The Pathologic and Physiologic Role of SOCS-3 in Liver Metabolism. Allison M. Gaudy. Submitted in Partial Fulfillment. of the

Transcription

1 The Pathologic and Physiologic Role of SOCS-3 in Liver Metabolism by Allison M. Gaudy Submitted in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy Supervised by Professor Dr. Robert A. Mooney Department of Pharmacology and Physiology The School of Medicine and Dentistry University of Rochester Rochester, New York 2010

2 ii Dedication I dedicate this thesis to my grandfathers William O Brien and Vincent Gaudy. The hard work and love you provided for your family was an inspiration. You were always extremely proud of my accomplishments. I know that your pride now would be without comparison.

3 iii Curriculum Vitae The author was born in Buffalo, New York on April 14 th, She attended the State University of New York at Geneseo from 2000 to 2004 and graduated with a Bachelor of Science in biochemistry in She came to the University of Rochester School of Medicine and Dentistry in the fall of 2004 and began graduate studies in the pharmacology and physiology department. Under the guidance of Dr. Robert A. Mooney, she pursued the study of how SOCS-3 modulates hepatic metabolism and received the Master of Science degree in 2006.

4 iv Acknowledgements First and foremost, I thank my thesis advisor, Dr. Robert Mooney. I would never have succeeded if it was not for his support, guidance, and motivational speeches. His positive attitude and dedication to science provided encouragement that I have been very grateful for over the past five years. I would also like to thank my committee members Dr. Alan Smrcka, Dr. David Yule, and Dr. Janet Sparks. Their guidance, suggestions, and support influenced my project, and me, as a scientist. Additionally, I need to thank my lab mates. The work performed by Dr. Bingrong Zhang greatly influenced my project, and his assistance with adenovirus production allowed me to carry on with my project in his absence. I am extremely grateful for my friendship with Dr. Alicia Clementi, both in and outside of the lab. Her guidance and mentoring shaped my lab experience, and she was always there to help with whatever task I was undertaking. I would also like to thank the department of Pharmacology and Physiology, especially Keigan Park and Ryan Loy for their friendships and support throughout this process. To my family and friends, thank you for your continued support throughout the years. Mom and dad, I could not ask for better parents. You have taught, and continue to teach me so much. Your continued support, encouragement and most importantly love, has shaped who I am today. Thank you for everything.

5 v Publications Gaudy, A.M., Clementi, A.H., Campbell, J.S., Smrcka, A.V., and Mooney, R.A., Suppressor of Cytokine Signaling-3 is a Glucagon-Inducible Inhibitor of PKA Activity and Gluconeogenic Gene Expression in Hepatocytes, In preparation. Clementi, A.H., Gaudy, A.M., van Rooijen, N., Pierce, R.H., and Mooney, R.A., Loss of Kupffer cells in diet-induced obesity is associated with increased hepatic steatosis, STAT-3 signaling, and further decreases in insulin signaling, Biochim. Biophys. Acta (2009) Nov;1792(11):

6 vi Abstract Suppressor of cytokine signaling (SOCS-3) is a signal transducer and activator of transcription (STAT-3) induced negative regulator of janus kinase (JAK)/STAT signaling. Obesity-related increases in circulating IL-6 increase hepatic SOCS-3 levels, and are associated with the development of insulin resistance. Recently, SOCS-3 has been shown to be induced by a camp-dependent pathway involving Epac (exchange protein directly activated by camp). This thesis examines the physiologic and pathologic role of SOCS-3 in regulating hepatic glucose metabolism. SOCS-3 inhibits the insulin signaling pathway by inhibition and degradation of insulin receptor substrates (IRS-1 and IRS-2). SOCS-3 contains a src homology (SH-2) domain that binds to tyrosine phosphorylated IRS proteins, and a C-terminal SOCS box that contains E 3 ligase activity that mediates ubiquitination and degradation of IRS. There are conflicting reports, however, on the mechanism by which SOCS-3 mediates its inhibition in the various insulin responsive tissues. To investigate the role of SOCS-3 in modulating hepatic insulin signaling, hepatic suppression of SOCS-3 was attained by adenovirus delivery of a shrna construct to the liver of both lean and obese mice. SOCS-3 suppression increased IRS-1 and AKT phosphorylation both basally and after an insulin bolus in lean and obese mice. IRS-1 and IRS-2 protein levels were unaltered in lean mice. In contrast, IRS-1 protein levels increased 80% in obese animals, without changes in messenger RNA levels. IRS-2 phosphorylation and protein levels were unaltered. These data demonstrate that SOCS-3 differentially regulates IRS-1 and IRS-2 protein in the obese mouse, and

7 vii that SOCS-3 is capable of modulating insulin signaling in the absence of IRS protein loss in the lean mouse. To determine the mechanism of SOCS-3 degradation of IRS-1 and IRS-2, mouse primary hepatocytes were infected with a SOCS-3 adenovirus construct. Surprisingly, SOCS-3 alone was insufficient to cause degradation of IRS-1 or IRS-2. To recapitulate conditions of hyperinsulinemia, cells were treated with insulin overnight. Chronic insulin treatment decreased IRS-1 mass by 60% in primary hepatocytes infected with SOCS-3, but had no affect on LacZ infected controls. Similar to our in vivo data, IRS-2 mass was unchanged. These data are consistent with the model that insulin treatment induces tyrosine phosphorylation and serine phosphorylation of IRS-1. Serine phosphorylation of IRS-1 has been reported to lead to IRS-1 protein degradation. To determine the role of serine phosphorylation in IRS degradation, primary mouse hepatocytes were treated with the pro-inflammatory cytokine TNF-α, which is known to cause serine phosphorylation of IRS proteins. TNF-α dramatically decreased IRS-1 protein, with or without SOCS-3, but had no effect on IRS-2. These data suggest that SOCS-3 specifically degrades IRS-1 in hepatocytes, and that serine phosphorylation plays a role in this regulation. Glucagon is a catabolic hormone that regulates liver metabolism in the fasted state primarily through camp signaling. In fasted mice, Socs3 mrna was increased 4-fold, compared to refed mice, suggesting a physiologic role for SOCS-3 in the fasted state. Treating primary hepatocytes with glucagon also resulted in a 4-fold increase in Socs3 mrna levels. Activation of hepatocytes with the selective camp

8 viii analog 8-4-(chlorophenylthio)-2 -O-methyladenosine-3, 5 -monophosphate, acetoxymethyl ester (cptome) increased Socs3 expression comparably, indicating Socs3 induction is dependent on Epac activation. Adenoviral expression of SOCS-3 in primary hepatocytes decreased 8-br-cAMP induced PKA phosphorylation of the transcription factor CREB by 50%, and induction of the gluconeogenic genes Ppargc1a, Pck1 and G6pc were suppressed nearly 50%. Induction of Pck1 and G6pc by glucagon was also reduced with ectopic SOCS-3 expression. Hepatocytes from liver-specific SOCS-3 knockout mice responded to 8-br-cAMP with a 200% increase in Ppargc1a and Pck1 expression, and a 30% increase in G6pc expression relative to wild type cells. Suppression of SOCS-3 by shrna in primary hepatocytes resulted in a 60% increase in G6pc and Pck1 expression relative to control cells. Primary hepatocytes expressing SOCS-3 had a 46% reduction in camp-dependent PKA activity, which corresponds to the magnitude of SOCS-3 inhibition of CREB phosphorylation. These data support the hypothesis that camp activates two opposing signaling pathways, in which Epac-mediated SOCS-3 induction negatively regulates PKA activity.

9 ix Table of Contents Page Dedication Curriculum Vitae Acknowledgements List of Publications Abstract Table of Contents List of Figures List of Abbreviations ii iii iv v vi ix xii xiv Foreword 1 1. Introduction Insulin Signaling Insulin Action Glucose Metabolism Lipid Metabolism Protein Synthesis Glucagon Signaling Glucagon Action Glycogenolysis Gluconeogenesis Lipolysis and β-oxidation 13

10 x 1.5. Obesity and Inflammation Insulin Resistance Serine Kinase Activation IL-6 and SOCS SOCS SOCS Epac and SOCS Experimental Objectives Suppressor of Cytokine Signaling-3 Selectively Affects IRS-1 in the Liver Introduction Results Discussion Epac Induction of SOCS-3: A Negative Regulator of PKA Mediated Hepatic Gluconeogenesis 3.1. Introduction Results Discussion Conclusions and Perspectives 4.1. The role of SOCS-3 in insulin signaling in the liver 112

11 xi SOCS-3 inhibition of insulin signaling SOCS-3 degradation of IRS proteins The role of camp mediated SOCS-3 induction in the liver Glucagon/Epac induction of SOCS SOCS-3 inhibition of gluconeogenesis SOCS-3 modulation of glucose metabolism SOCS-3 inhibition of PKA Epac/ PKA crosstalk Implications of SOCS-3 inhibition of PKA in other pathways 124 Appendix A. Materials and Methods 128 Appendix B. References 134

12 xii List of Figures Figure Title Page 1.1 Insulin receptor signaling Glucagon receptor signaling Gluconeogenesis Serine kinase activation inhibits insulin signaling SOCS-3 induction by IL-6 inhibits insulin signaling in 34 obesity 1.6 Epac-mediated SOCS-3 induction in COS1 cells Suppression of SOCS-3 in lean mice Suppression of SOCS-3 in high fat diet-induced obese 55 Mice 2.3 IRS-1 and IRS-2 protein and mrna levels in livers of 58 lean and obese mice as a function of SOCS-3 expression 2.4 SOCS-3 expression does not alter IRS protein levels SOCS-3 selectively decreases IRS-1 protein levels in 62 primary hepatocytes 2.6 TNF-α decreases IRS-1 protein levels independent of 64 SOCS-3 expression 2.7 TNF-α degradation of IRS-1 protein is independent of 66 SOCS TNF-α does not induce Socs1 or Socs3 expression in 68 primary hepatocytes

13 xiii 2.9 Ectopic SOCS-3 expression does not alter insulin 70 responsiveness in mouse primary hepatocytes 3.1 Glucagon-dependent induction of SOCS-3 in primary 88 Hepatocytes 3.2 Socs3 expression is elevated in fasted mice Glucagon-dependent induction of SOCS-3 is mediated 93 by Epac 3.4 PLCε activation is not required for Epac mediated 95 SOCS-3 induction. 3.5 Epac activation inhibits PKA-mediated CREB 97 phosphorylation and gluconeogenic gene expression 3.6 SOCS-3 expression inhibits CREB phosphorylation and 99 gluconeogenic gene expression 3.7 Loss of Socs3 expression increases gluconeogenic gene 101 expression 3.8 SOCS-3 knockout hepatocytes have increased 103 gluconeogenic gene expression in response to camp 3.9 SOCS-3 inhibits PKA-mediated Ser1756 phosphorylation 105 of the IP3R 3.10 SOCS-3 inhibits PKA activity SOCS-3 does not inhibit the translocation of the PKA 109 catalytic subunit to the nucleus 4.1 Model depicting Epac induction of SOCS-3 and inhibition 126 of PKA by SOCS-3

14 xiv Abbreviations ACC acetyl-coa carboxylase AKAP A kinase anchoring protein Akt PKB AMPK 5' AMP-activated protein kinase AP-1 activator protein 1 BMI body mass index camp cyclic AMP C/EBP CCAAT/enhancer-binding protein CBP CREB-binding protein CIS cytokine-inducible SH2 protein CPT carnitine palmitoyltransferase CRE camp-response element CREB camp response element-binding DAG diacylglycerol EGF epidermal growth factor Epac exchange protein directly activated by camp Epo Erythropoietin ERK extracellular signal-related kinase FAS fatty acid synthase FBPase fructose-1,6-bisphosphatase FFA free fatty acid FOXO Forkhead transcription factor G-6-Pase glucose-6-phosphatase GK glucokinase GLUT glucose transporter GP glycogen phosphorylase GS Glycogen synthase GSK glycogen synthase kinase

15 xv HUVEC IGF IGF1R IκB IKK IL IP 3 IP3R IRS JAK JNK LIF LPS MAPK MCP mtor NF-κB OSM PDE PEPCK PGC-1α PH PIAS PI3K PKA PKC PLC PTB S6K human umbilical vein endothelial cells insulin growth factor insulin-like growth factor 1 receptor inhibitor of nuclear factor kappa B IκB kinase interleukin inositol 1,4,5-trisphosphate IP 3 receptor insulin receptor substrate janus kinase c-jun N-terminal kinase Leukemia inhibitory factor lipopolysaccharide mitogen-activated protein kinase monocyte chemoattractant protein Mammalian target of rapamycin nuclear factor kappa-light-chain-enhancer of activated B cells Oncostatin M phosphodiesterase phosphoenolpyruvate carboxykinase peroxisome proliferator-activated receptor-gamma coactivator 1 alpha pleckstrin homology Protein inhibitor of activated STAT phosphatidylinositol 3-kinase cyclic AMP-dependent protein kinase protein kinase C phospholipase C phosphotyrosine binding p70-s6 Kinase

16 xvi SH2 Src homology domain 2 SHP SH2-domain-containing tyrosine phosphatase SOCS suppressor of cytokine signaling SREBP1c sterol regulatory element binding protein-1c STAT signal transducer and activator of transcription TLR Toll-like receptor TNF-α tumor necrosis factor

17 1 Foreword I would like to acknowledge the work completed by Dr. Bingrong Zhang in my thesis. His work presented in Figures 2.1 and 2.2 of chapter 2 provided a background for my early work in the lab. I also contributed to research published by Dr. Alicia Clementi, however, I did not include that work in this thesis.

