ROLE OF CALCIUM-INDEPENDENT PHOSPHOLIPASE A2 IN INSULIN-
STIMULATED GLUCOSE UPTAKE IN 3T3-L1 ADIPOCYTES 
 
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. 
 
 
_____________________________________ 
Juan Yang 
 
 
 
 
Certificate of Approval: 
 
 
_______________________________ 
Margaret C. Craig-Schmidt 
Professor 
Nutrition & Food Science 
 
_______________________________ 
Suresh Mathews 
Assistant Professor 
Nutrition & Food Science 
 
_______________________________ 
    Kevin W. Huggins, Chair 
    Assistant Professor 
    Nutrition & Food Science 
 
_______________________________ 
    Robert L. Judd 
    Associate Professor 
    Anatomy, Physiology, Pharmacology 
 
_____________________________________ 
                                   George T. Flowers 
                                   Dean 
                                   Graduate School 
  
ROLE OF CALCIUM-INDEPENDENT PHOSPHOLIPASE A2 IN INSULIN-
STIMULATED GLUCOSE UPTAKE IN 3T3-L1 ADIPOCYTES 
Juan Yang 
 
A Dissertation 
Submitted to 
the Graduate Faculty of 
Auburn University 
in Partial Fulfillment of the 
Requirements for the 
Degree of 
Doctor of Philosophy 
 
 
Auburn, Alabama 
December 18, 2009 
 
 iii
 ROLE OF CALCIUM-INDEPENDENT PHOSPHOLIPASE A
2
 IN INSULIN-
STIMULATED GLUCOSE UPTAKE IN 3T3-L1 ADIPOCYTES 
 
 
Juan Yang 
 
Permission is granted to Auburn University to make copies of this dissertation at its 
discretion, upon request of individuals or institutions and at their expense. The author 
reserves all publication rights. 
 
 
 
 
 
 
                                                                                  ______________________________ 
Signature of Author 
 
______________________________
Date of Graduation 
 
 
 
 
 
 
 
 
 
 iv
VITA 
 
 
 Juan Yang, daughter of Daizhang Yang and Shuyun Liu, was born in a small town 
named Shimen in southern China. After she graduated from a local high school in her 
hometown, she continued her study in the Life Science School of Wuhan University.  
 During her seven years in Wuhan University, she got her Bachelor of Science 
degree in Biology and then an M.S. degree in Microbiology. Before Juan enrolled as a 
graduate student in Auburn University in 2005, she had worked in the National Center 
For Gene Research in Shanghai, China for three years. At Auburn, she pursued a Ph.D. in 
Nutrition and a M.S. degree in Statistics.  
 
 
 
 
 
 
 
 
 
  
 
 v
DISSERTATION ABSTRACT 
 
ROLE OF CALCIUM-INDEPENDENT PHOSPHOLIPASE A2 IN INSULIN-
STIMULATED GLUCOSE UPTAKE IN 3T3-L1 ADIPOCYTES 
 
Juan Yang 
Doctor of Philosophy, December 18, 2009  
(M.S., Wuhan University, 2001)  
(B.S., Wuhan University, 1999)  
 
156 Typed Pages  
 
Directed by Kevin W. Huggins 
 
 
Calcium-independent phospholipase A
2
 (iPLA
2
) is a member of the phospholipase 
A
2
 family that catalyzes the hydrolysis of glycerophospholipid at the sn-2 position to 
liberate free fatty acid and lysophospholipid. Recent studies have suggested that iPLA
2
 
may be important for adipocyte biology. The purpose of this study was to determine the 
role of iPLA
2
 in insulin-stimulated actions on glucose uptake in 3T3-L1 adipocytes. 
The 3T3-L1 adipocytes were pretreated with bromoenol lactone (BEL, 10 or 
50 ?M), a chemical inhibitor of iPLA
2
, for 30 minutes. After insulin (100 nM) 
stimulation, glucose uptake was determined by measuring intracellular incorporation of 
[
3
H]-2-deoxyglucose. With 10 and 50 ?M BEL treatment, insulin-stimulated glucose 
uptake was decreased by 30% and 45%, respectively, compared to DMSO vehicle-treated 
cells. Further investigations confirmed that iPLA
2
 activity was responsive to the BEL
 
 vi
 inhibitory effects, and that iPLA
2
? and iPLA
2
? were both involved in this process. 
Additionally, exogenous arachidonic acid (AA, 100 ?M) restored the reduction of 
insulin-stimulated glucose uptake induced by BEL treatment. Small interfering RNA 
(siRNA) technique was utilized to selectively inhibit iPLA
2
? and iPLA
2
? gene expression 
in 3T3-L1 adipocytes. Consistent with the BEL treatment results, the 3T3-L1 adipocytes 
transfected with iPLA
2
? or iPLA
2
? siRNA (50 nM) exhibited impaired glucose uptake 
upon insulin administration, showing approximate 40% inhibition compared to scrambled 
siRNA treated cells. To elucidate the underlying mechanism for the effects of iPLA
2
 
inhibition on insulin-stimulated glucose uptake, insulin-stimulated phosphorylation of 
insulin receptor (IR) and Akt was assessed by Western blot analysis. There was no 
significant difference between BEL-treated and vehicle-treated cells in the 
phosphorylation levels of IR and Akt. Fraction analysis of insulin-responsive GLUT4 
translocation was also performed in adipocytes. Compared to vehicle control, BEL 
treatment noticeably inhibited the incorporation of GLUT4 into plasma membrane and 
lipid raft fractions in response to insulin.  
 These results demonstrate that insulin-stimulated glucose uptake was decreased 
by iPLA
2
 inhibition, and that this effect was mediated via attenuation of the insulin-
dependent GLUT4 translocation into plasma membrane. However, the early steps of 
insulin signaling were not affected by iPLA
2
 inhibition. It is conclude that insulin-
stimulated glucose uptake is mediated, at least in part, by iPLA
2
, and that iPLA
2
 may 
represent a novel therapeutic target for the treatment of insulin resistance related diseases, 
such as obesity, type 2 diabetes, and other metabolic diseases. 
 
 vii
ACKNOWLEDGMENTS 
 
 
 Foremost, I would like to express my sincere gratitude to my advisor Dr. Kevin, 
Huggins. Without his guidance and encouragement, this work would not have been 
accomplished. He has been abundantly helpful in all the time of research and writing of 
this dissertation. 
 I am also indebted to Dr. Craig-Schmidt for her constant help and support for my 
research, study and life in Auburn.  I equally wish to extend my appreciation and thanks 
to Dr. Mathews and Dr. Judd, the rest of my committee members, for their valuable help, 
support, interest and stimulating hints for my research. 
I would like to thank the faculties in the Department of Nutrition and Food 
Science who have helped and inspired me during my doctoral study: Dr. White, Dr. 
Fellers, Dr. Kim, Dr. Keith, Dr. Huang, Dr. Gropper, Dr. Bell, and Dr. Zizza. 
Many thanks go to the graduate students studying and working with me in 
Auburn. In particular, I am grateful to Yuan Kang and my former colleagues Kathryn 
Colbert and Carmen Teodorescu, for their friendship and help in my research, study and 
life.  
I can not end the acknowledgements without my deepest appreciation to my 
family, my father Daizhang Yang, mother Shuyun Liu, and fianc?e Xuan Li, on whose 
constant encouragement and love I have relied throughout my time at Auburn. 
 
 
 viii
Style manual or journal used: The Journal of Nutrition  
Computer software used: Microsoft Word, Microsoft PowerPoint, Microsoft Excel, 
Minitab, Adobe Acrobat Professional, LabWorks Image Acquisition Software v.4.6.  
 
 ix
TABLE OF CONTENTS 
 
LIST OF TABLES ........................................................................................................... xiii 
LIST OF FIGURES ......................................................................................................... xiv 
CHAPTER 1. INTRODUCTION ........................................................................................1 
CHAPTER 2. REVIEW OF LITERATURE .......................................................................4 
   2.1. PHOSPHOLIPASE A
2
...............................................................................................4 
   2.1.1. Classification of Phospholipase A
2
 ......................................................................5 
   2.1.2. Secreted Phospholipase A
2
 ..................................................................................5 
   2.1.3. Cytosolic Calcium-Dependent Phospholipase A
2
................................................6 
   2.1.4. Platelet-Activating Factor Acetylhydrolase .........................................................7 
   2.1.5. Other Phospholipase A
2
 .......................................................................................7 
   2.2. CALCIUM-INDEPENDENT PHOSPHOLIPASE A
2
 ..............................................8 
   2.2.1. Identification and Characterization of Calcium-Independent Phospholipase 
             A
2
 .........................................................................................................................8 
   2.2.2. Regulations of Calcium-Independent Phospholipase A
2
 Activation .................12 
   2.2.3. Inhibition of Calcium-Independent Phospholipase A
2
 ......................................13 
   2.2.4. Cellular Functions of Calcium-Independent Phospholipase A
2
 ........................17 
2.2.4.1. Phospholipid Remodeling ..........................................................................17 
2.2.4.2. Arachidonic Acid Metabolism ...................................................................18 
2.2.4.3. Control  of Calcium Entry .........................................................................19
2.2.4.4. Secretion and Exocytosis ...........................................................................20 
2.2.4.5. Other Cell Signaling Functions ..................................................................21 
   2.2.5. Calcium-Independent Phospholipase A
2
 in Metabolism ...................................21 
2.2.5.1. Glucose-Stimulated Insulin Secretion .......................................................22 
 
 x
2.2.5.2. Adipocyte Differentiation ..........................................................................23 
2.2.5.3. Hepatic Lipogenesis ...................................................................................24 
2.2.5.4. Mitochondrial Functions ............................................................................25 
 2.3. INSULIN SIGNALING ..........................................................................................25 
       2.3.1. Phosphatidylinositol 3-Kinase Pathway ...........................................................26 
       2.3.2. Cbl/CAP Pathway .............................................................................................27
       2.3.3. Mitogen-Activated
 
Protein Kinase Pathway .....................................................28 
   2.4. INSULIN-STIMULATED GLUT4 TRANSPORT .................................................31 
       2.4.1. Insulin Signaling Regulators .............................................................................32 
       2.4.2. Newly Identified Akt Substrates Involved in GLUT4 Exocytosis ...................34 
       2.4.3. Actin Remodeling .............................................................................................35 
       2.4.4. Calcium Influx ..................................................................................................36 
   2.5. OBESITY, TYPE 2 DIABETES AND INSULIN RESISTANCE .........................38 
   2.5.1. Epidemic of Obesity and Type 2 Diabetes ........................................................38 
   2.5.2. Molecular Mechanism of Insulin Resistance .....................................................40 
   2.5.3. Adipose Tissue and Insulin Resistance ..............................................................43 
CHAPTER 3. CALCIUM-INDEPENDENT PHOSPHOLIPASE A
2
 IS INVOLVED      
IN INSULIN-STIMULATED GLUCOSE UPTAKE IN 3T3-L1 ADIPOCYTES ...........46 
3.1. ABSTRACT ............................................................................................................46 
3.2. INTRODUCTION ...................................................................................................47 
3.3. MATERIALS AND METHODS .............................................................................49 
       3.3.1. Materials ...........................................................................................................49 
       3.3.2. Cell Culture of 3T3-L1 Fibroblasts and Adipocytes .........................................50 
       3.3.3. Reverse Transcription and Quantitative PCR ...................................................51 
       3.3.4. Insulin-Stimulated Glucose Uptake Assay .......................................................52 
       3.3.5. Preparation of protein samples from 3T3-L1 cells ...........................................53 
 
 xi
       3.3.6. Immunoprecipitation and Western Blotting......................................................53 
       3.3.7. Fractionation Analysis of the Subcellular Distribution of GLUT4 ..................54 
       3.3.8. Detection of GLUT4 in Lipid Raft Fractions ...................................................55 
       3.3.9. Statistical Analysis ............................................................................................58 
3.4. RESULTS ................................................................................................................58 
    3.4.1. Gene Expression of iPLA
2 
is Increased during 3T3-L1 Adipocyte 
              Differentiation ...................................................................................................58 
    3.4.2. Insulin-Stimulated Glucose Uptake in 3T3-L1 Adipocytes is Decreased by  
              BEL-Induced iPLA
2
 Inhibition .........................................................................59 
    3.4.3. Both iPLA
2
? and iPLA
2
? Contribute to Glucose Uptake in Insulin-Stimulated  
              Adipocytes ........................................................................................................60 
    3.4.4. Exogenous Arachidonic Acid Reverses the BEL-Inhibitory Effect in Insulin- 
              Stimulated Glucose Uptake ..............................................................................61 
    3.4.5. Insulin-Stimulated Glucose Uptake in L6-GLUT4 Myotubes is Decreased by   
              BEL-Induced iPLA2 Inhibition ........................................................................62 
3.4.6. Inhibition of iPLA
2
 by BEL Does Not Impair Insulin-Stimulated  
              Phosphorylation of IR and Akt .........................................................................63 
    3.4.7. Inhibition of iPLA
2
 by BEL Impacts the Insulin-Stimulated GLUT4   
              Translocation in 3T3-L1 adipocytes .................................................................63 
3.4.8. Inhibition of iPLA
2
 by BEL Alters GLUT4 Incorporation in Lipid Rafts .......64 
3.5. DISCUSSION ..........................................................................................................65 
CHAPTER 4. SMALL INTERFERING RNA KNOCKDOWN OF CALCIUM-
INDEPENDENT PHOSPHOLIPASE A
2
 INHIBITS INSULIN-STIMULATED 
GLUCOSE UPTAKE IN 3T3-L1 ADIPOCYTES ............................................................83 
4.1. ABSTRACT .............................................................................................................83 
4.2. INTRODUCTION ...................................................................................................84 
4.3. MATERIALS AND METHODS .............................................................................85 
 
 xii
       4.3.1. Materials ...........................................................................................................85 
       4.3.2. Cell Culture of 3T3-L1 Fibroblasts and Adipocytes .........................................85 
       4.3.3. Transfection of 3T3-L1 Adipocytes by Electroporation ...................................86 
       4.3.4. Evaluation of Transfection Efficiency ..............................................................87 
       4.3.5. Reverse Transcription and Quantitative PCR ...................................................88 
       4.3.6. Insulin-Stimulated Glucose Uptake Assay .......................................................89 
    4.3.7. Statistical Analysis ............................................................................................90 
4.4. RESULTS AND DISCUSSION ..............................................................................90 
    4.4.1. Electroporation Introduces Foreign Plasmids into 3T3-L1 Adipocytes ...........90 
    4.4.2. SiRNA Reduces the Amount of mRNA Levels of iPLA
2
? and iPLA
2
? in  
              3T3-L1 Adipocytes ...........................................................................................91 
    4.4.3. Pretreatment of siRNAs Targeting iPLA
2
? or iPLA
2
? Inhibits Insulin- 
              Stimulated Glucose Uptake in 3T3-L1 Adipocytes ..........................................92 
CHAPTER 5. SUMMARY AND FUTURE STUDIES ....................................................98 
REFERENCES ................................................................................................................103 
APPENDIX I ABBREVIATIONS ..................................................................................131 
APPENDIX II Protocol of Glucose Uptake Assay ..........................................................135 
APPENDIX III Protocol of Lipid Rafts Separation .........................................................138 
APPENDIX IV Protocol of siRNA Transfection Using Electroporation ........................140 
 
 xiii
 
LIST OF TABLES 
 
Table 1. Calcium-independent Group VI phospholipase A
2
 .............................................11 
Table 2. Calcium-independent phospholipase A
2
 inhibitors..............................................16 
Table 3. Oligonucleotide primer sequences for RT-PCR ..................................................70 
Table 4. Oligonucleotide siRNA sequences ......................................................................93 
 
 
 
 
 
 
 
 
 
 
  
 
 xiv
LIST OF FIGURES 
 
Figure 1. Three pathways initiated by insulin signaling ....................................................30 
Figure 2. Procedure for preparation of subcellular fractionations .....................................57 
Figure 3. mRNA levels of cPLA
2
, iPLA
2
? and iPLA
2
? in 3T3-L1 cells during hormone- 
              stimulated differentiation .....................................................................................71 
Figure 4. Effects of BEL on insulin-stimulated glucose uptake in 3T3-L1 adipocytes .....72 
Figure 5. Insulin-stimulated glucose uptake in 3T3-L1 adipocytes treated with  
              propranolol ...........................................................................................................73 
Figure 6. Effects of phospholipase A
2
 inhibitors on insulin-stimulated glucose uptake in  
               3T3-L1 adipocytes ..............................................................................................74 
Figure 7. Exogenous arachidonic acid effects on insulin-stimulated glucose uptake in  
               BEL pretreated 3T3-L1 adipocytes .....................................................................75 
Figure 8. Exogenous arachidonic acid effects on insulin-stimulated glucose uptake in  
               3T3-L1 adipocytes ..............................................................................................76 
Figure 9. BEL effects on insulin-stimulated glucose uptake in L6-GLUT4 myotubes .....77 
Figure 10. Effects of BEL on insulin-dependent insulin receptor phosphorylation ..........78 
Figure 11. Effects of BEL on insulin-dependent Akt phosphorylation .............................79 
Figure 12. BEL effects on insulin-induced GLUT4 translocation to plasma membrane ..80 
Figure 13. Distribution of proteins between lipid raft and non-raft fractions....................81
 
 xv
Figure 14. BEL effects on insulin-induced GLUT4 incorporation into lipid rafts ............82 
Figure 15. 3T3-L1 adipocytes after electroporation ..........................................................94 
Figure 16. Electroporation efficiency in 3T3-L1 adipocytes .............................................95 
Figure 17. Effects of siRNA transfection on the mRNA levels of iPLA
2
? and iPLA
2
? in  
                 3T3-L1 Adipocytes ...........................................................................................96 
Figure 18. Effects of siRNA gene silencing of iPLA
2
? and iPLA
2
? on insulin-stimulated  
                  glucose uptake in 3T3-L1 adipocytes ..............................................................97 
 
 1
  CHAPTER 1. INTRODUCTION 
?
Over the past few decades, obesity has represented one of the most serious public 
health problems on a global scale. According to recent reports, there are at least 400 
million obese adults around the world, and the incidence of obesity keeps rising at an 
alarming rate (1-3). In correlation with the rise in obesity, the prevalence of type 2 
diabetes, cardiovascular diseases and other pathological consequences is also increasing 
(4, 5). Numerous studies have demonstrated that obesity, and obesity-related metabolic 
diseases are characterized with abnormal energy storage and utilization, which is closely 
linked to adipose tissue dysfunction (6-8). As a central place for calorie storage, as well 
as an important secretory organ, adipose tissue plays critical roles in modulating whole 
body lipid and glucose homeostasis. A better comprehension of the physiological 
functions of adipocytes is essential for dissecting the pathophysiological origin and 
exploring possible therapies for obesity and associated diseases.  
Phospholipase A
2
 (PLA
2
) is a diverse group of acyl-hydrolases that catalyze the 
cleavage of the sn-2 fatty acyl bond of glycerophospholipids, resulting in the liberation of 
free fatty acid and lysophospholipid. These two products are both precursors for signaling 
molecules, which are particularly important for various physiological processes. Based on 
the activity dependence on calcium ion and sequence homology, the mammalian PLA
2
 
 2
superfamily are categorized into five principal types, namely the secreted PLA
2
 (sPLA
2
), 
the cytosolic Ca
2+
-dependent PLA
2
 (cPLA
2
), the platelet-activating factor acetylhydrolase 
(PAF-AH), lysosomal PLA
2
 (LPLA
2
), and the cytosolic Ca
2+
-independent PLA
2
 (iPLA
2
) 
(9). Most recently, a calcium-dependent intracellular PLA
2
 was identified in white 
adipose tissue, and denoted as adipose PLA
2 
(AdPLA) (10). 
In the PLA
2
 superfamily, iPLA
2
 enzymes are among the most recently described 
and least well-characterized lipases. It is generally accepted that the iPLA
2 
enzymes have 
multiple biological functions in homeostatic glycerophospholipids membrane remodeling, 
cell growth and proliferation, eicosanoid metabolism, apoptosis, gene expression, 
chemotaxis, signaling transduction and calcium entry (11-14). Importantly, there is an 
increasing body of evidence indicating the important roles of iPLA
2
 in whole body 
metabolism, including adipocytes differentiation (15), insulin secretion (16-21), glucose 
homeostasis (22, 23), hepatic adipogenesis (24, 25), and mitochondrial functioning (26, 
27). Based on these data, various investigations have postulated the connections between 
iPLA
2
 and obesity, type 2 diabetes, fatty liver disease and other manifestations of the 
metabolic syndrome (25).  
It is noteworthy that as 3T3-L1 fibroblasts differentiate into adipocytes, 
dramatically increased gene expression occurs in two members of iPLA
2
 family: iPLA
2
? 
(iPLA
2
-GVIE) and iPLA
2
? (iPLA
2
-GVID) (28). Similar results have also been found in 
other iPLA
2
 members: iPLA
2
? (iPLA
2
-GVIA) and iPLA
2
? (iPLA
2
-GVIB) (15). More 
interesting, Su and coworkers have demonstrated that the gene knockdown of iPLA
2
? and 
iPLA
2
? inhibits hormone-induced adipocyte differentiation by preventing peroxisome 
 3
proliferator-activated receptor ? (PPAR?) and the CCAAT/enhancer-binding protein ? 
(C/EBP?) expression (15). In addition, the protein levels of iPLA
2
? and iPLA
2
? were 
found both significantly increased in white adipose tissue of genetically obese fa/fa rats, 
compared to the lean controls (15). All of these results have underlined the importance of 
iPLA
2
 in adipocyte metabolism. However, the roles of iPLA
2
 in adipocyte biology, 
particularly the response to insulin, are currently unknown.  
 The objective of this study was to investigate the possible cellular functions of 
iPLA
2
 in insulin-stimulated adipocytes. In this study, we demonstrate a role for iPLA
2
 in 
glucose transport in the presence of insulin. To elucidate the iPLA
2
 function in insulin-
stimulated glucose uptake, we utilized a specific inhibitor of iPLA
2
,
 
Bromoenol lactone 
(BEL) and small interfering RNA (siRNA) technique to selectively inhibit iPLA
2
 in 3T3-
L1 adipocytes. We further examined the effects of BEL-induced iPLA
2 
inhibition in 
GLUT4 translocation and insulin signaling. Our studies are the first to demonstrate the 
significance of iPLA
2
 in insulin action of fully differentiated adipocytes. 
 4
CHAPTER 2. REVIEW OF LITERATURE 
?
2.1. PHOSPHOLIPASE A
2
  
