig. 13). Consistent with the ZDF rat, the ob/ob mouse demonstrated significantly increased phosphorylation of fetuin, compared with the ob/+, lean littermates. No significant changes in fetuin-A phosphorylation was observed in the db/db mice (Fig. 14). We did not observe any significant differences of total plasma fetuin levels between the groups (Fig. 15). Taking into account that both leptin-signaling impaired ZDF rats and leptin-deficient ob/ob mice demonstrated increased plasma phospho-fetuin levels, we postulated that leptin might play a role in the regulation of fetuin?s phosphorylation status. This hypothesis was tested in a streptozotocin (STZ) model of type 1 diabetes. Streptozotocin (STZ) type 1 diabetic rat model: STZ-treated male Wistar rats, a model for type 1 diabetes, were injected with leptin (5 ?g/day/10 days) icv to ascertain the effects of in vivo leptin treatment on plasma phospho-fetuin and total fetuin levels in type 1 diabetes. Glucose and insulin levels of 36 STZ-treated rats indicated that STZ-treatment induced a type 1 diabetic phenotype (Fig. 16, 17). In addition, central leptin administration to STZ-treated animals completely normalized blood glucose levels without altering insulin levels. STZ-treated animals exhibited decreased levels of phospho-fetuin, while leptin administration to STZ-treated animals seemed to further repress phosphorylation. Similarly, leptin administration to control animals significantly decreased fetuin phosphorylation (Fig. 18), consistent with the ob/ob and ZDF models. Total plasma fetuin levels were significantly elevated in the STZ animals compared to control and leptin-treated STZ animals. Likewise, total plasma fetuin levels were elevated in the leptin-treated control group, albeit not to a significant degree (Fig. 19). High fat-fed rat model: Rats fed a high fat diet exhibit hyperinsulinemia, hyperlipidemia, hyperleptinemia, and impaired insulin signaling and glucose metabolism (29). Therefore, our goal was to distinguish the differences in levels of phospho-fetuin and total plasma fetuin in animals fed a high fat or low fat diet. Rats on a 40% high-fat diet for six weeks demonstrated increased leptin levels and normal glucose levels but elevated insulin levels, suggesting insulin resistance as indicated by an elevated HOMA index (Table 1). Rats fed the high fat diet also demonstrated significantly decreased levels of phospho- fetuin while total plasma fetuin levels were unaltered compared to a low fat diet (Fig. 20, 21). Because phosphorylated fetuin inhibits IR-TK activity, we hypothesized that the high fat-fed rats, which exhibit decreased fetuin-A phosphorylation, would demonstrate no change in or increased IR-TK activity compared to chow-fed rats. Insulin receptors 37 from whole liver tissue were purified in wheat-germ agglutinin columns, and IR-TK activity was assayed. Extreme care was taken to preserve the integrity and phosphorylation status of the purified receptors by addition of protease and phosphatase inhibitors to all buffers and carrying out the entire purification procedure at 4?C. Similar studies have been conducted by other investigators to assay the phosphorylation status of insulin receptors (29; 115). While the results were not significant, the high fat-fed rats demonstrated a trend toward increased basal IR-TK activity and insulin-stimulated IR-TK activity compared to chow-fed rats (Fig. 22), indicating that in high-fat feeding a counter regulatory mechanism occurs, possibly in order to enhance insulin action. Effects of insulin- and leptin-treated Hep3B cells: The human hepatocellular carcinoma cell line, Hep3B, has been shown to express a functional leptin receptor (OB- R) (117) as well as synthesize and secrete fetuin in its phosphorylated form. In an attempt to corroborate our in vivo findings in intact cells, we treated Hep3B cells with different concentrations of leptin or insulin. Western blot analysis of fetuin secreted into the cell media indicated that leptin (1 and 100 nM) and insulin (0.01 and 1.0 ?M) considerably abated the amount of phospho-fetuin without any appreciable change in fetuin levels (Fig. 23). 38 DISCUSSION: A considerable amount of evidence has shown fetuin to act as an inhibitor of insulin receptor autophosphorylation and tyrosine kinase activity (90; 92). Phosphorylation of fetuin has been shown to be necessary for its inhibitory actions (23; 92). Recently, human fetuin was shown to be phosphorylated on 120 Ser and 312 Ser. These sites are conserved in human, pig, cow, sheep, rat, and mouse (82; 83; 86; 23; 87; 25; 88), suggesting these sites may be critical for fetuin?s physiological activity. Human data show fetuin levels are increased with elevated BMI, plasma insulin and triacylglycerides, insulin resistance, and metabolic syndrome (33-36); however, no data exist that evaluate phosphorylation status in these conditions. Accordingly, we have addressed whether fetuin phosphorylation is altered in animal models of obesity, insulin resistance, and diabetes. Here, we report that insulin and leptin downregulate fetuin phosphorylation in vivo and in intact cells. The ZDF rat is an animal model of impaired leptin signaling and extreme insulin resistance due to a mutation in the long form of the leptin receptor (118). In the ZDF rat, we observed an increase in plasma phospho-fetuin at 6 and 8 (data not shown) weeks of age. However, the magnitude of the increase in plasma phospho-fetuin at 8 weeks was not as great as at 6 weeks and was completely normalized by 10 weeks. The ZDF rats are overtly insulin resistant and demonstrate impaired leptin action, resulting in increased levels of plasma phospho-fetuin. At 6 weeks of age, the ZDF rat demonstrates euglycemia, similar to the high fat fed rat; however, these