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An investigation of the potential role of PKC isoforms in the regulation of acetyl-CoA carboxylase-1 Collins, Susan Elizabeth 2001

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AN INVESTIGATION OF THE POTENTIAL ROLE OF PKC ISOFORMS IN THE REGULATION OF ACETYL-CoA CARBOXYLASE-1 by SUSAN ELIZABETH COLLINS B.Sc, The University of Guelph, 1998 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE FACULTY OF GRADUATE STUDIES DEPARTMENT OF : BIOCHEMISTRY AND MOLECULAR BIOLOGY We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA April 2001 © Susan Elizabeth Collins, 2001 I n p r e s e n t i n g t h i s t h e s i s i n p a r t i a l f u l f i l m e n t o f t h e r e q u i r e m e n t s f o r an advanced degree a t t h e U n i v e r s i t y o f B r i t i s h C o l u m b i a , I agree t h a t t h e L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r r e f e r e n c e and s t u d y . I f u r t h e r agree t h a t p e r m i s s i o n f o r e x t e n s i v e c o p y i n g o f t h i s t h e s i s f o r s c h o l a r l y p u r p o s e s may be g r a n t e d by t h e head o f my department o r by h i s o r h e r r e p r e s e n t a t i v e s . I t i s u n d e r s t o o d t h a t c o p y i n g o r p u b l i c a t i o n o f t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l n o t be a l l o w e d w i t h o u t my w r i t t e n p e r m i s s i o n . Department o f O C J L C U M ^ W -The U n i v e r s i t y o f B r i t i s h C olumbia Vancouver, Canada Da ABSTRACT Acetyl-CoA carboxylase (ACC) catalyzes the conversion of acetyl-CoA to malonyl-CoA, the first committed step in de novo fatty acid synthesis. Although the enzyme is activated in response to insulin, the mechanism of activation is unclear. In adipose tissue, ACC is phosphorylated on a distinct site, the T-site', in a PI3K-dependent manner following insulin stimulation but the kinase responsible has not yet been identified. This thesis describes work done to test the role of the atypical PKC isozymes as potential ACC kinases. Analysis of kinase expression revealed that PKC isozymes delta, zeta, and mu (PKD) were highly expressed in rat white adipose tissue. Of nine purified PKC isozymes tested in vitro, PKC-zeta phosporylated ACC most efficiently, but the stoichiometry of phosphorylation was still very low. In adipocytes, the PKC inhibitor Ro 31-8220 accelerated the activation of ACC in response to insulin, suggesting that a target of the compound is normally involved in the inhibition of ACC activation. Another PKC inhibitor, Go 6983, had no effect. The two compounds have similar potency against PKC isozymes, with the exception of PKC-betal and -betall which are more sensitive to Ro 31-8220. AMPK, which has been reported to inhibit ACC in adipose tissue, is also inhibited by Ro 31-8220 in vitro, but probably not in adipocytes. This indicates that some kinase other than AMPK is responsible for inhibition of ACC in adipocytes. The delay in ACC activation following insulin stimulation may represent an important control in glucose utilization. In addition, the more rapid activation of ACC following treatment of adipocytes with Ro 31-8220 indicates that a target of this compound is responsible for the delay. ii TABLE OF CONTENTS Abstract ii Table of Contents iii List of Tables vi List of Figures vii List of Abbreviations viii Acknowledgements x 1.0 INTRODUCTION 1 1.1 INSULIN SIGNALING 1 1.1.1 Insulin Receptor Structure 2 1.1.2 Insulin Receptor Tyrosine Kinase Activity 2 1.1.3 Insulin Receptor-Associated Proteins 2 1.1.4 Phosphatidylinositol-3-Kinase 3 1.1.5 PDK1 4 1.1.6 PKB/Akt 5 1.1.7 P70S6 kinase 6 1.1.8 Atypical PKCs (zeta, lambda/iota) 6 1.2 METABOLIC EFFECTS OF INSULIN SIGNALING 8 1.2.1 Protein Synthesis 8 1.2.2 Glucose Transport 9 1.2.3 Glycogen Synthesis 10 1.2.4 Lipid Metabolism 11 1.3 ACETYL-CoA CARBOXYLASE 13 1.3.1 Structural and Functional Properties of ACC 13 1.3.2 ACCIsoforms 14 1.3.3 Polymerization of ACC 15 1.3.4 Long-term Regulation of ACC 17 1.3.5 Short-term Regulation of ACC 17 1.3.5.1 Allosteric Factors 17 1.3.5.2 Protein Regulator 18 1.3.5.3 Hormone Controls 18 1.3.6 ACC Kinases 19 1.3.6.1 Insulin-stimulated ACC Kinase 20 1.3.6.2 AMPK 20 1.3.6.3 PKA 21 1.3.6.4 CaMK 21 1.3.6.5 Casein Kinase 2 22 1.3.6.6 PKC 23 1.4 PKC FAMILY 24 1.4.1 PKC Isozyme Structure 24 1.4.2 PKCmu/PKD 26 1.4.3 PKC Isoform Specificity 27 1.5 THESIS INVESTIGATION 28 iii 2.0 EXPERIMENTAL PROCEDURES 29 2.1 MATERIALS : 29 2.2 METHODS 30 2.2.1 Tissue Preparation 30 2.2.1.1 Isolation 30 2.2.1.2 Adipose Tissue Incubations 30 2.2.1.3 Homogenization 31 2.2.2 Preparation of Adipocytes 31 2.2.3 Purification of Acetyl-CoA Carboxylase 32 2.2.3.1 Arnmonium Sulfate Precipitation 32 2.2.3.2 Dephosphorylation 33 2.2.3.3 Monomeric Avidin Column 33 2.2.3.4 Avidin Affinity Chromatography 33 2.2.4 ACC Activity Assays 34 2.2.5 PKCzeta Immunoprecipitation 35 2.2.6 Kinase Assays 36 2.2.6.1 PKC 37 2.2.6.2 AMPK 37 2.2.6.3 PKA 38 2.2.7 SDS-PAGE, Western Blotting and Phosphoimaging 38 2.2.7.1 SDS-PAGE 38 2.2.7.2 Western Blotting 39 2.2.7.2.1 PKCzeta 39 2.2.7.2.2 ACC 39 2.2.7.3 Phosphoimaging 40 2.2.8 Multi-kinase Analysis 40 2.2.9 Glycogen Synthase Assays 41 2.2.10 Protein Determination 41 2.2.11 Statistical Analysis 41 3.0 RESULTS AND DISCUSSION 42 3.1 KINASE EXPRESSION IN RAT WHITE ADIPOSE TISSUE 42 3.1.1 Comparison of Fat Pads, Adipocytes, and 3T3-L1 Cells 42 3.1.2 Comparison of Primary and 3T3-L1 Adipocytes 46 3.2 PHOSPHORYLATION OF PURIFIED ACC BY PKC ISOFORMS 48 3.2.1 Screening PKC Isoforms 48 3.2.2 ACC Phosphorylation by PKCzeta 53 3.2.3 Immunoprecipitation of PKCzeta from Adipose Tissue 56 3.2.4 PKCzeta Membrane Translocation 59 3.3 EFFECTS OF PKC INHIBITORS IN INTACT TISSUES AND CELLS 61 3.3.1 Choice of PKC Inhibitors 61 3.3.2 Effect of PKC Inhibitors on Purified ACC 62 3.3.3 Effect of Inhibitors in Fat Pads and Adipocytes 63 3.3.4 Effect of PKC Inhibitors on ACC Activity in Adipocytes 67 iv 3.4 FURTHER CHARACTERIZATION OF PKC INHIBITORS 72 3.4.1 Inhibition of PKC Isoforms by Ro 31 -8220 and Go 6983 72 3.4.2 Effect of PKC Inhibitors on Known ACC-Inhibitory Kinases 77 3.4.2.1 Inhibition of AMPK and PKA 77 3.4.2.2 AMPK Activation in Adipocytes : 80 4.0 CONCLUSIONS 84 5.0 REFERENCES 86 v LIST OF TABLES Table 1.1 Phosphorylation of Acetyl-CoA Carboxylase 19 Table 3.1 Comparison of Kinase Expression in Primary and 3T3-L1 Adipocytes 47 Table 3.2 Reported IC 5 0 (uM) Values for PKC Isozyme Inhibition 61 Table 3.3 Summary of Inhibition of PKC Isoforms by Ro 31 -8220 and Go 6983 (IC50 values in uM) 74 LIST OF FIGURES Figure 1.1 Insulin Signaling Pathways 7 Figure 1.2 Control Points for Lipid Biosynthesis in White Adipose Tissue 12 Figure 1.3 Polymerization of Acetyl-CoA Carboxylase Dimers 16 Figure 1.4 Structure of the PKC Isozyme Families 25 Figure 3.1 Multi-Kinase Analysis 45 Figure 3.2 ACC Phosphorylation by PKC Isoforms 50 Figure 3.3 ACC Phosphorylation with Increasing Amounts of PKC-zeta 54 Figure 3.4 ACC Phosphorylation by Immunoprecipitated PKC-zeta/-lambda 57 Figure 3.5 PKC-zeta Does Not Translocate to the Plasma Membrane Following Insulin Stimulation 60 Figure 3.6 PKC Inhibitors Do Not Directly Affect ACC Activity 62 Figure 3.7 Effects of PKC Inhibitors on Glycogen Synthase Activity in Extracts of Rat Fat Pads or Adipocytes 65 Figure 3.8 Structures of the Calbiochem Compounds Ro 31-8220 and Go 6983 66 Figure 3.9 Effect of PKC Inhibitors on ACC Activity in Rat Adipocytes 68 Figure 3.10 Inhibition of PKC Isoforms by Ro 31-8220 and Go 6983 73 Figure 3.11 Inhibition of AMPK and PKA by Ro 31-8220 and Go 6983 79 Figure 3.12 Time Course of AMPK Activity Following Insulin Stimulation 81 Figure 3.13 Baseline ACC Activity in Adipocytes 82 vii LIST OF ABBREVIATIONS ACC Acetyl-CoA carboxylase ACC-1 Acetyl-CoA carboxylase-1 (also ACC-265, ACCa) ACC-2 Acetyl-CoA carboxylase-2 (also ACC-280, ACCP) ADP Adenosine 5'-diposphate AICAR 5 -amino-4-imidazolecarboxamide riboside AKAP A-Kinase Anchoring Protein AMP Adenosine 5'-monophosphate AMPK AMP-activated protein kinase AMPKK AMP-activated protein kinase kinase aPKC Atypical protein kinase C ATP Adenosine 5'-triphosphate BSA Bovine serum albumin CaM Calmodulin CaMK Calcium/calmodulin-dependent protein kinase CaMKK Calcium/calmodulin-dependent protein kinase kinase cAMP 3',5'-cyclic adenosine monophosphate CAP c-Cbl-associated protein Cdk Cyclin-dependent kinase CK Casein kinase CoA Coenzyme A CPT Carnitine palmitoyltransferase DAG Diacylglycerol DTT Dithiothreitol 4E-BP eIF4E-binding protein EDTA Ethylenediaminetetraacetic acid EGTA Ethylene glycol-bis(2-aminoethyl ether)N,N,N',N',-tetraacetic acid elF eukaryotic initiation factor GLUT4 Glucose transporter 4 GRK G protein-coupled receptor kinase GSK Glycogen synthase kinase GS Glycogen synthase GTP Guanosine 5'-triphosphate HC03" Bicarbonate HEPES N-[2-hydroxyethyl]piperazine-N'-[2-ethanesulfonic