              The superfamily of phospholipase A
2
 (PLA
2
) is composed of a group of 
enzymes catalyzing the cleavage at the sn-2 position of glycerophospholipids. This 
hydrolysis reaction results in the release of free fatty acids and lysophospholipids (lyso 
PL), and both products serve as important precursors for bioactive molecules that can 
exert a multitude of biological functions (9). As a typical PLA
2
-derived free fatty acid, 
arachidonic acid (AA) is particularly important, because it can be converted by varied 
downstream metabolic enzymes, such as prostaglandin synthases, cytochrome P450 
proteins, and lipoxygenases, into numerous biologically active lipophilic compounds 
called eicosanoids, including prostaglandins (PGs), leukotrienes (LTs) and lipoxins (29). 
Through binding to specific G-protein receptors, these eicosanoids play critical roles in a 
wide range of physiological and pathological processes such as sleep regulation, immune 
responses, inflammation, and pain perception (30, 31). Lysophospholipids  
lysophosphatidic acid (LPA) and lysophosphatidylcholine (LPC), or their metabolites 
such as platelet activating factor (PAF), are lipid mediators that are essential for cell 
proliferation, survival and migration (32, 33). Since all metabolites mentioned above are 
 5
            greatly modulated by a variety of extracellular stimuli, and highly involved in the stimuli-
mediated signaling pathways, the enzymes catalyzing the initial step, PLA
2
s, are             
important for these signaling processes. As well, PLA
2
s often play vital roles in cell 
membrane homeostasis by contributing to the recycling of fatty acids moieties within 
membrane phospholipids and assisting phospholipid mass regulation (11).  
2.1.1. Classification of Phospholipase A
2
 
             There are more than 27 different proteins exerting PLA
2
 enzymatic activities that 
have been identified and cloned in mammalian systems. Initially, these proteins are 
grouped by a numbering system which is based on the nucleotide and amino acid 
sequence criteria (13, 34-36). However, the most commonly used classification of PLA
2
 
is based on the biochemical commonalities. Regarding the disparities of catalytic 
mechanism, as well as the difference in functional and structural features, the superfamily 
of PLA
2
 are traditionally grouped into five primary types, named the secreted PLA
2
s 
(sPLA
2
), the cytosolic calcium-dependent PLA
2
s (cPLA
2
), the platelet-activating factor 
acetylhydrolases (PAF-AH), the lysosomal PLA
2
, and cytosolic calcium-independent 
PLA
2
s (iPLA
2
) (37). Most recently, a new group of PLA
2
s has been identified and 
characterized in white adipose tissue (10). Because of its unique expression in adipose 
tissue, this 18 kDa protein was termed as Adipose PLA
2 
(AdPLA) (10). 
            2.1.2. Secreted Phospholipase A
2
 
  The secreted PLA
2
s (sPLA
2
s) group is the largest and first discovered PLA
2
 type, 
which have been isolated from a variety of sources such as bacteria, plants, insects, 
 6
reptiles and mammals (38). In mammalian system, there are 10 sPLA
2
 member proteins, 
which are ubiquitously expressed in all studied tissues with group specificities (13). They 
are proteins with small molecular weights around 14?18 kDa, and structurally 
characterized with multiple disulfide bonds, highly conserved Ca
2+
-binding loop, as well 
as catalytic histidine sites (9, 13).  The sPLA
2
s require comparably high concentrations of 
Ca
2+
 (?M levels) for their catalytic activities (9, 38). One of the primary functions of 
sPLA
2
s is to release AA from cellular membrane. The generated AA subsequently enters 
the eicosanoids synthesis cascades (39, 40); therefore, the sPLA
2
s appear to be involved 
play a role in several inflammatory diseases, such as rheumatoid arthritis, adult 
respiratory distress syndrome (ARDS), inflammatory bowel disease, pancreatitis and 
sepsis (41, 42). Furthermore, sPLA
2
s were suggested to play a role in the hydrolysis of 
low-density lipoprotein (LDL) and thus, may contribute to the development of 
atherosclerosis (9, 42). Other biological functions of sPLA
2
s, as reviewed by Kudo (13), 
include digestion, exocytosis, antibacterial action, and anticoagulation. 
            2.1.3. Cytosolic Calcium-Dependent Phospholipase A
2
 
 Cytosolic calcium-dependent PLA
2
 (cPLA
2
) are a group of large cytosolic 
proteins having variable protein masses which range from 61 to 114 kDa. At present, 6 
intracellular PLA
2
s are assigned to this group based on the sequence similarity. Generally, 
the cPLA
2
s contain an active serine site and a Ca
2+
 dependent lipid binding C2 domain, 
and both domains are important for a full activation of these enzymes (37). Among those 
serine acylhydrolases, cPLA
2
? (GIVA-PLA
2
) is the most extensively studied enzyme, 
and it is widely expressed in mammalian cells (43). The cPLA2? is the key enzyme that 
 7
mediates the release of arachidonic acid for the production of eicosanoids in response to 
extracellular stimuli. Thus its activation is important in physiological processes under 
various conditions in both health and disease (44). Numerous lines of evidence have 
demonstrated that cPLA2? contributes to the pathogenesis of a variety of inflammation 
diseases such as allergic reactions, acute lung injury, pulmonary fibrosis, brain injury, 
arthritis, bone resorption (44). This enzyme is also involved in the regulation of apoptosis, 
ulceration, tumorgenesis, pregnancy and labor (13). 
2.1.4. Platelet-Activating Factor Acetylhydrolase 
Platelet-activating factor acetylhydrolases (PAF-AHs) catalyze the acylhydrolysis 
at the sn-2 position of PAF (13). This group is composed of four serine enzyme assigned 
to two numbering groups GVII and GVIII (9). Lipoprotein-associated PLA
2
 is known to 
be one of GVII PLA
2
s, and it is the only secretory member in this group; the other three 
are all cytosolic proteins (9). PAF-AHs are showed to have effects in inhibiting the 
progression of atherogenesis, protecting cells against oxidative stress, and regulating 
brain development (9). 
2.1.5. Other Phospholipase A
2
 
The last two groups of PLA
2
s, lysosomal GXV phosphoslipase A
2
 (LPLA
2
) and 
adipose GXVI phosphoslipase A
2
 (AdPLA) are recently identified, and their biological 
functions remain unclear. LPLA
2
 is a protein localized to lysosome with Ca
2+
-
independent PLA
2
 and transacylase activities (45, 46). AdPLA contains a histidine lipase 
motif and possesses a Ca
2+
-dependent PLA
2
 activity
 
(10).  
 8
2.2. CALCIUM-INDEPENDENT PHOSPHOLIPASE A
2
  
Calcium-independent PLA
2
s (iPLA
2
) are high-molecular weight cytosolic 
proteins utilizing a serine active site. They do not require Ca
2+
 for catalysis. At present, 
up to 6 proteins from different mammalian sources have been recognized as iPLA
2
 family 
members. 
2.2.1. Identification and Characterization of Calcium-Independent Phospholipase 
A
2
 
             The first iPLA
2
 from a mammalian source was identified as a protein with a 
molecular weight about 85 kDa in P388D1 macrophages (47, 48), then purified, 
characterized and further cloned from Chinese hamster ovary (CHO) cells (47, 49, 50). It 
is the most extensively studied enzyme of this group, designated as group VIA PLA
2
 
(iPLA
2
-VIA) (47, 50, 51). iPLA
2
-VIA includes at least 5 splice variants with similar 
structural features with ankyrin repeats (13, 25, 50). Due to exon-skipping and intron-
insertion in gene translation process, three of the variants, iPLA
2
-VIA-1, -2, -3, possess 
both eight ankyrin repeated domains and the N-terminal catalytic serine, while other two, 
iPLA
2
-VIA-Ankyrin-1, and -Ankrin-2 are enzymatically inactive because of the lack of 
catalytic serine (52, 53). It is reported that each splice variant of iPLA
2
-VIA exhibits 
specific tissue distribution, and that this enzyme is ubiquitously expressed in a wide range 
of cells and tissues (52, 53). 
             In 2000, another mammalian protein possessing iPLA
2
 enzymatic activity was 
identified, and named as iPLA
2
-VIB by searching nucleic acid data base (54). Compared 
 9
to iPLA
2
-VIA, this enzyme has several highly conserved sequences in the C-terminal, but 
little homology at the N-terminal (54). It is also ubiquitously distributed in all tissues 
examined, and presumably membrane-bound, since it is predominantly presented in the 
membrane fraction when transfected into cells (54, 55). Besides the serine catalytic site, 
iPLA
2
-VIB is rich in serine and threonine residues, providing many potential 
phosphorylation sites for protein kinases A and C, proline-directed kinases, and mitogen-
activated protein kinase (MAPK) (54). These putative phosphorylation sites suggest 
possible mechanisms for regulatory controls on the activities of iPLA
2
-VIB enzyme (13). 
There are 4 methionine residues acting as potential translation initiation sites in iPLA
2
-
VIB, which produce distinct
 
sizes of its protein (56); the expression
 
of the individual 
splice variant of iPLA
2
-VIB is unique
 
in different cell types, as well as different cellular 
compartments (56, 57).              
Recently, there are four novel iPLA
2
 family members identified and termed as 
iPLA
2
-VIC, -VID, -VIE, and-VIF, respectively with the terminology of group numbering 
system (9). The iPLA
2
-VIC is an integral membrane protein expressed in neurons of 
human and mice, sharing some sequence similarity to iPLA
2
-VIA. It was previously 
reported to act as a neuropathy target esterase (NTE) (58, 59). In addition to the esterase 
role in brain development, iPLA
2
-VIC can first hydrolyze the sn-2 fatty acid from 
phosphatidylcholine (PC), and then release the fatty acid at the sn-1 site, which might be 
important for membranes homeostasis in neurons (60). Other three novel iPLA
2
s had 
been found in adipose tissue with activities other than phospholipase (28). Jenkins and 
coworkers have shown that these iPLA
2
s are capable of catalyzing the hydrolysis of both 
 10
linoleic acid (LA) and AA at the sn-2 position in the absence of free Ca
2+
 (28). In 
addition to the phospholipase activity, they also possess enzymatic activities such as 
triacylglycerol lipase and acylglycerol transacylase (28). It is interesting that, in some 
papers, cPLA
2
-IVC (cPLA
2
?) is also classified as one iPLA
2
 family member, since its 
PLA
2
 activity is not dependent on free Ca
2+  
(61). 
In literature, these enzymes are also referred to as iPLA
2
?, -?, -?, -?, -?, and -? 
(iPLA
2
-? being used for paratin, a non-mammalian enzyme structurally similar to cPLA
2
-
?) (13). Table 1 provides a brief summary for the classification of iPLA
2
 family. Due to 
the simplicity of this name system, these notations will be used in the following parts of 
this dissertation. 
 11
Table 1. Calcium-Independent Group VI phospholipase A
2
 
 
Group 
 
Molecular Mass 
(kDa) 
 
Features 
 
Alternative 
Names 
 
 
iPLA
2
-VIA 
 
 
47-90 
 
Ankyrin repeats 
 
iPLA
2
? 
iPLA
2
-VIB 
 
60-91 Membrane-bound 
iPLA
2
? 
iPLA
2
-VIC 
 
 
146 Integral membrane 
protein 
iPLA
2
?, 
neuropathy target 
esterase  
(NTE) 
iPLA
2
-VID 
 
 
53 Acylglycerol 
Transacylase, 
triacylglycerol 
lipase 
iPLA
2
?, 
adiponutrin 
iPLA
2
-VIE 
 
 
57 Acylglycerol 
Transacylase, 
triacylglycerol 
lipase 
iPLA
2
?, 
transport secretion 
protein-2.2 
(TTS-2.2) 
iPLA
2
-VIF 
 
 
28 Acylglycerol 
Transacylase, 
triacylglycerol 
lipase 
iPLA
2
?, 
gene sequence 2 
(GS2) 
 
 
 
The table has been adapted from (9) and modified based on (13, 28). 
  
 12
2.2.2. Regulations of Calcium-Independent Phospholipase A
2
 Activation 
Although the activity of iPLA
2
 is calcium independent, it is showed that Ca
2+
 is 
still highly involved in the regulation of iPLA
2
 activities (11, 14). The iPLA
2
? is reported 
to be activated with the depletion of intracellular Ca
2+
 concentration or the suppression of  
interaction of calmodulin with target binding proteins, resulting in AA release in resting 
smooth muscle cells (62). Similar results can be also found in research on pancreatic 
islets (63). Recently, Jenkins and coworkers identified the calmodulin binding site in 
iPLA
2
? and verified this physically interaction: the activity of this enzyme can be 
inhibited or activated via the binding or separation of calmodulin, respectively (64).  
Several studies have also suggested the involvement of protein kinase C ? (PKC?) in the 
regulation of iPLA
2
? activity (65-67). There are several possible underlying mechanisms:  
1.) The regulatory effects on iPLA
2
 are realized via the phosphorylation of an associated 
regulatory protein. 2.) The protein kinases may serve as a co-factor for iPLA
2
 activation 
without direct phosphorylation (68). Other lines of evidence have suggested that PKC 
affects the association of iPLA
2
 to membrane compartments, resulting in the activation of 
iPLA
2 
(65, 69). Other kinases, such as p38 MAPK is showed to be implicated in the 
thrombin-stimulated cellular regulation on iPLA
2
 activity (70). 
Under certain experimental circumstances, adenosine triphosphate (ATP) is 
reported to activate iPLA
2
 using in vitro assays, or to protect the enzyme from losing 
activity (53, 71). Moreover, reactive oxygen species (ROS), including superoxide anion, 
hydroxyl radical and hydrogen peroxide have been known to potentiate iPLA
2
 activity 
(72-74). When the uterine stromal cells were stimulated by hydrogen peroxide, a 
 13
significant increase in iPLA
2
 was detected via the increased calcium-independent AA 
release (73). Similarly, in RAW 264.7 cells or mouse peritoneal macrophages, both 
hydrogen peroxide and superoxide anion treatments led to increases in iPLA
2
 activity 
(75). The underlying mechanism was proposed by Balboa and Balsinde: the oxidative 
stress or damage induced lipid peroxide accumulation, subsequently changed the physical 
state of membrane substrate for iPLA
2
, leading to the enzyme activation (76). 
Furthermore, other regulatory factors influencing iPLA
2
 activities include 
proteolytic processing (50, 77, 78), and translational/posttranslational modifications (26, 
54, 79).  
2.2.3. Inhibition of Calcium-Independent Phospholipase A
2
 
As indicated above, the presence of catalytic serine in iPLA
2
 indicates that it is 
readily inhibited by hydrophobic serine site-directed inhibitors. At present, several 
chemical inhibitors are available, including methyl arachidonyl fluorophosphonate 
(MAFP) (51, 80), fatty acyl trifluoromethyl ketones and tricarbonyls (e.g. arachidonyl 
trifluoromethyl ketone (AACOCF
3
)) (49), and bromoenol lactone (BEL) (49, 81, 82). As 
shown as Table 2, MAFP and AACOCF3 share a common chemical structure as an 
arachidonyl tail, which can be coupled to a Ser-reactive group.? Because of the similarity 
in reactive-site of iPLA
2
 and cPLA
2
, the inhibitors MAFP and AACOCF3 fail to 
distinguish iPLA
2
 and cPLA
2
. Additionally, recent study has shown that different iPLA
2 
isoforms have distinct inhibition profiles across specifies and tissues, and the inhibitory 
effects of MAFP on iPLA
2
? and membrane-associated iPLA
2
? are limited to the specific 
 14
species and tissues: the microsomal iPLA
2
s are not inhibited by MAFP in rabbit kidney 
and heart, and rat kidney, nevertheless, these enzymes are significantly inhibited by 
MAFP in rat heart and brain tissues (61).   
Among inhibitors mentioned above, BEL is the only one which selectively targets 
iPLA
2
 over other calcium-dependent PLA
2
s (49, 81, 82).  BEL serves as a mechanism-
based suicide inhibitor, which means that the compound has to first react with iPLA
2
 
enzyme to produce the inhibitory derivatives. The inhibitory BEL derivative results from 
an enzymatic rupture of the lactone ring present in the molecule (as shown in Table 2), 
which covalently binds at or near the active site of iPLA
2
 immediately after hydrolysis 
and prior to diffusion from the catalytic cleft, leading to an irreversible inactivation (81, 
83). Furthermore, all known iPLA
2
s have been shown to be inhibited by BEL (28, 84-86). 
It has also been demonstrated that BEL inhibits the triglyceride lipase activity of iPLA
2
?, 
-? and -? (28). Therefore, BEL is the most prominent inhibitor used to investigate the 
cellular functions of iPLA
2
.  
Unfortunately, BEL is also reported to react with other enzymes, such as the 
Mg
2+
-dependent phosphatidic acid phosphohydrolase (PAP-1, lipin) (84). So, it is 
necessary to rule out the roles of PAP-1 by using PAP-1 inhibitor propranolol at high 
concentrations (greater than 150 ?M) when BEL is utilized in iPLA
2
 studies (87-89).  
More recently, Jenkins et al. (54) identified two chiral specific enantiomers for 
BEL, (S)-BEL and (R)-BEL. They also found that the inhibiting preference of these two 
isomers is different: (S)-BEL was considerably more selective for iPLA
2
? in comparison 
 15
to iPLA
2
? whereas (R)-BEL inhibited iPLA
2
? much better than iPLA
2
? (54). These 
results make BEL a more powerful compound to distinguish between these two iPLA
2
 
enzymes.  
Besides the application of chemical inhibitors, selective inhibition on iPLA
2
s can 
be performed through targeted gene silencing strategies. Those studies using antisense 
oligonucleotide or small interfering RNA (siRNA) provide more compelling evidence for 
the involvement of iPLA
2
 in interested cellular process in vitro (15, 57, 90). The iPLA
2
 
knockout animal models are also available for investigators to more accurately determine 
the physiological roles of iPLA
2 
in vivo (18, 91). 
 16
Table 2. Calcium-Independent Phospholipase A
2
 Inhibitors 
Inhibitor Chemical Structure/Molecular Formula PLA
2
s inhibited 
 
MAFP, 
Methyl Arachidonyl 
Fluorophosphonate  
 
 
?
?
C
21
H
36
FO
2
P 
 
cPLA
2
,  
AdPLA,  
iPLA
2
  
AACOCF
3
,  
Fatty Acyl  
Trifluoromethyl 
Ketone 
 
??
??????????????????????????????????C
21
H
31
F
3
O 
cPLA
2
,  
AdPLA,  
iPLA
2
  
BEL, 
Bromoenol Lactone 
 
 
 
C
16
H
13
BrO
2
 
 
iPLA
2
 ?, ? 
(S)-BEL, 
(S)-Bromoenol 
Lactone 
 
 
C
16
H
13
BrO
2 
 
 
iPLA
2
 ? 
(R)-BEL, 
(R)-Bromoenol 
Lactone 
 
  
C
16
H
13
BrO
2
 
 
iPLA
2
 ? 
 17
2.2.4. Cellular Functions of Calcium-Independent Phospholipase A
2
  
Of the
 
iPLA
2
 family, iPLA
2
? and iPLA
2
? are predominantly responsive for the 
PLA
2
 activity, whereas other four members show lipase or transacylase in marked 
preference to PLA
2
 activity. Moreover, iPLA
2
? and iPLA
2
? are two iPLA
2
s that have 
been earlier identified and more extensively studied. For these reasons, the cellular 
functions of iPLA
2
 discussed in this section are based on studies about iPLA
2
? and 
iPLA
2
?. 
2.2.4.1. Phospholipid Remodeling 
As mentioned above, intracellular PLA
2
s promote the cleavage of pre-existing 
membrane PL, leading to the production of free fatty acids and 2-lyso PL. The generated 
free fatty acid may be reincorporated into different membrane PL molecules, and the lyso 
PL may be reacylated with a different fatty acid as well. In this case, PLA
2
s are highly 
involved in the ongoing deacylation/reacylation cycle essential for the maintenance of 
membrane homeostasis (12). As a major group of PLA
2
 ubiquitously expressed in 
mammalian system, iPLA
2
s across different tissue sources have been reported to play 
roles in phospholipids remodeling, especially function on the incorporation of long chain 
polyunsaturated fatty acids (LCPUFA) into the glycerophospholipids (92-96). The 
LCPUFA incorporation process is particularly important for inflammatory cells such as 
macrophages and neutrophiles. Macrophages present a high capacity in membrane AA 
incorporation, which is mostly maintained by iPLA
2
 activity. Inhibition on iPLA
2
? via 
BEL treatment or antisense oligonucleotide both lead to reduced AA incorporation into 
 18
PLs and cellular LPC levels in P388D1 macrophages (82). Similar results are also found 
in brain cell studies: it is showed that BEL treatment inhibits the release of 
docosahexaenoic acid from the membrane phospholipids of astrocytes upon ATP 
stimulation (96). In contrast, overexpression of iPLA
2
 ? and iPLA
2
? in HEK293 cells 
results in the accumulation of free fatty acid and LPC, and this event can be blunted by 
BEL (57). Taken together, these findings highlight the importance of iPLA
2
? in 
membrane housekeeping.  
2.2.4.2. Arachidonic Acid Metabolism  
Given that AA and lyso PL can be both metabolized into a range of biologically 
active compounds, PLA
2 
activity are essential for many cell signaling functions. Released 
AA can be directed to the cyclooxygenase (COX) and lipoxygenase pathways for 
eicosanoid synthesis (97). Previous studies have demonstrated that cPLA
2
 and sPLA
2
 are 
the most contributors to AA release in response to a variety of stimuli (34, 98). Recently, 
iPLA
2
 has been found to play a role in cellular signaling initiated by AA release and 
sequential metabolism under certain conditions (66, 99, 100). For example, in RAW261.7 
macrophages, BEL markedly restricted the nitric-oxide-induced AA mobilization (99). In 
zymosan-stimulated P388D1 macrophages, prostaglandin E
2
 (PGE
2
) generation is 
significantly attenuated by BEL treatment or iPLA
2
? antisense (67). Similar results can 
also be found in calcium-ionophore-treated neutrophiles and pancreatic ? cells exposed to 
glucose (89, 101).  These effects appear to be iPLA
2
? dependent.  In these cells, the 
cytosolic enzyme iPLA
2
? is translocated to the membrane fractions in response to the 
stimuli, and iPLA
2
? movement appears to be regulated by protein kinase C (102). Even 
 19
though iPLA
2
? is less well investigated, there is strong evidence showing that this 
enzyme is also involved in AA release that ultimately results in the formation and 
metabolism of eicosanoids in HEK 293 cells (57). 
2.2.4.3. Control of Calcium Entry 
 Agonist-induced depletion of intracellular Ca
2+
 store is known to activate 
channels and capacitative Ca
2+ 
entry (103). Although the mechanisms underlying this 
replenishment remain unclear, two major models are proposed: ?conformational 
coupling? and ?Calcium Influx Factor? (CIF) (98). In the CIF model, a diffusible 
molecule is generated with the diminution of calcium stores, which interacts with the 
store-operated channels sitting in plasma membrane and induces Ca
2+
 flux (98). 
Increasing evidence shows that iPLA
2
 is involved in this process. A possible link between 
iPLA
2
 and calcium signaling was first discovered in 1990?s by two research groups (63, 
104), who reported that the depletion of intracellular Ca
2+
 stores resulted in an increased 
iPLA
2
 enzymatic activity, leading to phospholipid hydrolysis and free fatty acids 
accumulation in rat smooth muscle cells (104) and pancreatic islets (63). More recently, 
several investigations have shown similar results that iPLA
2 
is activated when 
intracellular Ca
2+
 stores are depleted (64, 69, 105). Also, iPLA
2
 appears to serve as a 
regulator of Ca
2+ 
replenishment. In a variety of cells and tissues, such as smooth muscle 
cells (105), rat basophilic leukemia (RBL) cells (106), platelets (107), prostate cancer 
cells (108), inhibition of iPLA
2
? by (S)-BEL or antisence oligonucleotides blocks 
calcium release-activated channels (CRAC) calcium influx. More importantly, LPC can 
reverse the inhibitory effects of (S)-BEL on store-operated channels and restore the Ca
2+
 