two rat models exhibit a 39 dichotomous relationship regarding fetuin phosphorylation status. The increased phospho-fetuin levels in the ZDF rat could be attributed solely to the impairment of leptin action. The ZDF rat also demonstrates an attenuating progression of phospho-fetuin and an increasing trend in total plasma fetuin levels as it ages. The ob/ob mouse, a model of obesity and severe insulin resistance due to a mutation in the leptin gene, similarly exhibited increased plasma phospho-fetuin levels. An important result here is that both the ZDF and ob/ob animals, exhibiting impaired and no leptin signaling, respectively, demonstrate elevated levels of plasma phospho-fetuin. However, we observed no increase in phospho-fetuin levels in db/db mice, which, like the ZDF rats, also have a mutation in the long form of the leptin receptor. Pooled plasma samples from ob/ob and db/db mice were collected at 8 weeks of age; therefore, a possible explanation is that phospho-fetuin levels may have already been normalized by 8 weeks of age in the db/db mice. Our studies demonstrate that fetuin phosphorylation status in ZDF rats, which was elevated in 6 weeks, began to return to control levels beginning at 9 weeks of age. At 10 weeks of age, ZDF rats demonstrate significantly elevated blood glucose levels (559.67 ? 25.04 vs. 128.67 ? 27.06) and a failure of the ?- cells to cope with rising glucose levels. Similarly, in the db/db model, it may be possible that fetuin phosphorylation status returned to control levels by 8 weeks of age. Since we observed an increase in plasma phospho-fetuin levels in leptin-impaired animals, we investigated the effects of central leptin administration on fetuin phosphorylation. STZ-treated rats exhibited increased total plasma fetuin levels and decreased fetuin gene expression (data not shown), whereas central leptin administration normalized blood glucose, total plasma fetuin levels, and gene expression in STZ-treated 40 rats. The decrease in total plasma fetuin levels, as seen in the STZ-leptin-treated rats, could be attributed to the normalization of blood glucose levels, i.e., hyperglycemia can lead to nonenzymatic glycation of intracellular and extracellular proteins resulting in advanced glycation end products (AGEs) (119). AGEs are compounds that have been implicated in many of the deleterious symptoms of diabetes, affecting protein turnover, transcriptional factors, and gene expression, to name a few (119). We speculate this putative mechanism could also be the reason why we observed an increasing trend in the levels of total plasma fetuin in the ZDF rat. Despite an increase in total plasma fetuin, STZ-treated rats demonstrated decreased phospho-fetuin levels (P = 0.08), and central leptin administration further repressed phospho-fetuin levels. Similarly, control animals treated with leptin exhibited decreased phospho-fetuin levels to a significant degree (P = 0.017). These results, combined with data from the ob/ob and ZDF animals, suggest that leptin decreases plasma phospho-fetuin. The high fat-fed rats exhibited modest elevation of leptin and insulin levels, yet maintained euglycemia, suggesting a trend toward insulin resistance. While the increased levels of insulin, leptin, and HOMA were not significant in the high fat fed rats, there was a significant decrease in plasma phospho-fetuin levels. We speculate that in conditions of euglycemia and slightly elevated levels of insulin and leptin, as in the high fat-fed model, a compensatory mechanism represses fetuin-A phosphorylation in order to enhance insulin action. Phosphorylated fetuin has been shown to be an IR-TK inhibitor, thus it would be logical to assume animals that demonstrate decreased phosphorylated fetuin would exhibit increased IR-TK activity. Accordingly, we show that the high fat-fed rats demonstrate a trend toward increasing both basal and insulin-stimulated IR-TK activity. 41 To further characterize insulin and leptin?s role in regulating fetuin phosphorylation, Hep3B cells were treated with insulin or leptin. Confirming our animal data, both insulin and leptin treatment resulted in downregulation of fetuin phosphorylation. In this study, we report, for the first time, that both levels and phosphorylation status are altered in animal models of obesity, insulin resistance, and diabetes. As observed in the high fat-fed model, conditions of slightly elevated insulin and leptin levels, as in mild insulin resistance or in the pre-diabetic stage, fetuin phosphorylation is decreased. This is further confirmed by our experiments in intact cells, where insulin and leptin repress fetuin phosphorylation. The decreased fetuin phosphorylation, as observed in the high fat-fed rats, may result as a compensatory mechanism in an attempt to enhance insulin signaling. However, with increased or extreme insulin resistance or leptin signaling impairment, as in the ZDF and ob/ob models, molecular mechanisms in the downregulation of fetuin phosphorylation is impaired, leading to significantly elevated levels of phosphorylated fetuin. 