acid] Hpk Histidine protein kinase HRP Horseradish peroxidase IC50 Inhibitory Concentration at which enzyme activity = 50% IKK IkappaB kinase ILK Integrin-linked kinase IP Immunoprecipitation IRS Insulin receptor substrate MAPK Mitogen-activated protein kinase MEK Mitogen-activated protein kinase kinase MOPS 3 - [N-morpholino]propanesulfonic acid viii Mos Mitogen-activated protein kinase kinase kinase rriRNA Messenger ribonucleic acid Nek2 NIMA-related kinase 2 p85 PI3K regulatory subunit pllO PI3K catalytic subunit PAGE Polyacrylamide gel electrophoresis PAKct p21-activated protein kinase PDA Piperazine diacrylamide PDK Phosphoinositide-dependent kinase Pfk Phosphofructokinase PH Pleckstrin homology Pi Inorganic phosphate PI3K Phosphatidylinositol-3 kinase PKA cAMP-dependent protein kinase PKB Protein kinase B PKC Protein kinase C PKD Protein kinase D PKG Protein kinase G PKR dsRNA-dependent protein kinase PMSF Phenylmethylsulfonylfluoride PRK-2 PKC-related kinase 2 P70S6K 70 kDa ribosomal S6 protein kinase P90S6K 90 kDa ribosomal S6 protein kinase PTB Phosphotyrosine-binding PtdIns(4,5)P2 Phosphatidylinositol-4,5-bisphosphate PtdIns(3,4,5)P3 Phosphatidylinositol-3,4,5-trisphosphate PVDF Polyvinylidene difluoride Pyk2 Proline-rich tyrosine kinase 2 RACK Receptor for activated C-kinase ROK Rho-associated kinase Rsk Protein kinase p90 SAPK Stress-activated protein kinase SDS Sodium dodecyl sulfate Ser Serine SH2 Src-homology 2 SH3 Src-homology 3 She SH2-containing protein TBS-t Tris-buffered saline (+ Tween-20) Thr Threonine TOR Target of Rapamycin Tris Tris(hydroxymethyl)aminomethane UDP Uridine diphosphate ix ACKNOWLEDGEMENTS I'd like to take this opportunity to thank some of the people who have been helpful during the completion of this degree. First and foremost, I would like to thank Dr. Roger Brownsey for his supervision, encouragement and interesting discussions. I would also like to thank Drs. Adrienne Boone and Jerzy Kulpa for assistance in the lab. In addition I would like to thank Jeff Flemming and my family for their encouragement. The financial support of the Natural Sciences and Engineering Research Council of Canada (NSERC) and the Canadian Institutes of Health Research (CIHR) was also greatly appreciated. The contributions of Dr. Carsten Schmitz-Peiffer (Garvan Institute of Medical Research, Sydney, Australia) who provided purified PKCiota and Dr. Steve Pelech (Kinexus Bioinformatics Corp., Vancouver, BC) who provided the multi-kinase analysis are also gratefully acknowledged. 1.0 INTRODUCTION 1.1 INSULIN SIGNALING Following a meal, high circulating glucose levels in the blood induce insulin secretion from the pancreatic p cells. Insulin then binds to its receptor, initiating a cascade of signaling events that result in an overall anabolic response. The metabolic effects of insulin signaling include an increase in glucose uptake into muscle and adipose tissue, stimulation of lipogenesis, glycogen synthesis, and protein synthesis, as well as inhibition of the opposing catabolic pathways. The main target tissues are liver, muscle and adipose. Opposing effects are induced in response to adrenaline or glucagon signaling, which generally indicate a state of stress. Defects in insulin signaling are the basis of a serious disease, diabetes mellitus. The defect in signaling can occur at the level of insulin production (type I diabetes) and/or at the level of propagation of the insulin signal (type II diabetes). Approximately 90% of diabetes cases are type II. It is estimated that over two million Canadians have diabetes, though this is only a rough estimate as many cases go undiagnosed. An important step to understanding the complexities of insulin signaling is the determination of the structure and function of the insulin receptor. Structural studies by crystallography (Eck et al., 1996; Hubbard et al., 1994) and electron microscopy (Woldin et al., 1999) have helped to elucidate the insulin receptor structure. 1 1.1.1 Insulin Receptor Structure The insulin receptor is a heterotetrameric protein composed of two extracellular alpha subunits linked via disulphide bond to two membrane-spanning beta subunits. Insulin is bound by a site on the extracellular portion of the receptor, which leads to activation of a tyrosine kinase activity within the intracellular portion of the beta subunits and allows transphosphorylation of multiple tyrosine residues within the receptor by the catalytic kinase activity of the adjacent subunits. 1.1.2 Insulin Receptor Tyrosine Kinase Activity The phosphorylation and activation of the insulin receptor kinase domain serves two purposes. First, phosphotyrosine residues in the juxtamembrane region of the receptor serve as recognition sites for the insulin receptor substrate (IRS) proteins, which bind to the phosphorylated residues via phosphotyosine binding (PTB) domains. Second, the activated insulin receptor kinase domain can now phosphorylate the associated IRS proteins on tyrosine residues, creating recognition sites for SH2 domain-containing signaling proteins. Thus, the signal is transmitted from the insulin receptor to various intracellular signaling proteins via a network of phosphorylation reactions. 1.1.3 Insulin Receptor-Associated Proteins Several IRS proteins have been identified in various tissues. IRS1 was the first such protein to be discovered and is the prototype for this family of proteins (White et al, 1985; Sun et al, 1991). Subsequently, at least four other IRS proteins have been described: IRS2 (Sun et al, 1995), IRS3 (Lavan et al, 1997b; Smith-Hall et al, 1997), 2 IRS4 (Lavan et al, 1997a) and Gabl (Holgado-Madruga et al., 1996). Common features of the proteins in this family include an amino-terminal pleckstrin homology (PH) domain and SH2 or PTB domain. These proteins also display multiple tyrosine residues which can create SH2 binding sites, proline-rich regions which can be recognized by SH3 or WW domains, and serine/threonine-rich regions. These features of the IRS proteins allow them to interact with downstream signaling molecules by specific domain-mediated interactions. She proteins also interact with the insulin receptor and become phosphorylated, though they differ structurally from the IRS proteins (Kovacina and Roth, 1993). It is possible that additional proteins associate with the insulin receptor. 1.1.4 Phosphatidylinositol-3-Kinase One of the best studied proteins known to interact with the IRS proteins is a lipid kinase, phosphatidylinositol-3-kinase (PI3K), which phosphorylates the 3' position of the inositol ring, especially on PtdIns(4,5)P2 to produce PtdIns(3,4,5)P3. The lipid product of this reaction acts as a second messenger within the cell, and has been implicated in the activation of several downstream kinases involved in various insulin signaling pathways. The PI3K inhibitors wortmannin and LY294002 have been used extensively in determining downstream targets. As a result, the serine/threonine kinase PKB (also known as Akt) and some protein kinase C (PKC) isoforms have been demonstrated to lie downstream of PI3K. Interestingly, site-directed mutagenesis studies in which the lipid kinase activity of PI3K was abolished have demonstrated that this enzyme also exhibits protein kinase activity (Bondeva et al., 1998). This protein kinase activity is thought to be involved in 3 activation of the mitogen-activated protein kinase (MAPK) pathway, which is ultimately involved in regulating gene expression by phosphorylation of nuclear transcription factors. Since the MAPK pathway is neither particularly insulin-sensitive nor involved in the early metabolic responses of the cell to insulin, this discussion will focus instead on the pathways that are believed to be linked to the metabolic effects of insulin. 1.1.5 PDK1 Inhibiting PI3K activity by treating cells with wortmannin or LY294002 abolishes most of the metabolic effects of insulin, demonstrating the key role of this kinase in a wide range of cellular signaling processes. The precise mechanism of activation of downstream signaling proteins was unclear for many years, though the importance of the lipid product PtdIns(3,4,5)P3 was recognized. Although the lipid second messenger was clearly involved in the activation of the downstream kinase PKB, an absolute requirement for phosphorylation of key serine and threonine residues was also demonstrated. Identification of the kinase responsible for PKB phosphorylation, the phosphoinositide-dependent kinase 1 (PDK1), permitted a more detailed understanding of the mechanism of activation (Stokoe et al, 1997). PDK1 is a serine/threonine kinase which associates with the plasma membrane via a C-terminal PH domain. The catalytic domain of PDK1 is similar to those of PKA, PKB and PKC. Due to a high affinity interaction with PtdIns(3,4,5)P3, PDK1 is associated with the membrane even in the absence of an insulin signal. Although some researchers insist that PDK1 is constitutively active, others have demonstrated that the kinase is instead activated by PtdIns(3,4,5)P3 (Stokoe et al, 1997; Stephens et al, 1998). PDK1 has subsequently been shown to phosphorylate a number 4 of kinases involved in insulin signaling, including p70S6K (Alessi et al, 1998; Pullen et al, 1998), PKA (Cheng et al, 1998) and PKC isoforms £ and 5 (Le Good et al, 1998). 