18 2 Chapter 1 Introduction

19 3 1.1 Insulin Signaling Insulin is an anabolic hormone that regulates blood glucose levels and has extensive effects on metabolism. Postprandial elevations in blood glucose cause insulin release from pancreatic β-cells, stimulating glucose absorption and metabolism by muscle, adipose, and liver. Insulin mediates its effects by binding to the insulin receptor. The insulin receptor is a transmembrane heterotetramer, composed of two extracellular insulin binding α-subunits and two cytoplasmic β subunits. Insulin binding causes a conformation change in the receptor and activation of an intrinsic tyrosine kinase, which leads to tyrosine autophosphorylation on the cytoplasmic β subunit [1-2]. Tyrosine phosphorylation at residue 960 on the insulin receptor lies in an NPXpY motif that recruits and associates with an N-terminal phosphotyrosine binding (PTB) domain on insulin receptor substrates (IRS-1) and IRS-2. IRS-1 and IRS-2 also contain a pleckstrin homology (PH) domain, just N-terminal of the PTB domain, which targets the proteins to the cell membrane. Association with an activated insulin receptor leads to phosphorylation of multiple tyrosine residues on IRS-1 and IRS-2. Although IRS-1 and IRS-2 activation can elicit mitogenic and metabolic pathways, this thesis will concentrate on the metabolic effects of insulin. Tyrosine phosphorylated IRS-1 and IRS-2 associate with the p85 regulatory subunit of the lipid kinase phosphatidylinositol 3-kinase (PI3K) via its Src homology domain 2 (SH2) domain. PI3K phosphorylates phosphatidylinositol-4,5- bisphosphate (PI(4,5)P 2 ) yielding PI(3,4,5)P 3, causing translocation of PH domain-

20 4 containing proteins PDK and AKT to the cell membrane. Co-localization of AKT with PDK allows for PDK to phosphorylation AKT on threonine-308. Thr308 phosphorylation stabilizes AKT in an active conformation, allowing for serine-473 phosphorylation by the rapamycin-insensitive complex mtorc2 (mammalian target of rapamycin complex 2) [3]. Phosphorylation at both thr308 and ser473 is necessary for full activation of AKT. AKT activation regulates multiple insulin-responsive pathways, illustrated in Figure 1.1. IRS-1 and IRS-2 It is well established that IRS-1 and IRS-2 are mediators of insulin signaling. Whether they play analogous roles, however is controversial. IRS-1 and IRS-2 have an overall sequence identity of only 43%, with the highest homology in the PTB and PH domains. IRS-1 and IRS-2 have 21 and 22 tyrosine phosphorylation sites, respectively, 14 of which are conserved between the proteins. The conserved phosphorylation sites are required for binding SH2 domains of downstream effectors, allowing for corresponding insulin signal transduction [4]. Ablation studies demonstrate that IRS-1 and -2 are not interchangeable and insulin responsive tissues are affected differently by their loss [5-6]. Deletion of IRS- 1 or IRS-2 has been shown to cause insulin resistance, but the loss of IRS-1 primarily affected muscle metabolism, while loss of IRS-2 impaired muscle, adipose and liver metabolism [6]. It has been established that loss of insulin sensitivity in the muscle does not have a marked affect on circulating glucose levels, rather, hepatic glucose

21 5 production dictates glucose levels in an individual [7]. Given that IRS-2 deficiency led to significant glucose intolerance in the liver, IRS-2 is believed to play a critical role in glucose metabolism, and is considered the main mediator of insulin signaling in the liver [6, 8]. 1.2 Insulin Action Insulin is a primary regulator of blood glucose concentration, stimulating glucose uptake and anabolic metabolism by peripheral tissues, and regulating glucose production by the liver. Insulin stimulates glycogen and protein synthesis in the liver and muscle, and lipogenesis in fat, resulting in the storage of excess energy derived from glucose. Insulin inhibits catabolic metabolism by inhibiting glycogenolysis, and lipolysis. Insulin also suppresses hepatic glucose output by inhibiting gluconeogenesis. The details of insulin action are described below Glucose Metabolism Glucose uptake and glycolysis Peripheral clearance of glucose is directly mediated by glucose transporter-4 (GLUT4) in the muscle and adipose tissue, and GLUT2 in the liver. Muscle uptake of glucose accounts for ~75% of glucose clearance from the blood postprandially [9]. Insulin activation of AKT and atypical protein kinase C (PKC) promote GLUT4 translocation to the plasma membrane of myocytes and adipocytes, resulting in glucose uptake [10-11]. In the liver, glucose uptake occurs by facilitated diffusion

22 6 through GLUT2, which is constitutively associated with the hepatocyte plasma membrane. Upon entering the hepatocyte, glucose is converted to glucose-6- phosphate by glucokinase (GK), resulting in cellular retention. Expression of GK increases when insulin levels are high, and decreases when serum glucagon levels increase [12]. Glycolysis is the metabolic pathway that converts glucose to pyruvate. Free energy released in this pathway is used to form the high energy compounds adenosine triphosphate (ATP) and reduced nicotinamide adenine dinucleotide (NADH) [12]. Regulation of glycolysis is primarily controlled by inhibition and activation of enzymes involved in the pathway. Insulin stimulates the expression of glycolytic enzymes phosphofructokinase and pyruvate kinase, promoting the conversion of glucose to pyruvate [13]. Pyruvate can be used as a substrate for anaerobic or aerobic respiration to generate energy. Glycogen synthesis Glucose is stored as glycogen in the liver and muscle. The first step in glycogenesis is the conversion of glucose-6-phosphate into glucose-1-phosphate. Glucose-1-phosphate is then converted into UDP-glucose by the action of Uridyl Transferase. Glycogen synthase (GS) is an enzyme that catalyzes the conversion of glucose to glycogen by binding to UDP-glucose and adding it to a glycogen polymer [14]. Insulin mediated AKT activation results in the phosphorylation and inhibition of glycogen synthase kinase-3 (GSK-3), which inhibits GS by phosphorylation.

23 7 Insulin, therefore, indirectly promotes GS activity by inhibiting GSK-3 [15]. Branches are added to glycogen by branching enzyme, which transfers the end of a glycogen chain onto another chain via α-1:6 glucosidic bond [16]. Glycogen is stored until its breakdown is required to provide energy during exercise and short-term fasting. Inhibition of gluconeogenesis In times of hypoglycemia, the liver can provide a de novo source of glucose through gluconeogenesis. The regulation of hepatic glucose output by insulin is important in preventing hyperglycemia. Gluconeogenic gene expression determines the rate of gluconeogenesis. Forkhead transcription factor (FOXO)1 and the coactivator peroxisome proliferator-activated receptor-gamma coactivator 1 alpha (PGC-1α) are transcription factors that promote gluconeogenic gene expression. Insulin activation of AKT phosphorylates FOXO1, excluding it from nucleus, inhibiting association with PGC-1α. Disruption of this interaction decreases expression of key gluconeogenic genes, phosphoenolpyruvate carboxykinase (PEPCK) and glucose-6-phosphatase (G-6-Pase), inhibiting gluconeogenesis [17]. Insulin inhibition of gluconeogenic gene transcription is therefore, an important mechanism of decreasing hepatic glucose production. A detailed description of gluconeogenesis will be discussed later in this chapter.

24 Lipid Metabolism Lipogenesis is the process in which acetyl-coa is converted into fatty acids, beginning with the conversion of acetyl-coa to malonyl-coa by acetyl-coa carboxylase (ACC). Maloynl-CoA is then converted into the saturated fatty acid, palmitate, by fatty acid synthase (FAS). Fatty acids can be esterified into triglyceride and stored as lipid droplets in liver and adipose tissue, or can be secreted from hepatocytes as VLDL into the blood [18]. Insulin promotes the synthesis of lipids and inhibits their degradation. This is accomplished by insulin mediated induction of the transcription factor sterol regulatory element binding protein-1c (SREBP1c), which regulates lipogenic genes including ACC and FAS [19]. Insulin also promotes lipogenesis by indirectly activating ACC through inhibition of the inhibitory kinase 5' AMP-activated protein kinase (AMPK), which inhibits ACC by phosphorylation [20-21]. Degradation of lipids, or lipolysis, in adipocytes is controlled by the activation of hormone sensitive lipase (HSL) through camp-dependent protein kinase (PKA) phosphorylation [22]. Insulin impairs lipolysis by activating phosphodiesterase 3 (PDE3), which degrades camp levels and decreases PKA activity [23] Protein Synthesis Mammalian target of rapamycin (mtor) is an important regulator of growth in response to nutrient availability, and can be stimulated by insulin. mtor is associated with two distinct signaling complexes. mtorc1 contains mtor, G-

25 9 protein β-subunit-like protein (GβL), and raptor. mtorc2 contains mtor, GβL and rictor [24]. mtorc1 is sensitive to the drug rapamycin and is important in cell growth and protein synthesis. mtorc2 is rapamycin insensitive and has been implicated in phosphorylating AKT on Ser473, which increases AKT activity [25]. Activation of mtor in the mtorc1 complex leads to activation of p70-s6 kinase (S6K), which can phosphorylate and activate S6 ribosomal protein and increase translation [26]. Phosphorylation of 4E-BP1 (eukaryotic initiation factor 4E (eif4e) binding protein 1) by mtor releases the inhibition of eif4e and increases protein translation [27]. 1.3 Glucagon Signaling Glucagon is the primary catabolic hormone affecting the liver in the fasting state. Glucagon is processed from proglucagon in the alpha cells in the pancreatic islets of Langerhans, and is released into the bloodstream when circulating glucose is low. Glucagon receptor expression has been identified in the pancreas, small intestine, and kidney, but is minimal compared to levels in the liver [28]. Glucagon promotes glycogenolysis, and stimulates gluconeogenesis, to increase hepatic glucose output and raise blood glucose levels. Glucagon signals through a seven transmembrane G protein-coupled receptor via Gα s [29]. Receptor mediated activation of GTP bound G s α activates adenylate cyclase, which converts ATP to 3,5 -camp. Increases in camp activate campdependent protein kinase, PKA. PKA is a tetrameric holoenzyme consisting of two

26 10 regulatory (R) and two catalytic (C) subunits. PKA exists in an inactive state until camp binds to the regulatory subunits, causing the release of the catalytic subunits. Active PKA phosphorylates serine/threonine residues on its substrates at the consensus sequence Arg-Arg-X-Ser/Thr-X. There are two types of regulatory subunits, I and II, each having two subtypes, α and β. RIα and RIIα are ubiquitously expressed while the β isoforms are predominantly found in the brain [30-31]. The catalytic subunits (α, β and γ) are similar in expression and function, however each regulatory isoform possesses a unique functional phenotype [32]. RI subunits are mostly cytoplasmic, while RII subunits are typically associated with anchoring proteins (AKAPs), which target them to specific cellular compartments and organelles. The AKAPs display isoform specificity in their binding to the regulatory subunits [33-34]. The glucagon signaling pathway is shown in figure 1.2 and discussed in further detail below Glycogenolysis 1.4 Glucagon Action Glucagon can stimulate glycogenolysis and inhibit glycogen synthesis in the liver. Glucagon mediated activation of PKA phosphorylates and activates phosphorylase kinase, which phosphorylates and activates glycogen phosphorylase (GP). Activated GP cleaves glycogen at α-1-4 linkages, yielding the glycogen chain and glucose-1-phosphate [35]. When only four glucose molecules are left on a glycogen branch, a debranching enzyme transfers three glucose units to a 1,4 end of