42 CONCLUSION Diabetes affects more than 20 million adults in the U.S., and Alabama leads the nation with over 440,000 cases, resulting in an estimated $3 billion dollars annually in health care costs in Alabama alone. Several elements have been implicated in the development of diabetes, including obesity and genetics, and many physiological modulators such as free fatty acids, TNF?, MCP-1, leptin, adiponectin, etc. have been shown to regulate insulin action. Fetuin-A, also known as human AHSG, has been shown to be a negative regulator of insulin action by acting as a physiological inhibitor of the IR-TK. Phosphorylation status of fetuin-A has been shown to be critical for its inhibitory activity. Human data demonstrate that circulating levels of total fetuin-A positively correlate with BMI, insulin resistance, fatty liver, and an atherogenic blood profile. Furthermore, the gene for fetuin-A is localized on chromosome 3q27, a locus that has been shown to be associated with the development of type 2 diabetes. While previous human studies have investigated the associations of fetuin-A in disease conditions, there are no reports examining its phosphorylation status. Therefore, the aim of this study was to elucidate the phosphorylation status of fetuin-A in obesity, insulin resistance, and diabetes. We show that high fat-fed animals, which exhibit euglycemia and slightly elevated levels of insulin and leptin, repress fetuin phosphorylation as a possible compensatory mechanism to enhance insulin action. Conversely, ZDF and ob/ob animals are leptin-signaling impaired and, as indicated by HOMA, are extremely insulin 43 resistance and therefore are unable to repress fetuin-A phosphorylation, which would possibly exacerbate the diabetic symptoms. Since phosphorylation of fetuin has been shown to be critical for its activity as an inhibitor of insulin action, our studies indicating that both levels and phosphorylation status of fetuin are altered in obesity, insulin resistance, and diabetes may constitute an important element in the pathogenesis of obesity, insulin resistance, and diabetes. That fetuin levels are correlated with the metabolic syndrome or increased cardiometabolic risk in humans suggests that phosphorylated fetuin can be an anti-diabetic drug target. Development of antibodies, vectors, antisense oligonucleotides, siRNA, shRNA or a small molecule targeting fetuin and/or its phosphorylation status; or identifying potential kinases, phosphatases or upstream regulators of fetuin phosphorylation, etc., may serve to ameliorate the deleterious symptoms of insulin resistance, obesity, diabetes and cardiovascular diseases. 44 FIG. 4. Specificity of phospho-fetuin ( 312 Ser) antibody: AHSG secreted into the media from human HepG2 hepatoma cells was purified, and eluted fractions were assayed by Western blotting for phospho-AHSG (upper panel) and AHSG (lower panel).Treatment with calf intestinal alkaline phosphatase (CIAP) dephosphorylated phospho-AHSG up to 90%. Anti- 312 Ser-AHSG antibody specifically detected the phosphorylated form of AHSG, while pan-AHSG antibody detected both phosphorylated and dephosphorylated AHSG. Dephosphorylated AHSG (CIAP-treated) P-AHSG AHSG Eluates: 1 2 1 2 45 6 7 8 9 10 11 12 13 14 15 50 150 250 350 * * * * * * * * ZDF Lean Age in weeks B ody w e i ght ( g ) FIG. 5. Body weights of ZDF and lean control rats. Zucker diabetic fatty rats and its lean controls were fed standard rat chow. Values shown are weekly mean body weights (g) ? SEM for each group (n=6). * p < 0.05. 46 6 7 8 9 10 11 12 13 14 15 50 150 250 350 450 550 * * ** ZDF Lean Age in weeks G l uc o s e (mg/ d L ) FIG. 6. Glucose levels in ZDF rats. Glucose levels were assayed with a handheld Accu- Chek glucometer (Roche Diagnostics) starting at 6 weeks of age through 15 weeks of age. Values shown are bimonthly mean glucose readings (mg/dL) ? SEM for each group (n=6). * p < 0.05 47 6 8 10 12 14 0 2 4 6 8 10 12 * *** *** *** *** Lean ZDF Age in weeks In s u l i n ( n g / m L ) FIG. 7. Insulin levels in ZDF rats. Insulin levels were assayed by ELISA (Linco Research Inc.) using rat standards as a control. Values shown are bimonthly mean insulin readings (ng/mL) ? SEM for each group (n=6). * p < 0.05; *** p < 0.001 48 Lean ZDF 0 5 10 15 *** P h os ph o - f e t u i n ( 6 w eeks) FIG. 8. Phosphorylation of fetuin in insulin resistant, obese ZDF rats (6 weeks). Plasma samples were taken and separated on 4-20% SDS-PAGE, transferred to nitrocellulose membrane, and developed with antibodies against human 312 Ser-Fetuin-A (Affinity BioReagents). Values shown are mean densitometric quantification units from Western blot analysis ? SEM for each group (n=6) at 6 weeks. *** p < 0.001 49 Lean ZDF 0 2 4 6 8 10 12 P h os ph o- f e t u i n ( 14 w eeks) FIG. 9. Phosphorylation of fetuin in insulin resistant, obese ZDF rats (14 weeks). Plasma samples were taken and separated on 4-20% SDS-PAGE, transferred to nitrocellulose membrane, and developed with antibodies against human 312 Ser-Fetuin-A (Affinity BioReagents).Values shown are mean densitometric quantification units from Western blot analysis ? SEM for each group (n=6) at 14 weeks. 50 Lean ZDF 0 3 6 9 12 Pl a s m a f e t u i n ( 6 w eeks) FIG. 10. Total plasma fetuin levels in insulin resistant, obese ZDF rats (6 weeks). Plasma samples were taken and separated on 4-20% SDS-PAGE, transferred to nitrocellulose membrane, and developed with antibodies against human fetuin-A (INCSTAR). Values shown are mean densitometric quantification units from Western blot analysis ? SEM for each group (n=6) at 6 weeks. 51 Lean ZDF 0 3 6 9 12 P l a s m a fe tu i n ( 14 w e e ks) FIG. 11. Total plasma fetuin levels in insulin resistant, obese ZDF rats (14 weeks). Plasma samples were taken and separated on 4-20% SDS-PAGE, transferred to nitrocellulose membrane, and developed with antibodies against human fetuin-A (INCSTAR). Values shown are mean densitometric quantification units from Western blot analysis ? SEM for each group (n=6) at 14 weeks. 52 ob/c ob/ob db/db db/c 0 5 10 15 20 I n s u l i n ( ng/ m L ) FIG. 12. Insulin levels in ob/ob and db/db mice. Insulin levels were assayed by ELISA (Linco Research Inc.) using mouse standards as a control. Values shown are insulin levels (ng/mL) assayed from pooled plasma samples. 53 ob/c ob/ob db/c db/db 0 5 10 30 40 50 Le p t i n ( n g/ m L ) FIG. 13. Plasma leptin levels in ob/ob and db/db mice. Leptin levels were assayed by ELISA (Linco Research Inc.) using mouse standards as a control. Values shown are leptin levels (ng/mL) assayed from pooled plasma samples. 