1.1.6 PKB/Akt Activation of PKB in response to insulin is a complex, multi-step process which requires both phosphorylation of key residues and interaction with PtdIns(3,4,5)P3. Even in the absence of stimulation, the kinase is constitutively phosphorylated on several serine/threonine residues by unidentified kinases. These phosphorylated residues do not activate PKB, but are more likely required for proper folding of the kinase. Following insulin stimulation of the cell, PI3K produces PtdIns(3,4,5)P3 which recruits PKB to the membrane via an interaction with the PKB PH domain. Translocation of the kinase to the membrane appears to serve two functions. First, lipid interaction with the PH domain changes PKB conformation such that the PH domain folds away from and unmasks key phosphorylation sites in the activation loop and kinase domain. Second, membrane association of PKB brings it into proximity with PDK1, which can then phosphorylate the unmasked site in the activation loop, Thr308. Full activation requires phosphorylation of an additional site, Ser473, in the kinase domain by an unknown kinase that has been tentatively called PDK2. Delcommenne et al. (1998) have shown that the integrin-linked kinase (ILK) can phosphorylate Ser473 of PKB, thus linking insulin and integrin signaling. Another group has suggested that PDK1 itself changes substrate specificity following association with a fragment from the PKC-related kinase-2 (PRK-2) and can then phosphorylate Ser473 (Balendran et al, 1999). A third explanation has been proposed, whereby this site is subject to autophosphorylation (Toker and Newton, 2000). 5 1.1.7 P70S6 kinase The ribosomal S6-kinase (p70S6 kinase) was the first kinase identified to be downstream of PB-kinase in vivo. Phosphorylation and activation of this kinase is a complex process involving phosphorylation of eight distinct residues (Pullen and Thomas, 1997). The contribution of the individual phosphorylation events has been difficult to distinguish. Site-directed mutagenesis studies have shown that removal of any of the phosphorylation sites impairs kinase activity. Phosphorylation of three sites (Thr-229, Thr-389, and Ser-371) is dependent on the PI3K and TOR pathways. Phosphorylation of Thr-229, which is in the activation loop of the kinase, is carried out by PDK1 (Alessi et al, 1998; Pullen et al, 1998). This phosphorylation event, however, does not require direct binding of PtdIns(3,4,5)P3 to p70S6 kinase which distinguishes it from PDK1-mediated phosphorylation of PKB. This may indicate that the activation of p70S6 kinase does not require membrane localization. The events leading to phosphorylation of the other sites in p70S6 kinase are unclear. 1.1.8 Atypical PKCs (zeta,lambda/iota) A subfamily of the protein kinase C isoenzymes has also been shown to be activated in response to insulin. These are the atypical PKC isoforms zeta and lambda/iota, which are calcium- and diacylglycerol-independent. The zeta isoform has an activation loop phosphorylation site that is analogous to the site in PKB and is similarly phosphorylated by PDK1 (Chou et al, 1998; LeGood et al, 1998). The phosphorylation and activation of PKC<^  by PDK1 requires PtdIns(3,4,5)P3, although the nature of phosphoinositide-dependency is unclear. 6 Figure 1.1: Insulin Signaling Pathways Protein Synthesis Schematic representation of the main signaling pathways downstream of the insulin receptor. The MAPK pathway is independent of PI3K and leads to increased expression gene transcription. The metabolic branch of insulin signaling leads to increased glycogen, protein, and lipid synthesis as well as increased glucose transport. See text for more details. 7 1.2 METABOLIC EFFECTS OF INSULIN SIGNALING Insulin is an anabolic hormone, resulting in a general growth and energy storage response. Following a meal, high circulating glucose concentrations signal the pancreatic islets to produce and release insulin. The major targets of the hormone are the liver, muscle and adipose tissue. Some of the known responses to insulin include stimulation of glucose uptake into responsive tissues, stimulation of glycogen, lipid and protein synthesis. Parallel inhibition of catabolic processes occurs, such that lipolysis, glycogenolysis, and gluconeogenesis are decreased in response to insulin. 1.2.1 Protein synthesis Two PtdIns(3,4,5)P3-activated serine/threonine kinases have been demonstrated to be involved in the stimulation of protein sythesis in response to insulin: PKB and p70S6 kinase. Protein synthesis is stimulated when the activated p70S6 kinase phosphorylates the ribosomal protein S6, which mediates translation of mRNA transcripts containing a 5' polypyrimidine tract. These transcripts generally code for ribosomal proteins and elongation factors. PI3K-dependent activation of p70S6 kinase occurs via a PKB—»mTOR—»p70S6K pathway, as demonstrated by inhibitor studies. Inhibition of mTOR by rapamycin prevents p70S6K activation (Sehgal, 1995). The activation of mTOR, in turn, appears to be dependent on PKB since activating PKB promotes and dominant negative PKB prevents mTOR activation (Scott et al., 1998; Gingras et al., 1998). It should be noted, however, that p70S6 kinase can be activated even when cells are treated with wortmannin, though activation is delayed (Somwar et al., 1998). There must, therefore, be another, PtdIns(3,4,5)P3-independent pathway of p70S6K activation. 8 Additional control of protein synthesis is mediated by the regulation of initiation factors. Insulin stimulation leads to the activation of eIF2B, which leads to an increase in general protein synthesis. Another initiation factor, eIF4E, has been implicated in the insulin-stimulated increase in translation of mPJSfAs with extensive secondary structure in the 5'-untranslated region. The binding of eIF4E to mRNAs is inhibited by binding proteins (4E-BPs) and this inhibition is removed following insulin stimulation which results in 4E-BP phosphorylation and decreased binding to eIF4E. Thus, the insulin-stimulated increase in protein synthesis is subject to complex control by multiple pathways. 1.2.2 Glucose transport One of the most extensively studied effects of insulin signaling is the stimulation of glucose uptake into muscle and adipose tissue. Glucose uptake in these tissues is dependent on the glucose transporter, GLUT4, which is translocated to the plasma membrane in response to insulin. The precise pathway leading to GLUT4 translocation has been the subject of much discussion. There is strong evidence to support the requirement of PKB in this process, as constitutively active PKB induces GLUT4 translocation (Kohn et al., 1996) and dominant negative PKB prevents translocation (Cong et al., 1997). However, equally strong evidence supports the role of atypical PKC isoforms in this process (Kotani et al., 1998; Kitamura et al., 1998). The relative roles of these pathways in stimulation of GLUT4 translocation remains to be resolved. An interesting element to stimulation of GLUT4 translocation has been recently elucidated. Given that insulin-stimulated glucose transport is inhibited by the PI3K 9 inhibitor wortmannin, GLUT4 translocation was determined to be dependent on PtdIns(3,4,5)P3. Addition of a cell-permeable analog of this lipid second messenger, in the absence of insulin, was therefore expected to stimulate GLUT4 translocation. Surprisingly, Jiang et al. (1998) found that the stimulation of glucose transport required an additional, PI3K-independent signal that was stimulated by insulin. Recently, the CAP-Cbl pathway has been proposed as the second signal required for GLUT4 translocation (Baumann et al., 2000). The Cbl protein binds to the insulin receptor-associated CAP protein via an amino-terminal SH3 domain in CAP, allowing Cbl to be phosphorylated by the insulin receptor tyrosine kinase (Ribon et al, 1998; Ribon and Salteil, 1997). Baumann et al. (2000) propose that phosphorylation of Cbl and subsequent translocation of the CAP-Cbl complex to the membrane generates a second, PI3K-independent signal that is essential for insulin-stimulated glucose transport. 