27 11 glycogen. The debranching enzyme converts the last 1,6 glucose molecule into free glucose. Glucose-1-phosphate is converted into glucose-6-phosphate, which is converted to glucose by glucose-6-phosphatase [29, 36-38]. GS catalyzes the transfer of UDP-glucose to a glycogen molecule. PKA inhibits glycogen synthesis in the liver by an inhibitory phosphorylation. GS can be phosphorylated on multiple serine and threonine residues, with each phosphorylation causing an additive inhibition [29, 39] Gluconeogenesis As glycogen stores are depleted during fasting, de novo glucose production, or gluconeogenesis, is required to maintain normoglycemia. Gluconeogenesis converts non-carbohydrate carbon substrates such as lactate, pyruvate and glycerol, into glucose. Transcriptional regulation is crucial to the regulation of the enzymes required in this metabolic pathway. Activation of the transcription factor camp response element-binding (CREB) is a main determinant of gluconeogenic gene expression. PKA phosphorylates and activates CREB, resulting in transcriptional activation at the camp-response element (CRE). The N-terminus of CREB contains the activation region, while nuclear localization signals and DNA binding motifs are present in the C-terminus [40]. CREB binds to a conserved TGACGTCA sequence in the promoter of camp responsive genes [41]. It is believed that CREB is constitutively bound to the CRE site and that PKA dependent serine-133 phosphorylation of CREB causes the recruitment of the coactivators, CREB-binding

28 12 protein (CBP), CREB regulated transcription coactivator 2 (CRTC2; also known as TORC2) and p300 [42-47]. CBP interacts with RNA helicase protein, promoting transcriptional activation [48]. CREB activation leads to the upregulation of key gluconeogenic genes PGC- 1α, PEPCK and G-6-Pase [29, 49]. Increases in the transcription coactivator PGC-1α further potentiate the induction of PEPCK and G-6-Pase by binding to coactivators hepatic nuclear factor-4α (HNF-4α) and FOXO1. It has also been reported that PGC- 1α coactivates with the glucocorticoid receptor [17, 50]. Figure 1.3 illustrates the gluconeogenic pathway from pyruvate to glucose. Unidirectional enzymes PEPCK, fructose-1,6-bisphosphatase (FBPase) and G-6-Pase determine the rate of gluconeogenesis. PEPCK catalyzes the conversion of oxaloacetate to phosphoenolpyruvate, an early rate-limiting step in the gluconeogenic pathway. FBPase converts fructose-1,6-bisphosphate into fructose-6-phosphate and is regulated allosterically [29]. In the final step of glucose production, G-6-Pase hydrolyzes glucose-6-phosphate generating free glucose. Glucose generated in the liver by glycogenolysis and gluconeogenesis enters the bloodstream through GLUT 2 via facilitated diffusion, and is delivered to cells throughout the body. Glucose delivery to the brain is especially important since the brain s primary source of energy is glucose.

29 Lipolysis and β-oxidation Lipids are stored as triglyceride in adipose tissue and liver, and can be used as a source of energy. PKA activation in the adipose tissue activates HSL, releasing free fatty acids and glycerol into the bloodstream [22]. Free fatty acids are taken up by peripheral tissues by fatty acid transporters such as CD36, and undergo β-oxidation [51]. The conversion of fatty acids into acyl-coa facilitates entry into the mitochondria via carnitine palmitoyltransferase 1 (CPT1) where β-oxidation yields acetyl-coa, which can then enter the Krebs cycle. 1.5 Obesity and Inflammation During the last twenty years there has been a dramatic increase in obesity in the United States. According to the World Health Organization, 32% of adults and 35% of children in the United States are obese, or have a body mass index (BMI) of 30 kg/m 2 or greater. Obesity is a major risk factor for cardiovascular disease, certain types of cancer, and type 2 diabetes. For example, a study in woman reported the risk of developing type 2 diabetes increased 11-fold when BMI increased from 20.1 kg/m 2 to 29.9 kg/m 2 [52]. The development of type 2 diabetes begins with the onset of insulin resistance, which is correlated with the expansion of visceral fat mass [53]. Insulin resistance begins with the impairment of insulin signal transduction in peripheral tissues. To maintain normal glucose levels, insulin production from the pancreatic β-

30 14 cells increases. Chronic secretion of insulin can cause β-cell failure, resulting in decreased insulin secretion, hyperglycemia, and the development of type 2 diabetes. The correlation between obesity and the development of insulin resistance is discussed below. Adipose tissue mediated inflammation The role of adipose tissue is no longer viewed simply as energy storage, but also as an endocrine organ. Adipocyte-derived hormones (adipokines) such as leptin and adiponectin, and proinflammatory cytokines are some of the various protein factors secreted from adipose tissue. Obesity is associated with increased release of the proinflammatory cytokines tumor necrosis factor (TNF)-α and interleukin (IL)-6 from adipose tissue, which leads to a state of chronic inflammation [54-55]. Contributing to this inflammation in obesity is increased macrophage infiltration of adipose tissue. Activated macrophages account for a significant amount of TNF-α and IL-6 release from obese adipose tissue [56]. Although the exact mechanisms are still under investigation, increased expression of monocyte chemoattractant protein-1 (MCP-1), and increased adipocyte necrosis in obesity have been linked to recruitment of macrophages to the adipose tissue [57-59]. In addition to adipokine production, obesity can be associated with increased circulating free fatty acids (FFAs), resulting in fat accumulation in non-adipose tissues such as liver and muscle [60-61]. Fat accumulation in these tissues has been mechanistically linked to the development of insulin resistance [60, 62-64]. This

31 15 evidence indicates that obesity-mediated increases in adipose tissue production of proinflammatory cytokines and FFAs can lead to inadequate insulin signal transduction, and subsequent insulin resistance. The mechanisms of this inhibition are discussed below. 1.6 Insulin Resistance In this section, two mechanisms of insulin resistance will be discussed: activation of serine kinases by TNF-α and FFAs and induction of suppressor of cytokine signaling (SOCS-3) by IL Serine Kinase Activation Activation of serine kinases by TNF-α and FFA can impair insulin signal transduction by phosphorylating serine residues on IRS-1 and IRS-2 [65-69]. Serine phosphorylation inhibits IRS-1 and IRS-2 association with the insulin receptor, resulting in their decreased tyrosine phosphorylation [70-71]. Serine phosphorylation is also implicated in the degradation of IRS-1 and IRS-2 [72]. These mechanisms of inducing insulin resistance are discussed below. JNK, IKK, PKC θ TNF-α activates the NF-κB and MAPK signaling pathways. TNF-α activation of its receptor, TNFR1, recruits the adaptor protein TRADD to the cytoplasmic domain of TNFR1. TRADD association with TRAF2 recruits and activates inhibitor

32 16 of nuclear factor kappa B (IκB) kinase (IKK). IKK phosphorylates IκB, targeting it for proteosomal degradation. IκB is an inhibitory protein that is bound to NF-κB, preventing its translocation into the nucleus. Degradation of IκB by IKK allows NFκB to enter the nucleus where it induces transcription of many proteins associated with survival and proliferation [73-74]. TRAF2 can also activate of the stress related MAPK pathway by activating mitogen-activated protein kinase kinase kinase 1 (MEKK1). MEKK1 activation leads to the phosphorylation of c-jun N-terminal kinase (JNK). JNK is a serine kinase that increases the transcription of proliferation, differentiation, and apoptotic genes by enhancing the transcription activity of activator protein 1 (AP-1) [73-74]. TNF-α activation of IKK and JNK has been directly linked to serine phosphorylation of IRS-1 and IRS-2. Serine phosphorylation of IRS-1 and IRS-2 decreases their insulin-stimulated tyrosine phosphorylation and increases their degradation. This contributes to the development of insulin resistance [65, 68-70]. FFAs have also been implicated in the development of insulin resistance by activating the serine kinases JNK and IKK [66-67]. Recent investigations demonstrate that FFAs are capable of activating Toll-like receptor 4 (TLR4), which has been well-characterized for its recognition of lipopolysaccharide (LPS) in gramnegative bacterial cell walls [75]. The TLR family of receptors activate proinflammatory signaling pathways in response to microbial pathogens [66, 75]. Activation of TLR4 causes activation of MyD88, an adaptor protein bound to the cytoplasmic domain of the receptor. MyD88 recruits the kinase IRAK-4, which

33 17 phosphorylates IRAK-1, allowing it to associate with TRAF-6. TRAF-6 activates two pathways, the first being the MAPK pathway involving JNK and AP-1 activation. Another pathway increases IKK activation and NF-κB nuclear translocation [76]. FFAs also activate the novel PKC isoforms PKC-θ and δ in the muscle and liver respectively, by increasing diacylglycerol (DAG) concentrations. These PKC s have been associated with serine phosphorylation of IRS-1/2 and insulin resistance [67, 77-78]. Loss of PKC-θ in mice inhibits the ability of lipid to induce skeletal muscle insulin resistance [79]. Figure 1.4 illustrates the signaling pathways involved in IRS-1/2 serine phosphorylation. mtor and S6K As mentioned above, insulin-mediated activation of mtor leads to phosphorylation and activation of the serine/threonine kinase S6K, which ultimately increases protein synthesis. mtor and S6K have also been implicated in negative regulation of IRS-1 by inducing its serine phosphorylation. It has been demonstrated that S6K phosphorylates serine 307 on IRS-1 [80-82] while mtor itself can phosphorylate Ser-636/639 [81, 83-84]. Serine phosphorylation by mtor can also result in IRS-1 protein degradation [81-82]. Acutely, insulin induced IRS-1 serine phosphorylation could represent negative feedback of the normal insulin signaling pathway, however chronic activation of mtor has been implicated in insulin resistance [85-86]. Nutrient overload, as seen in obesity, increases mtor activation

34 18 [84, 86]. Therefore, obesity induced activation of mtor and S6K can cause impaired insulin signaling via IRS-1 serine phosphorylation IL-6 and SOCS-3 IL-6 is a pleiotropic cytokine that is secreted by T-cells and macrophages to stimulate an immune response, and is an important mediator of hepatic acute phase response [87]. IL-6 is also produced by adipose tissue, liver and muscle [55, 88-92]. IL-6 is produced by the muscle in response to increased muscle contraction and exercise, [88] and has been shown to stimulate energy mobilization by stimulating lipolysis and β-oxidation in the liver and adipose tissue [93-94]. IL-6 signaling therefore plays an important role in liver. IL-6 binding to its receptor, IL-6R, forms a complex with gp130, a signal transducing transmembrane glycoprotein. IL-6R activates Janus kinases (JAKs), which tyrosine phosphorylate gp130 and recruit signal transducer and activator of transcription (STAT-3). Tyrosine phosphorylation of the transcription factor STAT-3 by JAKs cause STAT-3 dimerization and translocation to the nucleus increasing gene transcription [95]. Signaling from gp130 is subject to negative regulation by several mechanisms. SHP-2 is a tyrosine phosphatase that can negatively regulate gp130, JAK and STAT [96]. Protein inhibitor of activated STAT (PIAS-3) binds to STAT-3 and prevents it from binding to DNA [97]. IL-6 induction of SOCS-3 prevents STAT-3 activation by binding to tyrosine phosphorylated gp130 via its SH2 domain, and with lower affinity to JAKs [98-99].

35 19 Serum levels of IL-6 are increased with increased adiposity and are strongly associated with the development of type 2 diabetes [55, 90]. IL-6 induction of SOCS- 3 is an important mediator in developing insulin resistance, and is discussed in more detail in the next section. 1.7 SOCS SOCS proteins negatively regulate cytokine and hormone signaling. There are eight proteins in the SOCS family, SOCS-1-7 and cytokine-inducible SH2 protein (CIS). Each SOCS protein has three conserved regions: a variable N-terminal region, a central SH2 and a C-terminal SOCS box that contains a functional BC- box [ ]. Elongin binding to the BC-box results in ubiquitination and subsequent degradation of SOCS-3 target proteins [ ]. The N-terminal region in SOCS-1 and -3 consist of a kinase inhibitory domain (KIR) [103]. CIS and SOCS-1-3 have an N-terminal domain that is shorter than that of SOCS-4-7. Only in SOCS-1 and -3, however, has a functional role for the N-terminus been identified [104]. CIS and SOCS-1-3 have been extensively studied due to their rapid induction upon stimulation by a variety of cytokines and hormones. CIS is induced by, and in turn inhibits IL-2, IL-3, growth hormone, and erythropoietin [99]. SOCS-2 has been shown to bind to the cytoplasmic domain of insulin-like growth factor 1 receptor (IGF1R). SOCS-2 knockout mice are larger than wild-type controls. Thus SOCS-2 is thought to be critically involved in the regulation of IGF1R-mediated cell signaling [ ].