 20
influx, indicating that the iPLA
2
 activity-dependent LPC generation is required for the 
activation of store-operated channels. According to these observations, intracellular Ca
2+
 
store exhaustion induces the production of calcium influx factor, which interferes with 
the combination of calmodulin and iPLA
2
?. This dissociation with calmodulin triggers 
the enzymatic activity of iPLA
2
? leading to the production of lysophospholipids, which 
sequentially activate the store-operated channels and capacitative Ca
2+
 influx. Upon 
refilling of the stores and cessation of calcium-influx factor production, calmodulin 
would reassociate with iPLA
2
?, and terminate repletion of Ca
2+
 (105). Additionally, some 
other investigations propose another target regulated by iPLA
2
 activity affecting the Ca
2+
 
homeostasis, transient receptor potential (TRP) channels (108, 109). For example, in the 
Chinese hamster ovary cells, the response of cold-sensitive channel transient receptor 
potential (melastatin)-8 (TRPM8) to exogenous stimuli were abolished by inhibition of 
the iPLA
2
. Both lysophospholipids and  polyunsaturated fatty acids (PUFAs) are involved 
in regulating the activity of these channels (109). 
2.2.4.4. Secretion and Exocytosis 
Upon activation of PLA
2
, the
 
accumulation of lysophospholipids and free fatty 
acids may occur in local areas of membrane, and consequently change the membrane 
fluidity or promote non-bilayer structures, which eventually encourage the fusion of 
biological membranes. An accumulating body of evidence has confirmed the linkage of 
iPLA
2
 activity and secretion functions. For example, the lysozyme secretory responses to 
stimuli was diminished by iPLA
2
? antisense oligonucleotide treatment in U937 cells, and  
reduced levels of LPC were also observed (110). However, reconstitution of LPC 
 21
ultimately restored the lysozyme secretion, indicating that iPLA
2
? -mediated generation 
of LPC is important for full secretion to take place (110). Similar effects of iPLA
2 
inhibition in secretion can be also established in other studies, including glucose-
stimulated insulin secretion in pancreatic ? cells (89, 111, 112), exocytosis of amylase 
from parotid acinar cells (113), and lysosome-mediated interleukin-1? (IL-1?) secretion 
(114). In many exocytosis processes, increased cytosolic calcium is known to be 
required. BEL treatment has been shown to inhibit such processes, including release of 
neurotransmitter (115) and mast cell granule (116). Therefore, the regulatory roles of 
iPLA
2
 on calcium entry mentioned above may explain some aspects of its importance in 
secretion. 
2.2.4.5. Other Cell Signaling Functions 
Other cellular signaling functions of iPLA
2
 consist of cell growth and 
proliferation (117, 118), apoptosis (119-121), chemotaxis (122, 123), gene expression (35, 
124, 125), endothelium-dependent vascular relaxation (126), and development of cardiac 
ischemia (127, 128), none of these will be discussed in detail in this review. 
2.2.5. Calcium-Independent Phospholipase A
2
 in Metabolism 
Recently, several studies established the emerging roles of iPLA
2
 in whole body 
metabolism, including the adipocytes differentiation (15), glucose-stimulated insulin 
secretion (16-21), hepatic adipogenesis (24, 25), glucose homeostasis (22, 23), and 
cardiac myocytes mitochondrial functioning (26, 27). These findings spotlight iPLA
2
 as a 
 22
possible therapeutic target for obesity, type II diabetes, fatty liver disease and other 
manifestations of the metabolic syndrome (25).  
2.2.5.1. Glucose-Stimulated Insulin Secretion 
Pancreas dysfunction is a typical pathological characteristic of diabetes mellitus; 
thus, proper pancreatic ? cell function is essential for the whole body homeostasis. It is 
well understood that glucose-stimulated insulin secretion in pancreatic ?-cells and related 
cell lines is associated with increased hydrolysis of phospholipids (89, 111): glucose 
stimulation results in both insulin secretion and AA release, and inhibitors of 
phospholipase suppress the secretion of insulin. Involvement of iPLA
2
 in such a process 
was initially suggested by Ramanadham et al. in 1993.  BEL, the selective inhibitor of 
iPLA
2
 completely suppressed insulin secretion, as well as phospholipid hydrolysis (111). 
More recent studies extend these findings by using the INS-1 cells (rat insulinoma cell) 
transfected with iPLA
2
?. In the iPLA
2
? overexpressed cells, improvement was observed 
in both glucose responsiveness and insulin secretion in response to cyclic adenosine 
monophosphate (cAMP)-elevating agents (112, 129), which induce the translocation of 
iPLA
2
? to a perinuclear region facilitating the phospholipid hydrolysis in endoplasmic 
reticulum (ER) membrane. Released AA is postulated to serve as a signaling molecule for 
insulin secretion. Consistent with this hypothesis, inhibition of the insulin secretion was 
restored by exogenous AA (15). Studies in vivo provide supplementary proof for the 
roles of iPLA
2
? in this process. The iPLA
2
? knockout mice showed insufficient insulin 
secretion in response to glucose, developed severe glucose intolerance response to high-
fat diet, and exhibited more sensitivity to the streptozotocin (STZ) treatment (22, 23). On 
 23
the contrary, iPLA
2
? transgenic mice have a lower fasting glucose and higher insulin 
levels compared to wild type controls (23).  
2.2.5.2. Adipocyte Differentiation 
The hormone-stimulated differentiation of pre-adipocytes into mature adipocytes 
is effectively controlled by CCAAT/enhancer-binding protein (C/EBP) family and 
peroxisome proliferator-activated receptor ? (PPAR?), which are also important for 
glucose and lipid metabolism (130, 131). Recently, Su et al. (15) reported the 
indispensable roles of iPLA
2 
in 3T3-L1 adipocyte differentiation, as well as the 
relationship between iPLA
2 
activity and PPAR? regulation.  In this study, iPLA
2
? and 
iPLA
2
? were upregulated during the differentiation procedure, while cPLA
2
 was reduced 
to background level in mature adipocytes. Loss of iPLA
2
 ? and iPLA
2
? by BEL treatment 
or small interfering RNA inhibited the hormone-induced adipocyte differentiation via 
preventing PPAR? and C/EBP? gene expression. However, when the cells were treated 
by a PPAR? agonist, trigolitazone, the adipogenesis was restored. The authors speculated 
that LPC generated from iPLA
2
-catalyzed phospholipid hydrolysis, derivate LPA or other 
bioactive metabolites may serve as activators of PPAR? (15).  In addition, the protein 
levels of iPLA
2
? and iPLA
2
? were found both significantly increased in white adipose 
tissue of genetically obese fa/fa rats, compared to the lean controls (15). Another study 
also reported that during the differentiation of 3T3-L1 cells into adipocytes, there was 
dramatically upregulated gene expression in two other members of iPLA
2
 family: iPLA
2
? 
and iPLA
2
? (132). All these results suggest that iPLA
2
 may play important roles in the 
energy homeostasis in adipocytes.  
 24
2.2.5.3. Hepatic Lipogenesis 
Besides PPAR? and C/EBP?, sterol regulatory element binding proteins 
(SREBPs) are other key modulators for lipogenesis (133, 134). Calcium-independent 
PLA
2
 is also shown to be associated with the regulation of SREBP. It is well-established 
that PUFAs are capable of influencing SREBPs processing and translocation, resulting in 
the inhibition on fatty acid synthase, acetyl CoA carboxylase, and other lipogenic genes 
associated with triglyceride synthesis (135, 136). Based on these observations, Wilkins 
research group initiated a study on the regulatory effects of iPLA
2 
in SREBP (98). In this 
study, iPLA
2
 inhibition and overexpression in HepG2 cells resulted in the increases and 
decreases of the transcriptional activity of SREBP expression, respectively. These results 
indicate a potential role of iPLA
2 
in hepatic adipogenesis. 
2.2.5.4. Mitochondrial Functions 
It has been demonstrated that iPLA
2
? is a membrane-associated protein which is 
localized to mitochondria (137-139). In recent studies, iPLA
2
? has been reported to play 
an essential role in maintaining proper mitochondrial functions (26, 27). In isolated rabbit 
renal cortex
 
mitochondria, inhibition of iPLA
2
? by (R)-BEL accelerates lipid
 
peroxidation 
and swelling, and blocks mitochondrial permeability transition, which is critical for cell 
apoptosis (140, 141). More importantly, the iPLA
2
 knockout mice perform reduced 
Complex IV-mediated oxygen consumption, decreased cold tolerance, diminished 
exercise capacity but low capacity to gain weight (26, 27). Taken together, these findings 
suggest that the loss iPLA
2
 ?
 
induced whole body energy unbalance. It is intriguing that 
 25
in iPLA
2
?-overexpressed myocytes or iPLA
2
? transgenic animals, however, the 
mitochondria exhibit reduced respiratory control quotient (26). Although future studies 
are necessary for better understanding of the regulatory effects of iPLA
2
? in these 
procedures, these findings underscore the significance iPLA
2
? in energy homeostasis. 
2.3. INSULIN SIGNALING 
Insulin is the most influential hormone secreted by pancreas. This hormone 
lowers the blood glucose level by stimulating glucose influx into peripheral tissues, and 
promoting the fat deposition in adipocytes, glycogen synthesis in muscle and liver, as 
well as inhibiting the production and release of glucose from the liver (142). As a result, 
the key actions of insulin include modulating the whole body energy homeostasis, 
promoting proper metabolism, and helping body to keep normal body weight (143). 
Those actions are performed through a series of intracellular signal transduction. 
 When insulin is bond to the alpha-subunit of the insulin receptor (IR) residing on 
cell membrane, a transmembrane conformational change occurs, and the beta-subunit of 
IR undergoes a rapid autophosphorylation (addition of a PO4 group from adenosine 
triphosphate) reaction, leading to the phosphorylation at multiple tyrosine (Tyr) residues, 
which is necessary for the appropriate recognition of IR substrates and the activation of 
IR tyrosine kinase (IRK) activity. Subsequently, the downstream cytosolic moieties are 
tyrosine phosphorylated by, which include insulin receptor substrate family proteins 
(IRS-1, to -6), growth factor receptor bound 2 (Grb2)-associated binder-1 (Gab 1), Src 
homologous and collagen (Shc) proteins, signal-regulated proteins (SIRPs), Cbl, and 
 26
adapter protein with pleckstrin homology and Src homology domains (APS) (144). These 
proximal intracellular phosphorylation targets serve as docking sites for downstream 
effector proteins containing Src homology 2 (SH2) and phosphotyrosine binding (PTB) 
domains (145). Three major signaling pathways are propagated (Figure 1): 
phosphatidylinositol 3-kinase (PI3K) pathway, Cbl/CAP pathway and the mitogen-
activated
 
protein kinase (MAPK) pathway (144, 146).  
2.3.1. Phosphatidylinositol 3-Kinase Pathway 
Among those three pathways activated by insulin, PI3K cascade is the best 
established. In addition to glucose transport, it also mediates other metabolic functions of 
insulin. The IRS family in the PI3K cascade has attracted considerable attention due to 
their key roles in triggering insulin signaling (147). Upon tyrosine phosphorylation, the 
IRS proteins are associated with the 85-Kda regulatory subunit of phosphoinositide-3-
kinase (PI3K) thereby activate PI3K. Analogous to p85 PI3K subunit, other SH2 domain-
containing proteins, such as Nck, Fyn, Grb-2, and SHP2, also bind to IRS proteins and 
mediate the various aspects of insulin action (142, 148). PI3K regulates several signaling 
pathways through the generated lipid messenger, phosphotidyl-3, 4, 5-triphosphophate 
(PIP3), which stimulates phosphoinositide-dependent kinase (PDK-1) activity and 
initiates the activation of its downstream effectors, such as protein kinase B(Akt/PKB), 
mammalian target of rapamycin (mTOR), p70 S6 kinase (S6K1), and atypical protein 
kinase C isoforms (aPKC?/?) (149-153). Akt is one of the critical targets playing pivotal 
roles in mediating the insulin-responsive metabolic actions (154).The full activation of 
Akt can only be achieved via a dual phosphorylation on the both serine (Ser) and 
 27
threonine (Thr) residues. It is showed that PDK1 phosphorylats Thr
308
 (155), while the 
kinase for Ser
473
 phosphorylation is referred to as either PDK2 (156) or DNA-dependent 
protein kinase (156, 157). It appears that Ser
473
 phosphorylation is required for the 
phosphorylation of Thr
308
 (155). Once activated, Akt detaches from the plasma 
membrane and promotes the translocation of GLUT4 from intracellular compartments to 
cell surface, facilitating the glucose uptake into adipocytes or skeletal muscle cells (154). 
Another target of Akt is glycogen synthase kinase-3? (GSK-3?), which is a negative 
regulator of glycogen synthesis. Ser-phosphorylation of GSK-3? by Akt activates 
glycogen synthase and boosts the glucose anabolism in skeletal muscle cells (158, 159). 
Akt- GSK-3? pathway has also been proposed to mediate the anti-inflammatory effects of 
insulin (158, 159). Some other actions of the activation of Akt include promoting lipid 
deposition and protein anabolism, and regulating cell survival and growth (160-162).  
2.3.2. Cbl/CAP Pathway 
The Cbl/CAP pathway is another pathway mediating glucose uptake upon insulin 
administration. Research evidence for Cbl/CAP cascade remains controversial; however, 
increasing numbers of key effectors in this pathway have been identified in recent studies.  
As a substrate for IR phosphorylation, Cbl undergoes a tyrosine phosphorylation assisted 
by the associated protein substrate, APS (163). And then Cbl-associated protein (CAP) 
targets Cbl to recruit with IR in lipid raft domains, making the tyrosine phosphorylated 
Cbl serve as docking sites for the CrkII/C3G complex (164-166). CrkII (chicken tumor 
virus no.10 (CT10) regulator of kinase II) is an adaptor protein assisting the binding of 
Cbl to Crk SH3-binding guanine nucleotide-releasing factor (C3G), and C3G functions as 
 28
a guanine nucleotide exchange factor for the small GTP binding protein TC10, a protein 
essential for GLUT4 translocation (167), which is also thought to contribute to the 
maintenance of actin structure in 3T3-L1 adipocytes (167, 168). It is proposed that the 
last step of this pathway is associated with actin dynamics and linked to GLUT4 
translocation (169-171). Interestingly, in contrast to the original idea that Cbl/CAP and 
PI3K pathways are independent, some investigations have demonstrated that Cbl can also 
activate PKC?/? in a way paralleled to PI3K pathway (172, 173), and TC10 activation 
was linked to PI3P production, which may affect the GLUT4 translocation independent 
of Cbl/CAP cascade (174).  
2.3.3. Mitogen-Activated
 
Protein Kinase Pathway 
The mitogen-activated
 
protein kinase cascade mediates the insulin receptor 
signaling and triggers the mitogen-activated
 
protein kinase (MAPK) isoform 
p42MAPK/p44MAPK (ERK1/ERK2) cascade, which is not required for insulin's 
metabolic
 
effects but essential for mitogenic signaling manipulating gene expression in 
various cellular processes, like proliferation, differentiation and apoptosis (175). Upon 
the autophosphorylation on tyrosine residues of the insulin receptor, a Shc-Grb2 complex 
is formed, and recruited to the plasma
 
membrane, where it binds to son-of-sevenless 
(Sos), a nucleotide exchange protein that increases the rate of exchange of GTP for
 
GDP 
on Ras (a family of small GTP binding proteins), in turn activates Raf /MAP/ERK kinase 
cascade (176).  This leads to activation of a serine/threonine-tyrosine kinase, MEK, 
which phosphorylates and activates extracellular signal-related kinase ERK1/2. One 
important downstream element phosphorylated
 
by ERKs is p90 ribosomal
 
S6 kinase 
 29
(RSK2) (177). The proposed cellular functions of RSK include regulating gene 
expression via interaction with transcriptional regulators including c-Fos (a cellular 
proto-oncogene belonging to the immediate early gene family of transcription factors), 
estrogen receptor, nuclear factor-?B (NF?B) /inhibitor of NF?B (I?B?), cAMP-response 
element-binding protein (CREBP), controlling cell cycle, modulating protein synthesis, 
and phosphorylating the Ras GTP/GDP-exchange factor, Sos leading to feedback 
inhibition of the Ras-ERK pathway (178).  
 30
 
 
 
 
 
Figure 1. Three pathways initiated by insulin signaling.? Insulin binding to alpha-
subunit of the insulin receptor (IR) results in a rapid autophosphorylation in beta-subunit 
of IR, leading to the phosphorylation and activation of IR substrates. Subsequently, three 
downstream signaling cascades are propagated to mediate the insulin actions. 
 
 31
2.4. INSULIN-STIMULATED GLUT4 TRANSPORT 
The ability of insulin in clearing the blood glucose is mainly achieved by the 
regulation of glucose transporters in insulin-sensitive cells. Under basal conditions, these 
transporters are impounded in an intracellular compartment, while under the stimulation 
of insulin, they are rapidly driven to translocate into the plasma membrane, resulting in 
the glucose uptake of the cells from the plasma (179, 180). Currently there are 13 
members of this family identified including glucose transporter GLUT-1-12, and the H
+
-
myo-inositol transporter HMIT1 (181). Each of them exhibits different tissue 
distributions, kinetic properties and sugar specificity. GLUT-1 to 4, the class I glucose 
transporters, are the best-characterized members of this family (142). Among them, 
glucose transporter 4 (GLUT4) is the predominant one responding to the insulin signaling 
in the fat or muscle cells. 
In the resting state, GLUT4 cycles slowly between the vesicles compartments and 
cell surface, with the vast majority of the transporters localized in the intracellular pool. 
Upon insulin stimulation, the activation of signaling cascades substantially increases the 
rate of GLUT4 exocytosis. Simultaneously, the endocytosis of GLUT4 is attenuated. 
Collectively, the net effect of these steps leads to a dramatic redistribution of GLUT4 to 
the plasma membrane (PM) (179, 182, 183).  
Despite intensive investigation for several years, the insulin-responsive shift of 
GLUT4 in the distribution from intracellular compartments to the PM has not been fully 
deciphered. According to the basic trafficking process of GLUT4 in response to insulin, 
 32
posttranslational regulatory modalities are mainly identified in two biological systems ? 
signal transduction and vesicular transport. 
2.4.1. Insulin Signaling Regulators  
PI3K pathway and Cbl/CAP pathway represent two distinct branches contributing 
to glucose uptake response to insulin stimulation. Therefore, the signaling molecules 
participating in these two cascades must intensively impact the initiation of GLUT4 
translocation.  
As the key factor for the initiation of PI3K cascade, IRS proteins are required for 
insulin-dependent GLUT 4 translocation, which is supported by siRNA and gene 
knockout studies (172, 184). There are also extensive results proving that the activation 
of PI3K and consequent generation of PIP3 are indispensable for GLUT4 translocation. 
Overexpression of p110? subunit of PI3K in 3T3-L1 adipocytes induced an 
approximately 14-fold increase in the basal glucose transport rate, even greater than that 
observed in the stimulated control (185). Similar results were found in study with rat 
adipose cells (186). In opposition, inhibition of PI3K by pharmacological inhibitors (187, 
188), dominant-interfering mutants (187), endogenous inhibitory kinase (189), prevented 
the GLUT4 translocation to the PM, while exogenous PIP3 rescued the GLUT4 fusion 
with PM (188). However, the exogenous PIP3 dependent GLUT4 translocation was 
inadequate to fulfill the glucose uptake (188). Further investigation showed that an 
unmasked GLUT4 COOH terminus was needed to achieve the full function of the 
transporter, and the unmasking process was proposed to be controlled by PI3K activity 
 33
(188) (190), indicating a potential molecular mechanism for regulation on GLUT4 
activity(191). As the key downstream mediator of the PI3K pathway, Akt is directly 
correlated with GLUT4 translocation. The overexpression of a constitutively active, 
membrane-bound form of Akt in NIH3T3 adipocytes promoted GLUT4 translation (192). 
In contrast, downregulation on Akt by dominant-negative mutant (150), competing 
peptides (193), or gene silencing (194-196) inhibited GLUT4 translocation. PKC?/? is 
another downstream effector also involved in GLUT4-mediated glucose transport. The 
activation of PKC?/? has shown to contribute significantly to insulin-stimulated glucose 
uptake (173, 197, 198). For example, PKC? transgenic skeletal cells exhibited an 
enhanced glucose uptake (199), whereas aPKC selective inhibitory peptide significantly 
reduced the glucose transport in insulin-stimulated L6 cells (173), without affecting Akt 
(200, 201). However, the dependence of insulin-stimulated GLUT4 translocation on 
aPKC?/? remains controversial (196), and requires further investigation excluding the 
lurking factors (191). 
In spite of intensive exploration, downstream effectors of Cbl/CAP pathway 
exhibit controversial effects. Overexpressing mutants of CAP and TC10 impede the 
GLUT4 translocation, documenting their significance in this process (167). However, 
gene silencing of CAP, Cbl isoforms, and CrkII fail to impact in the insulin-dependent 
GLUT4 translocation in 3T3L1 adipocytes (202), indicating that they are not required 
components of insulin signaling to GLUT4 transporters. In addition, APS knockout mice 
exhibit increased insulin sensitivity and hypoinsulinemia compared to wild type control 
or APS overexpressed mice, though the transgenic model did not show any negative 
 34
alterations in insulin signaling and glucose uptake (203). Further studies are required to 
explain these observed discrepancies. 
In contrast to insulin-induced ?turning on? actions, GLUT4 translocation can be 
turned off by negative regulation on insulin signaling.  For example, serine/threonine 
phosphorylation of the IRS results into the dissociation of IR:IRS and IRS:PI3K complex, 
eventually blocks the insulin activated GLUT4 translocation (152, 162, 204). On the 
other hand, dephosphorylation of specific kinases by corresponding phosphatases also 
diminishes the insulin signaling. IRS-1 or Akt/ PKC have shown to be the inactivated by 
protein tyrosine phosphatase 1B (PTP1B) (205, 206)  or serine/threonine protein 
phosphatase 2A (PP2A) (207), respectively, resulting in the negative control toward 
GLUT4. In addition, lipid phosphatases, such as tumor suppressor gene phosphatase and 
tensin homolog deleted on chromosome 10 (PTEN) (208, 209) and SH2-containing 
inositol phosphatase 2(SHIP2) (210-212), are thought to negatively modulate insulin 
signaling controlling the duration of signaling. Overexpression of these proteins in 3T3-
L1 adipocytes leads to reduced PIP3 levels, inactivated Akt/PKC, and subsequently 
inhibits GLUT4 translocation (210-212). 
2.4.2. Newly Identified Akt Substrates Involved in GLUT4 Exocytosis 
More recently, a 160 KDa protein was identified in both adipocyte and skeletal 
muscle cells as an Akt substrate and denoted as AS160 (213). AS160 contains six 
consensus Akt phosphorylation domains as well as a Rab GTPase activating protein 
(GAP) domain (214). Since Rab proteins represent the largest branch of the Ras 
 35
superfamily of small GTP-binding proteins, and they regulate several steps of membrane 
transport, including vesicle budding, motility, tethering and fusion (215, 216). It is 
supposed that AS160 could serve as an mediator connecting insulin signaling 
transduction to GLUT4 translocation. Based on current data from AS160 studies, a 
working model has been postulated (217, 218): in the rest cells, AS160 maintains a GAP 
activity and inhibits the downstream Rab protein; after being phosphorylated on multiple 
Akt phosphomotifs in response to insulin signaling, the GAP activity of AS160 is blunted 
by phosphorylation or dissociation, leading to the transition of downstream Rab protein 
from GDP-bound form to a GTP-bound activate form, eventually facilitating GLUT4 
translocation (217, 218). 
SNAREs are membrane-bound partners for the soluble NSF attachment proteins 
(SNPs), and highly involved in the vesicle fusion machinery. Two potential SNAREs are 
demonstrated to be correlated with GLUT4 vesicle fusion: Vesicle-associated membrane 
proteins 2 and 3 (VAMP2 and VAMP3) (145). As a SNAER-associated protein, synip is 
another recent identified Akt substrate, which is supposed to bind syntaxin 4, a target-
SNARE (t-SNARE) localized to the cell surface and VAMP2, a v-SNARE on vesicle, 
and then regulate the docking and fusion of GLUT4 vesicles (219). 
2.4.3. Actin Remodeling 
With regard to insulin action and glucose transport, an increasing body of data 
demonstrates that actin plays a critical role in GLUT4 translocation (220-226). It is 
observed that under insulin stimulation, actin cytoskeleton perform a PI3K-dependent 
 36
remodeling in both adipocytes (225) and muscle cells (226). Additionally, the PI3K 
products, such as PIP3 or PIP2 (phosphotidyl-4, 5-biphosphophate) have been shown to 
bind to the cytoskeletal protein profilin, serving as an organizational site for actin 
remodeling (227). It has been reported that disruption of cortical actin filament formation 
by pharmaceutical inhibitors, inhibits insulin-stimulated GLUT4 translocation (220, 224, 
228). There are also some actin binding proteins indentified to mediate the insulin 
regulator effects, such as actin regulatory protein N-WASP (Neuronal Wiskott-Aldrich 
syndrome protein) (229). Moreover, TC10 is associated with actin dynamics and linked 
to GLUT4 translocation (142, 170). Taken together, it has been postulated that actin 
remodeling may provide a scaffold that coordinates the production of PIP3, recruits other 
signaling molecules, or offers a platform directing the GLUT4 compartments to the 
plasma membrane (PM) (191). 
2.4.4. Calcium Influx 
At the plasma membrane, activated GLUT4 vesicles undergo docking and fusion 
through a process assisted by several SNARE proteins, including VAMP-2, Synip, and 
Syntaxin-4 (230). Such an exocytosis process is very similar to the release of 
neurotransmitter, a process that is generally induced by transient increases in the Ca
2+
 