54 ob/c ob/ob db/c db/db 0 10 20 30 40 50 *** Phos pho- f e t u i n FIG. 14. Phosphorylation of fetuin in obese, diabetic ob/ob and db/db mice. Plasma samples were taken and separated on 4-20% SDS-PAGE, transferred to nitrocellulose membrane, and developed with antibodies against human 312 Ser-Fetuin-A (Affinity BioReagents).Values shown are mean densitometric quantification units from Western blot analysis ? SEM from pooled plasma samples run in duplicates. *** p < 0.001. 55 FIG. 15. Total plasma fetuin levels in obese, diabetic ob/ob and db/db mice. Plasma samples were taken and separated on 4-20% SDS-PAGE, transferred to nitrocellulose membrane, and developed with antibodies against human fetuin-A (INCSTAR). Values shown are mean densitometric quantification units from Western blot analysis ? SEM from pooled plasma samples run in duplicates. ob/c ob/ob db/c db/db 0 5 10 15 20 Pl as m a f e t u i n 56 control STZ STZ + leptin Leptin 0 100 200 300 400 500 *** G l uco s e ( m g/ d L ) FIG. 16. Glucose levels in STZ-treated rats. Glucose levels were assayed with a handheld Accu-Chek glucometer (Roche Diagnostics). Values shown are mean glucose readings (mg/dL) ? SEM for each group (n=5). *** p < 0.001 57 control STZ STZ + leptin Leptin 0.0 0.5 1.0 1.5 *** In s u lin (n g / d L ) FIG. 17. Insulin levels in STZ-treated rats. Insulin levels were assayed by ELISA (Linco Research Inc.) using rat standards as a control. Values shown are mean insulin readings (ng/dL) ? SEM for each group (n=5). *** p < 0.001 58 Control STZ STZ+Leptin Leptin 0 3 6 9 12 15 p = 0.08 *** * # Control STZ STZ+Leptin Leptin pFetuin pho spho- f e t u i n [d e n s i to m e tri c u n i t s ] FIG. 18. Phosphorylation of fetuin in STZ-treated rats. Plasma samples were taken and separated on 4-20% SDS-PAGE, transferred to nitrocellulose membrane, and developed with antibodies against human 312 Ser-Fetuin-A (Affinity BioReagents). Values shown are mean densitometric quantification units from Western blot analysis ? SEM for each group (n=5). * p < 0.05 compared to control; # p < 0.05 compared to STZ+Leptin; *** p < 0.001 compared to control and STZ. 59 Control STZ STZ+Leptin Leptin 0 3 6 9 12 15 18 Control STZ STZ + Leptin Fetuin Leptin * # * Pl asm a f e t u i n FIG. 19. Total plasma fetuin levels in STZ-treated rats. Plasma samples were taken and separated on 4-20% SDS-PAGE, transferred to nitrocellulose membrane, and developed with antibodies against human fetuin-A (INCSTAR). Values shown are mean densitometric quantification units from Western blot analysis ? SEM for each group (n=5). * p < 0.05 compared to control and STZ+Leptin; # p < 0.05 compared to Leptin. 60 TABLE 1 Metabolic indices in high fat-fed and chow-fed rats Glucose Insulin HOMA Leptin n (mg/dL) (ng/mL) (ng/mL) Chow 5 139.2 ? 30.7 1.22 ? 0.09 9.5 ? 1.0 1.4 ? 0.9 High fat 5 154.0 ? 11.3 1.73 ? 0.27 16.2 ? 3.8 2.2 ? 1.0 Values shown are means ? SEM. Glucose levels were assessed with a glucometer. Insulin and leptin levels were determined using ELISA. HOMA, an index of insulin resistance, was calculated as: [glucose (mmol/L) x insulin (?U/mL)/22.5]. 61 Low fat High fat 0 2 4 6 8 10 12 14 p = 0.018 n = 15 n = 15 P hos pho- f e t u i n FIG. 20. Phosphorylation of fetuin in high fat-fed rats. Plasma samples were taken and separated on 4-20% SDS-PAGE, transferred to nitrocellulose membrane, and developed with antibodies against human 312 Ser-Fetuin-A (Affinity BioReagents). Values shown are mean densitometric quantification units from Western blot analysis ? SEM for each group (n=5). 62 Low fat High fat 0 2 4 6 8 10 12 n = 15 n = 15 P l a s m a fe tu in FIG. 21. Total plasma fetuin levels in high fat-fed rats. Plasma samples were taken and separated on 4-20% SDS-PAGE, transferred to nitrocellulose membrane, and developed with antibodies against human fetuin-A (INCSTAR). Values shown are mean densitometric quantification units from Western blot analysis ? SEM for each group (n=5). 63 Basal Insulin Basal Insulin 0 1000 2000 3000 4000 5000 Chow High Fat * * I R -T K a c t iv it y (c p m ) FIG. 22. Insulin receptor tyrosine kinase activity in high fat-fed rats. IR?s were partially purified from whole liver tissue of high-fat fed and chow-fed rats using wheat germ agglutinin (WGA)-agarose columns. IR autophosphorylation was carried in the presence or absence of insulin using [?- 32 P]ATP in combination with 5 mmol MnCl 2 , 50 ?mol/l ATP, 50 mmol/l HEPES, pH 7.6, and 0.1% Triton X-100. IR-TK activity was assayed by quantitation of phosphorylation on exogenous poly(Glu 80 Tyr 20 ). Values shown are the mean (blank) ? SEM in each group (n=6). * p < 0.05. 64 C ont r o l I n s 10 n M M ? In s 1 C ont r o l Le p 1 nM Le p 1 0 0 nM 0 10 20 30 40 Phospho-fetuin Fetuin Insulin - .01 1.0 - - - (?M) Leptin - - - - 1.0 100 (nM) P h os pho - f e t u i n FIG. 23. Regulation of fetuin phosphorylation in Hep-3B human hepatoma cells. Hep-3B cells were grown to confluence and starved overnight. Cells incubated in the appropriate concentrations of insulin or leptin for 24 hours in serum free media. Cells were then washed, and fresh media with or without insulin or leptin was reapplied. Media was collected 6 hours later and assayed for fetuin and phospho-fetuin. 