36 20 SOCS-1 and -3 have been extensively studied for inhibiting JAK activity in a variety of signaling pathways. Both proteins have been implicated in inhibiting IL-2, IL-3, IL-4, IL-6, growth hormone, LIF, Epo, oncostatin M (OSM), and interferon γ signaling [99]. SOCS-1 and SOCS-3 have been shown to be of particular importance in interferon γ and IL-6 signaling, respectively [107]. SOCS-3 is also induced by, and inhibits leptin signaling [108]. TNF-α induction of SOCS-3 has been reported in adipocytes. The mechanism of induction, however, is not clear [ ]. One explanation is that TNF-α stimulates production of IL-6, which then increases SOCS- 3 induction [109]. SOCS-1 and -3 have been implicated in the impairment of insulin sensitivity in the liver and adipose tissue [ ]. Fewer studies have investigated the function of SOCS-4-7. Gene studies demonstrate that SOCS-4 and SOCS-5 are similar, as are SOCS-6 and SOCS-7, in that they share increased sequence identity in their SH-2 and SOCS box domains [104]. SOCS-4 and -5 have been shown to associate with and inhibit epidermal growth factor (EGF) signaling [113]. SOCS-6 and -7 have been shown to bind to IRS-4 and p85, which are stimulated by insulin growth factor (IGF)-1 and insulin. Although SOCS-6 knockout mice displayed no alteration in insulin responsiveness, they did exhibit a 10% reduction in body weight. This may implicate a role in regulation in IGF-1 signaling [114]. SOCS-7 has also been associated with binding and inhibiting STAT-5 and STAT-3 during prolactin, growth hormone and leptin signaling [115].

37 SOCS-3 The inhibitory role of SOCS-3 in JAK/STAT signaling has been extensively studied. Additionally, SOCS-3 is implicated in decreasing insulin responsiveness (Figure 1.5) [110, ]. It has been demonstrated that diet-induced and genetically obese mice have elevated SOCS-3 protein levels [112, 119]. Overexpression of SOCS-3 in the liver is associated with insulin resistance, while suppression of SOCS-3 in the genetically obese Lep db mouse model improved insulin sensitivity [112]. SOCS-3 knockout mice die in utero during embryonic development. Therefore, a hepatocyte specific SOCS-3 deficient mouse model was used to investigate hepatic insulin responsiveness [120]. As anticipated, liver-specific SOCS-3 KO mice demonstrated increased insulin sensitivity [121]. Taken together, increased levels of hepatic SOCS-3 in obesity negatively affects hepatic insulin signaling. There are multiple proposed mechanisms thought to mediate the inhibitory effect of SOCS-3 on insulin signaling. The SH-2 domain of SOCS-3 has been reported to associate with phosphotyrosine residue 960 on the insulin receptor, inhibiting downstream signal transduction [118, ]. SOCS-3 has also been shown to bind to IRS-1 and -2, decreasing tyrosine phosphorylation by the insulin receptor, and inhibiting p85 association [110, , 121]. IRS-1 and IRS-2 proteosomal degradation has been reported with SOCS-3 expression as well as with prolonged insulin exposure. The relationship between these components is not clear [ ]. Some models suggest that serine phosphorylation may be required for

38 22 IRS-1 degradation as well [72, 82]. Insulin-induced IRS-1 degradation had been shown to be dependent on serine phosphorylation of human ser312 (equivalent to mouse ser307) [82, 118]. There is controversy as to whether the degradation of IRS-1 and IRS-2 are affected equally by SOCS-3 and serine phosphorylation. Some data support degradation of both IRS-1 and -2 [125, ], while others have found only IRS-1 degradation [72, 124, 126, 131]. The relationship between serine phosphorylation and SOCS-3-mediated IRS-1/2 degradation is not well understood, and the mechanism of action has yet to be elucidated SOCS-3 and Epac Recently, it has been demonstrated that a signaling pathway activated by camp, independent of PKA, can mediate SOCS-3 induction [ ]. The signaling pathway leading to SOCS-3 induction via exchange-protein-directly- activated-by-camp (Epac) was elucidated using human umbilical vein endothelial cells (HUVEC) and COS-1 cells. Epac Until recently, increases in camp levels were thought to mediate their effects exclusively through PKA. Another signal transduction pathway activated by camp has now been elucidated, involving Epac. camp activates Epac, a guanine nucleotide exchange factor that then activates the small GTPase Rap1. Epac promotes the activation of Rap1 by exchanging bound GDP with GTP. There are two

39 23 isoforms of Epac, Epac1 and Epac2. Epac1 is ubiquitously expressed with most prominent expression in the kidney, thyroid, ovary, and brain. Epac2 expression is predominantly found in the brain, adrenal glands and liver [ ]. Epac1 contains one high affinity camp binding domain, while Epac2 contains both a low-affinity domain of unknown function and a high-affinity domain with physiologic relevance [ ]. Although initial studies believed that a higher concentration of camp was required for Epac activation relative to PKA, it has now been reported that camp affinity for Epac and PKA are similar (K d ~2.9 µm) [141]. Epac activation mediates many cellular functions including control of cell adhesion, regulation of ion channel function, cellular calcium handling, and exocytosis [ ]. downstream targets. Epac-Rap activation has been shown to activate several Aromataris et al. demonstrated that camp induction by glucagon can activate Ca 2+ and Cl - channels in hepatocytes, and that channel activation was Epac and phospholipase C (PLC)-ε dependent [142]. Epac-Rap activation of PLC-ε is important for Ca 2+ signaling and the stimulation of K ATP channels [139, ]. Activation of the MAPK pathway, specifically ERK-1/2, by Epac-Rap1 has been reported and is implicated in cell proliferation, the regulation of ion channels, and gene expression [132, 140, ]. While many roles for Epac have been described in various cell types, there is still much to be learned. In particular, the interaction between Epac and PKA is an important area of interest.

40 24 Epac-mediated SOCS-3 induction The activation of Epac1, and subsequently Rap1, has been shown to induce SOCS-3 expression in HUVECs and COS-1 cells [ ]. In this model, PLCε activation was necessary for SOCS-3 induction via Epac. PLCε cleaves the phospholipid PI(4,5)P 2 into DAG and inositol 1,4,5-trisphosphate (IP 3 ). IP 3 diffuses through the cytosol and binds to the IP 3 receptor on the endoplasmic reticulum, releasing Ca 2+. SOCS-3 induction by camp in COS1 cells was dependent on increases in Ca 2+ and DAG. Conventional PKCs are activated by both Ca 2+ and DAG, while novel PKCs are activated by DAG alone. Experiments confirmed that the conventional and novel PKCs, PKCα and PKCδ, are necessary for SOCS-3 induction. Only PKCα was found to be activated by Epac, while basal activity of PKCδ was implicated in SOCS-3 induction [132]. Both PKCα and PKCδ can phosphorylate and activate ERK, which activates the transcription factor CCAAT/enhancer-binding protein (C/EBP)-β, responsible for camp-mediated SOCS-3 induction [132, 134]. Figure 1.6 illustrates the signaling pathway required for SOCS-3 induction in COS-1 cells elucidated by Borland et al. [132]. The role of SOCS-3 in liver metabolism has been primarily studied with respect to insulin resistance. Determining the physiologic role of SOCS-3 induction by camp could elucidate yet another role for SOCS-3 in liver metabolism.

41 Experimental Objectives This study was designed to investigate the pathologic and physiologic role of SOCS-3 in hepatic metabolism. The aims addressed in the following two chapters are: Aim 1 SOCS-3 mediates insulin resistance via its role in the association and degradation of IRS proteins. The objective of chapter 2 was to define the mechanism of SOCS-3-dependent IRS-1 and -2 degradation and inhibition of insulin signaling. Aim 2 Glucagon induction of SOCS-3 inhibits camp dependent hepatic gluconeogenesis. Chapter 3 defines a novel physiologic role for SOCS-3 by demonstrating that camp-mediated Epac induction of SOCS-3 can negatively regulate PKA-mediated gluconeogenic gene induction.

42 26 Figure 1.1 Insulin receptor signaling. Insulin activation of the insulin receptor causes tyrosine autophosphorylation and recruitment and activation of IRS-1 and IRS-2. Tyrosine phosphorylated IRS-1 and IRS-2 associate with PI3K. PI3K converts PI(4,5)P 2 to PI(3,4,5)P 3 which causes translocation of PDK and AKT. AKT activation by PDK regulates multiple insulin-responsive pathways including glucose uptake and metabolism, lipid metabolism, and protein synthesis.

43 27 Insulin PI3K PI(3,4,5)P 3 SOS GRB2 py IRS-1/2 py PDK ps pt AKT Ras Raf MEK ERK mtor Protein Synthesis GS Glycogen Synthesis FOXO1 Gluconeogenesis,

44 28 Figure 1.2 Glucagon receptor signaling. Glucagon signals through a seven transmembrane G protein-coupled receptor. Activation of Gα s increases camp production by activating adenylate cyclase. camp activation of PKA promotes glycogen breakdown by activating GP, and inhibits glycogen synthesis by inhibiting GS. PKA phosphorylation of CREB stimulates glucose production by increasing transcription of the gluconeogenic genes PGC-1α, PEPCK, and G-6-Pase.

45 29 G s α AC camp PKA Glycogenolysis GP Gluconeogenesis Glycogen Synthesis GS Creb PGC-1α PEPCK G-6-Pase

46 30 Figure 1.3 Gluconeogenesis. Gluconeogenesis increases hepatic glucose production by converting pyruvate to glucose. Unidirectional enzymes such as PEPCK, fructose- 1,6-bisphosphatase and G-6-Pase determine the rate of gluconeogenesis. PEPCK catalyzes the conversion of oxaloacetate to phosphoenolpyruvate, an early ratelimiting step in the gluconeogenic pathway. FBPase converts fructose-1,6- bisphosphate into fructose-6-phosphate and is regulated allosterically. In the final step of glucose production, G-6-Pase hydrolyzes glucose-6-phosphate generating free glucose.

47 31 Pyruvate Oxaloacetate PEPCK Phosphoenolpyruvate (PEP) 3-Phosphoglycerate 1,3-Bisphosphoglycerate Fructose-1,6-bisphosphate Fructose-1,6-bisphosphatase Fructose-6-phosphate Glucose-6-phosphate Glucose-6-phosphatase Glucose

48 32 Figure 1.4 Serine kinase activation inhibits insulin signaling. Obesity associated increases in TNF-α and FFA promote activation of the serine kinases JNK, PKCθ, and IKKβ. Serine phosphorylation of IRS-1 by these kinases inhibits IRS-1 activation and decreases downstream insulin signaling.

49 33 Insulin TNFR-1 TNF-α TLR-4 FFA py TRADD TRAF2 MyD88 DAG IRS-1/2 ps IKK JNK PKC

50 34 Figure 1.5 SOCS-3 induction by IL-6 inhibits insulin signaling in obesity. IL-6 activation of its receptor leads to the activation of JAKs, which tyrosine phosphorylate gp130 and recruit STAT-3. Tyrosine phosphorylation of STAT-3 by JAK causes STAT-3 dimerization and translocation to the nucleus increasing gene transcription of SOCS- 3. SOCS-3 is a feedback inhibitor of IL-6 signal transduction. SOCS-3 also inhibits insulin signaling by associating with, and degrading IRS proteins.

51 35 IL-6 Insulin py SOCS-3 SOCS-3 JAK STAT3 STAT3 IRS-1/2 SOCS-3 Feedback Inhibition STAT3 P P STAT3 Proteosomal Degradation SOCS-3 STAT3 P P STAT3

52 36 Figure 1.6 Epac-mediated SOCS-3 induction in COS1 cells. Borland et al. [132] elucidated the pathway from Epac activation to SOCS-3 induction in COS1 cells. Increased camp production activates Epac/Rap1, leading to activation of PLCε. Increases in Ca 2+ and DAG activate the conventional PKC, PKCα. PKCα phosphorylates ERK, which activates the transcription factor C/EBPβ causing SOCS-3 induction.

53 37 camp EPAC Rap1 PLC-ε IP 3 Ca 2+ DAG PKCα PKCδ perk C/EBPβ SOCS-3

54 38 Chapter 2 Suppressor of Cytokine Signaling-3 Selectively Affects IRS-1 in the Liver

55 Introduction Insulin signaling regulates glucose uptake and anabolic metabolism in three main tissues: liver, muscle and adipose. Insulin binding to its receptor causes a conformational change leading to tyrosine autophosphorylation. The activated receptor recruits and tyrosine phosphorylates insulin receptor substrates (IRS -1 and IRS-2). IRS-1 and IRS-2 bind and activate PI3K, leading to the activation of AKT and the regulation of multiple insulin responsive pathways [1]. While it is well established that IRS-1 and -2 are mediators of insulin signaling, whether they play redundant roles is controversial. IRS-1 and IRS-2 share similarities in their structure, but have an overall sequence identity of only 43%. Both contain a pleckstrin homology (PH) domain and a phosphotyrosine (PTB) binding domain in their N-terminus, targeting them to the cell membrane and insulin receptor. IRS-1 and IRS-2 have 21 and 22 tyrosine phosphorylation sites, respectively, 14 of which are conserved between the proteins. The conserved phosphorylation sites are required for binding to SH2 domains in downstream effectors [4]. Ablation of IRS-1 or IRS-2 causes peripheral insulin resistance, but affects insulin target tissues differently [5-6]. Deletion of IRS-1 primarily affected muscle metabolism, while loss of IRS-2 impaired muscle, adipose and liver metabolism [6]. Several studies have implicated IRS-2 as the main mediator of insulin signaling in liver [6, 8]. These data indicate that IRS-1 and IRS-2 do not have identical roles in insulin signal transduction.