concentration at the release sites (231-233). Additionally, previous studies have shown 
that molecules chelating Ca
2+
 calmodulin antagonists attenuate the insulin-stimulated 
glucose uptake, along with the reduced GLUT4 translocation in adipocytes (234) or 
skeletal muscle cells (235, 236). Furthermore, other studies have demonstrated that 
insulin stimulation activates the Ca
2+ 
influx in a PI3K dependent manner, leading to 
 37
significantly increased calcium concentration close to the plasma membrane (237, 238). 
Collectively, the fact that increase or decrease Ca
2+
 influx also increase or decrease 
insulin-mediated glucose uptake makes Ca
2+
  influx a potential regulator for insulin-
stimulated GLUT4 translocation (239), and it mainly acts on late steps in the insulin-
signaling pathway (234, 240). In the skeletal muscle cell, it was observed that inhibition 
of Ca
2+
 influx with 2-aminoethoxydiphenyl borate (2-APB) reduced the glucose uptake 
but had no influence the phosphorylation state of PKB or the MAP kinases ERK1/2 was 
not affected by (240). Whitehead, et al. also reported that in the 3T3-L1 adipocytes, 
Ca
2+
/calmodulin plays a fundamental role in eukaryotic vesicle docking and fusion (234). 
Thus, it is proposed that the late steps in insulin signaling might involve an intracellular 
microdomain with increased Ca
2+
 and Ca
2+
 sensing proteins, which directly or indirectly 
modulate GLUT4 exocytosis (230).  
This emerging role of Ca
2+
 influx has been further supported by several potential 
Ca
2+
 censored proteins correlated with this process. Synaptotagmin-VII,  a membrane 
protein in the synaptotagmin family recognized as Ca
2+
 sensors in exocytosis (232, 233), 
is proven to play roles in insulin-mediated glucose uptake and GLUT4 trafficking in 
adipocytes  by a study using synaptotagmin VII-deficient mice, which performed 
impaired insulin response (241). Ca
2+
-calmodulin-regulated actin-filament-attached 
motor protein, Myo1c is also demonstrated to join in this process. Several research 
groups observed that genetic knockdown of Myo1c in 3T3-L1 adipocytes inhibited 
insulin-stimulated GLUT4 translocation and glucose uptake (242, 243). It is assumed that 
in insulin-stimulated adipocytes, Myo1c translocates to the plasma membrane and 
 38
interacts with GLUT4 in a Ca
2+
-dependent manner. Chen et al. showed that the 
interaction between the motor protein Myo1c and the small GTPase RaIA was required 
for GLUT4 vesicles docking, and the association between Myo1c and RaIA is modulated 
by calmodulin in a Ca
2+
-dependent manner (243). 
2.5. OBESITY, TYPE 2 DIABETES AND INSULIN RESISTANCE 
2.5.1. Epidemic of Obesity and Type 2 Diabetes  
Obesity, defined by a body mass index (BMI) greater than 30kg/m
2
, was 
introduced to the international classification of diseases more than half a century ago. In 
the current 21st century, obesity represents an expanding problem that has reached 
epidemic proportions in the worldwide scale. As conservatively estimated by the 
International Obesity Taskforce and World Health Organization (WHO), at least 9.8% 
adults around the world are obese (1, 3, 244). In some western countries, the percentage 
is even greater: for example, in the United States, 32.2% adults are obese (245). In 
addition, near 2.3 billion of people in the world may be at a risk for diseases associated 
with being overweight. Current mortality rates due to the high BMI could double to 5 
million deaths per year by the year 2030 - unless urgent action is taken now (1, 3, 244). 
This global pandemic disease is polygenic, and it is mostly induced by 
malfunctions in energy homeostasis due to specific congenital or hereditary defects. The 
recent rapid increase in obesity occurrence is thought to be a result of the unbalance in 
energy homeostasis: when energy storage exceeds energy expenditure, accumulated fat 
leads to weight gain and eventually results into obesity (246-248). The commonly cited 
 39
factors include the large quantities of calorie-rich food that are readily accessible in 
modern society; eating habits adapted to fast-paced lifestyles; low levels of physical 
activity; and genetic programs that have evolved, especially in populations prone to 
famine, to favor the storage of excess calories (244).  
As a result of metabolic overload, obesity, along with insulin resistance, 
hyperglycemia, dyslipidemia, hypertension, and atherosclerosis developing together 
make up the metabolic syndrome (249, 250). Obesity has been identified as a major 
causative factor for the insulin resistance and hyperglycemia associated with type 2 
diabetes (251).  A growing body of evidence has also suggested the links of overweight 
and obesity to a range of other serious diseases from heart disease to cancer (3, 252). The 
high rates of obesity occurrence have led to dramatic increases in the obesity-associated 
diseases. For instance, the increasing prevalence of type 2 diabetes in the United States 
has doubled and closely paralleled the rising rate of obesity that has been observed over 
the past decade (5). 
Diabetes mellitus type 2 or type 2 diabetes is a clinical disorder that is 
characterized with hyperglycemia in the context of insulin resistance and relative insulin 
deficiency. According to the reports of WHO, the global incidence of diabetes is 
surmounting 180 million, and type 2 diabetes comprises 90% of the people with diabetes 
around the world (253). Furthermore, this huge number will be doubled to 350 million 
cases by the year 2030 (254).  
 40
As blood sugar levels remain persistently high over time, diabetes in the long run 
can induce the damages in the heart, blood vessels, eyes, kidneys, and nerves (253). The 
associated clinical complications consist of retinopathy, nephropathy, neuropathy and 
cardiovascular disease development, which increase the patient's morbidity and mortality. 
The overall risk of dying among patients with diabetes is at least double the risk of their 
peers without diabetes (253). Besides the severe health compromising effects, type 2 
diabetes and its complications impose significant economic consequences on individuals, 
families, health systems and countries. Over the next 30 years, the expenditure attributed 
to diabetes is estimated to reach $132 billion in United States alone (5, 245). 
Obesity, type 2 diabetes and other metabolic syndrome diseases are strongly 
associated with insulin resistance, which is a pathophysiological state exhibiting 
combined inability of muscle and adipose tissue to facilitate glucose uptake and of the 
liver to suppress glucose output in response to increasing amounts of insulin. The high 
incidence of obesity, type 2 diabetes and the seriousness of their clinic consequences 
make it imperative to understand the molecular basis of insulin signaling and insulin 
resistance. 
2.5.2. Molecular Mechanism of Insulin Resistance 
Insulin resistance is defined as the failure of nominal levels of insulin to trigger its 
downstream metabolic actions, and it is a hallmark characteristic for the development of 
type 2 diabetes, obesity, and other metabolic syndromes (255). Various studies have 
proved that insulin resistance is tightly correlated to the impairment of insulin signaling 
 41
resulting from negative modulation on downstream effector activities (256).  It is also 
becoming clear that obesity promotes a chronic inflammation state and eventually 
develops insulin resistance, which is due to changes in functions of adipocytes and 
macrophages (7). 
Control over insulin signaling can be accomplished by autoregulation (feedback 
controls from downstream signaling effectors) or signals from other paralleled signaling 
pathways. IR and IRS are the primary targets for those control mechanisms. Many insulin 
resistance inducers are observed to activate IRS kinases that negatively regulate IRS 
mediated insulin action. The activation of these IRS kinases induces the serine/threonine 
phosphorylation of the IRS, resulting in the dissociation of IR:IRS and IRS:PI3K 
complex, eventually block the insulin action (152, 204, 257). During the studies 
searching the candidate IRS kinases, several unrelated signaling pathways are found to 
contribute to the development of insulin resistance. Desensitization of insulin signaling 
can be elicited by two major cascades in response to inflammatory signals. One is JNK 
(c-Jun N-terminal kinase 1) mediated pathway, another is mediated by IKK? (I?B kinase 
?) (258). In response to tumor necrosis factor-? (TNF-?), JNK phosphorylates IRS-1 at a 
serine residue (Ser
307
 in rodents; Ser
312
 in humans). These residues are generally adjacent 
to the PTB domain (259, 260). Therefore, serine phosphorylation impedes the tyrosine 
phosphorylation of IRS-1, impairing the following insulin signals (259, 261). Support for 
this conclusion is derived from studies with JNK-deficient animal models; decreased 
adipocity and significantly improved insulin response were observed in these animals 
(262). On the contrary, in both genetic and dietary-induced obese animal models, an 
 42
increased JNK activity was detected in the liver, muscle, and adipose tissue (262). More 
interestingly, JNK activation in pancreatic ? cells rendered transcriptional inhibition in 
pivotal genes, including insulin gene (263). This negative modulating effect was mainly 
caused by a phosphorylation of both IRS-1 and IRS-2 (263). 
Similar to JNK, IKK? is a Ser/Thr protein kinase. It, however, works at a slightly 
different manner. There are two possible mechanisms for IKK? to affect insulin signaling. 
As a part of the IKK complex, IKK? phosphorylats the inhibitor of NF-?B (nuclear 
factor-?B), I?B, resulting in the degradation of I?B, hence activating NF-?B (264)  that 
stimulates the synthesis of pro-inflammatory molecules such as TNF-? and interleukin-6 
(IL-6), (265, 266). In genetic obese model ob/ob mice, heterozygous deletion of IKK? 
prevented the development of insulin resistance during the high fat diet (258, 267). 
Similar results were also derived from studies in mice with selectively IKK? ? deficiency 
in myeloid cells (268). Conversely, over-expression of IKK? in cultured 3T3-L1 
adipocytes was observed to attenuate insulin signaling, whereas its inhibition reversed 
insulin resistance (258). These findings support that IKK? exerts its effects both in 
globally and locally and has a central role in the induction of insulin resistance. Besides 
the effects through I?B, IKK? also have been observed to interact with IRS-1 through 
phosphorylation at the serine residue. Nakmori et al. demonstrated the formation IRS-1?
IKK? complex, which was assisted by NEMO (nuclear factor ?B essential modulator) 
and motor protein Myo1c promoted the TNF-??induced Ser
307
 phosphorylation of IRS-1, 
resulting in the attenuation of insulin signaling and glucose transport (269). 
 43
Impaired insulin signaling transduction can be also triggered by activation of 
PKC?, which is induced by the content of long-chain fatty acyl-CoA. The upregulated 
activity of PKC? leads to a decrease of IR tyrosine phosphorylation and reduced insulin 
sensitivity (270, 271); whereas the PKC? deletion enhanced insulin sensitivity in high fat 
diet fed mice (272). It is most likely that PKC? plays essential roles in the fatty-acid 
induced insulin resistance. At the molecular level, PKC? can also work as the activator of 
JNK and IKK? and indirectly inactivate IRS (272, 273). 
2.5.3. Adipose Tissue and Insulin Resistance  
In classical perception, adipose tissue is considered as an energy-storing depot. 
Nevertheless, work over the past several years has fundamentally replaced our 
understanding of the physiological functions of adipose tissue. Besides storing free fatty 
acids after food intake, adipose tissue serves as a key endocrine and secretory organ, 
releasing non-esterified fatty acids (NEFAs), glycerol, hormones (e.g. leptin, adiponectin, 
resistin, endothelin-1, retinol binding protein 4 (RBP4)), and proinflammatory cytokines 
(e.g. TNF-?, IL-6, IL-1? ) (7, 255). With the intensive investigation, more and more 
adipokines have been discovered and indentified in this rapidly expanding family, 
including chemerin, omentin, and vaspin (274). These bioactive molecules play a pivotal 
role in lipid and glucose metabolism in both normal and disease situations (8). As 
adipocity increases in obesity, the ability of adipocytes to function as endocrine cells is 
altered. The term ?adipocyte dysfunction? is denoted for this state of hypersecretion of 
pro-atherogenic, pro-inflammatory, and pro-diabetic bioactive molecules (e.g. TNF-?, IL-
6, resistin, and RBP4) accompanied with hyposecretion of anti-atherogenic, anti-
 44
inflammatory, and anti-diabetic adipocytokines (i.e. adiponectin, chemerin, omentin and 
vaspin) (7, 255, 274, 275). The abnormal secretion profile ensues to a pathological state, 
i.e. insulin resistance. Below is a summary for several of the most studied adipokines 
associated with insulin resistance. 
High circulating free fatty acids (FFAs) has been identified as a key mediator for 
insulin resistance in skeletal muscle and adipose tissue. This conclusion is supported by 
studies where elevated FFAs in circulation caused peripheral insulin insensitivity in both 
animals and humans (276, 277), as well as studies where pharmacologically lowering of 
FFAs enhanced insulin-stimulated glucose uptake in the periphery (278). They also affect 
downstream targets of the insulin receptor such as PI3-K activity in the skeletal muscle 
(115) or insulin signaling regulator PKC? as discussed above.  
Inflammatory cytokines, TNF-?, and interleukin-6 (IL-6), are the most widely 
studied cytokines, playing essential roles in the development of insulin resistance. In 
humans, adipose tissue TNF-? expression was shown to be correlated with BMI, 
percentage of body fat, and hyperinsulinemia, whereas weight loss decreased TNF-? 
level (279). In addition, targeted null mutation in the TNF-? and TNF-? receptors 
resulted in significantly improved insulin sensitivity in both diet-induced obese and ob/ob 
mice models (280). These results indicate that TNF-alpha is an important mediator of 
insulin resistance in obesity through its effects on several important sites of insulin action. 
Similarly, increased circulating IL-6 levels were observed in obese and insulin resistant 
subjects (281, 282). However, there are conflicting data regarding the role of IL-6 in 
insulin resistance. IL-6 was reported to impair insulin response by reducing insulin-
 45
dependent hepatic glycogen synthesis (283, 284) and glucose uptake in adipocytes (285), 
whereas enhance insulin signaling in myotubes by increasing insulin-dependent glucose 
uptake and  glycogen synthesis, concomitant with lipid oxidation (286). Nevertheless, 
both TNF-? and IL-6 are capable of activating the inflammation pathways JNK and IKK? 
pathways and demolishing insulin signaling (285).  
Unlike most other adipokines, plasma adiponectin levels were reduced in animal 
models of obesity and insulin resistance (287, 288), while the administration of 
adiponectin to obese and insulin resistant mice improves insulin sensitivity (289-291). 
The anti-diabetic metabolic effects of adiponectin are dependent on two distinct receptors 
termed adiponectin receptor 1 (AdipoR1) and adiponectin receptor 2 (AdipoR2) (292), 
and the expression of both receptors was also decreased in mouse models with insulin 
resistance (293, 294). Studies with transgenic or disrupted expression of AdipoR1 or 
AdipoR2 indicated that adiponectin exerted a potent insulin-sensitizing effect through 
binding to its receptors AdipoR1 and AdipoR2, leading to activation of AMPK, 
peroxisome proliferator-activated receptor ? (PPAR?), and presumably other yet-
unknown signaling pathways (294-297) . 
?  
 46
CHAPTER 3. CALCIUM-INDEPENDENT PHOSPHOLIPASE A
2
 IS INVOLVED 
IN INSULIN-STIMULATED GLUCOSE UPTAKE IN 3T3-L1 ADIPOCYTES 
?
3.1. ABSTRACT 
Calcium-independent phospholipase A
2
 (iPLA
2
) is a type of hydrolase that 
catalyzes the release of free fatty acid from the sn-2 position of glycerophospholipids. 
Recent studies have shown that iPLA
2
 expression is upregulated during the 
differentiation of 3T3-L1 fibroblast into adipocytes, and that its activity is required for 
hormone-stimulated differentiation of 3T3-L1 adipocytes. The purpose of the present 
study was to determine the role of iPLA
2
 in insulin-stimulated glucose uptake in 3T3-L1 
adipocytes. 
Bromoenol lactone (BEL), a selective inhibitor of iPLA
2
 was utilized to treat 3T3-
L1 adipocytes. In pretreated adipocytes, insulin-stimulated glucose uptake was 
determined by measuring intracellular incorporation of [
3
H]-2-deoxyglucose. With 10 
and 50? ?M BEL treatment, insulin-stimulated glucose uptake was decreased by 30% and 
45%, respectively, compared to vehicle-treated cells. BEL is also reported to inhibit 
Mg
2+
-dependent phosphatidate phosphohydrolase-1 (PAP-1). Propranolol, the specific 
inhibitor of PAP-1, failed to inhibit insulin-stimulated glucose uptake in 3T3-L1 
adipocytes, ruling out the involvement of PAP-1 in this process. Additionally, methyl 
 47
arachidonyl fluorophosphonate (MAFP), a non-specific inhibitor for PLA
2
, exhibited 
similar inhibitory effects as BEL on insulin-stimulated glucose uptake in 3T3-L1 
adipocytes, indicating that the BEL inhibitory effects resulted from iPLA
2
 inhibition. 
Furthermore, BEL treatment also dramatically impaired the insulin-stimulated glucose 
uptake in L6-GLUT4 myotubes.  
To evaluate the effects of iPLA
2
 inhibition on insulin signaling, insulin-stimulated 
phosphorylation of insulin receptor (IR) and Akt was assessed by western blot analysis, 
and GLUT4 translocation was analyzed by subcellular fraction analysis in BEL-
pretreated cells. There was no significant difference between BEL-treated and vehicle-
treated cells in the phosphorylation levels of IR and Akt response to insulin. However, 
there was a reduced level of insulin-responsive GLUT4 incorporated in plasma 
membrane and lipid raft fractions of BEL-treated cells versus vehicle-treated controls. 
 These results demonstrate that insulin-stimulated glucose uptake is decreased by 
BEL-induced iPLA
2
 inhibition, and that this effect is mediated via decreased 
incorporation of GLUT4 into plasma membrane, without affecting insulin signaling up to 
Akt phosphorylation.  
3.2. INTRODUCTION 
Phospholipase A
2
 is a diverse group of acyl-hydrolases that catalyze the cleavage 
of the sn-2 fatty acyl bond of glycerophospholipids, resulting in the liberation of free 
fatty acid and lysophospholipids. Both products of this reaction are precursors for 
signaling molecules, which are particularly important for various physiological processes 
 48
(9). Based on the activity dependence on calcium ion as well as sequence homology, 
mammalian PLA
2
s are categorized into five principle types, namely the secreted PLA
2
s 
(sPLA
2
), the cytosolic Ca
2+
-dependent PLA
2
s (cPLA
2
s), the platelet-activating  factor 
(PAF) acetylhydrolases, the lysosome PLA
2
s, and the cytosolic Ca
2+
-independent PLA
2
s 
(iPLA
2
) (9).  
Compared to other types of PLA
2
s, iPLA
2
s are unique in that they do not require 
Ca
2+
 for their enzymatic activity (298). It is generally accepted that the iPLA
2
s have 
multiple biological functions in homeostatic phospholipid (PL) remodeling, cell growth, 
eicosanoid metabolism, apoptosis, gene expression, chemotaxis, and calcium entry (11). 
In addition, accumulating evidence indicates that these special enzymes also play roles in 
signal transduction. The inhibition of iPLA
2
 with the inhibitor BEL suppresses diverse 
signaling pathways, such as parathyroid-induced generation of AA in rat proximal 
tubules (299), superoxide generation in neutrophiles (300, 301), stimulated production of 
inducible nitric oxide synthase (iNOS) protein and nitric oxide in cardiac myocytes (302), 
and glucose induced insulin secretion in ?-pancreatic cells (303). 
Recently, it was reported that when 3T3-L1 cells differentiate into adipocytes, 
iPLA
2
? (iPLA
2
-GVIA) and iPLA
2
? gene expression was dramatically upregulated 
(iPLA
2
-GVIB) (15). In addition, Su and coworkers have demonstrated that the gene 
knockdown of iPLA
2
? and iPLA
2
? inhibits hormone-induced adipocyte differentiation by 
preventing PPAR? and C/EBP? expression. Also, the protein levels of iPLA
2
? and 
iPLA
2
? were both found significantly increased in white adipose tissue of genetically 
 49
obese fa/fa Zucker rats, compared to the lean controls (15). All these findings have 
underlined the significance of iPLA
2
 in adipocyte metabolism. 
In addition to be a place for fatty acid storage, adipocytes serve as important 
secretory cells and release various bioactive adipokines, which are essential for whole 
body homeostasis (7, 255). The pivotal role of adipose tissue is fulfilled through the high 
responsiveness of adipocytes to insulin. Previous studies have confirmed that insulin 
action enhance the activity of PLA
2
 in adipocytes (304). However, the potential role of 
adipocytes iPLA
2
 in insulin signaling is still unclear. The goal of this study was to 
elucidate the role of iPLA
2
 in adipocytes response to insulin treatment.  
3.3. MATERIALS AND METHODS 
3.3.1. Materials 
3T3-L1 cells were purchased from ATCC (Manassas, VA). Fetal calf serum 
(FCS), fetal bovine serum (FBS), glutamine, penicillin streptomycin and Dulbecco?s 
modified Eagle?s medium (DMEM) were obtained from Invitrogen, Inc. (Carlsbad, CA). 
Phosphate-buffered saline (PBS) was purchased from Mediatech, Inc. (Manassas, VA). 
Reagents for RNA extraction, reverse transcription and quantitative polymerase chain 
reaction (PCR) were obtained from Bio-Rad (Hercules, CA). Oligonucleotide primer 
pairs were ordered from Integrated DNA Technologies, Inc (Coralville, IA). 
Radiolabeled 2-deoxyglucose was obtained from Perkin Elmer (Waltham, MA). BEL, (S) 
or (R)-BEL, MAFP were purchased from Cayman (Ann Arbor, MI). Anti-GLUT4 
antibody, anti-phospho-Akt (Thr
308
) antibody, and anti-Akt antibody were obtained from 
 50
Cell signaling (Boston, MA). Anti-insulin receptor antibody (C-19) was obtained from 
Santa Cruz Biotechonology, Inc. (Santa Cruz, CA), and anti-phosphotyrosine antibody 
was obtained from Upstate (Millipore, Billerica, MA). Anti-flotillin-1 antibody and anti-
caveolin-1 were obtained from BD Biosciences (San Jose, CA). Anti-?-actin antibody 
was purchased from Sigma (St. Louis, MO). Horseradish peroxidase linked anti-rabbit 
and anti-mouse IgG and western blotting detection reagents were obtained from GE 
Healthcare (Piscataway, NJ). All other chemicals were obtained from Sigma except those 
as indicated. 
3.3.2. Cell Culture of 3T3-L1 Fibroblasts and Adipocytes 
 3T3-L1 fibroblasts were grown in DMEM containing 4.5 g/L D-glucose, 10% FCS, 
1% L-glutamine, and 1% penicillin streptomycin at 37 ?C and 5% CO
2
. The medium was 
changed every other day until 100% confluent. Two days after confluence (Day 0), cell 
differentiation into adipocytes was introduced by changing the medium to DMEM 
containing 5% D-glucose, 10% FBS, 1% L-glutamine, 1% penicillin streptomycin, 4 
?g/ml insulin, 0.5 mM 3-isobutyl-1-methylxanthine (IBMX), and 0.25 mM 
dexamethasone. Subsequent medium changes contained 4 ?g/mL insulin and occurred 
every three days until the cells were fully differentiated and harvested for succeeding 
experiments. Prior to use, cells were washed three times in sterile PBS buffer and serum-
starved in serum free DMEM with 0.1% bovine serum albumin (BSA) (Fisher, Waltham, 
MA) for 2 hours. 
 51
3.3.3. Reverse Transcription and Quantitative PCR 
Following the protocol provided by the manufacturer of RNA extraction kit from 
Qiagen (Valencia, CA), total RNA was extracted from 3T3-L1 fibroblasts (Day 0), 
preadipocytes (Day 2,4, and 6) and adipocytes (Day 8), and the RNA concentration and 
purity were assayed by determining the absorbency of the RNA sample at 260 nm and 
280 nm using DV? 530 Life Science UV/Vis spectrophotometer (Beckman, Fullerton, 
CA).Complementary DNA (cDNA) was synthesized from 1?g of the total RNA of each 
sample according to the manufacturer?s protocol using an iScript
TM
 cDNA Synthesis Kit 
(Bio-Rad). The cDNA synthesis was initiated by incubating the reaction mixture in 25?C 
for 5 minutes, extended by 42?C incubation for 30 minutes and then inactivated by 85?C 
incubation for 5 minutes.  
 Quantitative real-time reverse transcriptase polymerase chain reaction (RT-PCR) 
was performed for 40 PCR cycles by utilizing iQ
TM
 SYBR
?
 Green Supermix (Bio-Rad). 
Mouse gene specific primers were designed from Primer Bank (http://pga.mgh.harvard. 
edu/primerbank/citation.html), and constructed by Integrated DNA Technologies, Inc. 
(IDT, Inc., Coralville, IA). Oligonucleotide primer sequences are listed as Table 2. 
Relative quantification of target gene expression was assayed using the comparative 
threshold (C
T
) and computed by the 2
???C
T
 method described by the manufacturer (Bio-
Rad). Changes in messenger RNA (mRNA) levels of specific genes were calculated 
according to the reference gene, 36B4. 
 52
3.3.4. Insulin-Stimulated Glucose Uptake Assay 
3T3-L1 adipocytes at 8-10 days after differentiation initiation were used for 
insulin-stimulated glucose uptake assay. The steps followed the protocol previously 
described (305). Briefly, serum-starved adipocytes were washed and incubated for 15 
minutes in Kreb?s Ringer phosphate HEPES (KRPH) buffer (136 mM NaCl, 20 mM 
HEPES, 5 mM sodium phosphate buffer, 4.7 mM KCl, 1 mM MgSO
4
, 1mM CaCl
2
, pH 
7.4) and then incubated with or without insulin (100 nM) for 30 minutes. Glucose uptake 
was initiated by adding [
3
H]-2-deoxyglucose (2-DOG) to a final assay concentration for 
10 minutes and stopped by washing the cells three times with ice-cold PBS buffer. Air-
dried cells were then solublized in 0.2 N NaOH solution. The intracellular incorporation 
of [
3
H]-2-deoxyglucose was measured by counting the radioactivity in half of the cell 
lysate with Packard liquid scintillation analyzer (Meriden, CT). The rest of the lysate was 
subjected for protein concentration determination by using BCA protein assay kit (Bio-
Rad) with BSA as standard. The level of non-specific glucose uptake was determined in 
cells pretreated with cytochalasin B before insulin stimulation. For each group of cells, 
the glucose uptake values were denoted as ?pmol radioactive 2-deoxyglucose taken up 
per minute and per mg protein?, and the amount of [
3
H]-2-deoxyglucose taken up was 
calculated according to the formula: 
(Observed cpm? cytochalasin B cpm) ? 2/total cpm ? 25
10 ? mg protein per mL
 