1.2.3 Glycogen synthesis Once transported into the cell, glucose can be incorporated into glycogen. Glycogen synthesis is carried out by the cytosolic enzyme glycogen synthase, which is subject to complex control. Glycogen synthase is activated by the net removal of phosphate from inhibitory sites. To accomplish this, glycogen synthase kinase 3(3 must be inhibited to prevent further phosphorylation of the synthase and protein phosphatase 1G must be activated to facilitate dephosphorylation. Inhibition of GSK3P is achieved by PKB-mediated phosphorylation, leading to glycogen synthase activation in response to insulin (Cross et al., 1995). 10 There is some evidence of an additional, GSK3-independent, mechanism of glycogen synthase activation (Lavoie et al., 1999; Skurat et al., 2000). This alternate pathway has not been well characterized, and the relative contribution to glycogen synthase activation remains to be determined. 1.2.4 Lipid metabolism Although much research attention has been focussed on elucidating insulin signaling pathways, relatively little is known about the mechanisms by which lipogenesis is stimulated in response to insulin. Three main control points are thought to be involved in the regulation of de novo fatty acid synthesis. First, glucose uptake via the GLUT4 transporter provides fuel for synthesis in adipose tissue. This glucose transporter translocates to the plasma membrane following insulin signaling. This point of control is not relevant in liver, in which GLUT4 is not expressed appreciably. A second control point is pyruvate dehydrogenase, a mitochondrial enzyme which converts pyruvate to acetyl-CoA. Pyruvate dehydrogenase is activated by dephosphorylation mediated by a specific phosphatase (Denton et al., 1989). The third point of control is provided by the cytosolic enzyme acetyl-CoA carboxylase (ACC), which converts acetyl-CoA to malonyl-CoA. This reaction is the first committed step in de novo fatty acid synthesis, and as such plays an important role in the regulation of the rate of lipogenesis. Regulation of this enzyme is complex, and involves phosphorylation, allosteric factors and association with a regulator protein. ACC is activated following insulin stimulation, but the mechanism of activation is incompletely understood. 11 Figure 1.2: Control Points for Lipid Biosynthesis in White Adipose Tissue glucose glucose pyruvate pyruvate acetyl-CoA—• citrate Mitochondria Plasma Membrane citrate Fatty Acid Synthesis Schematic diagram showing the major control points for lipid biosynthesis in white adipose tissue. Control is exerted at the level of glucose uptake into the cell (1), which depends on translocation of the glucose transporter GLUT4 to the cell membrane. Conversion of pyruvate to acetyl-CoA in the mitochondria by pyruvate dehydrogenase (2) is a second point of control. The third point is the conversion of acetyl-CoA to malonyl-CoA in the cytoplasm by acetyl-CoA carboxylase (3). Control of glycolysis by Pfk-1, Pfk-2 and pyruvate kinase can also contribute to control of de novo fatty acid biosynthesis, though this is more relevant to liver than adipose tissue. 12 1.3 ACETYL-CoA CARBOXYLASE 1.3.1 Structural and Functional Properties of ACC Acetyl-CoA carboxylase is a biotin-containing enzyme which, catalyzes the first committed step in de novo fatty acid synthesis. As such, it represents an important control point in the regulation of this metabolic pathway. The ACC reaction occurs in two steps, the first of which is the transfer of a carboxyl group from bicarbonate to a biotin prosthetic group in the enzyme at the expense of one ATP (a). The second step of the enzyme reaction is the transfer of the carboxyl group from biotin to acetyl-CoA to produce malonyl-CoA (b). (a) ACC-biotin + HC03" + ATP -> ACC-biotin-C02" + ADP + Pi (b) ACC-biotin-C02" + acetyl-CoA ->ACC-biotin + malonyl-CoA ACC is composed of three distinct functional domains: the biotin carboxylation domain and the carboxy-transferase domain, with a biotin arm which can access both active sites. In many bacteria the ACC reaction is mediated by an enzyme complex in which the three functional domains are encoded in four separate proteins. The most extensively studied bacterial form of ACC is found in Escherichia coli. The structures of the separate polypeptide components of the enzyme complex have been resolved (Waldrop et al, 1994; Roberts et al, 1999). Through gene fusion, ACC has evolved into a single enzyme as currently found in most eukaryotes. The single polypeptide form of ACC has some interesting properties, which will be discussed. Unfortunately, due to the large size of the single-polypeptide form of ACC, resolution of the enzyme three-dimensional structure has not been possible. 13 1.3.2 ACC Isoforms There are two isoforms of mammalian ACC, with molecular weights of 265 and 280 kDa. The smaller isoform (ACC-1, ACCa, ACC-265) was the first characterized. ACC-2 (ACC(3, ACC-280) is slightly larger, mainly due to an additional N-terminal sequence, and was identified more recently (Thampy, 1989). The two isoforms display very different tissue distribution patterns. ACC-1 is the only isoform expressed in rat adipose tissue, as well as testes, bladder, uterus and placenta. Both isoforms are expressed in liver, lactating mammary tissue and pancreas, though 75% of the enzyme in liver is ACC-1. In heart and skeletal muscle, ACC-2 is the predominant isoform. The tissue distribution pattern of the two isoforms was the first indication that they might have different roles in the regulation of lipid metabolism. ACC-1, which is mainly expressed in lipogenic tissues, is involved in the de novo synthesis of fatty acids by catalyzing the first committed step: the conversion of acetyl-CoA to malonyl-CoA. In heart and skeletal muscle, however, very little lipid synthesis occurs, due to low levels of fatty acid synthase. The role of ACC-2, which is mainly expressed in these non-lipogenic tissues, is to inhibit fatty acid oxidation. This occurs through the inhibition of carnitine palmitolyl transferase (CPT) by malonyl-CoA, which prevents the transport of fatty acids to the mitochondrial matrix, the site of fatty acid oxidation. The N-terminal extension found in ACC-2, which appears to encode a membrane targeting sequence, may facilitate the role of this isoform in the regulation of fatty acid oxidation by localization of ACC in close proximity to CPT in the mitochondrial membrane (Ha et al., 1996). Overexpression of the N-terminal regions of the ACC 14 isoforms provided evidence of the importance of this region in the determination of ACC function. Overexpression of the ACC-1 N-terminus stimulated fatty acid synthesis whereas overexpression of the ACC-2 N-terminus stimulated fatty acid oxidation (Kim et al, 1998). Further evidence of the mitochondrial localization of ACC-2 was provided by immunofluorencence studies which demonstrated the colocalization of the ACC-2 fragment with carnitine palmitolytransferase 1 (CPT1) on the mitochondrial membrane (Abu-Elheiga et al, 2000). The same studies demonstrated that ACC-1 was a cytosolic enzyme, which is consistent with its role in fatty acid synthesis. 1.3.3 Polymerization of ACC One of the unique features of mammalian ACC-1 is its ability to polymerize. The inactive enzyme is a dimer, composed of two identical subunits. When the enzyme is activated, ten to twenty dimers come together to form the active, polymeric ACC. Due to its large size, polymeric ACC can be separated from dimers by gel-filtration chromatography, and the proportion of ACC in the polymeric form can be used as an indication of the degree of activation of the enzyme (Borthwick et al., 1987). The polymerization of ACC is influenced by a number of factors including the phosphorylation state of the enzyme and the presence of allosteric modulators such as citrate (Figure 1.3). For ACC-2, which does not undergo polymerization, activity is associated with the dimeric form of the enzyme (Boone, 2000). This is another example of the differences in regulation between the two ACC isoforms. 15 Figure 1.3: Polymerization of Acetyl-CoA Carboxylase Dimers Active Polymers Insulin, Citrate Catecholamines, Malonyl-CoA, Fatty-acyl-CoA Inactive Dimers Liver and adipose ACC exist in an equilibrium between relatively inactive homodimers and highly active polymers. The polymerization state of the enzyme correlates with enzyme activity, and activators of ACC promote polymerization whereas inhibitors promote depolymerization. Feed forward activation by citrate and feedback inhibition by malonyl-CoA and fatty acyl-CoA promote polymerization and depolymerization, respectively. Hormone signaling can promote polymerization (insulin) or depolymerization (glucagon or epinephrine) by influencing the phosphorylation state of ACC. 16 1.3.4. Long-term Regulation of ACC Long term regulation of ACC is mediated by changes in the level of expression of the protein. Enzyme expression is repressed in situations of starvation, high-fat diet or diabetes and the repression can be reversed by re-feeding, low-fat diet or administration of insulin, respectively (Allmann et al., 1965; Wieland and Eger-Neufeldt, 1963). There are at least three ACC promoters, and only one of them is sensitive to hormone and dietary signals (Lopez-Casillas and Kim, 1989). 