56 40 Obesity is a major health concern linked to the onset of insulin resistance and type 2 diabetes. The development of obesity-related insulin resistance is associated with chronic inflammation. Adipose tissue production of the proinflammatory cytokines IL-6 and TNF-α have been implicated in insulin resistance models [110, , ]. Proposed mechanisms of proinflammatory cytokine-induced insulin resistance include TNF-α stimulated serine phosphorylation of IRS-1 and IRS- 2, and IL-6 induction of suppressor of cytokine signaling-3 (SOCS-3). Both serine phosphorylation and SOCS-3 are associated with the degradation and inhibition of IRS-1 and IRS-2, resulting in the development of insulin resistance. IL-6 serum levels increase with increasing adiposity and are strongly associated with the development of type 2 diabetes [55, 90]. IL-6 signaling begins with IL-6 activation of its receptor, IL-6R, which is complexed with gp130, the signal transducing transmembrane glycoprotein. IL-6R activates constitutively bound janus kinase (JAK2) that subsequently tyrosine phosphorylates gp130. Recruitment and tyrosine phosphorylation of STAT-3 by JAKs cause STAT-3 dimerization and translocation to the nucleus, where STAT-3 induces gene transcription [95]. STAT-3 induces SOCS-3 as a mechanism of feedback inhibition of IL-6 signaling. SOCS-3 inhibits IL-6 signaling by binding to tyrosine phosphorylated gp130, and with lower affinity to JAKs, inhibiting downstream activation of STAT-3 [98-99]. SOCS proteins are a family of negative regulators of cytokine signaling, but are also known antagonists of growth hormone, leptin and the insulin receptor pathway [110, 117, ]. There are eight proteins in the SOCS family, SOCS-1-

57 41 7 and CIS. Each SOCS protein has three conserved regions: a variable N-terminal region, a central Src homology domain 2 (SH2) and a C-terminal SOCS box that contains a functional BC- box which targets substrates for ubiquitin-mediated proteosomal degradation [ ]. The N-terminal regions in SOCS-1/3 are kinase inhibitory regions (KIR) and are functionally equivalent [103]. SOCS-3 is functionally associated with proteosomal degradation of IRS-1 and IRS-2, linking SOCS-3 to decreased insulin responsiveness [ , 128]. SOCS-3 expression has also been implicated in decreasing insulin-mediated IRS and AKT phosphorylation [110, 117]. TNF-α production by adipocytes has been shown to induce SOCS-3. The ability of TNF-α signal transduction to serine phosphorylate IRS-1, however, is widely accepted as TNF-α s major role in insulin resistance [65, 68, , 130, 153]. TNF-α can activate many serine kinases, including c-jun-nh2-terminal kinase (JNK), IκB kinase (IKK), AKT and mammalian target of rapamycin (mtor) [154]. TNF can also stimulate lipolysis, which results in increased serum free fatty acids [38, 155]. Free fatty acids can also increase serine phosphorylation of IRS-1 by activating JNK and IKK [38]. Insulin-mediated AKT activation of mtor and p70s6kinase (S6K) has been shown to promote serine phosphorylation of IRS-1. Nutrient overload, as seen in obesity, can also increase mtor activation [84, 86]. While insulin-mediated serine phosphorylation of IRS-1 may have a physiological role as a mechanism of negative

58 42 feedback of insulin action, chronic activation of mtor has been implicated in insulin resistance [81, 85-86]. Serine phosphorylation of IRS-1 and IRS-2 has been shown to decrease their tyrosine phosphorylation by the insulin receptor, decreasing downstream insulin signaling. Degradation of IRS proteins may also be accelerated by serine phosphorylation, but the mechanism is unclear [72, 82]. Insulin-mediated serine phosphorylation of ser307 has been determined to be an important mediator of IRS-1 degradation [82, 118]. The relationship between serine phosphorylation of IRS-1/2 and SOCS-3-mediated IRS-1/2 degradation is not well understood, and the mechanism of action has yet to be elucidated. To address the role of SOCS-3 in hepatic insulin signaling, SOCS-3 was knocked down in the liver of lean and diet induced obese (DIO) mice by a shrna construct. In this study we demonstrate that suppression of hepatic SOCS-3 increases insulin responsiveness in both lean and obese mouse models, and that SOCS-3 selectively modulates IRS-1. From this data we investigated the mechanism of SOCS-3-mediated differential inhibition and degradation of IRS-1. SOCS-3 degraded IRS-1 only in the presence of chronic insulin treatment or serine phosphorylation, while SOCS-3 inhibition of insulin signaling could not be recapitulated in primary hepatocytes. These data suggest that SOCS-3 modulation of IRS-1 is complex and requires multiple factors. These results support the hypothesis that SOCS-3 is a mediator of insulin resistance, however, the exact mechanism remains elusive.

59 Results To determine if SOCS-3 modulates hepatic insulin responsiveness, SOCS-3 was suppressed in the liver of lean mice by an adenovirus shrna construct. Basal and IL-6-induced Socs3 mrna and protein were suppressed by 60% (Fig. 1A, B). Surprisingly, basal and insulin stimulated tyrosine phosphorylation of IRS-1 increased 30% in mice with SOCS-3 suppression, compared to control infected mice (Fig. 1C). Hepatic suppression of SOCS-3 also increased basal and insulinstimulated AKT phosphorylation (Fig. 1D). To examine the role of SOCS-3 in an insulin resistant state, SOCS-3 was suppressed in mice fed a high fat diet. SOCS-3 protein and mrna message levels were suppressed approximately 65-70% (Fig. 2A, B). SOCS-3 suppression in diet induced obese (DIO) mice resulted in an increase in basal and insulin stimulated IRS- 1 and AKT phosphorylation, compared to control mice (Fig. 2C, E). In contrast to IRS-1, IRS-2 phosphorylation was unaffected by SOCS-3 suppression (Fig. 2D). Inhibition of insulin signaling is associated with SOCS-3-mediated degradation of IRS-1/2, however, neither IRS-1 nor IRS-2 protein levels varied in lean mice with SOCS-3 suppression (Fig 3A). These results indicate that insulin signaling in the liver of lean animals can be modulated by physiological levels of endogenous SOCS-3, independent of IRS protein levels. In contrast to lean animals with SOCS-3 suppression, IRS-1 protein was increased 80% in DIO mice (Figure 3B). IRS-2 protein levels were unchanged. IRS-1 protein increases were not due to an increase in transcription, as IRS-1 and IRS-2 mrna expression was unaffected by

60 44 SOCS-3 suppression (Fig. 3A and 3B). Taken together, these data suggest that SOCS-3 selective modulation of IRS-1 can alter insulin responsiveness by decreasing activation of IRS-1, and subsequently its downstream target, AKT. To determine the mechanism of SOCS-3-mediated regulation of IRS-1 and IRS-2, SOCS-3 was expressed in primary hepatocytes using a SOCS-3 adenoviral construct. Surprisingly, ectopically expressing SOCS-3 alone did not alter IRS-1 or IRS-2 protein levels (Fig. 4A, B). To recapitulate conditions of hyperinsulinemia seen in insulin resistance, cells were treated with insulin overnight. Chronic exposure to insulin caused a 60% decrease in IRS-1 protein levels in cells expressing SOCS-3. Control cells were unchanged. As seen in vivo, IRS-2 was unaffected by all cell conditions (Figure 5A, B). Insulin exposure causes serine phosphorylation, which has been implicated in the degradation of IRS-1 [72, 81-82]. To determine the role of serine phosphorylation in IRS-1 degradation in primary hepatocytes, serine kinases were activated by TNF-α. Control and SOCS-3 infected cells were also treated with IL-6 to induce endogenous SOCS-3. TNF-α caused a 60-70% decrease in IRS-1 protein levels, independent of IL-6 and adenoviral SOCS-3 expression (Fig. 6A). IRS-2 was unaffected under all conditions. IRS-1 degradation by TNF-α and TNF-α plus IL-6 demonstrates that TNF-α mediates degradation of IRS-1 independent of SOCS-3 induction by IL-6 (Fig. 6B). To confirm that TNF-α-mediated degradation of IRS-1 is independent of SOCS-3, a liver-specific SOCS-3 knockout mouse was used. Primary hepatocytes

61 45 from liver-specific SOCS-3 knockout and littermate controls were treated with IL-6 and Socs3 expression was measured. IL-6 increased Socs3 expression 7-fold in control hepatocytes, but failed to induce Socs3 in the SOCS-3 knockout hepatocytes (Fig. 7A). TNF-α treatment of SOCS-3 knockout hepatocytes decreased IRS-1 levels after 24 hours of exposure. TNF-α appeared to induce serine phosphorylation of IRS- 2 as reflected in a molecular weight shift, but it had no affect on IRS-2 degradation (Fig. 7B). These data suggest that TNF-α can selectively degrade IRS-1 independent of SOCS-3. TNF-α has been shown to induce SOCS-3 in adipocytes. It has not been reported in hepatocytes [ ]. It is possible that TNF-α is inducing SOCS-1 in SOCS-3 knockout hepatocytes, which leads to IRS-1 degradation. To test this hypothesis, primary hepatocytes were treated with TNF-α and Socs induction was examined. TNF-α treatment did not increase either Socs3 or Socs1 message levels when compared to IL-6 (Fig. 8). Socs1 induction by IL-6 occurred at a later time point, and was not as robust as Socs3 induction, which is consistent with previous findings [81]. These data indicate that TNF-α cannot induce Socs1 or Socs3 in hepatocytes, and that TNF-α-mediated IRS-1 degradation does not require Socs1 or Socs3 induction. Suppression of SOCS-3 in lean mice led to an increase in insulin responsiveness without changes in IRS protein levels (Fig 1, 3). SOCS-3 ectopic expression has been shown to inhibit insulin signaling in various cell lines, though the mechanism of inhibition is not well understood [ , 128]. To elucidate the

62 46 structural role of SOCS-3 inhibition of insulin signaling, wild-type SOCS-3 and SOCS-3 constructs containing loss of function mutations in their conserved domains were to be used. These experiments required robust inhibition of insulin signaling by ectopic SOCS-3 expression. However, insulin treatment of primary hepatocytes infected with SOCS-3 did not alter insulin responsiveness when compared to controls. IRS-1 and IRS-2 tyrosine phosphorylation, as well as AKT phosphorylation, was unaltered by SOCS-3 expression (Fig. 9A, B). Thus, conditions could not be established to test the SOCS-3 domain mutants. Although insulin signaling was enhanced with SOCS-3 suppression in vivo, these results could not be recapitulated in vitro. Multiple factors appear to contribute to the inhibition of insulin signaling by SOCS-3, making it difficult to recapitulate its effects in hepatocytes in culture.

63 Discussion Increases in SOCS-3 are seen in inflammation and obesity, and are associated with the development of insulin resistance. SOCS-3 is implicated in inhibiting IRS-1 and IRS-2 signaling, and causing their degradation by the proteosome. The mechanism of SOCS-3 action, however, is poorly understood. The current study investigated the in vivo role of SOCS-3 in modulating hepatic insulin signaling, as well as the mechanism of SOCS-3 action in vitro. Hepatic suppression of SOCS-3 was attained by adenovirus delivery of a shrna construct to the liver of both lean and obese mice. In the obese mouse model, suppression of hepatic SOCS-3 nearly doubled IRS-1 protein levels, without changes in messenger RNA levels. In contrast to changes in IRS-1, IRS-2 was unaffected. Primary hepatocytes were isolated to investigate the mechanism of SOCS-3 mediated IRS-1 and IRS-2 degradation. SOCS-3 was found to be necessary for insulinmediated IRS-1 degradation, however, IRS-2 was unaltered. These results agree with our in vivo data, demonstrating that SOCS-3 selectively degrades IRS-1. This differential regulation of IRS-1 conflicts with reports demonstrating expression of SOCS-3 (or SOCS-1) causes degradation of both IRS-1 and IRS-2 [125, ]. Rui et al. [128] demonstrated that SOCS-1 and SOCS-3 can degrade both IRS-1 and IRS-2 by SOCS-mediated ubiquitination. Using a adipose tissue SOCS-3 transgenic mouse model, Shi et al. [127] demonstrated that SOCS-3 impaired adipose tissue insulin signal transduction by decreasing protein levels and tyrosine phosphorylation of IRS-1 and IRS-2.