65 BIBLIOGRAPHY 1. National and state medical expenditures and lost earnings attributable to arthritis and other rheumatic conditions--United States, 2003. MMWR Morb Mortal Wkly Rep 56:4-7, 2007 2. Calle EE, Kaaks R: Overweight, obesity and cancer: epidemiological evidence and proposed mechanisms. Nat Rev Cancer 4:579-591, 2004 3. Wellen KE, Hotamisligil GS: Inflammation, stress, and diabetes. J Clin Invest 115:1111-1119, 2005 4. Boden G, Shulman GI: Free fatty acids in obesity and type 2 diabetes: defining their role in the development of insulin resistance and beta-cell dysfunction. Eur J Clin Invest 32:14-23, 2002 5. Yamauchi T, Kamon J, Waki H, Terauchi Y, Kubota N, Hara K, Mori Y, Ide T, Murakami K, Tsuboyama-Kasaoka N, Ezaki O, Akanuma Y, Gavrilova O, Vinson C, Reitman ML, Kagechika H, Shudo K, Yoda M, Nakano Y, Tobe K, Nagai R, Kimura S, Tomita M, Froguel P, Kadowaki T: The fat-derived hormone adiponectin reverses insulin resistance associated with both lipoatrophy and obesity. Nat Med 7:941-946, 2001 6. Dandona P, Aljada A, Bandyopadhyay A: Inflammation: the link between insulin resistance, obesity and diabetes. Trends Immunol 25:4-7, 2004 7. Hotamisligil GS, Shargill NS, Spiegelman BM: Adipose expression of tumor necrosis factor-alpha: direct role in obesity-linked insulin resistance. Science 259:87-91, 1993 8. Hotamisligil GS, Spiegelman BM: Tumor necrosis factor alpha: a key component of the obesity-diabetes link. Diabetes 43:1271-1278, 1994 9. Sartipy P, Loskutoff DJ: Monocyte chemoattractant protein 1 in obesity and insulin resistance. Proc Natl Acad Sci U S A 100:7265-7270, 2003 10. Taylor SI Accili D, Imai Y: Insulin resistance or insulin deficiency: which is the primary cause of NIDDM? Diabetes 43:735-740, 1994 11. Polonsky KS, Sturis J, Bell GI: Non-insulin-dependent diabetes mellitus-A genetically programmed failure of the beta cell to compensate for insulin resistance. N Engl J Med 334:777-783, 1996. 12. White MF: The insulin signalling system and the IRS proteins. Diabetologia 40 Suppl 2:S2-17, 1997 13. Ottensmeyer FP, Beniac DR, Luo RZ, Yip CC: Mechanism of transmembrane signaling: insulin binding and the insulin receptor. Biochemistry 39:12103-12112, 2000 14. Avruch J: Insulin signal transduction through protein kinase cascades. Mol Cell Biochem 182:31-48, 1998 15. Fruebis J, Tsao TS, Javorschi S, Ebbets-Reed D, Erickson MR, Yen FT, Bihain BE, Lodish HF: Proteolytic cleavage product of 30-kDa adipocyte complement-related 66 protein increases fatty acid oxidation in muscle and causes weight loss in mice. Proc Natl Acad Sci U S A 98:2005-2010, 2001 16. Shimomura I, Hammer RE, Ikemoto S, Brown MS, Goldstein JL: Leptin reverses insulin resistance and diabetes mellitus in mice with congenital lipodystrophy. Nature 401:73-76, 1999 17. Lehmann JM, Moore LB, Smith-Oliver TA, Wilkison WO, Willson TM, Kliewer SA: An antidiabetic thiazolidinedione is a high affinity ligand for peroxisome proliferator- activated receptor gamma (PPAR gamma). J Biol Chem 270:12953-12956, 1995 18. Kletzien RF, Clarke SD, Ulrich RG: Enhancement of adipocyte differentiation by an insulin-sensitizing agent. Mol Pharmacol 41:393-398, 1992 19. Sandouk T, Reda D, Hofmann C: Antidiabetic agent pioglitazone enhances adipocyte differentiation of 3T3-F442A cells. Am J Physiol 264:C1600-1608, 1993 20. Taniguchi CM, Emanuelli B, Kahn CR: Critical nodes in signalling pathways: insights into insulin action. Nat Rev Mol Cell Biol 7:85-96, 2006 21. Le Cam A, Le Cam G: A new negatively regulated acute-phase phosphoprotein synthesized by rat hepatocytes. Biochem J 230:603-607, 1985 22. Le Cam A, Magnaldo I, Le Cam G, Auberger P: Secretion of a major phosphorylated glycoprotein by hepatocytes. Characterization of specific antibodies and investigations of the processing, excretion kinetics, and phosphorylation. J Biol Chem 260:15965-15971, 1985 23. Auberger P, Falquerho L, Contreres JO, Pages G, Le Cam G, Rossi B, Le Cam A: Characterization of a natural inhibitor of the insulin receptor tyrosine kinase:cDNA cloning, purification and anti-mitogenic activity. Cell 58:631-640, 1989 24. Haasemann M, Nawratil P, Muller-Esterl W: Rat tyrosine kinase inhibitor shows sequence similarity to human alpha 2-HS glycoprotein and bovine fetuin. Biochem J 274:899-902, 1991 25. Dziegielewska KM, Brown WM, Casey SJ, Christie DL, Foreman RC, Hill RM, Saunders NR: The complete cDNA and amino acid sequence of bovine fetuin. Its homology with alpha 2HS glycoprotein and relation to other members of the cystatin superfamily. J Biol Chem 265:4354-4357, 1990 26. Christie DL, Dziegielewska KM, Hill RM, Saunders NR: Fetuin:The bovine homologue of human ?HS glycoprotein. FEBS Lett 214:45-49, 1987 27. Dziegielewska KM, Mollgard K, Reynolds ML, Saunders NR: A fetuin-related glycoprotein (? 2 HS) in human embryonic and fetal development. Cell Tissue Res 248:33- 41, 1987 28. Mathews ST, Chellam N, Srinivas PR, Cintron VJ, Leon MA, Goustin AS, Grunberger G: ?2-HSG, a specific inhibitor of insulin receptor autophosphorylation, interacts with the insulin receptor. Mol Cell Endrocrinol 264:87-98, 2000 67 29. Mathews ST, Singh GP, Ranalletta M, Cintron VJ, Qiang X, Goustin AS, Jen KL, Charron MJ, Jahnen-Dechent W, Grunberger G: Improved insulin sensitivity and resistance to weight gain in mice null for the Ahsg gene. Diabetes 51:2450-2458, 2002 30. Mathews ST, Rakhade S, Zhou X, Parker GC, Coscina DV, Grunberger G: Fetuin- null mice are protected against obesity and insulin resistance associated with aging. Biochem Biophys Res Commun 350:437-443, 2006 31. Kissebah AH, Sonnenberg GE, Myklebust J, Goldstein M, Broman K, James RG, Marks JA, Krakower GR, Jacob HJ, Weber J, Martin L, Blangero J, Comuzzie AG: Quantitative trait loci on chromosomes 3 and 17 influence phenotypes of the metabolic syndrome. Proc Natl Acad Sci U S A 97:14478-14483, 2000 32. Lavebratt C, Wahlqvist S, Nordfors L, Hoffstedt J, Arner P: AHSG gene variant is associated with leanness among Swedish men. Hum Genet 117:54-60, 2005 33. Stefan N, Hennige AM, Staiger H, Machann J, Schick F, Krober SM, Machicao F, Fritsche A, Haring HU: Alpha2-Heremans-Schmid glycoprotein/fetuin-A is associated with insulin resistance and fat accumulation in the liver in humans. Diabetes