1.3.5 Short-term Regulation of ACC Acetyl-CoA carboxylase is also subject to short term control, which is the primary focus of this thesis. In response to certain stimuli, enzyme activity is stimulated or inhibited rapidly, which enables rapid control of the rate of fatty acid synthesis. Regulators of ACC activity can be divided into three categories: those that modify allosterically, those that affect the phosphorylation state of the enzyme, and other proteins which associate with ACC. 1.3.5.1 Allosteric factors Citrate is a strong allosteric activator of ACC (Martin and Vagelos, 1962). Physiologically, citrate is the cytosolic precursor to acetyl-CoA and represents a measure of the concentration of substrate for ACC. Similarly, glutamate activates ACC which may indicate that amino acids are an important signal of nutritional status (Boone et al., 2000). Conversely, ACC is subject to feedback inhibition by malonyl-CoA and fatty acyl-CoA esters (Gregolin et al, 1966; Goodridge, 1972). The carboxylase is also 17 inhibited by free CoA, chloride, and MgATP (Moule et al, 1992; Alfred and Roehrig, 1980; Gregolin et al, 1966). Allosteric modulators influence the polymerization of ACC. Activators of the enzyme promote polymerization and inhibitors promote depolymerization (Figure 1.3). 1.3.5.2 Protein regulator ACC activity can also be influenced by association with other proteins. ACC regulator proteins have been identified in both adipose tissue (Heesom et al., 1998) and liver (Quayle et al, 1993) of rats. The adipose and liver activator proteins are 130 and 75 kDa, respectively, and whether these two proteins are related remains to be determined. Association of ACC with the regulator proteins enhances enzyme activity. The mechanism by which the regulator proteins modify ACC activity is unclear, but the stimulatory effect may be due to the ability of the protein to facilitate ACC polymerization. 1.3.5.3 Hormone controls ACC is activated in response to insulin and inhibited in response to adrenaline/epinephrine or glucagon. These hormones exert their effects on ACC activity by phosphorylation which in turn influences the state of polymerization of the enzyme. 18 1.3.6 ACC Kinases The regulation of ACC by phosphorylation is complex. It has been well established that ACC is inactivated by phosphorylation following adrenaline/epinephrine or glucagon signaling. The inhibition can be partially reversed by removal of inhibitory phosphate, but this does not result in full activation of ACC. Studies of the in vivo phosphorylation of this enzyme following insulin stimulation demonstrated phosphorylation of a specific serine residue in a tryptic peptide termed the T-peptide'. The phosphorylation coincides with activation of ACC, but may not be sufficient to exert the effect. In vitro phosphorylation studies have demonstrated that phosphorylation of the I-site, by itself, does not activate ACC. Table 1.1: Phosphorylation of Acetyl-CoA Carboxylase Kinase Kinase Regulation Phosphorylation Site(s) Effect Reference T-peptide kinase' insulin (+) T-peptide' activation ? Brownsey and Denton, 1982. AMPK AMP (+) insulin (-) Ser-79,-1200, -1215 inhibition Munday et al, 1988; Lopez-Casillas et al, 1988 CK2 Ser-25 none Haystead et al, 1988; Lopez-Casillas et al, 1988 PKC DAG, PS, CaI+ (+) Ser-77, -95 ? Hardie et al, 1986; Haystead and Hardie, 1988 PKA catecholamines, cAMP (+) Ser-77, -1200 inhibition? Munday et al, 1988; Lopez-Casillas et al, 1988 CaMK Ca"+/CaM (+) Ser-29 none Hardie et al, 1986; Lopez-Casillas et al, 1988 19 1.3.6.1 Insulin-stimulated ACC Kinase The identity of the T-peptide' kinase is currently unknown, despite extensive research efforts. Inhibition of PI3K by Wortmannin or LY294002 prevents both the phosphorylation and activation of ACC. The I-peptide kinase does not lie downstream of p70S6K, as treatment with rapamycin does not inhibit ACC activation by insulin. Other potential kinases included both PKB/Akt and the atypical PKC isozymes, demonstrated downstream targets of PI3K activation. Previous studies in this lab have shown that PKB does not phosphorylate ACC (Zhande, 1998). The potential role of the atypical PKC isozymes has not yet been investigated. 1.3.6.2 AMPK The AMP-activated protein kinase (AMPK) is a heterotrimer made up of catalytic (a) and regulatory (P and y) subunits (Mitchelhill et al., 1994). Full activation of the kinase requires co-expression of the regulatory subunits (Dyck et ah, 1996). AMPK is stimulated by AMP (Stapleton et al., 1996) and by phosphorylation of Thr-172 of the alpha subunit by AMPK kinase (AMPKK) (Hawley et al, 1996). The role of AMPK in phosphorylation and inhibition of ACC-1 has been extensively investigated. This kinase phosphorylates ACC-1 on several serine residues. Site directed mutagenesis of ACC-1 was used to show that inhibition of carboxylase activity is accomplished by phosphorylation of Ser79, and the other sites do not appear to have any direct effect on ACC-1 activity. Activation of AMPK in cells by treatment with AICAR was also shown to inhibit ACC-1 activity by phosphorylation of the same serine residue, confirming the role of this kinase in ACC-1 inhibition. 20 1.3.6.3 PKA The cAMP-dependent protein kinase (PKA) is a heterotetramer, composed of two regulatory and two catalytic subunits (R2C2). PKA is activated by cAMP, which is produced by adenylate cyclase following glucagon or catecholamine signaling. Each regulatory subunit of PKA binds two molecules of cAMP, which causes the dissociation of the regulatory subunits from the catalytic subunits and relieves inhibition of kinase activity. PKA can be associated with the membrane, via an interaction of the regulatory subunits with anchoring proteins known as AKAPs (Rubin et al, 1979; Lohmann et al., 1984). Some regulatory subunits do not interact with AKAPs, and PKA containing these subunits is cytosolic. PKA phosphorylates ACC-2 much more efficiently than ACC-1 (Winz et al, 1994; Boone, 2000), which implicates it in the regulation of ACC activity in heart and skeletal muscle. The sites of phosphorylation have been incompletely defined for ACC-2, but phosphorylation occurs only on serine residues (Boone, 2000). The sites of phosphorylation of ACC-1 have been better characterized and they are Ser-77 and Ser-1200. 1.3.6.4 CaMK The calcium/calmodulin-dependent protein kinase (CaMK) family includes several isoforms. As the name suggests, this kinase is activated by calcium/calmodulin binding. CaMKII and CaMKIV become phosphorylated following Ca /CaM binding and develop autonomous activity. While CaMKI is also phosphorylated, this isoform 21 does not become independent of Ca2 +/CaM. PKA phosphorylates and inhibits CaMKI and CaMKIV, which links cAMP and Ca2+ signaling pathways. The consensus sequence for substrates of CaMK is very similar to that of the substrates of AMPK, and CaMKII has been shown to phosphorylate ACC-1 on Ser-29 (Hardie et al, 1986; Lopez-Casillas et al, 1988) but phosphorylation at this site does not have any effect on carboxylase activity in vitro (Hardie et al., 1986). CamKII can also phosphorylate ACC-2 on several sites, again without any apparent effect on carboxylase activity (Boone, 2000). 1.3.6.5 Casein Kinase 2 Casein kinase (CK2) is a tetrameric enzyme composed of two catalytic alpha subunits and two regulatory beta subunits. Despite much research, it is not clear if, or how, this kinase might be controlled by second messengers or phosphorylation although activation by insulin has been documented (Diggle et al, 1991). CK2 appears to be constitutively active, and has been demonstrated to phosphorylate at least 160 substrate proteins (Pinna and Meggio, 1997). Substrate specificity is determined by the beta subunit (Bidwai et al, 1992; Meggio et al, 1992) and CK2 can use either ATP or GTP as a phosphate donor (Tuazon and Traugh, 1991). CK2 phosphorylates ACC-1 on Ser-25 in vitro, without any effect on carboxylase activity, so the significance of the phosphorylation is unclear. Ser-25 is phosphorylated in intact cells, and may have some role in vivo. Since the phosphorylation does not affect ACC activity directly, it may instead be involved in mediating interaction with other proteins. 