64 48 Surprisingly, adenoviral expression of SOCS-3 in primary hepatocytes did not alter protein levels of IRS-1 or IRS-2 unless chronically treated with insulin. Serine phosphorylation of IRS-1 has been shown to promote degradation, and is often associated with selective degradation of IRS-1 [65, 72, 81-82]. Insulin has been shown to cause degradation of IRS-1 by inducing serine phosphorylation mediated by mtor [81]. Our data suggest that both SOCS-3 and serine phosphorylation is required to degrade IRS-1. Reports by both Ishizuka et al. [109] and Shi et al. [130] support this mechanism, demonstrating that both TNF-α-mediated serine phosphorylation of IRS-1, and SOCS-3 is required for IRS-1 degradation. To investigate the above hypothesis in primary hepatocytes, cells were treated with TNF-α with or without expression of SOCS-3. Unlike insulin mediated degradation of IRS-1, TNF-α treatment alone was sufficient to degrade IRS-1. IRS-2 protein levels were unaltered. TNF-α treatment of SOCS-3 deficient primary hepatocytes also decreased IRS-1 protein levels. We have seen an increase in SOCS- 1 transcription in SOCS-3 deficient primary hepatocytes. Therefore, it is possible that SOCS-1 is compensating for the loss of SOCS-3 and is mediating the degradation of IRS-1 in SOCS-3 deficient hepatocytes. To rule out this possibility, SOCS-1 expression will need to be suppressed in conjunction with SOCS-3 suppression. How TNF-α is capable of IRS-1 degradation without SOCS-3 induction is not clear. TNFα has been shown to increase SOCS-3 messenger RNA and protein levels in adipocytes [ , 130], however, we demonstrate that TNF-α fails to induce SOCS-3 and SOCS-1 in primary hepatocytes. It is possible that TNF-α causes serine

65 49 phosphorylation of IRS-1 more readily, or on different residues than insulin, allowing for protein degradation without increases in SOCS proteins. While considerable literature link TNF-α to insulin resistance, it is controversial whether TNF circulates systemically to influence the liver. The role of TNF-α in IRS-1 degradation needs to be further investigated [55, 148]. Insulin responsiveness was increased in lean mice without changes in IRS-1 and IRS-2 protein levels. These data suggest that there are multiple mechanisms by which SOCS-3 modulates insulin signal transduction. A report by Torisu et al. [121] supports our data that SOCS-3 can modulate hepatic insulin signaling without altering IRS protein levels. In their study, liver-specific knockout mice had increased insulindependent phosphorylation of IRS-1 and AKT relative to wild type controls, while IRS-1 protein levels remained unchanged. Ueki et al. [111] observed decreases in insulin-mediated IRS-1 and -2 tyrosine phosphorylation in the liver of mice with SOCS-1 and SOCS-3 adenovirally expressed. They demonstrate that insulin signaling can be modulated by SOCS-3 without a loss in IRS protein levels, however, conflict with our data that demonstrates IRS-1 signaling is selectively affected. To further explore the mechanism of SOCS-3 inhibition of insulin signaling, an initial aim of this thesis was to explore the structure-function relationships of SOCS-3 as they relate to insulin signaling. These studies would require ectopic SOCS-3 expression to inhibit insulin signaling in primary hepatocytes as we have previously shown in hepatoma cell lines. Unexpectedly, ectopic SOCS-3 expression had no affect on insulin signaling in primary hepatocytes. Tyrosine phosphorylation

66 50 of IRS proteins remained unchanged compared to control cells. Phosphorylation of AKT was also unaltered in SOCS-3-infected cells. Insulin signaling was also unaltered in primary hepatocytes from liver-specific SOCS-3 knockout mice when compared to wild-type controls (data not shown). Expression of SOCS-3 has been previously shown to decrease insulin mediated activation of IRS-1 and AKT in vitro [110, 117], however these studies were carried out in various cell types. Results demonstrated in hepatoma cells lines and adipose tissue most likely do not represent the signaling pathways present in primary hepatocytes. The mechanism of SOCS-3- mediated IRS degradation and inhibition of insulin signaling has proven to be a complex process, due in part to the tissue specificity of SOCS-3 action. Renstrom et al. [131] found that IRS-2 levels were unaffected in human adipocytes exposed to high glucose and insulin, while IRS-2 was degraded in primary rat hepatocytes. Based on our current study, SOCS-3 differentially modulates IRS-1 and IRS-2 protein degradation, and responsiveness to insulin signaling in the liver. Our observations demonstrate that SOCS-3 promotes IRS-1 degradation without affecting IRS-2. SOCS-3 also selectively alters insulin-mediated tyrosine phosphorylation of IRS-1 without changes in IRS-1 protein levels. This demonstrates SOCS-3 modulates insulin signaling by multiple mechanisms. It is possible that SOCS-3 is complexed with IRS-1 even under basal conditions, rendering complexed IRS-1 inactive. A second stimulus, serine phosphorylation, is then required for protein degradation. In summary, the current data support our hypothesis that SOCS-3 is a modulator of hepatic insulin action. Recapitulating the physiologic conditions that

67 51 contribute to the actions of SOCS-3 in vivo has proved to be a complex process, not allowing for the exact mechanisms of SOCS-3 action to be established. Multiple factors must be involved in SOCS-3 mediated inhibition of insulin signaling, making it difficult to recapitulate physiologic conditions present in vivo, in primary hepatocytes in culture.

68 52 Figure 2.1 Suppression of SOCS-3 in lean mice. A, B)Male C57Bl/6 mice were injected with 5 x viral particles of adeno-shsocs-3 or adeno-shluciferase (control). Following a 12 h fast, mice were i.p. injected on day 10 with IL-6 (5 µg/kg) or saline. Livers were harvested 90 min later. Socs3 message levels were quantified by qrt- PCR (A) and protein levels assessed by Western blot analysis (B). C) Livers were harvested 10 min after an i.p. injection of insulin (1.5 U/kg) or saline. Lysates were immunoprecipitated for IRS-1 and tyrosine phosphorylation was assessed by Western blot analysis. D) Akt activation was assessed by Western blot analysis using a phosphoakt (S473) antibody. Data are normalized to insulin-treated, control mice and represent the mean ± s.e.m., n=6-8. * = p< 0.05; ** = p< 0.01.

69 53 A. SOCS3 mrna Relative Expression Control shsocs3 Control 1 IL-6 2 B. SOCS-3 β-actin shluciferase shso C S IL

70 54 C. C o n tr o l s h S O C S 3 Insulin IP:IRS1 IB:pY IB:IRS1 IRS-1 Tyrosine Phosphorylation (arbitrary units) Control shsocs3 ** D. 0 Insulin p-a kt A kt Basal Insulin C ontrol shs O C S Akt(S473) Phosphorylation (arbitrary units) Control shsocs3 * ** 0 Basal 1 Insulin 2

71 55 Figure 2.2 Suppression of SOCS-3 in high fat diet-induced obese mice. A, B) Diet-induced obese male C57Bl/6 mice were injected with shsocs-3 or control shrna and livers were harvested on day 10. SOCS-3 protein levels were assessed by Western blot analysis (A) and Socs3 message was quantitated by qrt-pcr (B). C, D) Following a 12 h fast, mice were i.p. injected on day 10 with insulin (1.5 U/kg) or saline. Livers were harvested after 10 min and lysates were immunoprecipitated for IRS-1 (C) and IRS-2 (D), and tyrosine phosphorylation was assessed by Western blot analysis. E) Akt activation was assessed by Western blot analysis using a phosphoakt (S473) antibody. Data are normalized to control mice and represent the mean ± s.e.m., n=6-8. ** = p< 0.01.

72 56 A. Control shrna SOCS3 β-actinβ B. R elative S O CS 3 mr N A E xpression Cont 1 shsocs3 C. Insulin IP:IRS-1 IB:pY IB:IRS-1 Control shsocs IR S-1 Tyrosine Phosphorylation (arbitra ry un its) Control shsocs3 ** ** 1 2 Basal Insulin

73 57 D. Control shsocs3 Insulin IP:IRS2 IB:pY IB:IRS2 E. Control shsocs3 Insulin pakt Akt Akt (S473) Phosphorylation (arbitrary units) Control shsocs3 ** ** 0 Basal Insulin

74 58 Figure 2.3 IRS-1 and IRS-2 protein and mrna levels in livers of lean and obese mice as a function of SOCS-3 expression. IRS-1 and IRS-2 protein levels were determined by Western blot analysis in tissue lysates from lean (A) and obese (B) mouse livers. Representative experiments are shown with β-actin as a loading control. mrna expression levels of IRS-1 and IRS-2 in livers from lean (A) and obese (B) mice were determined by qrt-pcr. Data are normalized to control mice in each pair and represent the mean ± s.e.m., n=6. ** = p<0.01

75 59 A. Control shsocs3 IRS -1 IRS -2 β-actin IRS Protein (arbitrary u nits) shsocs3 Control shsocs3 IRS m RNA (AU) Control shsocs3 IRS-1 IRS-2 IRS-1 IRS-2 B. Control shsocs3 IRS -1 IRS -2 β-actin IRS Protein (arbitrary units) ** Control shsocs3 IRS-1 1 IRS-2 2 IRS mrna (AU) Control shsocs3 IRS-1 1 IRS-2 2

76 60 Figure 2.4 SOCS-3 expression does not alter IRS protein levels. A) SOCS-3 was ectopically expressed in primary hepatocytes from male C57/Bl6 mice using adenovirus infection (LacZ control) at varying m.o.i.s. After 24 h, cells were harvested and protein levels were assessed by Western blot analysis. β-actin was used as a loading control. B) Primary hepatocytes were infected with adeno-socs-3 (200 m.o.i.) for 24 h and then harvested and assessed for IRS-2 and SOCS-3 protein levels by Western blotting analysis.

77 61 A. IRS -1 SOCS -3 B- Actin LacZ SOCS -3 MOI B. IRS -2 SOCS -3

78 62 Figure 2.5 SOCS-3 selectively decreases IRS-1 protein levels in primary hepatocytes. Primary hepatocytes were infected with an adenovirus expressing SOCS-3 or (A) LacZ (200 m.o.i.) for 24 h. (B). After a 24 h insulin (100nM) treatment, IRS-1 and IRS-2 protein levels were measured by Western blot analysis. Representative blots are shown. Data represent the mean ± S.D., n = 6.

79 63 A. IRS-1 IRS-2 Insulin IRS Protein (AU) SOCS Insulin B. IRS-1 IRS-2 Insulin IRS Protein (AU) SOCS-3 Insulin

80 64 Figure 2.6 TNF-α decreases IRS-1 protein levels independent of SOCS-3 expression. A) Primary hepatocytes were infected with an adenovirus expressing SOCS-3 or LacZ (200 m.o.i.) for 24 h. Hepatocytes were treated with TNF-α (20ng/ml) or IL-6 (20ng/ml) for an additional 24 h, and then harvested. Protein levels were assessed by Western blot analysis. A representative experiment is shown with β-actin as a loading control. B) Time dependent degradation of IRS-1 in response to TNF-α and IL-6, or TNF-α alone, was quantified by densitometry from Western blot analysis. Data represent the mean ± S.D., n = 4-6.

81 65 A. LacZ SOCS-3 TNF -α IL IRS -1 IRS -2 SOCS-3 NS β - actin B. IRS-1 Relative Degradation TNF- α TNF- α and IL Hours

82 66 Figure 2.7 TNF-α degradation of IRS-1 protein is independent of SOCS-3. A) Primary hepatocytes from liver specific SOCS-3 knockout mice and their littermate controls (WT) were treated with IL-6 (20ng/ml) for 90 min. Socs3 message was measured by qrt-pcr. B) Time dependent degradation of IRS-1 and IRS-2 by TNF-α (20ng/ml) was measured by Western blot analysis in liver specific SOCS-3 knockout primary hepatocytes and WT controls. A representative experiment is shown with β-actin as a loading control. Data represent the mean ± S.D., n = 6.

83 67 A. SOCS-3 Relative Expression No Treatment IL-6 WT SOCS-3 KO B. WT TNF -a (hrs) IRS -1 IRS -2 B -actin

84 68 Figure 2.8 TNF-α does not induce Socs1 or Socs3 expression in primary hepatocytes. Time dependent increases in Socs3 (A) and Socs1 (B) expression in response to IL-6 (20ng/ml) and TNF-α (20ng/ml) were measured by qrt-pcr. Data represent the mean ± S.D., n = 4.

85 69 A. Relative Expression SOCS-3 TNF-α IL Hours B. 3 SOCS-1 TNF-α IL-6 Relative Expression Hours

86 70 Figure 2.9 Ectopic Socs3 expression does not alter insulin responsiveness in mouse primary hepatocytes. Primary hepatocytes were infected with an adenovirus expressing SOCS-3 and LacZ (m.o.i 200) for 24 h. A) Cells were harvested after a 10 min insulin (100nM) treatment and lysates were immunoprecipitated for IRS-1 and IRS-2. Tyrosine phosphorylation was assessed by Western blot analysis. B) Akt activation was assessed by Western blot analysis using a phosphoakt (S473) antibody. Data represent the mean ± S.D., n = 6.