Glucose uptake assay in GLUT4
myc
 L6 cells (kindly provided by Dr. Suresh 
Mathews, Auburn University) were performed in the same manner with slight 
 53
modifications that the insulin incubation was performed at 37?C but not at room 
temperature. 
For the assay associated with chemical inhibitor treatment, 3T3-L1 adipocytes 
were incubated with chemical inhibitors: BEL (10 or 50 ?M), (S)-BEL (50 ?M), (R)-
BEL (50 ?M), propranolol (150 ?M), or MAFP (50 ?M), respectively, or dimethyl 
sulfoxide (DMSO) vehicle for 30 minutes prior to the addition of insulin.
 
3.3.5. Preparation of protein samples from 3T3-L1 cells 
The cell monolayer, with or without insulin or BEL treatments, was washed with 
ice-cold PBS buffer and scraped into ice cold lysis buffer (20mM Tris, 150 mM NaCl, 
1mM EDTA, 1% Triton X-100) with protease inhibitor cocktail (1 tablet per 10 ml, 
Roche Applied Science). The cell lysate was homogenized on ice for 20 seconds for three 
times utilizing 60 Sonic Dismembrator (Fisher).  The cell homogenate was spun at 
10,000 g at 4 ?C for 5 minutes to remove the insoluble material.  Sample protein 
concentrations were assessed using Bio-Rad Protein Assay Reagent. The whole cell 
lysates were either subjected to immunoprecipitation or sodium dodecyl sulfate-
polyacrylamide gel (SDS-PAGE) separation directly, or stored at -80 ?C for future use. 
3.3.6. Immunoprecipitation and Western Blotting  
For immunoprecipitation with IR?, 1 mL whole-cell lysates were precleared by 
addition of 100 ?L protein A agrose beads (Invitrogen). Then 50 ?L precleared protein 
sample was incubated and rotated with 4 ?g anti- IR? subunit antibody overnight at 4?C. 
 54
The resulted immune complexes were precipitated by rotation with 100 ?L protein A 
agrose beads for 2 hours at 4?C. After pulse micro centrifugation, the pellets were washed 
three times in ice-old PBS buffer, and ready for subsequent protein electrophoresis. 
 Whole cell lysates or immunoprecipitates mixed with sample loading buffer (5% 
SDS, 5% 2-mercaptoethanol, 62.7 mM Tris-HCl, pH 6.8, 10% glycerol, 0.003% 
bromphenol blue) were boiled at 95?C for 5 minutes, separated in 10% SDS-PAGE gels, 
and then transferred to a nitrocellulose membrane (Amersham Biosciences) by the wet or 
semi-dry transfer method using transfer buffer (25 mM Tris, 192 mM Glycine, 20% 
Methanol).  
Membranes were blocked in either 5% non-fat dry milk (NFDM) or 5% BSA 
(Fisher Scientific, Fairlawn, NJ) in Tris Buffered Saline (TBS) with Triton X 100 (TBS-T 
buffer, 10 mM Tris pH 7.4, 100 mM NaCl, 0.1% Tween-20). The blocked membranes 
were incubated with specific primary antibodies (diluted to 1:1000 in TBS-T containing 5% 
NFDM) as indicated, and then corresponding secondary antibodies (diluted to 1:2000 in 
TBS-T containing 5% NFDM). The membrane was developed using an enhanced 
chemiluminescent substrate for 5 minutes.  The blots were imaged and analyzed using the 
UVP Bioimaging System and LabWorks software package (UVP, Upland, CA).  
3.3.7. Fractionation Analysis of the Subcellular Distribution of GLUT4  
The procedure for subcellular fractionation analysis of GLUT4 was described 
previously by Elmendorf (306). A schematic graph of this fractionation procedure is 
showed in Figure 2.  All steps were performed on ice or at 4?C in a cold room. 3T3-L1 
 55
adipocytes at 10 days after withdrawal from differentiation medium, with or without 
specific treatments as indicated, were washed and scraped into HEPES-EDTA-sucrose 
(HES) buffer (20 mM HEPES, 1 mM EDTA, 255 mM sucrose, pH 7.4) containing 
protease inhibitors. The homogenate was prepared by passing the cell solution through a 
22-gauge needle for 10 times on ice. After homogenization, the cell lysates were 
centrifuged at 19,000 g in a fixed-angle rotor (JA20 rotor, Beckman) for 20 minutes. The 
supernatant was subjected to another centrifugation for 20 minutes at 41,000 g to yield 
the high density microsome (HDM) fraction pellet. The resulting supernatant was 
removed and centrifuged at 180,000 g for 75 minutes to pellet the low density microsome 
(LDM) fraction.  For plasma membrane (PM) fraction separation, the pellet resulted from 
the first 19,000 g centrifugation was suspended and washed in HES buffer by 
centrifugation at 19,000 g for 20 minutes, then resuspended in 5 mL of HES buffer, and 
layered onto a 6.3 mL sucrose cushion (38.5% sucrose, 20 mM HEPES, 1 mM EDTA, 
pH 7.4), followed by a centrifugation for 60 minutes at 100,000 g with a swing-out rotor 
(SW41, Beckman). The PM fraction was collected from the white fluffy band on the top 
of the sucrose cushion, resuspended in HES buffer, and repelleted by centrifugation for 
20 minutes at 40,000 g. All pellets were finally suspended in appropriate volumes of HES 
buffer, and the LDM and PM fractions were subjected to SDS-PAGE and Western 
blotting for GLUT4 analysis. 
3.3.8. Detection of GLUT4 in Lipid Raft Fractions 
Lipid raft fractions were separated following the method developed by Macdonald 
and Pike (307). 3T3-L1 adipocytes, with or without BEL treatment, were treated for 30 
 56
minutes in medium containing insulin. Cells were then washed and scraped in base buffer 
(20mM Tris-Hcl, pH7.8, 250mM sucrose) containing 1mM CaCl
2
, 1mM MgCl
2
 and 
protease inhibitors. The cells were then lysed by passage through a 22 g?3'' needle 20 
times on ice. After centrifugation at 1,000g for 10 min, 2 ml of the postnuclear 
supernatants were mixed with equal volume of base buffer containing 50% Optiprep 
(Axis-Shield, Oslo, Norway), placed to the bottom of 12 ml ultracentrifuge tube 
(Beckman), and followed with a continuous gradient of 5% to 20% Optiprep in base 
buffer. Gradients were centrifuged for 90 min at 52,000 g using a SW-Ti55 rotor. 
Gradients were then fractionated into 12 fractions, and the lipid rafts fractions were 
verified by the distribution of lipid raft marker proteins, as well as protein and cholesterol 
concentrations. GLUT4 protein level was assessed in combined lipid raft factions 1-2, 3-4 
and non-raft fractions 11-12 from cells with different treatment by western blotting 
analysis.  
 
 57
 
?
Figure 2. Procedure for preparation of subcellular fractionations. The figure has 
been modified based on (306).
 58
3.3.9. Statistical Analysis 
Data are presented as mean ? S.E. Statistical significance difference between 
treatments was determined using the Student?s t tests, and statistical significance level is 
set as 0.05. 
3.4. RESULTS 
3.4.1. Gene Expression of iPLA
2 
Is Increased during 3T3-L1 Adipocyte 
Differentiation 
It has been previously shown that iPLA
2
? and iPLA
2
? are required for the 
adipogenic program, which differentiates 3T3-L1 fibroblast into adipocytes under 
hormone stimulation (15).  Since the upregulated gene expression of iPLA
2
? and iPLA
2
? 
predicts their possible importance in 3T3-L1 adipocytes, we sought to confirm the 
alterations in the mRNA levels of iPLA
2
? and iPLA
2
? during the differentiation of 3T3-
L1 cells. In addition, we sought to determine the expression of another intracellular PLA
2
, 
cPLA
2
, which has not been shown to be involved in 3T3-L1 differentiation (15). Total 
RNA samples were isolated from the cells at day 0, 2, 4, 6, and 8 days post-
differentiation and reverse transcribed into cDNA.  
The results from quantitative real-time PCR analysis demonstrated that the 
mRNA levels of iPLA
2
? and iPLA
2
? were both increased dramatically during the 
differentiation program. At day 8, the amounts of mRNA encoding iPLA
2
? and iPLA
2
? 
were increased approximately 8 and 7.2 folds respectively (Figure 3). However, mRNA 
 59
level of cPLA
2
 was reduced more than 80% during the differentiation of 3T3-L1 
preadipocytes. These results are consistent with a previous study (15), and suggest that in 
3T3-L1 adipocytes, iPLA
2
? and iPLA
2
? may be important for specific physiological 
functions in 3T3-L1 adipocytes. 
3.4.2. Insulin-Stimulated Glucose Uptake in 3T3-L1 Adipocytes Is Decreased by 
BEL-Induced iPLA
2
 Inhibition 
For adipocytes, proper response to insulin is essential to maintain normal cellular 
functions. To investigate the possible physiological roles of iPLA
2
 in 3T3-L1 adipocytes, 
we first initiated a study to determine whether iPLA
2
 has effects on insulin stimulated 
cellular glucose uptake. Since the functional significance of intracellular iPLA
2
 can most 
easily be investigated using selective inhibitors, we utilized BEL, a chemical inhibitor 
specific for iPLA
2
 to block iPLA
2
 activity in vitro.  
Fully differentiated 3T3-L1 adipocytes (day 8-10) were incubated with two 
different concentrations of BEL (10 ?M or 50 ?M) for 30 min at 37 ?C, and the basal and 
insulin-stimulated glucose uptake were assessed by measuring intracellular incorporation 
of [
3
H]-2-deoxyglucose. 
As seen in? Figure 4, insulin stimulation significantly increased to almost 14- fold 
of the basal 2-deoxyglucose uptake in 3T3-L1 cells. Compared to cells incubated in 
vehicle DMSO alone, 3T3-L1 adipocytes pretreated with BEL exhibit decreased 
intracellular incorporation of [
3
H]-2-deoxyglucose under insulin stimulation. At 10 and 
50?M BEL, insulin-stimulated glucose uptake was decreased approximately 30 and 50%, 
 60
respectively. There was no significant difference in basal glucose uptake with BEL 
treatment.  
Since high concentrations of BEL have been shown to also inhibit the enzyme, 
Mg
2+
-dependent phosphatidate phosphohydrolase-1 (PAP-1) (84), glucose uptake assays 
were performed in the presence of the PAP-1 inhibitor, propranolol (150 ?M). There was 
no apparent difference in insulin-stimulated glucose uptake in propranolol-treated cells 
versus vehicle controls (Figure 5), excluding the possibility that PAP-1 contributes to the 
observed inhibitory effects of BEL.  
3.4.3. Both iPLA
2
? and iPLA
2
? Contribute to Glucose Uptake in Insulin-Stimulated 
Adipocytes 
Previous research has demonstrated the specificity of the (S) - and (R)- 
enantiomers for preferentially inhibiting iPLA
2
? and iPLA
2
?, respectively (132). To find 
out which iPLA2 was involved in the insulin-stimulated glucose uptake, 3T3-L1 
adipocytes were pretreated with 50 ?M (S) - or (R) - BEL, separately, and the basal and 
insulin-stimulated glucose uptake was compared to that in cells incubated in vehicle 
alone. As shown by Figure 6, the measured [
3
H]-2-deoxyglucose in insulin-stimulated 
adipocytes was reduced approximately 35% by either (S) - or (R) - BEL. This finding 
demonstrates that both iPLA
2
? and iPLA
2
? contribute similarly to the insulin-stimulated 
glucose uptake in 3T3-L1 adipocytes.  
To further verify that PLA
2
 activity was implicated in the insulin-stimulated 
glucose uptake, we used the PLA
2
 inhibitor, methyl arachidonyl fluorophosphonate 
 61
(MAFP), which is reported as a suicide inhibitor for iPLA
2
, cPLA
2
 and AdPLA (10). As 
expected, MAFP treatment markedly reduced about 30% of the incorporation of [
3
H]-2-
deoxyglucose in insulin-stimulated 3T3-L1 adipocytes (Figure 6). Compared to BEL 
treatment, MAFP administration did not exhibit strong effects in insulin stimulated 
glucose, which may suggest that other PLA
2
s like cPLA
2
 and AdPLA are not included in 
this process. Together with the results above, it is suggested that the inhibitory effects of 
BEL on insulin-stimulated glucose uptake we observed in 3T3-L1 adipocytes are induced 
by inhibition of iPLA
2
 activity, implicating a role for iPLA
2 
in insulin-stimulated glucose 
uptake.  
3.4.4. Exogenous Arachidonic Acid Reverses the BEL-Inhibitory Effect in Insulin-
Stimulated Glucose Uptake  
Arachidonic acid (AA) is one of the products of phospholipid hydrolysis 
catalyzed by iPLA
2
, which has been implicated in mediating some of the physiological 
effects of iPLA
2
. Recent studies have also established the relationship between AA 
treatment and adipocyte glucose uptake (308, 309). To determine whether BEL-
suppressed arachidonic acid (AA) liberation was involved in insulin-stimulated glucose 
uptake, BEL-pretreated adipocytes were incubated with medium containing AA (10 or 
100 ?M) for 30 min, and subjected to insulin-stimulated glucose uptake assay. As 
demonstrated in Figure 7, 10 ?M AA treatment did not exhibit significant effects on 
insulin-stimulated glucose uptake in BEL-pretreated cells, however, addition of 100 ?M 
exogenous AA increased approximately 30% of insulin-stimulated uptake in BEL-
pretreated adipocytes, and restored the inhibitory effect of BEL treatment. Previous 
 62
studies have shown that long-term AA administration influences the glucose uptake in 
both resting and insulin-stimulated adipocytes (309, 310). With this in mind, we further 
tested the effects of AA incubation on adipocytes not treated with BEL. There was a 
dose-dependent increase in both basal and insulin-stimulated glucose uptake induced by 
AA incubation, however, the increase was not statistically significant (Figure 8). Taken 
together, these results indicate that iPLA
2
-stimulated release of AA is at least partially 
contributing to the insulin-stimulated glucose transport in 3T3-L1 adipocytes. 
3.4.5. Insulin-Stimulated Glucose Uptake in L6-GLUT4 Myotubes Is Decreased by 
BEL-Induced iPLA
2
 Inhibition 
Analogous to adipocytes, skeletal muscle cells are sensitive to insulin and perform 
glucose uptake through the rapid translocation of GLUT4 glucose
 