Care 29:853-857, 2006 34. Ix JH, Shlipak MG, Brandenburg VM, Ali S, Ketteler M, Whooley MA: Association between human fetuin-A and the metabolic syndrome: data from the Heart and Soul Study. Circulation 113:1760-1767, 2006 35. Kalabay L, Cseh K, Pajor A, Baranyi E, Csakany GM, Melczer Z, Speer G, Kovacs M, Siller G, Karadi I, Winkler G: Correlation of maternal serum fetuin/alpha2-HS- glycoprotein concentration with maternal insulin resistance and anthropometric parameters of neonates in normal pregnancy and gestational diabetes. Eur J Endocrinol 147:243-248, 2002 36. Mori K, Emoto M, Yokoyama H, Araki T, Teramura M, Koyama H, Shoji T, Inaba M, Nishizawa Y: Association of serum fetuin-A with insulin resistance in type 2 diabetic and nondiabetic subjects. Diabetes Care 29:468, 2006 37. Mathews ST, Deutsch DD, Iyer G, Hora N, Pati B, Marsh J, Grunberger G: Plasma alpha2-HS glycoprotein concentrations in patients with acute myocardial infarction quantified by a modified ELISA. Clin Chim Acta 319:27-34., 2002 38. Haglund AC, Ek B, Ek P: Phosphorylation of human plasma alpha2-Heremans- Schmid glycoprotein (human fetuin) in vivo. Biochem J 357:437-445, 2001 39. Olshansky SJ: Projecting the future of U.S. health and longevity. Health Aff (Millwood) 24 Suppl 2:W5R86-89, 2005 40. Li Z, Bowerman S, Heber D: Health ramifications of the obesity epidemic. Surg Clin North Am 85:681-701, v, 2005 41. Rocchini AP: Childhood obesity and a diabetes epidemic. N Engl J Med 346:854-855, 2002 42. Gimeno RE, Klaman LD: Adipose tissue as an active endocrine organ: recent advances. Curr Opin Pharmacol 5:122-128, 2005 68 43. Tilg H, Moschen AR: Adipocytokines: mediators linking adipose tissue, inflammation and immunity. Nat Rev Immunol 6:772-783, 2006 44. La Cava A, Matarese G: The weight of leptin in immunity. Nat Rev Immunol 4:371- 379, 2004 45. Yang Q, Graham TE, Mody N, Preitner F, Peroni OD, Zabolotny JM, Kotani K, Quadro L, Kahn BB: Serum retinol binding protein 4 contributes to insulin resistance in obesity and type 2 diabetes. Nature 436:356-362, 2005 46. Hotamisligil GS, Murray DL, Choy LN, Spiegelman BM: Tumor necrosis factor alpha inhibits signaling from the insulin receptor. Proc Natl Acad Sci U S A 91:4854- 4858, 1994 47. American Diabetes Association: Diagnosis and classification of diabetes mellitus. Diabetes Care 30:S42-S47, 2007 48. Ottensmeyer FP, Beniac DR, Lou RZT, Yip CC: Mechanism of transmembrane signaling: insulin binding and the insulin receptor. Biochem 39:12103-12112, 2000 49. White MF: Insulin signaling in health and disease. Science 302:17101711, 2003 50. White MF: IRS proteins and the common path to diabetes. Am J Physiol Endocrinol Metab 283:E413-422, 2002 51. Cai D, Dhe-Paganon S, Melendez PA, Lee J, Shoelson SE: Two new substrates in insulin signaling, IRS5/DOK4 and IRS6/DOK5. J Biol Chem 278:25323-25330, 2003 52. Taniguchi CM, EManuelli B, Kahn CR: Critical nodes in signalling pathways: insights into insulin action. Nat Rev Mol Cell Biol 7:85-96, 2006 53. Kern PA, Saghizadeh M, Ong JM, Bosch RJ, Deem R, Simsolo RB: The expression of tumor necrosis factor in human adipose tissue. Regulation by obesity, weight loss, and relationship to lipoprotein lipase. J Clin Invest 95:2111-2119, 1995 54. Ozes ON, Mayo LD, Gustin JA, Pfeffer SR, Pfeffer LM, Donner DB: NF-kappaB activation by tumour necrosis factor requires the Akt serine-threonine kinase. Nature 401:82-85, 1999 55. Yuan M, Konstantopoulos N, Lee J, Hansen L, Li ZW, Karin M, Shoelson SE: Reversal of obesity- and diet-induced insulin resistance with salicylates or targeted disruption of Ikkbeta. Science 293:1673-1677, 2001 56. Miller BS, Shankavaram UT, Horney MJ, Gore AC, Kurtz DT, Rosenzweig SA: Activation of cJun NH2-terminal kinase/stress-activated protein kinase by insulin. Biochemistry 35:8769-8775, 1996 57. Taniguchi CM, Ueki K, Kahn R: Complementary roles of IRS-1 and IRS-2 in the hepatic regulation of metabolism. J Clin Invest 115:718-727, 2005 58. Todoric J, Loffler M, Huber J, Bilban M, Reimers M, Kadl A, Zeyda M, Waldhausl W, Stulnig TM: Adipose tissue inflammation induced by high-fat diet in obese diabetic mice is prevented by n-3 polyunsaturated fatty acids. Diabetologia 49:2109-2119, 2006 59. Ropelle ER, Pauli JR, Prada PO, de Souza CT, Picardi PK, Faria MC, Cintra DE, Fernandes MF, Flores MB, Velloso LA, Saad MJ, Carvalheira JB: Reversal of diet- 69 induced insulin resistance with a single bout of exercise in the rat: the role of PTP1B and IRS-1 serine phosphorylation. J Physiol 577:997-1007, 2006 60. Hevener AL, Reichart D, Olefsky J: Exercise and thiazolidinedione therapy normalize insulin action in the obese Zucker fatty rat. Diabetes 49:2154-2159, 2000 61. Wang C, Mao X, Wang L, Liu M, Wetzel MD, Guan KL, Dong LQ, Liu F: Adiponectin sensitizes insulin signaling by reducing p70 S6 kinase-mediated serine phosphorylation of IRS-1. J Biol Chem, 2007 62. Dib K, Whitehead JP, Humphreys PJ, Soos MA, Baynes KC, Kumar S, Harvey T, O'Rahilly S: Impaired activation of phosphoinositide 3-kinase by insulin in fibroblasts from patients with severe insulin resistance and pseudoacromegaly. A disorder characterized by selective postreceptor insulin resistance. J Clin Invest 101:1111-1120, 1998 63. Ahmad F, Azevedo JL, Cortright R, Dohm GL, Goldstein BJ: Alterations in skeletal muscle protein-tyrosine phosphatase activity and expression in insulin-resistant human obesity and diabetes. J Clin Invest 100:449-458, 1997 64. Wu X, Hoffstedt J, Deeb W, Singh R, Sedkova N, Zilbering A, Zhu L, Park PK, Arner P, Goldstein BJ: Depot-specific variation in protein-tyrosine phosphatase activities in human omental and subcutaneous adipose tissue: a potential contribution to differential insulin sensitivity. J Clin Endocrinol Metab 86:5973-5980, 2001 65. Ueki K, Kondo