87 71 A. LacZ SOCS-3 Insulin py IRS -1 IRS -1 py IRS -2 IRS -2 SOCS -3 NS 25 IRS-1 IRS-2 LacZ SOCS-3 IRS py/irs protein (AU) Basal Insulin Basal Insulin

88 72 B. LacZ SOCS-3 Insulin pakt AKT AKT pakt (ser 473)/ AKT Mass (AU) Control SOCS-3 Basal Insulin

89 73 Chapter 3 Epac Induction of SOCS-3: A Negative Regulator of PKA Mediated Hepatic Gluconeogenesis

90 Introduction Glucagon is a catabolic hormone that is synthesized and released by α cells in the pancreatic islets in response to hypoglycemia [29]. Glucagon maintains normoglycemia by increasing hepatic glucose output through mobilization of glucose from glycogen (glycogenolysis) and increasing de novo glucose production (gluconeogenesis) [12, 29]. Receptor expression has been found in the pancreas, small intestine, and kidney, but is minimal compared to levels in the liver [28]. The glucagon receptor is a classic Gα s coupled receptor. Receptor-ligand coupling results in activation of adenylate cyclase, camp production, and protein kinase A (PKA) activation. In a reaction that is critical to gluconeogenesis, activated PKA phosphorylates camp response element-binding protein (CREB) leading to its transcriptional activation at the camp-responsive element (CRE) in the promoter region of key genes including peroxisome proliferator-activated receptor-gamma coactivator 1 alpha (Ppargc1a), phosphoenolpyruvate carboxykinase (Pck1) and glucose-6-phosphatase (G6pc) [29, 49]. PEPCK catalyzes the rate limiting step in gluconeogenesis, while G-6-Pase hydrolyzes glucose-6-phospate to generate free glucose that can be released from the hepatocyte into the circulation. PGC-1α coactivates hepatic nuclear factor-4α (HNF-4α) and the forkhead transcription factor FOXO1, robustly increasing transcription of Pck1 and G6pc [17, 29, 50]. PKA is a tetrameric holoenzyme consisting of two regulatory (R) and two catalytic (C) subunits. PKA exists in an inactive state until camp binds to the regulatory subunits, causing the release of the catalytic subunits. Active PKA

91 75 phosphorylates serine/threonine residues on its substrates at the consensus sequence Arg-Arg-X-Ser/Thr-X. There are two types of regulatory subunits, I and II, each having two subtypes, α and β. RIα and RIIα are ubiquitously expressed while the β isoforms are predominantly found in the brain [30-31]. While the catalytic subunits (α, β and γ) are similar in expression and function, each regulatory isoform possesses a unique functional phenotype [32]. RI subunits are mostly cytoplasmic, while RII subunits are typically associated with anchoring proteins (AKAPs) that target PKA to specific cellular compartments and organelles. The AKAPs display isoform specificity in their binding to the regulatory subunits [33-34]. PKA dependent Serine-133 phosphorylation of CREB in the nucleus does not alter the binding of CREB to the Cre site in the PEPCK promoter, however it promotes the recruitment of the coactivators, CREB-binding protein (CBP), CREB regulated transcription coactivator 2 (CRTC2; also known as TORC2) and p300 to a transcriptional complex [42-46]. CBP interacts with RNA helicase protein, promoting transcriptional activation [48]. This transcription activation pathway is negatively regulated by dephosphorylation of CREB at ser133 by the serine/threonine phosphatases PP-1 and PP-2A, with PP-2A being the predominate phosphatase mediating this reaction in hepatocytes [ ]. SOCS (suppressor of cytokine signaling) proteins are a family of negative regulators of signal transduction pathways. There are eight members of this protein family that are defined by a variable N-terminal region, a central Src homology domain 2 (SH2) and a C-terminal SOCS box that contains a functional BC box that

92 76 targets substrates for proteosomal degradation [ ]. SOCS-3 is a known antagonist of the pro-inflammatory cytokine IL-6, growth hormone, leptin and the insulin receptor pathway [110, 117, ]. Our lab and others have shown that SOCS-3 can antagonize insulin action by proteosomal degradation of the insulin receptor substrates-1/2 (IRS-1/2) as well as inhibit phosphorylation of IRS-1/2 and AKT [ , 117, , 128]. Taken together these data demonstrate that SOCS-3 is not only a negative regulator of cytokine signaling in liver, but is involved in the direct regulation of hepatic metabolic signal transduction pathways. Recently it was reported that SOCS-3 can be induced in human umbilical vein endothelial cells (HUVEC) by a camp dependent pathway that is independent of PKA [ ]. This PKA-independent pathway involves a camp-dependent activation of the Epac family of exchange factors. Yarwood et al. demonstrated that activation of Epac by camp leads to the downstream activation of the small G protein Rap1 causing increased induction and activation of CCAAT/enhancer-binding proteins (C/EBPs). Activation of C/EBPβ via Epac in COS1 cells appears to require activation of PKCα with subsequent activation of ERK [132]. Glucagon mediated increases in camp have now been implicated in the activation of Epac in hepatocytes. Aromataris et al. demonstrated that glucagon can alter hepatic ion channel activity by camp activation of Epac in rat hepatocytes [142]. We now demonstrate that glucagon induces SOCS-3 in hepatocytes via Epac activation, and identify a mechanism of inhibitory crosstalk between Epac and PKA

93 77 that is mediated by SOCS-3 and is critical for regulation of gluconeogenic gene expression.

94 Results To determine if SOCS-3 expression is regulated by camp in hepatocytes, primary mouse hepatocytes were treated with 8-bromo-cAMP (8-br-cAMP). Expression levels of Socs3 increased approximately 10-fold with 8-br-cAMP treatment. In comparison, IL-6 increased Socs3 expression by approximately 7 fold (Fig. 1A). A time course of glucagon-dependent induction showed peak expression of 3.5 fold occurring at 1 h, with a return to baseline at 3-4 h (Fig. 1B). In contrast, expression of the gluconeogenic genes Pck1 and G6pc peaked at 2 h (Fig. 1B). Concentration-dependent induction of Socs3 and the gluconeogenic genes by glucagon was comparable (Fig. 1C). The above data suggested that SOCS-3 may be regulated physiologically by glucagon. To determine if SOCS-3 expression is altered physiologically, male C57/Bl6 mice were fasted 24 h and half subsequently refed for 4 h. Hepatic Socs3 expression was nearly 4-fold higher in fasted mice compared to refed mice (Fig. 2). Pck1 expression was increased in fasted mice as expected. Taken together glucagon induces Socs3 in vitro, and Socs3 expression in the liver is higher under metabolic conditions that favor higher glucagon levels. It has been previously shown that SOCS-3 induction via camp is mediated by Epac in mouse embryonic fibroblasts and COS1 cells [132, ]. To demonstrate that SOCS-3 is induced by the PKA-independent Epac pathway in hepatocytes, primary mouse hepatocytes were exposed to glucagon in the presence of the PKA specific inhibitor, H-89. Inhibition of PKA had no effect on Socs3

95 79 induction, but decreased induction of Pck1 as expected (Fig. 3A, B). The Epac selective camp analog 8-4-(chlorophenylthio)-2 -O-methyladenosine-3, 5 - monophosphate, acetoxymethyl ester (cptome) induced Socs3 similarly to glucagon (Fig. 3A). Rap1 has been implicated as the downstream mediator of Epac action. To examine this in primary hepatocytes, RapGAP1 was ectopically expressed using adenoviral delivery. RapGAP1, which increases the rate of Rap1 bound GTP hydrolysis, significantly decreased glucagon induction of Socs3 (Fig. 3C). These data argue that glucagon induction of SOCS-3 in primary hepatocytes is mediated by Epac activation and involves Rap1. Rap1 activation of PLCε has been implicated in the Epac mediated signaling pathway that leads to SOCS-3 induction [132]. To determine if SOCS-3 induction is mediated by PLCε, primary hepatocytes from PLCε knockout mice were treated with glucagon and cptome. Socs3 expression in response to glucagon and cptome was comparable in PLCε knockout and wild-type hepatocytes (Fig. 4A). Activation of extracellular signal-related kinase (ERK1/2) has been shown to be required for Epac induction of SOCS-3 in COS1 and HUVEC cells [132, 135]. Primary hepatocytes, however, treated with 8-br-cAMP actually decreased ERK phosphorylation compared to non-treated controls (Fig 4B). These data indicate that SOCS-3 induction in primary hepatocytes is not dependant on PLCε and ERK activation. The Epac pathway appears to have distinct differences between cell types. Given that SOCS-3 is a negative feedback inhibitor of several signal transduction pathways, SOCS-3 inhibition of glucagon/camp signaling was

96 80 investigated. Cells were pretreated with cptome for one h to induce SOCS-3. Cyclic AMP analogs were then added for either 2 h to examine gluconeogenic gene expression, or 30 min for CREB phosphorylation. Pretreatment with the Epacspecific analog cptome markedly suppressed subsequent ser133 phosphorylation of CREB (Fig 5A). Similarly, pretreatment with cptome decreased subsequent induction of Ppargc1a, G6pc, and Pck1 by approximately 20% (Fig 5B). Epac activation therefore, appears to negatively regulate PKA activation of gluconeogenic gene expression at the level of CREB phosphorylation. Since the camp analog, spcamp is highly resistant to cyclic nucleotide phosphodiesterases, this effect of Epac appears to be independent of phosphodiesterases. To determine if SOCS-3 is involved in the Epac-dependent inhibition of CREB phosphorylation and gluconeogenic gene expression, SOCS-3 was expressed in primary hepatocytes using a SOCS-3 adenoviral construct. SOCS-3 decreased 8- br-camp-mediated phosphorylation of CREB by nearly 40%, and caused a 47%, 45%, and 32% suppression in Ppargc1a, G6pc and Pck1 expression, respectively, compared to LacZ infected controls (Fig 6A, B). Glucagon-dependent induction of G6pc and Pck1 was suppressed by SOCS-3. SOCS-3 plus the PKA inhibitor H-89 produced a modest increase in the suppression of gluconeogenic gene expression (Fig 6C). To further establish a role for SOCS-3 in regulating gluconeogenic gene expression, SOCS-3 expression was knocked down with shrna in primary hepatocytes. Basal expression was decreased 40% and induction by 8-br-cAMP was

97 81 blunted by 55% (Fig. 7A). Under the condition of suppressed SOCS-3 expression, gluconeogenic genes G6pc and Pck1 were increased by approximately 60% (Fig. 7B). Ppargc1a expression was unaltered. Liver-specific SOCS-3 knockout mice were used to further examine the role of SOCS-3 in hepatic camp signaling. Primary hepatocytes from SOCS-3 knockout mice (Cre + ) and littermate controls (Cre - ) were treated with IL-6 and 8-br-cAMP. As expected, SOCS-3 was induced in the WT control hepatocytes, but not in the SOCS-3 deficient hepatocytes (Fig. 8A). Treatment with 8-br-cAMP increased Ppargc1a, Pck1, and G6pc expression in SOCS-3 deficient hepatocytes by 200%, 225%, and 30%, respectively, relative to littermate controls (Fig. 8B). To investigate whether inhibition of CREB phosphorylation by SOCS-3 is unique to this one nuclear PKA substrate or is a shared effect by other PKA substrates, the effect of SOCS-3 on camp-dependent phosphorylation of the inositol 1,4,5-triphosphate receptor (IP3R) was examined. Treatment of hepatocytes with 8- br-camp increased phosphorylation of IP3R at Ser1756 almost 3-fold. Ectopic expression of SOCS-3, however, reduced pip3r by 30% (Fig. 9). These data demonstrate that SOCS-3 does not selectively inhibit CREB phosphorylation, but rather, may be a more general effect on PKA substrates. To test the hypothesis that SOCS-3 inhibits PKA activity, PKA activity was measured in primary hepatocytes treated with 8-br-cAMP, with or without cptome pretreatment. Stimulation with 8-br-cAMP increased PKA activity approximately 3- fold. Cells pretreated with cptome, however, had significantly reduced PKA

98 82 activity (Fig. 10A). Ectopic expression of SOCS-3 in primary hepatocytes blunted camp-dependent PKA activity by 80% compared to LacZ infected controls (Fig. 10B). There data argue that SOCS-3 decreases the PKA pathway by inhibiting PKA s ability to phosphorylate downstream targets. PKA exists as an inactive holoenzyme until camp binds to the regulatory subunits causing release of the catalytic subunits. The regulatory subunits remain cytoplasmic, or bound to AKAPs that target them to specific cellular compartments [31-32]. To determine if SOCS-3 inhibits PKA activity by inhibiting the separation of the R and C subunits, nuclear localization of the catalytic subunit as a surrogate assay for the free catalytic subunit was performed. The catalytic subunit of PKA was targeted to the nucleus after camp treatment with and without SOCS-3 expression. Localization to the nucleus was modestly increased with SOCS-3 expression. This demonstrates that the catalytic subunit is released from the R subunits and translocates to the nucleus despite its decreased phosphorylation of CREB (Fig. 11). SOCS-3 is also found in the nucleus. These results may suggest a model in which SOCS-3 is associating with the catalytic subunit to inhibit PKA signaling.