22 1.3.6.6 PKC The ability of PKC to phosphorylate ACC has been demonstrated both in vitro (Vaartjes et al., 1987; Hardie et al., 1986) and by phorbol ester activation of the kinase in intact cells (Vaartjes et al, 1987; Haystead and Hardie, 1988). The phosphorylation in cells has been suggested to activate ACC in hepatocytes (Vaartjes et al., 1987), but is without effect in adipocytes (Haystead and Hardie, 1988). Interestingly, the site of phosphorylation was different when purified PKC was used to phosphorylate ACC in vitro. This may indicate that the effects of phorbol ester were not mediated by PKC. The effect of the phosphorylation, in response to phorbol esters or purified PKC, on carboxylase activity has been reported as inhibitory by some (Hardie et al., 1988) and stimulatory by others (Vaartjes et al., 1987). Investigation of the sites of phosphorylation by PKC demonstrated ACC phosphorylation on Ser-77 and Ser-95. As previously discussed, PKA also phosphorylates Ser-77. The contrasting results on ACC activity observed following PKC-mediated phosphorylation may be due to differences in the purity of the kinase preparation used. At the time, little was known about the different PKC isoforms, and therefore no effort was made to define the role of the individual isozymes in ACC phosphorylation. 23 1.4 PKC FAMILY At least eleven PKC isoforms exist and they are subdivided into three families, based on cofactor requirements. The classical PKC isozymes (a, (31, (311, y) require calcium, diacylglycerol and phosphatidylserine for optimal activation. The novel family of PKC isozymes (5,s, n) have the same lipid requirements, but do not require calcium for activation. The third family, the atypical PKC isozymes (C„ x/X), require phosphatidylserine but not diacylglycerol or calcium for activation. PKC-iota is the murine homolog of PKC-lambda. Another kinase which is sometimes considered part of the PKC family is PKD (PKC-mu). This kinase differs significantly from the other PKC isozymes both in structure and substrate specificity. 1.4.1 PKC Isozyme Structure PKC isozymes have a modular organization, composed of an N-terminal regulatory domain and a C-terminal catalytic domain (Figure 1.4). The classical isozymes contain four conserved regions (C1-C4). The novel and atypical isozymes differ in cofactor requirements due to differences in the regulatory domain structure. The classical PKCs possess four conserved regions which are involved in binding DAG (Cl), Ca 2 + (C2), ATP (C3), and substrate (C4). The lipid binding region contains two cysteine-rich zinc fingers which are involved in the binding of DAG or phorbol esters (Hurley et al., 1997). The C2 domain binds Ca 2 + , which induces a conformational change that exposes the binding site for acidic phospholipids (Grobler et al., 1996). 24 Figure 1.4: Structure of the PKC Isozyme Families a) classical (a, pi, pil, y) |— Regulatory domain — | | — CI C2 N DAG, Ca z , phorbol ester phosphatidyl-serine Catalytic domain C3 C4 b) novel (8, s, 0, n) Regulatory domain C2-like CI N * phosphatidyl-serine DAG, phorbol ester Catalytic domain C3 C4 c) atypical (£, X) Regulatory domain C2-like Cl-like N phosphatidyl-serine Catalytic domain C3 C4 ^ ^Substrate^^ Q The modular structure of PKC isozymes from the classical (a), novel (b), and atypical (c) subfamilies. The catalytic domain contains ATP-binding (C3) and substrate-binding (C4) sites. The regulatory domains differ between subfamilies. Classical PKC isozymes contain a diacylglycerol-binding site with two zinc finger motifs (CI) which is also the site of action of phorbol esters. Classical PKC isozymes also contain another region where calcium and phosphatidylserine bind (C2). In novel and atypical PKC isozymes, the C2 region is modified and binds only phosphatidylserine (C2-like). In the atypical isozymes, which do not bind diacylglycerol or phorbol esters, the CI region contains only one zinc finger motif. 25 The novel PKC isozymes are DAG and phorbol ester sensitive, due to the presence of a Cl domain. These isozymes are Ca 2 + insensitive as they possess a modified 'C2-like' domain which lacks residues involved in Ca 2 + binding. The third family, the atypical PKC isozymes, also have a C2-like domain that does not bind Ca . In addition, the Cl domain of these isozymes contains only one zinc finger motif, rendering this family insensitive to DAG and phorbol esters. Only the atypical PKC isozymes are insensitive to phorbol esters, and this has allowed some assessment of functional significance of these isozymes. 1.4.2 PKCmu/PKD Protein kinase D (also known as PKC mu) is related to the PKC family. This kinase is similar to the novel PKC isozymes, in that it is insensitive to Ca 2 + and stimulated by phorbol esters. However, PKD has structural and functional properties which distinguish it as a separate class of enzyme, distinct from the PKC family. It contains a putative transmembrane segment and a PH domain, which suggest that PKD interacts directly with membranes. The preferred substrate sequence for this kinase also differs dramatically from the PKC family. There is evidence to suggest that the activation of PKD occurs via a PKC-dependent mechanism, as inhibition of PKC by Ro 31-8220 prevents PKD activation in intact cells but does not directly inhibit the kinase in vitro (Paolucci and Rozengurt, 1999). 26 1.4.3 PKC Isoform Specificity The large number of PKC isoforms that have been identified suggests that they mediate distinct cellular functions. The preferred substrate sequences have been determined for several PKC isozymes (Nishikawa et al., 1997). While some variation exists, the isozymes are able to phosphorylate serine/threonine residues within very similar motifs, and differences in function are unlikely to be mediated simply by different substrate specificity. Similarly, the differences in cofactor requirements are insufficient to explain the differences in isoform functions. Differing tissue distribution and subcellular localization are likely to play a major role in the divergent roles of individual PKC isozymes. Information on the tissue distribution of the isozymes is incomplete, though some information does exist. Many researchers use transfection studies in a tissue culture system to assess the role of PKC isozymes in cellular processes. In the absence of evidence that the isozymes are normally expressed in the tissue of interest, the results of these transfection studies should be interpreted with caution. The localization of PKC isoforms is largely accomplished through association with other proteins, known as RACKs (Receptors for Activated C-Kinase). These proteins selectively bind the individual PKC isozymes by unique peptide sequences in the regulatory domains and are thought to mediate isozyme-specific functions by targeting the isozymes to their site of action (Mochly-Rosen et al, 1991). Thus the RACKs are thought to mediate specificity by bringing individual PKC isozymes in proximity to their lipid activators as well as their substrate proteins. 27 1.5 THESIS INVESTIGATION It is known that exposure of fat cells to insulin leads to site-specific phosphorylation of ACC-1. This phosphorylation is associated with ACC activation but the relevant kinase has not yet been identified. Although the identity of the ACC-1 'I-peptide' kinase has been elusive, some of its properties have been established. Based on inhibitor studies, it is known that activation of the I-peptide kinase is dependent on PI3K activity. Previous studies in this lab have demonstrated that PKB does not directly phosphorylate ACC-1 (Zhande, 1998). The atypical PKC isozymes zeta and iota have also been demonstrated to lie downstream of PI3K in insulin signaling, and their ability to phosphorylate ACC has not yet been determined. The initial hypothesis was that one of the atypical PKC isozymes is involved in the insulin-mediated phosphorylation and activation of ACC-1. Two approaches were taken to assess the role of PKC in ACC phosphorylation and activation. First, individual PKC isozymes were examined for the ability to phosphorylate rat liver ACC, in vitro, using either commercially available or immunoprecipitated kinase. Second, PKC inhibitors were used to inhibit PKC in intact cells and the effect on ACC activity was determined. This thesis describes an investigation of the potential role of PKC isozymes in the insulin-mediated phosphorylation and activation of ACC. 28 2.0 EXPERIMENTAL PROCEDURES 2.1 MATERIALS Male Wistar rats were obtained either from the University of British Columbia animal care facility or from Charles River Laboratories, usually at a weight of 140-180g. Laboratory chemicals and solvents were generally obtained from Fisher Scientific or BDH Chemicals Canada Ltd. HEPES, Na 3V0 4, glycine, EDTA, EGTA, PMSF, collagenase, insulin, D-glucose-6-phosphate, p-nitrophenylphosphate, NaF, isoproterenol, Kemptide, AMP and DTT were from Sigma Chemical Co. MOPS, Tris, BSA, ATP and glycogen were from USB. Fatty-acid free BSA was from ICN. Coenzyme A, Pefablock-SC, and UDP-glucose were from Boehringer Mannheim. The peptide inhibitors Pepstatin A and leupeptin were from Peptides International. Radioisotopes, streptavidin-HRP, ACS scintillation fluid, and pre-stained SDS-PAGE high molecular weight rainbow markers were from Amersham Pharmacia Biotech. PVDF membrane and Ultrafree-15 centrifugal filter device (Biomax-50) were from Millipore/Amicon. Affigel-10 and electrophoresis reagents were from BioRad. Glutathione was from Aldrich. The anti-PKCC, (C-20) antibody and protein A-agarose were from Santa Cruz. The SAMS peptide was from the Nucleic Acid and Protein Service Laboratory (U.B.C.) Protein kinase C isozyme panel, PKC inhibitors (Ro 31-8220 and Go 6983), histone HI, and the epsilon peptide were from Calbiochem. PKC^ was also obtained from Upstate Biotechnology. PKCi was provided by Carsten Schmitz-Peiffer (Garvan Institute of Medical Research, Sydney, Australia). Avidin was provided by Canadian Inovatech (Abbotsford, B.C.). PKA and AMPK were purified by Adrienne Boone (Boone, 2000). 