transporters to the 
plasma membrane. We also tested the effects of BEL treatment in insulin-stimulated 
glucose uptake in L6-GLUT4
myc 
myotubes. BEL administration dramatically reduced the 
glucose uptake in insulin-activated L6 cells. In cells treated with 10 ?M BEL, the glucose 
uptake upon insulin administration was inhibited about 45% compared to the vehicle 
control (Figure 9). However, there was a nearly 30% reduction in basal glucose uptake 
observed in these cells, indicating that BEL influences myotubes in a way partially 
different from that in adipocytes. The results also showed that high concentration of BEL 
(50 ?M) inhibited approximately 80% of basal glucose uptake and 90% of insulin-
stimulated glucose uptake in L6-GLUT4
myc 
myotubes. 
 63
3.4.6. Inhibition of iPLA
2
 by BEL Does Not Impair Insulin-Stimulated 
Phosphorylation of IR and Akt  
One possible mechanism by which BEL treatment inhibited insulin-stimulated 
glucose uptake is through alteration in insulin signaling pathways. In 3T3-L1 adipocytes, 
insulin-stimulated phosphorylation of receptor (IR) and Akt are important for the 
initiation and transduction of insulin signaling (194). Therefore, we assessed the activity 
of IR and Akt by measuring their phosphorylation levels. Western blot analysis showed 
that insulin treatment increased the amount of phosphorylated-IR and phosphorylated-
Akt dramatically, however, there was no significant difference in the phosphorylation 
levels of IR and Akt between BEL-treated and vehicle-treated cells (Figure 10 and Figure 
11). These results indicate that the insulin signaling up to forehead of Akt is not affected 
by BEL treatment in 3T3-L1 adipocytes. 
3.4.7. Inhibition of iPLA
2
 by BEL Impacts the Insulin-Stimulated GLUT4 
Translocation in 3T3-L1 adipocytes 
In response to insulin, GLUT4 translocates to plasma membrane from the 
intracellular storage compartments, leading to the increased glucose transport into 
adipocytes (147). Since BEL treatment decreased the insulin-stimulated glucose transport 
in adipocytes, we next examined the effects of BEL treatment on the cellular localization 
of GLUT4. 3T3-L1 adipocytes with or without BEL (50 ?M) treatment were stimulated 
by insulin and then subjected to subcellular fractionation. The GLUT4 levels in plasma 
membrane (PM) and low density microsome (LDM) fractions were assessed by western 
 64
blotting. In the absence of insulin, LDM showed the majority of GLUT4, while PM 
fraction contained a low level of background GLUT4. Upon insulin stimulation, an 
approximately 1.5-fold increase in the protein level of GLUT4 was observed in PM 
fraction. In contrast, in BEL-treated cells, there was a nearly 30% decrease in plasma 
membrane incorporated GLUT4 versus vehicle-treated cells (Figure 12).   
3.4.8. Inhibition of iPLA
2
 by BEL Alters GLUT4 Incorporation in Lipid Rafts  
Lipid rafts are specialized compartments of the plasma membrane enriched in 
cholesterol and glycerolsphingolipids. Various studies have established the associations 
between GLUT4 and caveolae (substructure of lipid rafts), and suggested that lipid rafts 
are required for efficient localization of GLUT4 into plasma membrane (PM). Previously, 
we detected some effects of BEL on lipid raft structures and distributions of raft protein 
markers (data not shown). For this reason, we next examined whether these effects of 
BEL impact GLUT4 incorporation in lipid rafts. With or without BEL pretreatment, we 
subjected basal and insulin-stimulated 3T3-L1 adipocytes to non-detergent 
homogenization and gradient fractionation as described by Pike (307). As shown in 
Figure 13, throughout the generated 12 fractions, the first four fractions (Fraction1-4) 
were enriched in lipid raft marker proteins caveolin-1 and flotillin-1, which were 
identified as lipid raft (LR) fraction, whereas the non-raft (NR) protein ?-actin was 
localized in the bottom fractions (Fraction 8-12). For GLUT4 incorporation detection, we 
combined LR and NR fractions according to the description above. Western blot analysis 
with anti-GLUT4 antibody showed an increase in GLUT4 protein presence in the LR 
fractions was observed under the insulin stimulation, however, the presence of GLUT4 
 65
was noticeably reduced in LR fractions when adipocytes were pretreated by BEL (Figure 
11). With 10 ?M BEL treatment, the GLUT4 protein in the first combined LR fraction 
(original fractions 1 and 2) was significantly attenuated; with 50 ?M BEL treatment, 
levels of GLUT4 in both of the LR factions were both reduced to background level.  
Along with the distribution change in GLUT4 proteins, the localization of lipid raft 
marker protein caveolin-1 and flotillin-1 was also altered by BEL treatment. For both 
caveolin-1 and flotillin-1, BEL treatment introduced the shift of the proteins from the first 
(original fractions 1 and 2) to the second combined LR fractions (original fractions 3 and 
4), as well as a reduction in the total protein levels in LR fractions (Figure 14). These 
findings imply that the BEL treatment impacts interactions of GLUT4 with lipid raft 
domains in plasma membrane. 
3.5. DISCUSSION 
Recently, numerous studies have indicated the essential roles of iPLA
2
 in whole 
body metabolism, including adipocyte differentiation (15), insulin secretion (16-21, 23), 
glucose homeostasis (22, 23), hepatic adipogenesis (24, 25), and cardiac myocytes 
mitochondrial functioning (26, 27). These data have linked iPLA
2
 to obesity, type 2 
diabetes, fatty liver disease and other manifestations of the metabolic syndrome. 
However, the prospective significance of iPLA
2
 in whole body homeostasis remains a 
puzzle. Su et.al (15) originally reported that iPLA
2
 exhibited much higher gene 
expression compared to the background expression level of cPLA
2
. With this in mind, we 
have hypothesized that iPLA
2
 plays important roles in adipocyte biology. Propagating 
signals from insulin, as it relates to energy metabolism, is one of the major functions of 
 66
adipocytes. The objective of this study was to investigate the possible functions of iPLA
2
 
in insulin action within the adipocytes.  
Using the selective inhibitor for iPLA
2
, BEL, we established the involvement of 
iPLA
2
 in insulin-stimulated glucose uptake in fully differentiated 3T3-L1 adipocytes. Our 
results also indicate the comparable contribution of iPLA
2
? and iPLA
2
? in this process. 
Earlier studies have already described the ?insulin-like? effects of phospholipase on 
glucose and amino acid uptake in adipocytes (311, 312), and previous studies have also 
shown that insulin enhances the phospholipase A
2
 activities in adipocyte membranes 
resulting in global enrichment of lyso deviates and  membrane fatty acids (304). The 
major types of phospholipase A
2
 responsive to those events were not specified in these 
studies. However, our results suggest that iPLA
2
 could represent the most prospective 
candidate. Among the PLA
2
 family, cPLA
2
 performs a Ca
2+
 dependent translocation into 
cellular membrane (13); iPLA
2
 ?, although originally purified from the cytosolic source, 
can be preferentially distributed in membrane fractions (313, 314); and iPLA
2
? is 
reported to be a membrane-associated protein (54). Recently, a novel member of PLA
2
, 
AdPLA, was identified in adipocytes, and found to be ubiquitously localized in both 
membrane and cytoplasmic compartments (10). In this study, we utilized another PLA
2
 
inhibitor, MAFP, parallel to BEL. In contrast to iPLA
2
, cPLA
2
 and AdPLA are efficiently 
inhibited by MAFP, but not BEL.  Our results show that MAFP treatment does not 
exhibit extra effects on the insulin-induced glucose uptake compared to BEL, which may 
rules out the participation of cPLA
2
s and AdPLA in this process.  
 67
As one of the primary products of reactions catalyzed by iPLA
2
, AA has been 
reported to influence glucose uptake in adipocytes. In 2001, Nugent and coworkers 
demonstrated that exposure of fully differentiated 3T3-L1 adipocytes to 0.8 mM AA 
significantly enhanced the insulin-stimulated glucose uptake, and the maximum effects 
occurred at 4 hours post treatment when membrane phospholipid content of AA was 
markedly increased (309). In the present study, we observed similar but not significant 
trend of AA effects on the insulin-stimulated glucose uptake in 3T3-L1 cells with a lower 
concentration (100 ?M) and shorter exposure time (30 minute). Most importantly, we 
found that the addition of exogenous AA successfully restored the reduced glucose 
uptake resulting from iPLA
2
 inhibition by BEL, indicating that the AA, released through 
iPLA
2
 enzymatic activity, is highly involved in insulin action on adipocyte glucose 
uptake. It is intriguing that another product of phospholipid hydrolysis, lysophospholipids, 
and its metabolites are also involved in adipocytes glucose transport. Two recent studies, 
performed by Yea and colleagues (315, 316) reported that lysophosphatidic acid (LPA) 
and lysophosphatidyl-serine (LPS) promoted glucose uptake in both L6 GLUT4
myc
 
myotubes and 3T3-L1 adipocytes in a dose- and time-dependent manner. These studies 
provide supplementary evidence for the roles of iPLA
2
 activity in glucose regulation. 
Furthermore, similar inhibitory effects of BEL were detected in L6-GLUT4
myc
 myotubes, 
which provides additional support for the conclusion that iPLA
2
 is partially mediating 
insulin-stimulated glucose uptake. 
To further evaluate the mechanism that iPLA
2
 impacts adipocyte glucose 
transport under insulin stimulation, we attempted to dissect the molecular events 
 68
underlying the inhibitory effects of BEL.  It is well known that insulin-stimulated glucose 
uptake in adipose tissue is mediated by the translocation of insulin-sensitive GLUT4 from 
intracellular storage vesicles to the plasma membrane. We further investigated the 
GLUT4 translocation in BEL-treated adipocytes to elucidate the mechanism by which 
BEL decreases the insulin-stimulated uptake. Our studies showed that BEL reduced the 
presence of GLUT4 in the PM fraction. Therefore, these results potentiate the 
relationship between iPLA
2
 activity and GLUT4 translocation.  
Additionally, specific membrane microdomains, known as lipid rafts, serve as 
sites for assembly of signaling complexes that play an important role in insulin action, 
especially the Cbl/CAP cascade (317).  The insulin-responsive presence of GLUT4 in 
lipid rafts has been verified by previous studies (317). Our results also suggest similar 
findings: upon insulin stimulation, GLUT4 protein presence increases in the lipid raft 
fractions of 3T3-L1 adipocyte, and BEL treatment prevents the incorporation of GLUT4 
in lipid rafts. It is also noteworthy that the distributions of caveolin-1 and flotillin-1 are 
also altered by BEL treatment. Previous studies have showed that caveolin and flotillin 
are colocalized in distinct subdomains of the plasma membrane, and that insulin induces 
the translocation of Cbl to these caveolin- and flotillin-enriched compartments through its 
interaction with CAP (168). Together with results provided in this study, BEL treatment 
could affect the GLUT4 translocation via the possible influence on the Cbl/CAP pathway. 
To initiate the insulin-responsive GLUT4 translocation, insulin-activated 
phosphorylation cascade of the downstream components is imperative. Control over 
insulin signaling can be achieved by regulating the phosphorylation levels of several key 
 69
molecules, such as IR, IRS, PI3K and Akt (191). In the current study, we tried to 
determine whether the Akt kinase pathway, a pathway essential for glucose transport 
responsive to insulin stimulation, is affected by BEL-induced iPLA
2
 inhibition in 3T3-L1 
adipocytes. Interestingly, the immunoblotting showed that BEL pre-incubation did not 
influence the insulin-responsive phosphorylation of IR and Akt in 3T3-L1 adipocytes. 
Therefore, these results suggest that inhibition of iPLA
2
 does not affect insulin-initiated 
signal transduction pathway leading to Akt kinase activation and works on the later 
stages of insulin action on glucose uptake. 
Collectively, these findings in the present study demonstrate that iPLA
2
, at least 
partially, mediating the insulin-stimulated glucose uptake in 3T3-L1 adipocytes. It is 
involved in the GLUT4 translocation without affecting Akt kinase pathway upon insulin 
stimulation. This novel function of iPLA
2
 in 3T3-L1 adipocytes makes it a promising 
target for strategies for prevention and treatment of obesity, type 2 diabetes and other 
linked metabolic diseases. 
 70
Table 3. Oligonucleotide Primer Sequences for RT-PCR 
 
Gene 
 
Sequence 
 
iPLA
2
? 
 
Fwd 5?-GCC TCG TCA ACA CCC TCA G -3? 
Rev 5?-CCT TCA CCC GGA ATG GGT TC -3? 
 
iPLA
2
? Fwd 5?-AGC CTT TTC CAT TAC ACA TAC GG-3? 
Rev 5?-CCT GGA GAA CTG GAT TTC CTT TC -3? 
 
cPLA
2
 Fwd 5?-CAG CAC ATT ATA GTG GAA CAC CA -3? 
Rev 5?- AGT GTC CAG CAT ATC GCC AAA-3? 
 
36B4 Fwd 5?-AGA TTC GGG ATA TGC TGT TGG C-3? 
Rev 5?-TCG GGT CCT AGA CCA GTG TTC -3? 
  
 
 71
  
Figure 3. mRNA Levels of iPLA
2
?, iPLA
2
? and cPLA
2
 in 3T3-L1 cells during 
hormone-stimulated differentiation. 3T3-L1 fibroblasts were cultured and subjected to 
hormone-induced differentiation. During this process, total RNA was collected at day 0, 
2, 4, 6, and 8 after the initiation of differentiation. Total RNA (1 ?g) was then reverse 
transcribed to cDNA, followed by RT-PCR analysis. The mRNA levels of iPLA
2
?, 
iPLA
2
? and cPLA
2
 were determined using 36B4 as the internal reference gene. The 
relative mRNA levels are computed using  the mRNA levels at day 0 as internal standard, 
and the fold changes are represented as mean ? S.E. of three independent experiment. 
0
2
4
6
8
10
02468
Relative
?
mRNA
?
Levels
Days
iPLA2?beta
iPLA2?gamma
0
0.2
0.4
0.6
0.8
1
1.2
02468
Relative
?
mRNA
?
Levels
Days
cPLA2
 72
 
Figure 4. Effects of BEL on insulin-stimulated glucose uptake in 3T3-L1 adipocytes. 
Day 8-10 3T3-L1 adipocytes in multiple-well plates were serum starved for 2 hours, and 
then pretreated for 30 minutes with two concentrations of BEL (10 or 50 ?M) or vehicle 
DMSO, separately. Cells were then untreated (dark grey, left) or stimulated with 100 nM 
insulin (light grey, right) for 30 minutes and assayed for [
3
H]-2-DOG uptake over 10 
minutes in the presence of 10 or 50 ?M BEL or vehicle. Data are mean ? S.E. from three 
or more independent experiments performed in triplicate, normalized to insulin-
stimulated uptake in vehicle treated adipocytes. Statistical significance of treatments on 
basal or insulin-stimulated 2-DOG uptake was determined by comparing the means of 
basal or insulin-stimulated 2-DOG uptake of cells treated with BEL (10 or 50 ?M) to that 
from the  vehicle controls using 2-sample student?s t test (*, p<0.05). 
0
20
40
60
80
100
120
Vehicle +?10?uM?BEL? +?50?uM?BEL?
2
?
DOG
?
Uptake
(%
?
of
?
insulin
?
stimulated
??
2
?
DOG
?
uptake
?
in
?
negative
?
control)
? insulin? +?insulin?
*
*
 73
 
Figure 5. Insulin-stimulated glucose uptake in 3T3-L1 adipocytes treated with 
propranolol. Day 8-10 3T3-L1 adipocytes in multiple-well plates were serum starved for 
2 hours, and then pretreated for 30 minutes with 150 ?M propranolol or vehicle DMSO, 
separately. Cells were then untreated (dark grey, left) or stimulated with 100 nM insulin 
(light grey, right) for 30 minutes and assayed for [
3
H]-2-DOG uptake over 10 minutes in 
the presence of 150 ?M propranolol or vehicle. Data are mean ? S.E. from three or more 
independent experiments performed in triplicate, normalized to insulin-stimulated uptake 
in vehicle treated adipocytes. Statistical significance of treatments on basal or insulin-
stimulated 2-DOG uptake was determined by comparing the means of basal or insulin-
stimulated 2-DOG uptake of cells treated with propranolol to that from the vehicle 
controls using 2-sample student?s t test (*, p<0.05). 
0
20
40
60
80
100
120
Vehicle +?150?uM?propranolol?
2
?
DOG
?
Uptake
(%
?
of
?
insulin
?
stimulated
??
2
?
DOG
?
uptake
?
in
?
negative
?
control)
? Insulin
+?Insulin
 74
 
Figure 6. Effects of PLA
2
 inhibitors on insulin-stimulated glucose uptake in 3T3-L1 
adipocytes. Day 8-10 3T3-L1 adipocytes in multiple-well plates were serum starved for 
2 hours, and then pretreated for 30 minutes with vehicle DMSO  or three different PLA
2
 
inhibitors, (R)-BEL(50 ?M), (S)-BEL (50 ?M), and MAFP (50 ?M), separately. Cells 
were then untreated (dark grey, left) or stimulated with 100 nM insulin (light grey, right) 
for 30 minutes and assayed for [
3
H]-2-DOG uptake over 10 minutes in the presence of 
inhibitors or vehicle. Data are mean ? S.E. from three or more independent experiments 
performed in triplicate, normalized to insulin-stimulated uptake in vehicle treated 
adipocytes. Statistical significance of treatments on basal or insulin-stimulated 2-DOG 
uptake was determined by comparing the means of basal or insulin-stimulated 2-DOG 
uptake of cells treated with different inhibitors to that from vehicle-treated cells using 2-
sample student?s t test (*, p<0.05). 
 
0
20
40
60
80
100
120
Control +50??M?(R)?BEL +50??M?(S)?BEL +50??M?MAFP
2
?
DOG
?
Uptake
(%
?
of
?
insulin
?
stimulated
?
2
?
DOG
?
uptake
in
?
negative
?
control)
?Insulin +Insulin
*?
*
*
 75
 
Figure 7. Exogenous arachidonic acid effects on insulin-stimulated glucose uptake in 
BEL pretreated adipocytes. Day 8-10 3T3-L1 adipocytes in multiple-well plates were 
serum starved for 2 hours, and then subjected for four different treatment procedures: 
vehicle control, 30 minutes pretreatment with BEL (50 ?M), 30 minutes pretreatment 
with BEL (50 ?M) followed by 30 minutes AA (10 ?M) incubation, and 30 minutes 
pretreatment with BEL (50 ?M), 30 minutes pretreatment with BEL (50 ?M) followed by 
30 minutes AA (100 ?M) incubation. Cells were then untreated (dark grey, left) or 
stimulated with 100 nM insulin (light grey, right) for 30 minutes and assayed for [
3
H]-2-
DOG uptake over 10 minutes in the presence of BEL and/or AA or vehicle. Data are 
mean ? S.E. from three or more independent experiments performed in triplicate, 
normalized to vehicle insulin-stimulated uptake. Statistical significance of treatments on 
basal or insulin-stimulated 2-DOG uptake was determined by comparing the means of 2-
DOG uptake of cells treated with BEL or/and AA with that from vehicle controls using 2-
sample student?s t test (*, p<0.05). 
 
0.00
20.00
40.00
60.00
80.00
100.00
120.00
2
?
DOG
?
Uptake
(%
?
of
?
insulin
?
stimulated
?
2
?
DOG
?
uptake
?
in
?
negative
?
control)
?Insulin +Insulin
*
*
 76
 
Figure 8. Exogenous arachidonic acid effects on Insulin-stimulated glucose uptake 
in 3T3-L1 adipocytes. Day 8-10 3T3-L1 adipocytes in multiple-well plates were serum 
starved for 2 hours, and then incubated with AA (10, 50 or 100 ?M) for 30 minutes. Cells 
were then untreated (dark grey, left) or stimulated with 100 nM insulin (light grey, right) 
for 30 minutes and assayed for [
3
H]-2-DOG uptake over 10 minutes in the presence of 
AA (10, 50 or 100 ?M) or vehicle. Data are mean ? S.E. from three or more independent 
experiments performed in triplicate, normalized to vehicle insulin-stimulated uptake. 
Statistical significance of treatments on basal or insulin-stimulated 2-DOG uptake was 
determined by comparing the means of 2-DOG uptake of cells treated with AA to that 
from vehicle controls using 2-sample student?s t test. 
0.00
20.00
40.00
60.00
80.00
100.00
120.00
140.00
Vehicle +?10uM?AA +?50uM?AA +?100uM?AA
2
?
DOG
?
Uptake
(%
?
of
?
insulin
?
stimulated
??
2
?
DOG
?
uptake
in
?
negative
?
control
? Insulin +?insulin?
 77
 
 
Figure 9. BEL effects on insulin-stimulated glucose uptake in L6-GLUT4 myotubes. 
L6-GLUT4
myc
 myotubes were serum starved for overnight, and then incubated with BEL 
(10 or 50 ?M) for 30 minutes or vehicle DMSO, separately. Cells were then untreated 
(dark grey, left) or stimulated with 100 nM insulin (light grey, right) for 30 minutes and 
assayed for [
3
H]-2-DOG uptake over 10 minutes in the presence of 10 or 50 ?M BEL or 
vehicle. Data are mean ? S.E. from three or more independent experiments performed in 
triplicate, normalized to insulin-stimulated uptake in vehicle treated adipocytes. 
Statistical significance of treatments on basal or insulin-stimulated 2-DOG uptake was 
determined by comparing the means of basal or insulin-stimulated 2-DOG uptake of cells 
treated with BEL (10 or 50 ?M) to that from the vehicle controls using 2-sample 
student?s t test (*, p<0.05; **, p<0.01, ***, p<0.001). 
?
0
20
40
60
80
100
120
Vehicle +?10?uM?BEL? +?50?uM?BEL?
2
?
DOG
?
Uptake
(%
?
of
?
insulin
?
stimulated
??
2
?
DOG
?
uptake
?
in
?
negative
?
control)
? insulin? +?insulin?
*
*
***
**
 78
 
Figure 10. Effects of BEL on insulin-dependent insulin receptor phosphorylation. 
Day 8-10 3T3-L1 adipocytes in 60 mm dishes were serum starved for 2 hours, and then 
treated with or without BEL (50 ?M) for 30 minutes. After consequent insulin (100 nM) 
incubation for 30 minutes, insulin-dependent tyrosine phosphorylation of insulin receptor 
? subunit (IR?) levels were assessed by immunoprecipitation with anti-IR antibody and 
immunoblotting with anti-phosphotyrosine antibody. Shown are representative blot (top) 
and densitometry analysis (bottom) of independent experiments in which results represent 
the mean ? S.E. of phospho-tyrosine level relative to total IR level (relative phospho-IR? 
level), and relative phospho-IR? level in controls (+Insulin- BEL) was assigned a value 
of 1.  
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
Vehicle +BEL(50?M)
Relatvie
?
Phospho
?
IR
?
Levels
 79
 
Figure 11. Effects of BEL on insulin-dependent Akt phosphorylation. Day 8-10 3T3-
L1 adipocytes in 60 mm dishes were serum starved for 2 hours, and then treated with or 
without BEL (50 ?M) for 30 minutes. After consequent insulin (100 nM) incubation for 
30 minutes, insulin-dependent threonine phosphorylation of Akt was assessed by western 
blot analysis with anti-phospho-Akt antibody and anti-Akt antibody. Shown are 
representative blot (top) and densitometry analysis (bottom) of independent experiments 
in which results represent the mean ? S.E. of phospho-Akt level relative to total Akt level 
(relative phospho-Akt level), and relative phospho-Akt level in controls (+Insulin - BEL) 
was assigned a value of 1. 
 
0
0.2
0.4
0.6
0.8
1
1.2
1.4
Vehicle +BEL(50?M)
Relative
?
phosho
?
Akt
?
Levels
upon
?
insulin
?
stimulation
 80
 
?
 
Figure 12. BEL effects on insulin-induced GLUT4 translocation to plasma 
membrane. Day 8-10 3T3-L1 adipocytes in 100 mm dishes were serum starved for 2 
hours, and then treated with or without BEL (50 ?M) for 30 minutes. Pretreated cells 
were stimulated by insulin (100 nM) for 30 minutes, and subcellular membrane fractions 
were obtained by differential centrifugation procedure. To detected GLUT4 protein 
levels, equal amount of low-density microsomal protein (LDM) and plasma membrane 
protein (PM) were separated by SDS-PAGE and assessed by western blot analysis with 
anti-GLUT4 antibody. 
 81
 
Figure 13. Distribution of proteins between lipid raft and non-raft fractions. 
Differentiated 3T3-L1 adipocytes (day 8-10) were harvested and lysed in base buffer on 
ice. The cell lysate was mixed with equal volume of base buffer containing 50% 
Optiprep, placed to the bottom of 12 ml ultracentrifuge tube, and followed with a 
continuous gradient of 5% to 20% Optiprep in base buffer. Gradients were then 
fractionated into 12 fractions, and the lipid raft fractions were verified by the distribution 
of lipid raft marker proteins, as well as protein and cholesterol concentrations. GLUT4 
protein level was assessed in combined lipid raft factions 1-2, 3-4 and non-raft fractions 
11-12 from cells with different treatment by western blotting analysis. Equal amount of 
proteins were separated by SDS-PAGE gel and western blot analysis. Caveolin-1 and 
Flotillin-1 were utilized as lipid raft markers, while ?-actin was used as marker for non-
raft proteins. 
 