T, Kahn CR: Suppressor of cytokine signaling 1 (SOCS-1) and SOCS-3 cause insulin resistance through inhibition of tyrosine phosphorylation of insulin receptor substrate proteins by discrete mechanisms. Mol Cell Biol 24:5434-5446, 2004 66. Emanuelli B, Peraldi P, Filloux C, Chavey C, Freidinger K, Hilton DJ, Hotamisligil GS, Van Obberghen E: SOCS-3 inhibits insulin signaling and is up-regulated in response to tumor necrosis factor-alpha in the adipose tissue of obese mice. J Biol Chem 276:47944-47949, 2001 67. Ueki K, Kondo T, Tseng YH, Kahn CR: Central role of suppressors of cytokine signaling proteins in hepatic steatosis, insulin resistance, and the metabolic syndrome in the mouse. Proc Natl Acad Sci U S A 101:10422-10427, 2004 68. Kawazoe Y, Naka T, Fujimoto M, Kohzaki H, Morita Y, Narazaki M, Okumura K, Saitoh H, Nakagawa R, Uchiyama Y, Akira S, Kishimoto T: Signal transducer and activator of transcription (STAT)-induced STAT inhibitor 1 (SSI-1)/suppressor of cytokine signaling 1 (SOCS1) inhibits insulin signal transduction pathway through modulating insulin receptor substrate 1 (IRS-1) phosphorylation. J Exp Med 193:263- 269, 2001 69. Goding JW, Terkeltaub R, Maurice M, Deterre P, Sali A, Belli SI: Ecto- phosphodiesterase/pyrophosphatase of lymphocytes and non-lymphoid cells: structure and function of the PC-1 family. Immunol Rev 161:11-26, 1998 70 70. Maddux BA, Goldfine ID: Membrane glycoprotein PC-1 inhibition of insulin receptor function occurs via direct interaction with the receptor alpha-subunit. Diabetes 49:13-19, 2000 71. Maddux BA, Chang YN, Accili D, McGuinness OP, Youngren JF, Goldfine ID: Overexpression of the insulin receptor inhibitor PC-1/ENPP1 induces insulin resistance and hyperglycemia. Am J Physiol Endocrinol Metab 290:E746-749, 2006 72. Frittitta L, Spampinato D, Solini A, Nosadini R, Goldfine ID, Vigneri R, Trischitta V: Elevated PC-1 content in cultured skin fibroblasts correlates with decreased in vivo and in vitro insulin action in nondiabetic subjects: evidence that PC-1 may be an intrinsic factor in impaired insulin receptor signaling. Diabetes 47:1095-1100, 1998 73. Frittitta L, Youngren J, Vigneri R, Maddux BA, Trischitta V, Goldfine ID: PC-1 content in skeletal muscle of non-obese, non-diabetic subjects: relationship to insulin receptor tyrosine kinase and whole body insulin sensitivity. Diabetologia 39:1190-1195, 1996 74. Maddux BA, Sbraccia P, Kumakura S, Sasson S, Youngren J, Fisher A, Spencer S, Grupe A, Henzel W, Stewart TA, et al.: Membrane glycoprotein PC-1 and insulin resistance in non-insulin-dependent diabetes mellitus. Nature 373:448-451, 1995 75. Olivier E, Soury E, Ruminy P, Husson A, Paramentier F, Daveau M, Salier J: Fetuin- B, a second member of the fetuin family in mammals. Biochem J 350:589-597, 2000 76. Heremans F: Les Globulines Seriques du Systeme Gamma. Arcia, Brussels, 1960 77. Schmid K, Burgi W: Preparation and proliferation of the human plasma Ba-?2-HS glycoprotein. Biochim Biophys Acta 47:440-453, 1961 78. Pedersen K-O: Fetuin, a new globulin isolated from serum. Nature 154:575, 1944 79. Elzanowski A, Barker WC, Hunt LT, Seibel-Ross E: Cystatin domains in alpha-2- HS-glycoprotein and fetuin. FEBS Lett 227:167-170, 1988 80. Lee CC, Bowman BH, Yang FM: Human alpha 2-HS-glycoprotein: the A and B chains with a connecting sequence are encoded by a single mRNA transcript. Proc Natl Acad Sci U S A 84:4403-4407, 1987 81. Kellermann J, Haupt H, Auerswald EA, Muller-Ester W: The arrangement of disulfide loops in human alpha 2-HS glycoprotein. Similarity to the disulfide bridge structures of cystatins and kininogens. J Biol Chem 264:14121-14128, 1989 82. Yoshioka Y, Gejyo F, Marti T, Rickli EE, Burgi W, Offner GD, Troxler RF, Schmid K: The complete amino acid sequence of the A-chain of human plasma alpha 2HS- glycoprotein. J Biol Chem 261:1665-1671, 1986 83. Geyjo F, Chang J-L, Burgi W, Schmid K, Offner GD, Troxler RF, Van Halbeek H, Dorland L, Gerwig GJ, Vliegenthart JF: Characterization of the B-chain of human plasma alpha 2HS-glycoprotein. The complete amino acid sequence and primary structure of its heteroglycan. J Biol Chem 258, 1983 71 84. Araki T, Yoshioka Y, Schmid K: The position of the disulfide bonds in human plasma alpha 2 HS- glycoprotein and the repeating double disulfide bonds in the domain structure. Biochim Biophys Acta 994:195-199, 1989 85. Jahnen-Dechent W, Trindl A, Godovac-Zimmermann J, Muller-Esterl W: Posttranslational processing of human alpha 2-HS glycoprotein (human fetuin). Evidence for the production of a phosphorylated single-chain form by hepatoma cells. Eur J Biochem 226:59-69, 1994 86. Rauth G, P?schke O, Fink E, Eulitz, M., Tippmer S, Kellerer M, H?ring HU, Nawratil P, Haasemann M, Jahnen-Deschent W, M?ller-Esterl W: The nucleotide and partial amino acid sequences of rat fetuin. Identity with the natural tyrosine kinase inhibitor of the rat insulin receptor. Eur. J. Biochem 204: 523-529., 1992 87. Yang F, Chen ZL, Bergeron JM, Cupples RL, Friedrichs WE: Human alpha 2-HS- glycoprotein/bovine fetuin homologue in mice: identification and developmental regulation of the gene. Biochim Biophys Acta 1130:149-156, 1992 88. Brown WM, Dziegielewska KM, Saunders NR, Christie DL, Nawratil P, Muller- Esterl W: The nucleotide and deduced amino acid structures of sheep and pig fetuin. Common structural features of the mammalian fetuin family. Eur J Biochem 205:321- 331, 1992 89. Dziegielewska KM, Brown WM: Fetuin. Heidelberg, Springer-Verlag, 1995 90. Srinivas PR, Wagner AS, Reddy LV, Deutsch DD, Leon MA, Goustin AS, Grunberger G: Serum alpha 2-HS-glycoprotein is an inhibitor of the human insulin receptor at the tyrosine kinase level. Mol Endocrinol 7:1445-1455, 1993 91. Srinivas PR, Deutsch DD, Mathews ST, Goustin AS, Leon MA, Grunberger