29 2.2 METHODS 2.2.1 Tissue Preparation 2.2.1.1 Isolation All animal procedures were carried out according to the guidelines of the Canadian Council of Animal Care and with the approval of the UBC committee on Animal Care. Rats were maintained on a 12 hour light/12 hour dark cycle (dark from 8 am to 8 pm) at a controlled temperature with unlimited access to water and Purina rat chow. For experimental use, rats were killed either by stunning and cervical dislocation (170-220 g rats) or by CO2 asphyxiation (rats > 220 g). Liver was immediately removed onto ice, adipose tissue into pre-gassed (02:C02;95:5) Krebs-Henseleit buffer (25 mM NaHC0 3 , 1.2 mM K H 2 P 0 4 , 120 mM NaCl, 5 mM KC1, 1.2 mM M g S 0 4 , 1.3 mM CaCl2, and 11 mM glucose) pre-warmed to 37°C. 2.2.1.2 Adipose Tissue Incubations Epididymal and perirenal fat pads were rinsed once with warmed, pre-gassed Krebs-Henseleit buffer, then incubated with gentle shaking in fresh buffer for 20 minutes to counteract the effects of any residual hormones. The fat pads were then dissected into smaller pieces and subjected to treatment with insulin (0.5 ug/ml final concentration) for various times, as indicated in figure legends. To stop the incubations, tissues were rinsed with cold Krebs-Henseleit buffer and either homogenized immediately (immunoprecipitation experiments) or frozen in liquid nitrogen and stored at -70°C until homogenization (PKC inhibitor experiments). 30 2.2.1.3 Homogenization Tissues were homogenized in 3 volumes of homogenization buffer (20 mM MOPS (pH 7.2), 250 mM sucrose, 2 mM EDTA, 2 mM EGTA, 2.5 mM benzamidine, 3 uM Pepstatin A, 5 uM leupeptin, 2.5 mM glutathione, and 0.5 mM PMSF) using either a fitted teflon-glass Potter-Elvhj em homogenizer (liver) or a Polytron homogenizer (adipose tissue). All homogenization steps were carried out on ice, in the presence of the indicated protease inhibitors to minimize protein degradation and denaturation. 2.2.2 Preparation of Adipocytes Adipocytes were prepared from rat epididymal and perirenal fat pads by collagenase digestion essentially as described by Rodbell (1964). Fat pads were preincubated in Krebs-Henseleit buffer for 20 minutes, then dissected into smaller pieces and incubated in the presence of collagenase (0.5 mg/ml) in Krebs-Henseleit buffer containing fatty-acid free BSA (2% w/v) that had been pre-dialyzed overnight against saline plus CaCl2 (1.3 mM). The BSA was dialyzed overnight against the experimental concentrations of NaCl (120 mM) and CaCi2 (1.3 mM) particularly to prevent removal of calcium ions the Krebs-Henseleit buffer during experimental incubations. Collagenase digestion was carried out for 14-18 minutes, until fat pad pieces were approximately 30-40% digested by visual estimation. Adipocytes were recovered by passage through a nylon mesh filter, where isolated fat cells passed through freely but residual tissue was retained. The adipocytes were then washed three times with fresh Krebs-Henseleit buffer to remove collagenase, then resuspended in 12 ml Krebs-Henseleit containing 2% BSA and aliquoted into experimental samples. Adipocyte incubations were performed in a 31 37°C oven, in a 95%02:5%C02 atmosphere. Where indicated, the adipocytes were incubated in the presence of inhibitors for 30 minutes prior to insulin (0.5 pg/ml) treatment. To terminate incubations, media was removed and the cells were frozen in liquid nitrogen and stored at -70°C until homogenization. 2.2.3 Purification of Acetyl-CoA Carboxylase For ACC purification, tissues were isolated and homogenized as described above. The homogenate was then centrifuged at 2000 rpm to pellet cell debris and allow the fat cake to separate. The infranatent was filtered through glass wool and subjected to ultracentrifugation at 50000 rpm for one hour at 4°C. ACC was precipitated from the high-speed supernatent with ammonium sulfate. 2.2.3.1 Ammonium Sulfate Precipitation A saturated ammonium sulfate solution was slowly added to the high speed supernatent at 4°C, to give a final saturation of 40%. The mixture was stirred gently at 4°C for one hour, then the precipitate was recovered by centrifugation at 15000 rpm and the pellet was resuspended as indicated. In most cases, the pellet was resuspended in DE buffer (50 mM MOPS (pH 7.5), 250 mM sucrose, 2 mM EDTA, 2 mM EGTA, 5% (v/v) glycerol, 3 mM sodium azide, 3 uM Pepstatin A, 5 uM leupeptin, and 2 mM DTT) containing 0.5M NaCl. If dephosphorylation of ACC was desired, the pellet was instead resuspended in homogenization buffer containing 150 mM NaCl and 0.4 mM Pefablock. 32 2.2.3.2 Dephosphorylation Dephosphorylation of ACC was accomplished by an endogenous phosphatase that is present in liver ammonium sulfate fractions. The resuspended ammonium sulfate pellet was incubated at 30°C for 60-90 minutes for ACC dephosphorylation, then placed on ice and either stored at -70°C overnight or immediately subjected to avidin-agarose affinity chromatography. 2.2.3.3 Monomeric Avidin Column Due to the high affinity interaction between tetrameric avidin and biotin, binding of ACC to tetrameric avidin via a biotin prosthetic group can only be reversed under extremely denaturing conditions such as boiling in SDS buffer containing 2-mercaptoethanol. By monomerizing the avidin and saturating the highest affinity sites with free biotin, however, the affinity of the interaction can be reduced, and thus the "modest affinity" specific biotin-avidin interaction can be exploited for affinity purification of ACC (and other biotin-containing proteins). A tetrameric avidin column was first prepared by binding avidin to Affigel-10 (from BioRad), according to the manufacturer's instructions. The avidin was then monomerized using the procedure of Kohanski and Lane (1990). The dimensions of the column were 1cm x 8cm. 2.2.3.4 Avidin Affinity Chromatography For affinity purification of ACC, the resuspended ammonium sulfate pellet was slowly applied to the monomeric avidin-agarose column, previously equilibrated in DE buffer containing 0.5M NaCl. The sample was recirculated for approximately 1 hour to 33 allow binding of ACC, then the unbound proteins were washed from the column with > 10 column volumes of DE buffer containing 0.5M NaCl. ACC was then eluted with elution buffer (DE buffer containing 0.5M NaCl and 2 mM biotin). The eluate was concentrated using Biomax-50 centrifugal filter units (Millipore), and assayed for ACC activity. 2.2.4 ACC Activity Assays ACC activity was determined by the [14C]-bicarbonate fixation method (Martin and Vagelos, 1962; Halestrap and Denton, 1973). Unless otherwise indicated, the assay was initiated with a pre-incubation period (20 minutes), to allow ACC activation by polymerization. Subsequent to the preincubation, ACC was added to pre-warmed assay buffer containing I4C-labelled bicarbonate. For PKC inhibitor studies in fat pads and adipocytes, ACC activity was determined in crude cell extracts. The extracts were prepared by centrifugation of homogenized tissue at 12000 x g for 15 minutes, and the infranatent was retained. An aliquot of the crude extract (40 ul) was used to determine ACC activity, in duplicate, at various citrate concentrations. Pre-incubation of ACC Prior to Assay Samples were incubated at 37°C for preincubation, in buffer containing 2 mg/ml BSA, 20 mM MOPS pH 7.4, 0.5 mM EDTA, and ACC in a final volume of 110 ul. Where indicated, the allosteric activator citrate was also included in the preincubation mixture at various concentrations. 34 Assay of ACC To begin the assay, 50 pi of the preincubation mixture was transferred to 450 pi of prewarmed assay buffer (HEPES pH 7.2, 10 mM MgS04, 0.5 mM EDTA, 5 mM ATP, 7.5 mM glutathione, 2 mg/ml BSA, 150 uM acetyl-CoA and 15 mM [1 4C]-KHC03). The assays were terminated by addition of 200 ul of 5 M HC1. Following a brief centrifugation (12000 x g), 600 ul of the reaction mixture were transferred to a liquid scintillation vial and evaporated to dryness under a stream of air. Malonyl-CoA, the product of the ACC reaction, is acid stable and non-volatile and remains in the residue after evaporation of the liquid. The dried sample was then resuspended in 400 pi of water prior to addition of 5 ml of ACS liquid scintillation fluid (Amersham). The specific activity of 1 4 C in the assay buffer was determined, in triplicate, by counting 10 pi of buffer in 5 ml of ACS scintillation fluid containing 200 pi 2-phenylethylamine (a CO2" trapping compound). One unit of ACC activity is defined as the amount of ACC required to convert 1 pinole of acetyl-CoA to malonyl-CoA in one minute. When protein concentration was estimated based on ACC activity, 1 mU ACC activity was defined as 0.5 mg ACC protein. 