F
8
w
nM
gr
co
w
se
ut
igure 14. BE
-10 3T3-L1
ithout BEL 
 insulin fo
adients cen
mbined lip
ith differen
parated by 
ilized as lip
L effects o
 adipocytes 
pretreatmen
r 30 Minut
trifugation.
id raft (LR)
t treatment 
SDS-PAGE
id raft mark
n insulin-in
 in 100 mm
t (10 or 50 ?
es. The cell
 For each 
 factions 1-
by western
 gel and we
ers, while ?-
82
duced GLU
 dishes we
M) for 30 m
s were then
treatment, G
2, 3-4 and n
 blotting an
stern blot a
actin was u
T4 incorp
re serum st
inutes, the 
 harvested a
LUT4 pro
on-raft (NR
alysis. Equ
nalysis. Cav
sed as marke
oration into
arved for 2
cells were s
nd fractiona
tein level 
) fractions 
al amount 
eolin-1 and
r for non-ra
 lipid rafts.
 hours. Wi
timulated by
ted by Opt
was assesse
11-12 from 
of proteins 
 Flotillin-1 
ft proteins.
 
 Day 
th or 
 100 
iprep 
d in 
cells 
were 
were 
 83
CHAPTER 4. SMALL INTERFERING RNA KNOCKDOWN OF CALCIUM-
INDEPENDENT PHOSPHOLIPASE A
2
 INHIBITS INSULIN-STIMULATED 
GLUCOSE UPTAKE IN 3T3-L1 ADIPOCYTES 
?
4.1. ABSTRACT 
In our previous study, in order to determine the involvement of iPLA
2 
in 
adipocyte insulin actions on glucose uptake, we used BEL, a selective suicide inhibitor of 
iPLA
2
, to inhibit the iPLA
2
 activity in 3T3-L1 adipocytes. In the present study, we 
preformed a more selective approach, small interfering RNA to investigate the role of 
iPLA
2
 in 3T3-L1 adipocytes. 3T3-L1 adipocytes at day 6 post differentiation were 
transfected by 50 ?M siRNA against iPLA
2
? or iPLA
2
?, respectively, by electroporation. 
Our primary results showed that selective delivery of siRNA efficiently inhibited the 
gene expression of iPLA
2
? and iPLA
2
? by a ~70% reduction in the mRNA levels of 
iPLA
2
?, and a nearly 80% reduction in mRNA levels of iPLA
2
? in 3T3-L1 adipocytes. 
Consistent to the gene silencing effects, the glucose uptake under insulin stimulation in 
adipocytes transfected with siRNA selective to iPLA
2
? and iPLA
2
? was diminished also: 
the insulin-stimulated glucose uptake was reduced about 40% in 3T3-L1 adipocytes 
transfected by either iPLA
2
? or iPLA
2
? siRNAs. All these results further enforced the 
essential role of iPLA
2
 in insulin-stimulated glucose uptake. 
 84
4.2. INTRODUCTION 
Calcium-independent phospholipase A
2
 (iPLA
2
) is one of the major types of 
phospholipase hydrolase family catalyzing the release of free fatty acid from the sn-2 
position of membrane glycerophospholipids. In most iPLA
2 
activity
 
studies, chemical 
inhibitors facilitate the distinguishing of iPLA
2
 from other PLA
2 
types, and they are also 
widely used to assess the role of iPLA
2 
in particular cellular processes. BEL is the most 
prominent chemical inhibitor for iPLA
2
 since it is the only inhibitor that selectively 
targets iPLA
2
 over other calcium-dependent PLA
2
s, and inhibits all known iPLA
2
s (84-
86). Recently, increasing research groups started to using, targeted gene tools to selective 
inhibit iPLA
2
s (28, 84-86).  
Small interfering RNA is one of the important gene silencing strategies to 
decrease the expression and activity of almost any given enzyme (15, 57, 90). SiRNA 
cuts down the level of mRNA encoding a specific protein via an endogenous cellular 
mechanism. By combining and degrading the target protein mRNA, this technique ideally 
leads to a reduction of the protein level and enzymatic activity if applicable. In previous 
studies, siRNA selectively targets iPLA
2
? or iPLA
2
? were utilized to determine the roles  
of iPLA
2
 in numerous biological processes, including adipocytes differentiation (15), 
glucose- induced insulin secretion (90), gene expression (318),  AA release and 
eicosanoids metabolism (57). 
Our previous study has shown that BEL treatment decreased insulin-stimulated 
glucose uptake and such an effect is mediated via decreased translocation of GLUT4 to 
 85
PM. To further confirm the roles of iPLA
2
, in the present study, we applied siRNA in 
3T3-L1 to selectively downregulate the gene expression of iPLA
2
? and iPLA
2
? and 
examined whether the insulin-stimulated glucose uptake in the 3T3-L1 adipocyte with 
iPLA
2
? or iPLA
2
? knockdown was affected by gene silencing.  
4.3. MATERIALS AND METHODS 
4.3.1. Materials 
3T3-L1 cells were purchased from ATCC (Manassas, VA). Fetal calf serum 
(FCS), Fetal bovine serum (FBS), glutamine, penicillin streptomycin and Dulbecco?s 
modified Eagle?s medium (DMEM) were obtained from Invitrogen, Inc. (Carlsbad, CA). 
Phosphate-buffered saline (PBS) was purchased from Mediatech, Inc. (Manassas, VA). 
Reagents for RNA extraction, reverse transcription and quantitative polymerase chain 
reaction (PCR) were obtained from Bio-Rad (Hercules, CA). SiRNA oligonucleotides 
were constructed by Ambion (Austin, TX). CMV-LacZ plasmid and detection kit were 
kindly provided by Dr. Suresh Mathews (Auburn University). Radiolabeled 2-
deoxyglucose was obtained from Perkin Elmer (Waltham, MA). All other chemicals were 
obtained from Sigma except those as indicated. 
4.3.2. Cell Culture of 3T3-L1 Fibroblasts and Adipocytes 
 3T3-L1 fibroblasts were grown in DMEM containing 5% D-glucose, 10% FCS, 1% 
L-glutamine, and 1% penicillin streptomycin at 37 ?C and 5% CO
2
. The medium was 
changed every other day until 100% confluent. Two days after confluence (Day 0), cell 
 86
differentiation into adipocytes was introduced by changing the medium to DMEM 
containing 5% D-glucose, 10% FBS, 1% L-glutamine, 1% penicillin streptomycin, 4 
?g/ml insulin, 0.5 mM 3-isobutyl-1-methylxanthine (IBMX), and 0.25 mM 
dexamethasone. Subsequent medium changes contained 4 ?g/ml insulin and occurred 
every three days until the cells were fully differentiated and harvested for succeeding 
experiments. Prior to use, cells were washed three times in sterile PBS buffer and serum-
starved in serum free DMEM with 0.1% bovine serum albumin (BSA) (Fisher, Waltham, 
MA) for 2 hours. 
4.3.3. Transfection of 3T3-L1 Adipocytes by Electroporation  
 The procedure of 3T3-L1 adipocyte electroporation was described previously (319). 
Four 100 mm dishes of 3T3-L1 adipocytes (at day 5 or 6 postdifferentiation) were 
trypsinized and resuspended in 12 mL complete medium (DMEM containing 5% D-
glucose, 10% FBS, 1% L-glutamine without antibiotics). The cells were then pelleted by 
centrifugation at 200 g and room temperature for 5 minutes and washed in 40 mL sterile 
D-PBS for 3 times. The cell pellet was suspended in 1.5-2 mL sterile D-PBS to achieve a 
final cell concentration approximately at 2? 10
7 
cells per mL. 0.5 mL cell solution was 
transferred to an electroporation cuvet, and mixed with specific amount of CMV-Laz 
plasmid DNA (50 ?g) or siRNA (50 nM). The electroporation was performed by Bio-Rad 
Gene Pulse Xcell at the setting as 0.18 kV, and 950 ?F capacitance. Right after 
electroporation, cells were mixed with 2.5 mL fresh complete medium and incubated  for 
10 minutes at room temperature, than reseeded to 24- well plates (0.5 mL per well), and 
incubated at 37 ?C and 5% CO
2.
 The medium were changed to normal growth medium 
 87
containing 5% D-glucose, 10% FBS, 1% L-glutamine, and 1% penicillin streptomycin 12 
hours after electroporation.  
The siRNA directed against iPLA
2
? and iPLA
2
? were constructed employing the 
Ambion Silencer siRNA construction kit according to the protocol provided by 
manufacturer. The siRNA sequences were listed in Table 4. The siRNA (50 nM) was 
delivered into 3T3-L1 adipocytes through electroporation as described above. For all 
transfection experiments, scramble siRNA were used as negative controls. Fourth-eight 
hours after transfection, the cells were subjected to post-transfection analyses, including 
mRNA level analysis by reverse transcription and quantitative PCR, and glucose uptake 
assay.  
 4.3.4. Evaluation of Transfection Efficiency  
3T3-L1 adipocytes transfected with CMV-LacZ plasmid DNA were subjected to ?-
galactosidase staining 48 hours post electroporation. ?-galactosidase was visualized 
utilizing the In Situ ?-Galactosidase Staining kit (Stratagene, Ceder Creek, TX) according 
to the procedure provide by manufacturer. Briefly, cells were washed three times in ice-
cold PBS followed by fixation using fixing solution for 10 minutes. Next, the cells were 
stained with 5-bromo-4-chloro-3-indoyl-?-D-galactopyranoside (X-Gal) diluted in 
staining solution. Cells expressing ?-galactosidase appeared blue, which was visualized 
and captured using a Canon PowerShot S31S-attached Nikon TS100-F inverted 
microscope. 
 88
4.3.5. Reverse Transcription and Quantitative PCR 
Following the protocol provided by the manufacturer of RNA extraction kit from 
Qiagen (Valencia, CA), total RNA was extracted from 3T3-L1 adipocytes transfected by 
siRNA selectively targeting against iPLA
2
? or iPLA
2
?, or scramble siRNA.  The RNA 
concentration and purity were assayed by determining the absorbency of the RNA sample 
at 260 nm and 280 nm using DV? 530 Life Science UV/Vis spectrophotometer 
(Beckman, Fullerton, CA).Complementary DNA (cDNA) was synthesized from 1?g of 
the total RNA of each sample according to the manufacturer?s protocol using an iScript
TM
 
cDNA Synthesis Kit (Bio-Rad). The cDNA synthesis was initiated by incubating the 
reaction mixture in 25?C for 5 minutes, extended by 42?C incubation for 30 minutes and 
then inactivated by 85?C incubation for 5 minutes.  
 Quantitative real-time reverse transcriptase polymerase chain reaction (RT-PCR) 
was performed for 40 PCR cycles by utilizing iQ
TM
 SYBR
?
 Green Supermix (Bio-Rad). 
Mouse gene specific primers were designed from Primer Bank (http://pga.mgh.harvard. 
edu/primerbank/citation.html), and constructed by Integrated DNA Technologies, Inc. 
(IDT, Inc., Coralville, IA). Oligonucleotide primer sequences are listed as Table 2. 
Relative quantification of target gene expression was assayed using the comparative 
threshold (C
T
) and computed by the 2
???C
T
 method described by the manufacturer (Bio-
Rad). Changes in messenger RNA (mRNA) levels of specific genes were calculated 
according to the reference gene, 36B4. 
 89
4.3.6. Insulin-Stimulated Glucose Uptake Assay 
3T3-L1 adipocytes at 48 Hours after siRNA transfection were subjected to 
insulin-stimulated glucose uptake assay. The steps followed the protocol previously 
described (305). Briefly, serum-starved adipocytes were washed and incubated for 15 
minutes in Kreb?s Ringer phosphate HEPES (KRPH) buffer (136 mM NaCl, 20 mM 
HEPES, 5 mM sodium phosphate buffer, 4.7 mM KCl, 1 mM MgSO
4
, 1mM CaCl
2
, pH 
7.4) and then incubated with or without insulin (100 nM) for 30 minutes. Glucose uptake 
was initiated by adding [
3
H]-2-deoxyglucose (2-DOG) to a final assay concentration for 
10 minutes and stopped by washing the cells three times with ice-cold PBS buffer. Air-
dried cells were then solublized in 0.2 N NaOH solution. The intracellular incorporation 
of [
3
H]-2-deoxyglucose was measured by counting the radioactivity in half of the cell 
lysate with Packard liquid scintillation analyzer (Meriden, CT). The rest of the lysate was 
subjected for protein concentration determination by using BCA protein assay kit (Bio-
Rad) with BSA as standard. The level of non-specific glucose uptake was determined in 
cells pretreated with cytochalasin B before insulin stimulation. For each group of cells, 
the glucose uptake values were denoted as ?pmol radioactive 2-deoxyglucose taken up 
per minute and per mg protein?, and the amount of [
3
H]-2-deoxyglucose taken up was 
calculated according to the formula: 
(Observed cpm? cytochalasin B cpm) ? 2/total cpm ? 25
10 ? mg protein per mL
 
The 3T3-L1 adipocytes transfected by scrambled siRNA were used to be negative 
controls. 
 90
4.3.7. Statistical Analysis 
Data are presented as mean ? S.E. Statistical significance difference between 
treatments was determined using the Student?s t tests, and statistical significance level is 
set as 0.05. 
4.4. RESULTS AND DISCUSSION 
4.4.1. Electroporation Introduces Foreign Plasmids into 3T3-L1 Adipocytes 
The introduction of DNA or genes of interest into adipocytes by standard 
transfection protocols such as calcium phosphate, DEAE-dextran, and liposome-mediated 
transfection are very inefficient (319). It is also difficult that transgenes can be expressed 
in adipocytes, and this genetic tool is substantially more burdensome. Furthermore, the 
production of recombinant adenoviruses to use in infection of insulin-responsive tissues 
is comparative more time-consuming and requires very high titers of adenovirus. 
Therefore, it is relatively more efficient, simple, and fast to transfect adipocytes using 
electroporation (319). Differentiated 3T3-L1 adipocytes were electroporated to deliver 
the foreign nucleotides. A large number of 3T3-L1 cells were killed during the 
electroporation procedure, and a lipid-droplet-loss was observed in electroporated cells. 
However, the living cells successfully attached to the cell culture plates in 3~5 hours after 
the electroporation.   As showed in Figure 15, there were no noticeable changes in the 
adipocyte appearance before or after electroporation. To determine the efficiency of 
electroporation, we transfected 3T3-L1 adipocytes with CMV-LacZ plasmid to express ?-
Galactosidase, which is an enzyme that catalyzes the hydrolysis of ?-galactosides, 
 91
including lactose. Transfected cells expressing ?-galactosidase can cleave X-Gal to 
produce a blue stain. The transfection efficiency is determined by counting stained and 
unstained cells under a microscope and calculating the percentage of stained cells in the 
total population. Using 50 ?g of CMV-LacZ plasmid DNA, we obtained an 
electroporation efficiency of less than 30% (Figure 16), which is relatively low. 
According to the original protocol (319), the efficiency is closely correlated with the 
amount of plasmid used.  When 600 ?g of CMV-LacZ plasmid DNA was used, the 
transfected adipocytes displayed 80%-90% efficiency (319). Due to the source limitation, 
we did not utilize the huge amount of plasmid DNA in this study. However, the 30% 
transfection efficiency was reasonable considering the small amount of plasmid DNA 
used in the current study. 
4.4.2. SiRNA Reduces the Amount of mRNA Levels of iPLA
2
? and iPLA
2
? in 3T3-
L1 Adipocytes 
To test whether siRNA can induce gene-selective silencing in 3T3-L1 adipocytes, 
we tested mRNA and protein levels of iPLA
2
? and iPLA
2
? in transfected 3T3-L1 
Adipocytes. Results from RT-PCR showed that both genes were dramatically reduced by 
siRNA transfection (Figure 17): iPLA
2
? mRNA level was attenuated by ?70%, and the 
suppressed iPLA
2
? mRNA level was up to 80%, indicating that siRNA gene silencing 
technique effectively reduced the gene expression of iPLA
2
? and iPLA
2
? in 3T3-L1 
adipocytes. 
 92
4.4.3. Pretreatment of siRNAs Targeting iPLA
2
? or iPLA
2
? Inhibits Insulin-
Stimulated Glucose Uptake in 3T3-L1 Adipocytes 
Previous studies show that inhibition of iPLA
2
 activity utilizing chemical inhibitors 
significantly decreased the insulin-stimulated glucose uptake in 3T3-L1 adipocytes 
without affecting the basal glucose uptake levels (Chapter 3). Considering the intense 
diminution of iPLA
2
? and iPLA
2
? mRNA levels observed in transfected cells, we 
assessed effect of iPLA2? or iPLA2? siRNA transfection on the insulin-stimulated 
glucose uptake in the 3T3-L1 adipocyte. Compared to negative control electroporated 
with scrambled siRNA, selective attenuation of iPLA
2
? significantly reduced the insulin-
stimulated glucose uptake by approximately 40% (Figure 18). Similar inhibitory effects 
were observed in adipocytes transfected by iPLA
2
? siRNA. This results are consistent 
with previous studies utilizing (S)-and (R)-BEL (Figure 6 in Chapter 3). Collectively, our 
results have shown that selective knockdown of iPLA
2
? and iPLA
2
? inhibits insulin-
stimulated glucose uptake in 3T3-L1 adipocytes, indicating the important roles of iPLA
2 
in adipocyte biology. Combined with the results from previous studies using BEL, we 
conclude that insulin-stimulated glucose uptake is mediated, at least in part, by iPLA
2
 
enzymatic activity, which indicates that iPLA
2
 may represent a novel therapeutic target 
for the treatment of insulin resistance and associated diseases. 
 93
Table 4. Oligonucleotides siRNA sequences. 
Gene Sequence 
iPLA
2
?-1 Sense        5?- CGC  UGC AAC CAA AAC AUU Att-3? 
Antisense  5?- UAA UGU UUU GGU UGC AGC Ggg-3? 
 
iPLA
2
?-2 Sense        5?- GCC UGG  UCA UUA UCC AGC Utt-3? 
Antisense  5?- AGC UGG AUA AUG ACC AGG Cct-3? 
 
iPLA
2
?-1 Sense        5?- CAA GAG UGA GUA UUG AUA Att-3? 
Antisense  5?- UUA UCA AUA CUC ACU CUU Gca-3? 
 
iPLA
2
?-2 Sense        5?- GGC UGA GAC AAG UUA AGG Att-3? 
Antisense  5?- UCC UUA ACU UGU CUC AGC Cgt-3? 
 
 94
?
Figure 15. 3T3-L1 adipocytes after electroporation. Day 5 3T3-L1 adipocytes were 
electroporated with a Bio-Rad gene pulser system at the setting of 0.18kV and 960 ?F  
capacitance. The state of adipocytes after electroporation was compared to those without 
electroporation. (A) 3T3-L1 adipocytes with electroporation viewed at 400 ?; (B)  3T3-
L1 adipocytes with electroporation viewed at 200 ? ; (C) 3T3-L1 cells without 
electroporation viewed at 400 ?; (D) 3T3-L1 cells without electroporation at 200 ? under 
microscope. 
 95
 
Figure 16. Electroporation Efficiency in 3T3-L1 adipocytes. Day 5 3T3-L1 adipocytes 
were electroporated with 50 ?g CMV-LacZ plasmid DNA, and transfection/expression of 
?-galactosidase efficiency was determined by In Situ ?-galactosidase staining kit. Cells 
taking up the blue stain express ?-galactosidase, which is used as a measure of 
transfection efficiency. The stained and unstained cells in randomly selected fields were 
counted. The transfection efficiency is the percentage of stained cells in the total 
population. 
 
 
 96
?
Figure 17. Effects of siRNA transfection on the mRNA levels of iPLA
2
? and iPLA
2
? 
in 3T3-L1 Adipocytes. Day 6 3T3-L1 adipocytes were cultured and transfected with 50 
nM negative control siRNA, siRNA directed against iPLA
2
? and iPLA
2
? by 
electroporation. 48 hours after transfection, total RNA was collected and reverse 
transcribed to cDNA. RT-PCR analysis was performed to determine the mRNA levels of 
iPLA
2
? and ? utilizing 36B4 as the reference gene. The relative mRNA levels are 
computed using the mRNA levels in negative control cells (scrambled siRNA treated) as 
internal standard, and the fold changes are represented as mean ? S.E. of independent 
experiments. Statistical significance of siRNA treatments on mRNA levels was 
determined by comparing the mean of mRNA level of iPLA
2
? or iPLA
2
? of cells 
transfected by selective siRNA with that from cells treated with scramble siRNA using 2-
sample student?s t test (***, p<0.001). 
?
0
0.2
0.4
0.6
0.8
1
1.2
iPLA2?beta? iPLA2?gamma?
Relative
?
mRNA
?
Levels
Scrambled?siRNA
Selective?siRNA
***
***
 97
 
 
 
 
Figure 18. Effects of siRNA gene silencing of iPLA
2
? and iPLA
2
? on insulin-
stimulated glucose uptake in 3T3-L1 adipocytes. Day 6 3T3-L1 adipocytes were 
cultured and transfected with 50 nM negative control siRNA, siRNA directed against 
iPLA
2
? and ? by electroporation. 48 hours after transfection, cells were serum-starved, 
and then stimulated by 100 nM insulin for 30 minutes. 2-Deoxygluocse uptake in these 
adipocytes was measured over 10 minutes. Data are mean ? S.E. from three or more 
independent experiments performed in triplicate, normalized to negative control insulin-
stimulated uptake. Statistical significance of treatments on basal or insulin-stimulated 2-
DOG uptake was determined by comparing the mean of basal or insulin-stimulated 2-
DOG uptake of cells transfected by selective siRNA with that from cells treated with 
scramble siRNA using 2-sample student?s t test (*, p<0.05). 
  