99 Discussion Glucagon is an important catabolic hormone for regulation of metabolism in the fasted state, particularly in promoting glucose release from hepatocytes via gluconeogenesis and glycogenolysis. Glucagon signaling has long been associated with the stimulation of camp production and activation of PKA. Recently, glucagon-dependent camp production has been shown by Aromataris et al. [142] to also activate Epac. One consequence of camp-dependent Epac activation is induction of Socs3 expression [134]. The current study investigated the physiological role of camp-induced Socs3 induction in hepatocytes. Our data demonstrate for the first time that glucagon-dependent Socs3 induction via Epac negatively regulates the PKA pathway in hepatocytes. Inhibition of PKA activity by SOCS-3 leads to decreases in CREB phosphorylation and gluconeogenic gene induction. Induction of Socs3 via the Epac-dependent pathway in primary hepatocytes differs from that described in other cell systems. As a guanine nucleotide exchange protein, Epac is assumed to mediate its effects through activation of small G-proteins. Inhibition of Rap1 activity with expression of RapGap1 in primary hepatocytes decreased Socs3 induction following glucagon treatment. This observation is in agreement with studies implicating Rap1 as the downstream mediator and target of Epac activity [132, ]. Borland et al. [132] reported that Rap1-dependent activation of PLCε in COS1 cells is required for maximal Socs3 induction. This observation is supported by other studies demonstrating Rap1-dependent activation of PLCε [143, 145]. Our data demonstrate, however, that primary hepatocytes from

100 84 PLCε knockout mice induced Socs3 similarly to WT controls when stimulated with glucagon. Thus, PLCε is not an essential contributor to Epac-dependent Socs3 induction in hepatocytes. Borland et al. [132] also implicated the activation of ERK as the mechanism for C/EBPβ activation and induction of Socs3 in COS1 cells. As shown in this study, primary hepatocytes treated with 8-br-cAMP actually demonstrated decreased ERK phosphorylation. This has been previously reported in hepatocytes [158]. These data demonstrate that the mechanism of Socs3 induction differs between hepatocytes and COS cells. Epac2 is the predominant isoform of Epac in the liver [136], and is absent from COS1 cells [ ]. This may, in part, explain the differences in signaling pathways. Taken together, camp induction of Socs3 is mediated by Epac-Rap1 in hepatocytes; however, the remaining signaling steps responsible for Socs3 induction need to be elucidated. Treatment of hepatocytes with the Epac-selective analog, cptome, and ectopic expression of Socs3, suppressed camp and glucagon-dependent CREB phosphorylation and induction of Pck1, G6pc, and Ppargc1a. This directly implicates SOCS-3 as the mediator of these effects. Two loss-of-function approaches were used to further confirm the role of SOCS-3 as a negative modulator of the glucagon-camp-pka pathway. Sp-cAMP is highly resistant to cyclic nucleotide phosphodiesterases. Nonetheless, its ability to phosphorylate CREB and induce gluconeogenic genes was suppressed by SOCS-3 comparably to that of glucagon or 8-b-cAMP. This argues that the mechanism by which SOCS-3 negatively regulates PKA signaling is independent of phosphodiesterase activity. Decreased

101 85 phosphorylation of the IP3R by 8-br-cAMP indicates that the suppressive effect of SOCS-3 is not specific to CREB. This led us to test the hypothesis that SOCS-3 inhibits PKA activity. Using an in vitro PKA assay, we confirmed that PKA activity was suppressed by SOCS-3. SOCS-3 inhibition of C subunit dissociation from R subunits was ruled out considering nuclear translocation of the PKA catalytic subunit was not decreased by SOCS-3 expression. While requiring experimental validation, our data are consistent with a model in which SOCS-3 mediates its inhibitory effects by interacting with the catalytic subunit of PKA. The N- and C-terminal domains of the PKA catalytic subunit are important in mediating protein-protein interactions [32]. The C-terminus has a hydrophobic motif that allows it to bind to a hydrophobic pocket on PDK1, the high affinity inhibitor PKI, and the regulatory subunits [32, ]. The C-terminus also plays a role in recognizing protein substrates [32]. The N- terminus has been shown to undergo covalent modifications, as well as bind to the recently identified protein AKIP1, which targets the catalytic subunit to the nucleus [32, 162]. It is therefore feasible that SOCS-3 could be a binding partner of the PKA catalytic subunit. Interestingly, the KIR (kinase inhibitory region) domain of SOCS-3 contains an arginine residue -3 from a serine residue. This sequence is the minimal consensus sequence required for PKA catalytic subunit recognition and phosphorylation of its target proteins. It is possible that the N-terminal region of SOCS-3 containing the KIR domain plays a role in the inhibition of PKA activity. This is the first report demonstrating SOCS-3-dependent modulation of the glucagon signaling pathway and PKA signaling. SOCS-3 inhibition of

102 86 gluconeogenesis may be a mechanism for negatively modulating hepatic glucose production (HGP) to help prevent hyperglycemia in the fasted state. Interestingly, prolonged fasting has been shown to decrease activation of the CRE promoter, subsequently decreasing gluconeogenesis [163]. This correlates with an increase in circulating ketone bodies during the late protein-sparing phase of fasting [ ]. Liu et al. [163] attributed this metabolic switch to the degradation of CREB-regulated transcription coactivator 2 (CRTC2; also known as TORC2), which is a co-activator of CREB. SOCS-3 could potentially play an additional complementary role in prolonged fasting, as a mechanism to decrease gluconeogenesis and switch to fatty acid metabolism and the production of ketone bodies as an energy substrate. Given that insulin and glucagon are opposing hormones, it has been somewhat counter-intuitive that several previous studies indicated that glucagon enhances the suppressive effect of insulin on HGP [ ]. Here we report an Epac-mediated negative feedback mechanism by which glucagon may negatively modulate HGP. This mechanism may, in part, explain these previous reports. Insulin activation of AKT leads to phosphorylation of FOXO1, its exclusion from the nucleus, and inhibition of its association with PGC-1α. This is recognized as a dominant mechanism for inhibition of HGP. Epac-mediated SOCS-3 inhibition of PKA may also contribute to the inhibition of HGP by suppressing PGC-1α and gluconeogenic gene expression. It is possible that glucagon-mediated SOCS-3 induction accentuated the inhibition of HGP when administered along with insulin.

103 87 SOCS-3 may play differing roles in liver metabolism depending on the physiologic and pathologic conditions. Serum levels of IL-6 are increased in obesity and are associated with elevated hepatic SOCS-3 protein levels and the development of type 2 diabetes [55, 90, 112, 119]. SOCS-3 can decrease insulin responsiveness by inhibiting insulin-mediated IRS-1 and -2 tyrosine phosphorylation, as well as target IRS-1 and -2 for proteosomal degradation [110, , 121]. During the course of this study, insulin signaling was not affected by pretreatment with glucagon in primary hepatocytes. These data indicate that induction of SOCS-3 by Epac does not alter insulin signaling in vitro. We hypothesize that SOCS-3 induction by Epac specifically regulates glucagon-mediated PKA signal transduction, and does not alter insulin signaling in vivo. Perhaps, post-translational modifications of IRS1/2 or the insulin receptor under pathological conditions, such as with exposure to cytokines, promotes additional interactions involving SOCS-3. In summary, these data support the hypothesis that glucagon-dependent camp production activates two distinct signaling pathways in which the Epac-dependent pathway induces SOCS-3 and negatively regulates the PKA-dependent pathway. SOCS-3 appears to directly inhibit PKA activity, leading to decreased CREB phosphorylation and decreased gluconeogenic gene expression. This cross-talk may provide additional modulatory regulation of hepatic metabolism in the fastingfeeding cycle. SOCS-3 inhibition of PKA may also play an important role in other cell types and signaling pathways, making this inhibitory relationship important to investigate in the future.

104 88 Figure 3.1 Glucagon-dependent induction of Socs3 in primary hepatocytes. A) Primary hepatocytes were treated with IL-6 (20ng/ml) or 8-br-cAMP (0.1mM) for 2 h. Relative Socs3 expression was measured by qrt-pcr. Time dependent (B) and concentration dependent (C) expression of Socs3, Pck1, and G6pc in response to glucagon (10nM) were measured. Data represent the mean ± S.D., n=3-6. **p<0.01

105 89 A. Socs3 Relative Expression No Treatment IL-6 8-br-cAMP B. Socs3 Pck1 Relative Expression Relative Expression Hours of Glucagon Hours of Glucagon G6pc 120 Relative Expression Hours of Glucagon

106 90 C. Relative Expression SO C S G lucagon (nm ) Relative Expression PE PC K Glucagon (nm) Relative Expression G6Pase Glucagon (nm)

107 91 Figure 3.2 Socs3 expression is elevated in fasted mice. Socs3 and Pck1 mrna levels were measured in liver from mice fasted 24 h, or refed for 4h following a 24h fast. Data are normalized to fed mice. Data represent the mean ± S.D., n=6. **p<0.01

108 92 Socs3 Pck1 7 ** Refed Fasted Relative Expression **

109 93 Figure 3.3 Glucagon-dependent induction of Socs3 is mediated by Epac. Primary hepatocytes were treated with glucagon (10nM), the Epac-selective analog cptome (5 µm), or the PKA inhibitor H-89 (10 µm) for 2 h. mrna levels were determined for Socs3 (A) and Pck1 (B). C) Socs3 mrna was measured after a 2 h glucagon treatment in primary hepatocytes infected with RapGAP1-expressing adenovirus (AV-RapGap) (200 m.o.i.) or an adenovirus-gfp (AV-GFP) control. Bar graph in (C) is normalized to GFP with no treatment. Data represent the mean ± S.D., n=3-6. ** p< 0.01

110 94 A. Socs3 5 Relative Expression Glucagon H cptome B. Pck1 Relative Expression ** Glucagon H C. 5 Socs3 AV-GFP AV-RapGap Relative Expression ** 1 0 Glucagon

111 95 Figure 3.4 PLCε activation is not required for Epac-mediated Socs3 induction. A) Wild type (WT) and PLCε -/- primary hepatocytes were treated with glucagon (10 nm) or cptome (5 µm) for 2 h. Socs3 mrna levels were measured by qrt-pcr. B) WT primary hepatocytes were treated with 8-br-cAMP (0.1mM). Phosphorylation of p44/p42 MAP kinase (ERK) was detected by Western blot analysis with a phosphoerk antibody (Thr202/Tyr204). Data are normalized to no treatment controls. Data represent the mean ± S.D., n=5-8.

112 96 A. 4 Socs3 WT PLC -/- ε Relative Expression No Treatment Glucagon cptome B. 8-br-cAMP perk p44 p42 ERK

113 97 Figure 3.5 Epac activation inhibits PKA-mediated CREB phosphorylation and gluconeogenic gene expression. cptome (5 µm) was added to primary hepatocytes for 1 h followed by a 30 min (A) or 2 h (B) incubation with sp-camp (10 µm). A) CREB phosphorylation was determined by Western blot analysis using a phosphocreb-specific (Ser133) antibody. B) Ppargc1a, G6pc and Pck1 mrna levels were determined by qrt-pcr. Data are normalized to sp-camp treated controls. Data represent the mean ± S.D., n=6. * p<.05; ** p<0.01

114 98 A. sp -camp cptome pcreb Creb B. 1.2 sp-camp cptome + sp-camp Relative Suppression ** * * Ppargc1a G6pc Pck1

115 99 Figure 3.6 SOCS-3 expression inhibits CREB phosphorylation and gluconeogenic gene expression. Primary hepatocytes were infected with either adeno-socs-3 or adeno- LacZ (control) (200 m.o.i) for 24 h. A) After a 30 min 8-br-cAMP (0.1mM) treatment, CREB phosphorylation was measured by Western blot analysis. A representative blot is shown. Bar graph represents quantitation of pser133-creb relative to AV-LacZ untreated controls. B) Pck1, G6pc and Ppargc1a induction was measured by qrt-pcr after a 2 h 8-br-cAMP (0.1mM) treatment. Data are normalized to AV-LacZ cells treated with 8-br-cAMP. C) Pck1, G6pc and Ppargc1a induction was measured by qrt-pcr after a 2 h stimulation with glucagon (10nM) or glucagon plus H-89 (10 µm). Data are normalized to glucagon treated AV-LacZ controls. Data represent the mean ± S.D., n=6. *p<0.05; **p <.01

116 100 A. LacZ SOCS3 4 pcreb 8-br-cAMP pc R E B 3 2 ** CREB 1 SOCS3 0 LacZ SOCS br - camp B. Ppargc1a G6pc Pck1 1.2 AV LacZ AV SOCS-3 Relative Suppression ** ** ** 0 C. G6pc Pck1 Relative Suppression ** ** ** ** ** ** 0 AV-LacZ AV-SOCS3 Glucagon H