G: Recombinant human alpha 2-HS glycoprotein inhibits insulin-stimulated mitogenic pathway without affecting metabolic signalling in Chinese hamster ovary cells overexpressing the human insulin receptor. Cell Signal 8:567-573, 1996 92. Kalabay L, Chavin K, Lebreton JP, Robinson KA, Buse MG, Arnaud P: Human recombinant alpha 2-HS glycoprotein is produced in insect cells as a full length inhibitor of the insulin receptor tyrosine kinase. Horm Metab Res 30:1-6, 1998 93. Cheatham B, Vlahos CJ, Cheatham L, Wang L, Blenis J, Kahn CR: Phosphatidylinositol 3-kinase activation is required for insulin stimulation of pp70 S6 kinase, DNA synthesis, and glucose transporter translocation. Mol Cell Biol 14:4902- 4911, 1994 94. Kim T, Price JC, Sunkara R, Mathews ST: Fetuin inhibits insulin-stimulated glucose uptake and glycogen synthesis in L6-GLUT4myc skeletal muscle cells (Abstract) 1211-P 66 th Annual Scientific Sessions, Washington DC, American Diabetes Association 95. Mathews ST, Srinivas PR, Leon MA, Grunberger G: Bovine fetuin is an inhibitor of insulin receptor tyrosine kinase. Life Sci 61:1583-1592, 1997 72 96. Chen H, Srinivas PR, Cong LN, Li Y, Grunberger G, Quon MJ: Alpha2-Heremans Schmid glycoprotein inhibits insulin-stimulated Elk-1 phosphorylation, but not glucose transport, in rat adipose cells. Endocrinology 139:4147-4154, 1998 97. Shoelson SE, White MF, Kahn CR: Tryptic activation of the insulin receptor. Proteolytic truncation of the a-subunit releases the b-subunit from inhibitory control. J. Biol. Chem. 263:4852-4860., 1988 98. Vionnet N, Hani EH, Dupont S, Gallina S, Francke S, Dotte S, De Matos, F, Durand E, Lepretre F, Lecoeur C, Gallina P, Zekiri L, Dina C, Froguel P: Genomewide search for type 2 diabetes-susceptibility genes in French whites: Evidence for a novel susceptibility locus for early-onset diabetes on chromosome 3q27-qter and independent replication of a type 2-diabetes locus on chromosome 1q21-q24. Am J Hum Genet 67:1470-1480, 2000 99. Jahnen-Dechent W, Schinke T, Trindl A, Muller-Esterl W, Sablitzky F, Daiser S, Blessing M: Cloning and targeted deletion of the mouse fetuin gene. J Biol Chem 272:31496-31503, 1997 100. Osawa M, Umetsu K, Sato M, Ohki T, Yukawa N, Suzuki T, Takeichi S: Structure of the gene encoding human alpha 2 -HS glycoprotein (AHSG). Gene 196:121-125, 1997 101. Falquerho L, Patey G, Paquereau L, Rossi V, Lahuna O, Szpirer J, Szpirer C, Levan G, Le Cam A. Primary structure of the rat gene encoding an inhibitor of the insulin receptor tyrosine kinase. Gene 98:209-216, 1991 102. Vinson CR, Hai T, Boyd SM: Dimerization specificity of the leucine zipper- containing bZIP motif on DNA binding: prediction and rational design. Genes Dev 7:1047-1058, 1993 103. Grange T, Roux J, Riquad G, Pictet R: Cell-type specific activity of two glucocorticoid responsive units of rat tyrosine aminotransferase gene is associated with multiple binding sites for C/EBP and a novel liver-specific nuclear factor. Nucleic Acids Res 19:131-139, 1991 104. Woltje M Tschoke B, von Bulow V, Westenfeld R, Denecke B, Graber S, Jahnen- Dechent W: CCAAT enhancer binding protein ? and hepatocyte nuclear factor 3? are necessary and sufficient to mediate dexamethasone-induced up-regulation of alpha 2 HS- glycoprotein/fetuin-A gene expression. J Mol Endocrinol 36:261-277, 2006 105. Andrew RC, Walker BR: Glucocorticoids and insulin resistance: old hormones, new targets. Clin Sci (Lond) 96:513-23, 1999 106. Lin X, Braymer HD, Bray GA, York DA: Differential expression of insulin receptor tyrosine kinase inhibitor (fetuin) gene in a model of diet-induced obesity. Life Sci 63:145- 153, 1998 107. Osawa M, Umetsu K, Ohki T, Nagasawa T, Suzuki T, Takeichi S: Molecular evidence for human alpha2-HS glycoprotein (AHSG) polymorphism. Hum Genet 99:18- 21, 1997 108. Dahlman I, Eriksson P, Kaaman M, Jiao H, Lindrgen CM, Kere J, Arner P: ? 2 - Heremans-Schmid glycoprotein gene polymorphisms are associated with adipocyte insulin action. Diabetologia 47:1974-1979, 2004 73 109. Siddiq A, Lepretre F, Hercberg S, Froguel P, Gibson F: A synonymous coding polymorphism in the ?2-Heremans-Schmid glycoprotein gene is associated with type 2 diabetes in French Caucasians. Diabetes 54:2477-2481, 2005 110. Lavebratt C, Dungner E, Hoffstedt J: Polymorphism of the AHSG gene is associated with increased adipocyte ?2-adrenoceptor function. J Lipid Res 46:2278-2281, 2005 111. Wang JL, Chinookoswong N, Scully S, Qi M, Shi ZQ: Differential effects of leptin in regulation of tissue glucose utilization in vivo. Endocrinology 140:2117-2124, 1999. 112. Berg AH, Combs TP, Scherer PE: ACRP30/adiponectin: an adipokine regulating glucose and lipid metabolism. Trends Endocrinol Metab 13:84-89, 2002 113. Banerjee RR, Lazar MA: Resistin: molecular history and prognosis. J Mol Med 81:218-226, 2003 114. Winer J, Jung CK, Shackel I, Williams PM: Development and validation of real- time quantitative reverse transcriptase-polymerase chain reaction for monitoring gene expression in cardiac myocytes in vitro. Anal Biochem 270:41-49, 1999. 115. Zick Y, Grunberger G, Rees-Jones RW, Comi RJ: Use of tyrosine containing polymers to characterize the substrate specificity of insulin and other hormone-stimulated tyrosine kinase. Eur. J. Biochem 148:177-182, 1985 116. Bahary N, Leibel RL, Joseph L, Friedman JM: Molecular mapping of the mouse db mutation. Proc Natl Acad Sci U S A 87:8642-8646, 1990 117. Cohen B, Novick D, Rubinstein M: Modulation of insulin activities by leptin. Science 274:1185-1188, 1996 118. Tofovic SP, Kusaka H, Kost CK, Jr., Bastacky S: Renal function and structure in diabetic, hypertensive, obese ZDFxSHHF-hybrid rats. Ren Fail 22:387-406, 2000 119. Singh R, Barden A, Mori T, Beilin L: Advanced glycation end products: a review. Diabetologia 44:129-146, 2001