0
20
40
60
80
100
120
SiRNA?(iPLA2?Beta) SiRNA?(iPLA2?Gamma) Srambled?siRNA
2
?
DOG
?
Uptake
(%
?
of
?
insulin
?
stimulated
??
2
?
DOG
?
uptake
?
in
?
negative
?
control)
+?insulin? ? insulin?
*
*
 98
CHAPTER 5. SUMMARY AND FUTURE STUDIES 
 
In this dissertation, we demonstrate a role for iPLA
2
 in glucose transport in 
insulin-stimulated 3T3-L1 adipocytes. We found that adipocyte insulin-stimulated 
glucose uptake was significantly reduced with BEL-induced iPLA
2
 inhibition. In 
agreement with the BEL treatment results, selective knockdown of iPLA
2
? or iPLA
2
? 
using siRNA technique significantly decreased the insulin-stimulated glucose uptake in 
the 3T3-L1 adipocytes.  We further explored the mechanism for the inhibitory effect of 
BEL. The results showed that the impaired insulin action on glucose transport was not 
due to the alterations in insulin signaling pathways, but due to decreased incorporation of 
GLUT4 into the plasma membrane or lipid raft microdomains.  
Although the regulation of GLUT4 trafficking have been areas of intensive 
investigation since its identification, the mechanisms involved in our results remain to be 
established. As far as the biological functions of iPLA
2
 are concerned, three possible 
mechanisms by which insulin-stimulated glucose uptake is regulated by iPLA
2
 enzymatic 
activity are proposed here: 
1. Activation of PPAR?: PPAR? is believed to be a prominent effector controlling 
insulin sensitivity. Evidence is accumulating to indicate that PPAR? activation can affect 
insulin signaling in adipose tissue through upregulation of a number of downstream 
 99
molecules, including IRS proteins, the p85 subunit of PI3K and CAP - all of which might 
be predicted to enhance GLUT4 activity (320). Recent studies have shown that iPLA
2
 
could serve as PPAR? activator through providing lipids or lipid precursors during the 
adipocytes differentiation (15). Additionally, AA exposure potentiates the insulin-
stimulated glucose uptake by increasing GLUT4 level at the plasma membrane, and this 
potentiation is proved to be dependent on PPAR? activation (309). It is possible that 
iPLA
2
 inhibition prevents PPAR? activation, leading to the reduced insulin response in 
adipocytes.   It was unclear; however, whether short term treatments would be sufficient 
to cause such effects. The non-altered Akt and IR phosphorylation may let us exclude 
PPAR? in the present study. Long term BEL treatment or selective gene knockdown 
would help us to better understand this possible mechanism.  
2. Alterations of phospholipid membrane structure: One of the most important 
cellular functions of iPLA
2
 is controlling the membrane glycerophospholipid metabolism. 
By regulating the cleavage of pre-existing membrane PL, the reincorporation of 
generated fatty acids into membrane PL molecules, and reacylating the lysoPL with a 
different fatty acid, iPLA
2
s are highly involved in the ongoing deacylation/reacylation 
cycle critical for membrane homeostasis (12). More importantly, the accumulation of 
lysophospholipids and free fatty acids may increase the membrane fluidity or promote 
non-bilayer structures, and eventually stimulate the fusion of biological membranes. 
Recent studies have confirmed the linkage of iPLA
2
 activity and several exocytosis 
processes, including glucose-stimulated insulin secretion in pancreatic ? cells (89, 111, 
112), lysozyme secretion in U397 cells (110), exocytosis of amylase from parotid acinar 
 100
cells (113), and lysosome-mediated IL-1? secretion (114). Similar to these secretion or 
exocytosis procedures, GLUT4 translocation to the plasma membrane requires the 
formation and transport of vesicle, as well as the fusion of bilayers of vesicle and plasma 
membrane (218). Therefore, it is reasonable to postulate that GLUT 4 translocation is at 
least partly dependent on iPLA
2
 activity. In addition, alterations in the fatty acid 
composition of membrane phospholipids can affect specific membrane microdomains, 
such as lipid rafts (310), which serves as sites for assembly of signaling complexes that 
play an important role in insulin action, especially the Cbl/CAP cascade (317). To our 
knowledge, no studies to date have discussed the relation between iPLA
2 
activities or 
BEL effects on lipid raft structures; however, the observed alteration in GLUT4 
distributions indicates this possibility. 
3. Regulations of Ca
2+ 
homeostasis: Calcium-independent PLA
2
 has recently been 
shown to regulate Ca
2+
 entry by replenishing the depleted intracellular Ca
2+
 stores. It has 
been observed in a variety of cells and tissues that both chemical and genetic inhibition of 
iPLA
2
? activation result in the block of store-operated Ca
2+
 channels (SOC), and the Ca
2+
 
influx can be restored by LPC (105-108). These studies suggest that the iPLA
2
 activity is 
necessary for the activation of SOC. The iPLA
2
 is also reported to regulate the Ca
2+ 
influx by transient receptor potential (TRP) channels (108, 109). In many exocytosis 
processes, increased cytosolic calcium is known to be required. BEL treatment has been 
shown to inhibit such processes, including release of neurotransmitter (115) and mast cell 
granule (116). Therefore, the regulatory roles of iPLA
2
 on calcium entry may explain 
some aspects of its importance in secretion. 
 101
Similar to these proteins, the movement of GLUT4 has been shown to be 
controlled by Ca
2+
, and it appears to be caused by physically associated signaling 
molecules that sense local Ca
2+
 increases at the mouth of the channels (240, 321). The 
Ca
2+-
binding protein calmodulin has a central role in this process. As reviewed by Lanner 
(321), the late steps of insulin signaling contains an increase in the concentrations of  
intracellular Ca
2+
  and Ca
2+
 sensing proteins, which serve as modulators for GLUT4 
exocytosis in either direct or indirect manner. Some research groups found that using 
Ca
2+
 chelators or calmodulin antagonists in insulin-sensitive cells or tissues decreases the 
glucose uptake upon insulin administration and reduces the level of GLUT4 in plasma 
membrane (240, 322). Of note is the work of Lanner and coworkers in 2006 (240), who 
demonstrated that inhibition of Ca
2+
 influx by 2-aminoethoxydiphenyl borate (2-APB) 
decreased insulin-mediated glucose uptake in skeletal muscle but did not affect the 
phosphorylation state of PKB/Akt or the MAP kinase ERK1/2. In the current study, we 
also show that BEL treatment influence the insulin actions in 3T3-L1 adipocytes in the 
similar way as 2-APB. Consequently, possible Ca
2+
 influx blocking resulting from iPLA
2
 
inhibition contributes to the inhibitory effects of BEL on insulin action observed in this 
study. 
The present results constitute the first demonstration of an important role of 
iPLA
2
 in insulin action on glucose uptake. It is involved in some aspects of the GLUT4 
translocation in response to insulin without affecting insulin signal pathway up to 
forehead of Akt kinase. Although further studies are needed to identify the specific 
underlying mechanism, this novel function of iPLA
2
 in 3T3-L1 adipocytes makes it a 
 102
promising target for strategies for prevention and treatment of obesity, type 2 diabetes 
and other linked metabolic diseases. 
  
 103
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 131
APPENDIX I  
ABBREVIATIONS 
2-APB, 2-Aminoethoxydiphenyl Borate  
2-DOG, 2-Deoxyglucose 
AA, Arachidonic Acid  
AACOCF3, Arachidonyl 
Trifluoromethyl ketone (1,1,1-trifluoro-
6Z, 9Z, 12Z, 15Z-heneicosatetraen-2-
one) 
AdipoR1, Adiponectin Receptor 1 
AdipoR2, Adiponectin Receptor 2 
AdPLA, Adipose PLA2  
aPKC, Atypical Protein Kinase C  
APS, Adapter Protein with Pleckstrin 
Homology and Src Homology Domains 
ARDS, Adult Respiratory Distress 
Syndrome  
ATP, Adenosine Triphosphate  
BEL, Bromoenol Lactone ((E)-6-
(bromomethylene)-3-(1-naphthalenyl)-
2H-tetrahydropyran-2-one) 
BMI, Body Mass Index 
BSA, Bovine Serum Albumin 
C/EBP, CCAAT/Enhancer-Binding 
Protein  
C3G, Crk SH3-binding guanine 
nucleotide-releasing factor  
cAMP, Cyclic Adenosine 
Monophosphate  
CAP, Cbl-Associated Protein  
CHO, Chinese Hamster Ovary  
CIF, Calcium Influx Factor  
COX, Cyclooxygenase  
cPLA2, Cytosolic Ca2+-Dependent 
PLA2  
CRAC, Calcium Release-Activated 
Channels  
CREBP, cAMP-Response Element-
Binding Protein 
CrkII, Chicken Tumor Virus No.10 
(CT10) Regulator of Kinase II 
CT, Comparative Threshold 
CYT, Cytosol 
DMEM, Dulbecco?s Modified Eagle?s 
Medium  
DMSO, Dimethyl sulfoxide 
DNA, Deoxyribonucleic Acid 
ER, Endoplasmic Reticulum 
FBS, Fetal Bovine Serum 
FCS, Fetal Calf Serum  
FFA, Free Fatty Acids  
 132
Gab-1, Grb2-Associated Binder-1 
GAP, GTPase Activating Protein  
GLUT4, Glucose Transporter 4  
Grb2, Growth Factor Receptor Bound 2  
GS2, Gene Sequence 2 
GSK-3?, Glycogen Synthase Kinase-3?  
HDM, High Density Microsome 
HEPES, 4-(2-Hydroxyethyl)-1-
Piperazineethanesulfonic Acid  
HES, HEPES-EDTA-Sucrose 
HMIT, H+-Myo-Inositol Transporter 
IBMX, 3-isobutyl-1-methylxanthine 
IKK?, I?B Kinase ? 
IL-1, Interleukin-1  
IL-6, Interleukin-6  
iNOS, Inductible Nitric Oxide Synthase  
iPLA2, Cytosolic Ca2+-Independent 
PLA2 
IR, Insulin Receptor  
IRK, IR Tyrosine Kinase 
IRS, Insulin Receptor Substrate  
I?B?, Inhibitor of NF?B  
JNK, c-Jun N-Terminal Kinase 
KRPH, Kreb?s Ringer Phosphate 
HEPES  
LA, Linoleic Acid  
LCPUFA, Long Chain Polyunsaturated 
Fatty Acid 
LDL, Low-Density Lipoprotein 
LDM, Low Density Microsome 
LPA, Lysophosphatidic Acid  
LPC, Lysophosphatidylcholine 
LPLA2, Lysosomal PhosphoslipaseA2 
LR, Lipid Raft 
LT, Leukotriene 
Lyso PL, Lysophospholipid 
MAFP, Methyl Arachidonyl 
Fluorophosphonate (5Z, 8Z, 11Z, 14Z-
eicosatetraenyl-phosphonofluoride, 
methyl ester) 
MAPK, Mitrogen-Activated Protein 
Kinase 
MN, Mitochondrial Nuclei 
mTOR, Mammalian Target of 
Rapamycin  
NEFA, Non-Esterified Fatty Acid 
NEMO, Nuclear Factor ?B Essential 
Modulator 
NF?B, Nuclear Factor-?B  
NR, Non-Raft 
 133
NTE, Neuropathy Target Esterase  
N-WASP, Neuronal Wiskott-Aldrich 
Syndrome Protein 
PAF, Platelet-Activating Factor  
PAF-AH, Platelet-Activating Factor 
Acetylhydrolase  
PAP-1, Mg2+-dependent phosphatidic 
acid phosphohydrolase (Lipin) 
PBS, Phosphate-Buffered Saline 
PC, Phosphatidylcholine  
PDK, Phosphoinositide-Dependent 
Kinase  
PG, Prostaglandin 
PGE2, Prostaglandin E2  
PI3K, Phosphatidylinositol 3-Kinase  
PIP2, Phosphotidyl- 4, 5-
Biphosphophate  
PIP3, Phosphotidyl-3, 4, 5-
Triphosphophate  
PKB, Protein Kinase B(Akt) 
PKC, Protein Kinase C  
PL, Phospholipid 
PLA2, Phospholipase A2  
PM, Plasma Membrane  
PP2A, Serine/Threonine Protein 
Phosphatase 2A  
PPAR?, Peroxisome Proliferator-
Activated Receptor ?  
PPAR? , Peroxisome Proliferator-
Activated Receptor ?  
PTB, Phosphotyrosine Binding 
PTEN, Phosphatase and Tensin 
Homolog Deleted on Chromosome 10  
PTP1B, Protein Tyrosine Phosphatase 
1B  
PUFA, Polyunsaturated Fatty Acid 
(R)-BEL, (R)-(E)-6-(bromomethylene)-
3-(1-naphthalenyl)-2H-tetrahydropyran-
2-one 
RBP4, Retinol Binding Protein 4  
RNA, Ribonucleic Acid  
ROS, Reactive Oxygen Species  
RSK2, p90 Ribosomal S6 Kinase  
RT-PCR, Quantitative Teal-Time 
Reverse Transcriptase Polymerase Chain 
Reaction  
S6K1, S6 Kinase 1 
(S)-BEL, (S)-(E)-6-(bromomethylene)-
3-(1-naphthalenyl)-2H-tetrahydropyran-
2-one 
SDS-PAGE, Sodium Dodecyl Sulfate-
Polyacrylamide Gel  
SH2, Src Homology 2  
Shc, Src Homologous and Collagen  
 134
SHIP2, SH2-Containing Inositol 
Phosphatase 2 
siRNA, Small Interfering RNA  
SIRP, Signal-Regulated Protein  
SNARE, Soluble N-ethylmaleimide-
Sensitive Fusion protein (NSF) 
Attachment Protein Receptor  
SOC, Store-Operated Ca2+ Channel 
Sos, Son-of-Sevenless  
sPLA2, Secreted PLA2  
SREBP, Sterol Regulatory Element 
Binding Protein 
 
 
 
 
 
 
 
 
 
 
 
 
 
STZ, Streptozotocin  
TBS, Tris Buffered Saline  
TNF-?, Tumor Necrosis Factor-? 
TRP, Transient Receptor Potential 
TRPM8, Transient Receptor Potential 
(Melastatin)-8  
TTS-2.2, Transport Secretion Protein-2.2 
VAMP, Vesicle-Associated Membrane 
Proteins 
X-Gal, 5-Bromo-4-Chloro-3-Indoyl-?-
D-Galactopyranoside
 135
APPENDIX II  
Protocol of Glucose Uptake Assay  
The protocol is modified from the method described by Lakshmanan (305). 
Reagents: 
Serum Free DMEM 
DMEM containing 5% D-glucose, 1% L-glutamine, 1% penicillin streptomycin and 0.1% 
BSA 
KRPH Buffer  
Final concentration  Stock solution Volume (250 mL buffer) 
136 mM NaCl 5.0 M NaCl 6.8 mL 
20 mM HEPES 1.0 M HEPES 5.0 mL 
5 mM sodium phosphate  
buffer (pH 7.4) 
0.1 M sodium phosphate  
buffer (pH 7.4) 12.5 mL 
4.7 mM KCl 1 M KCl 1.18 mL 
1 mM MgSO
4
 1 M MgSO
4
 0.25 mL
1mM CaCl
2
 1 M CaCl
2
 0.25 mL 
Add ddH
2
O up to about 240 mL, adjust the pH to 7.4, and make up the volume to 250 mL 
with ddH
2
O. Prepare fresh. 
 
Cytochalasin B Stock and Working Solution 
 
Stock: 10 mM (4.796 mg in 1 mL 95% EtOH); stored at -20?C. 
Working: 20 ?M in KRPH buffer (add 8 ?L stock to 4 mL KRPH buffer). Prepare prior 
to use. 
 
Insulin Stock and Working Solution 
 
Stock: 335 ?M (2 mg/mL insulin in 3 mM HCl); filter sterilized, and stored at 4 ?C. 
Working: 10 ?M (dilute 100 ?L stock with 3250 ?L ddH
2
O). Prepare prior to use. 
 
KRPH-[
3
H]-2-Deoxyglucose Label 
 
Mix 25 ?L of 100 mM 2-DOG stock, 45.5 ?L of [
3
H]-2-DOG and 2.442 mL KRPH. 
Prepare prior to use. 
 
 136
Procedure: 
? For all incubation, the media volume added is depending on the cell culture dishes 
used for glucose assay: 1mL for 60 mm dish and 6-well plate, 500 ?L for 12-well 
plate, and 300 ?L for 24-well plate.  
? The final concentrations of the inhibitor, DMSO, insulin, cytochalasin B, and 
radiolabeled 2-DOG are the same. The adding volume is adjusted according to the 
media volume in one dish (60 mm dish) or well (multiple-well plate).  
? For the addition of each treatment (inhibitor, DMSO, insulin, cytochalasin B, 
orradiolabeled 2-DOG), the dishes or wells are treated in the same order and 
staggered at 1-minute intervals.  
? To check the effect of treatment on insulin-stimulated glucose uptake. The cells 
are grouped into treatment group(s), control group, and non-specific uptake 
group. For each group, it is divided into two sub-groups treated with or without 
insulin to determine the basal and insulin-stimulated glucose uptake. 
? 3T3-L1 adipocytes at 8-10 days post differentiation or L6-GLUT4
myc 
cells upon 
confluent are used for assays. 
? For each group of cells, the glucose uptake values were denoted as ?pmol 
radioactive 2-deoxyglucose taken up per minute and per mg protein?, and the 
amount of [
3
H]-2-deoxyglucose taken up was calculated according to the formula: 
(Observed cpm? cytochalasin B cpm) ? 2/total cpm ? 25
10 ? mg protein per mL
 
Step1. Wash cells twice with PBS and incubate them in serum-free DMEM at 37 ?C (2 
hours for adipocytes, and 5 hours for L6-GLUT4
myc 
cells). 
*Step2. If inhibitor treatment needed, change media to serum-free DMEM containing 
inhibitor with specific final concentration for dishes or wells in treatment group(s) and 
incubate at 37 ?C for 30 minutes.  Incubate cells in control and non-specific uptake 
groups in serum-free DMEM containing DMSO (The volume of DMSO is equal to the 
largest volume of inhibitor. For example, in experiment with both 10 ?M and 50 ?M 
BEL treatments, the control media contains DMSO with equal amount to BEL solution 
added to DMEM containing 50 ?M BEL). 
*Step3. Wash the inhibitor pretreated cells twice in KRPH buffer, and incubate them in 
KRPH buffer containing inhibitor with the treatment concentration for 15 minutes; wash 
and incubate cells in control and non-specific uptake groups with KRPH buffer 
containing DMSO. 
 137
* Step 2 and 3 can be skipped if no inhibitor treatment required. Wash and incubate 
serum-starved cells in KRPH buffer for 15 minutes. 
Step4. At the last 5 minutes of KRPH incubation, change the buffer with premixed 
KRPH- cytochalasin B for non-specific uptake wells.  
Step5. Add 100 ?M insulin at a 1:100 dilution rate (final concentration as 100 nM) to all 
insulin stimulation dishes or wells and incubate for 30 minutes. For adipocytes, incubate 
at room temperature; for L6 myotubes, incubate at 37 ?C. 
Step6. Add KRPH-[
3
H]-2-Deoxyglucose Label at a 1:20 dilution rate to all dishes or 
wells and incubate for 10 minutes at room temperature. 
Step7. Wash the cells three times with ice-cold PBS buffer. Air-dry cells for 15 minutes 
at room temperature. 
Step8. Solublize cells in 0.2N NaOH solution, mix cells by pipeting.  
Step9. Use half of lysate for radiolabel counting. Mixed the lysate with 4 mL of 
scintillation cocktail to determine the total counts per minutes (cpm). Use equal volume 
of 0.2N NaOH solution mixed with 4 mL of scintillation cocktail as blank, and 1/25 
volume of radiolabel added mixed with 4 mL of scintillation cocktail for the total cpm 
determination. 
Step10. Use the rest of the lysate for protein assay. 
 
  
 138
APPENDIX III  
Protocol of Lipid Rafts Separation 
The protocol is following the method described by Pike and Macdonald (307).  
All steps were carried out on ice. Centrifuge rotor and tubes, and gradient maker were 
pre-cold in 4?C. 
Reagents: 
Base buffer  
20 mM Tris-HCl, pH 7.8, 250 mM sucrose 
50% Opti-Prep in base buffer containing 1 mM CaCl
2
, 1 mM MgCl
2
 
20% OptiPrep in base buffer 
5% OptiPrep in base buffer 
 
Procedure: 
Step1. Wash and scrape four 100-mm dishes of cells into base buffer containing 1 mM 
CaCl
2
 and 1 mM MgCl
2
; 
Step2. Pellet cells by centrifugation for 2 min at 250g and resuspended in 1 mL of base 
buffer containing 1 mM CaCl
2
, 1 mM MgCl
2
, and protease inhibitor cocktail; 
Step3. Lyse cells by passage through a 22 g?3'' needle 20 times;  
Step4. Centrifuge cell lysates were centrifuged at 1,000g for 10 minutes, and collect the 
resulting postnuclear supernatant. Transfer the supernatant to a separate tube. 
Step5. Resuspend the pellet in 1 mL of base buffer containing 1 mM CaCl2, 1 mM 
MgCl2, and protease inhibitor cocktail, and lyse it again by sheering 20 times through a 
needle and syringe.  
Step6. Centrifuge at 1,000g for 10 minutes, combined the second postnuclear supernatant 
with the first. 
Step7. Add an equal volume (2 mL) 50% Opti-Prep in base buffer containing 1 mM 
CaCl
2
, 1 mM MgCl
2
 to the combined postnuclear supernatants (2 mL) and place the 
mixture at the bottom of a 12 ml centrifuge tube.  
 139
Step8. Layer an 8 mL continuous gradient of 5% to 20% OptiPrep in base buffer on the 
top of the lysate mixture (containing 25% of OptiPrep.) 
*Step9. Centrifuge the gradients for 90 minutes at 52,000g, 4?C, using an SW-41 rotor in 
a Beckman ultracentrifuge.  
*After centrifugation, cloudiness could be seen throughout the gradient. A distinct white 
band was apparent at the top of gradients. 
Step10. Fractionate gradients into 12 fractions (1mL/fraction).  
Step11. Assess the distribution of various proteins by Western blotting, the total protein 
in each fraction by Bio-Rad BCA protein assay kit, and the total cholesterol in each 
fraction using the Wako CII Total Cholesterol assay kit. 
  
 140
APPENDIX IV  
Protocol of siRNA Transfection Using Electroporation 
The protocol is modified from the method described by Okada (319). 
Reagents: 
Complete medium  
DMEM containing 5% D-glucose, 10% FBS, 1% L-glutamine without antibiotics) 
D-PBS Buffer  
PBS buffer without Mg 
2+
 and Ca
2+
) 
SiRNA Stock and Working Solution 
 
Stock: Disolve 5 nmol siRNA in 100 ?L RNase-free H
2
O (50?M) 
Working: Dilute 5 ?L siRNA stock with 20 ?L RNase-free H
2
O (10?M) 
 
Procedure: 
Step1. Add 2 mL/dish trysin-EDTA to four 100 mm dishes of 3T3-L1 adipocytes (at day 
5 or 6 postdifferentiation) and swirl gently. Aspirate the trysin-EDTA solution and 
incubate the cells at 37?C for 10 minutes.  
Step2. Resuspend all the trypsinized cells in 12 mL complete medium, and pipet up and 
down gently for 10 times in order to disperse the cell clumps.Transfer the cell solution to 
a 50 mL centrifuge tube.  
Step3. Centrifuge the cell suspension for 5 minutes at 200 g and room temperature. 
Carefully remove the supernatant. Resuspend  the pellet in 40 mL sterile D-PBS, disperse 
the cells by pipeting, and pellet the cells at 200 g and room temperature for 5 minutes. 
Repeat the wash in 40 mL sterile D-PBS for twice.  
Step4. Suspend the cell pellet in 1.5-2 mL sterile D-PBS to achieve a final cell 
concentration approximately at 2? 10
7 
cells per mL.  
Step5. Transfer 2.5 ?L siRNA (10?M) into the bottom of an electroporation cuvet. Add 
0.5 mL cell solution to the electroporation cuvet, and mix the cells and siRNA by gentle 
tapping.  
 141
Step6. Put the cuvet in the Bio-Rad Gene Pulse Xcell and electroporate the cells at the 
setting as 0.18 kV, and 950 ?F capacitance.  
Step7. Immediately after electroporation, add 1 mL fresh complete medium, and transfer 
the cell solution to a 15 mL centrifuge tube containing 1.5 mL fresh complete medium.  
Incubate for 10 minutes at room temperature. 
Step8. Reseed the cells to 24- well plates (0.5 mL per well), and incubate the cells at 
37 ?C and 5% CO
2.
 Change the medium to normal growth medium 12 hours after 
electroporation.  
Step9. Apply post-transfection assays after 48 hours post electroporation.