2.2.5 PKCzeta Immunoprecipitation Fat pads were homogenized in 3 volumes of PKC homogenization buffer (20 mM Tris (pH 7.5), 250 mM sucrose, 2 mM EDTA, 2 mM EGTA, 1 mM Na 3V0 4 , 150 mM NaCl, 1 mM NaF, 20 mM para-nitrophenyl phosphate, 20 ug/ml leupeptin, 20 ug/ml aprotinin, 1 mM DTT, 100 uM microcystin, 2.5 mM benzamidine, 2 ug/ml Pepstatin A, and 0.4 mM Pefablock) immediately after insulin treatment. The homogenate was then 35 centrifuged (12000 x g) for 20 minutes to generate a fat-free infranatent. For each treatment, 500 ag of total protein was added to 800 pi of IP buffer (same composition as PKC homogenization buffer except sucrose is omitted and Triton X-100 (1%, v/v) and Nonidet-P40 (0.5%, v/v) are added). The extracts were equilibrated with IP buffer for 30 minutes at 4°C prior to addition of 2 ug of anti-PKCzeta antibody (Santa Cruz). Antibody binding was allowed to occur for 2 hours (end-over-end incubation), following which 20 pi of ProteinA-agarose beads were added to capture the antibody. Incubations continued for another hour. The antibody-kinase complexes were recovered by brief centrifugation (12000 x g) and the supernatent was kept as the "unbound" fraction. The complexes were washed 3x5 minutes with IP (500 pi) buffer, then 1x10 minutes with kinase assay buffer (1 ml). Samples prepared for Western blotting were then boiled in SDS sample buffer for 10 minutes and stored at -20°C until use. Samples for kinase assays were resuspended in 20 pi of kinase assay buffer and used in kinase assays as described below. 2.2.6 Kinase Assays Phosphorylation of ACC was determined by SDS-PAGE followed by phosphoimaging. In some experiments, bands were cut out of the gel and the number of moles of P incorporated per mole of ACC subunit was determined. For histone HI and peptide phosphorylation, an aliquot of the reaction was spotted onto P81 phosphocellulose paper and immersed in 0.85% phosphoric acid. Unincorporated [y-PJ-ATP and free phosphate was removed by washing the filter papers 3x20 minutes in 36 0.85% phosphoric acid. The filter papers were counted in dH20, using the setting for 3 H (Cerenkov counting). The specific assay conditions for the different kinases used are indicated below. For inhibition curves, Ro 31-8220 or Go 6983 (dissolved in DMSO) were added to the incubations at the indicated concentration. For control reactions, DMSO was added. 2.2.6.1 PKC The activity of classical PKC isoforms (a, pi, pil,y) was determined by 3 2P incorporation into affinity purified rat liver ACC (5 ug/assay) or histone HI (200 ug/ml). For novel (5, s, n) and atypical (£, i) isozymes the modified s pseudosubstrate peptide (ERMRPRKRQGSVRRRV; 100 ug/ml) or affinity purified rat liver ACC (5 ug/assay) were used as substrates. The assay buffer contained 20 mM MOPS (pH 7.2), 5 mM MgCl2, 1 mM Na3V04, and 50 uM [y-32P]-ATP. For the immunoprecipitation experiments, 20 pg/ml aprotinin, 20 ug/ml leupeptin, 100 uM Na4P207, 100 uM NaF and 0.5 mM PMSF were also added to the assay buffer. 2.2.6.2 AMPK AMP-activated protein kinase activity was determined by P incorporation into the SAMS peptide (HMRSAMSGLHLVKRR), a synthetic peptide based on the sequence around Ser-79 of ACC-1. The assay buffer contained 50 mM MOPS (pH 7.4), 10 mM MgS04, 0.5 mM EDTA, 200 uM AMP, 80 uM SAMS peptide, and 50 uM [y-32P]-ATP. 37 2.2.6.3 PKA Protein kinase A activity was determined by 3 2P incorporation into Kemptide (LRRASLG). The assay buffer was essentially as indicated for AMPK, except for the omission of AMP and the use of 80 uM Kemptide instead of the SAMS peptide. In some experiments, affinity purified rat liver ACC (5ug) was used as the substrate. 2.2.7 SDS-PAGE, Western Blotting and Phosphoimaging 2.2.7.1 SDS-PAGE Discontinuous SDS-PAGE was performed (Laemmli, 1970), using a 4.5% separating gel/ 4.5% stacking gel system for ACC and a 7.5% separating/4.5% stacking mini-gel system for PKCzeta. A larger gel was used for ACC to allow better separation of ACC-1 and ACC-2. Due to the low percentage gels used, PDA (0.12%) was used as the crosslinker to strengthen the gels. Samples were heated for 5 minutes at 95°C in SDS sample buffer (pH 6.8, Tris (125 mM), SDS (10% w/v), sucrose (20% w/v), and bromophenol blue (0.02% w/v)) prior to loading into sample wells. Prestained high molecular weight markers were also run on each gel to allow estimation of protein molecular weight. Large gels were generally run overnight, using constant current (6 mA/gel), whereas mini-gels were run at constant voltage (200 V) until the dye front approached the bottom of the gel. If immunodetection was desired, proteins were transferred directly to PVDF membrane following electrophoresis. Otherwise, gels were stained with Coommassie blue. Gels were then placed between two sheets of cellulose film and dried overnight. Where required, equal loading of the lanes was determined by video densitometry using a Bio-Rad Model 620 densitometer. When quantitation of 3 2P 38 incorporation into ACC subunits was desired, the labelled protein was cut out and the gel was digested with H2O2 (Brownsey and Denton, 1982). 2.2.7.2 Western Blotting Following electrophoresis, gels were equilibrated for 20 minutes with transfer buffer (Towbin et al., 1979) containing Tris (48 mM) and glycine (39 mM). Proteins were transferred to PVDF membrane using a semi-dry transfer system (BioRad) using current limits of 3 mA/cm for large gels and 5.5 mA/cm for mini-gels. The membranes were briefly washed with TBS-t (20 mM Tris (pH 7.2), 137 mM NaCl, 0.1% Tween-20 (v/v)), then used for immunodetection as described below. 2.2.7.2.1 PKCzeta For detection of PKCzeta, PVDF membranes were first blocked for 1 hour in TBS-t containing 5% powdered milk. The membrane was then washed 3x10 minutes in TBS-t and incubated for 1 hour with an antibody directed against the C-terminus of PKCzeta (1/800 dilution in TBS-t containing 0.5% powdered milk). After washing in TBS-t, the membrane was incubated for 1 hour with the secondary antibody (1/1000 goat anti-rabbit, alkaline phosphatase-linked). A standard alkaline phosphatase detection assay was used. 2.2.7.2.2 ACC ACC was detected by binding of HRP-labelled streptavidin to the biotin moiety of the protein. The PVDF membrane was blocked for 1 hour in TBS-t, then incubated for 1 39 hour with streptavidin-HRP (1/5000 dilution in TBS-t containing 2% BSA). The membrane was washed 3x10 minutes with TBS-t, then subjected to chemiluminescent detection using the ECL detection system (Biorad). 2.2.7.3 Phosphoimaging For quantitation of 3 2P incorporation into ACC subunits, the dried SDS-PAGE gels were analyzed using a phosphoimager (Molecular Dynamics). This allowed an estimation of the relative efficiency of ACC phosphorylation in samples run on the same gel. 2.2.8 Multi-kinase Analysis Tissue samples for multi-kinase analysis were treated and homogenized as previously described, with the addition of the phosphatase inihibitors NaF (5 mM) and microcystin-LR (1 uM) to the homogenization buffer to preserve phosphorylation of kinases. For each sample, 400 ug of total protein was added to an equal volume of SDS sample buffer and heated for 5 minutes at 95°C. Sample analysis was performed by a local company, Kinexus Bioinformatics Corp. (Vancouver, B.C). The extracts were run on SDS-PAGE to separate proteins by molecular weight, then transferred to nitrocellulose membrane for Western blotting. A multi-channel blotting system was used, which allowed the simultaneous detection of many kinases of different molecular weights in each channel. 40 2.2.9 Glycogen Synthase Assays Glycogen synthase assays were performed based on the method of Thomas et al. (1968). A 50 pi aliquot of GS assay buffer (75 mM Mops (pH 7.4), 75 mM NaF, 10 mg/ml glycogen, 2 mM UDP-[14C]-glucose) was prewarmed for 5-10 minutes at 30°C. The reaction was started by addition of 25 pi of cell extract and was incubated at 30°C for 30 minutes. Assays were terminated by spotting 50 ul of the sample on Whatmann 3 MM chromatography paper (2x3 cm), which was then immediately immersed in 66% ethanol. The filters were washed 3 x 30 minutes in 66% ethanol, air-dried, and counted for 1 4 C in ACS scintillation fluid. The specific activity of the buffer was determined, in triplicate, by counting 10 pi of buffer in ACS scintillation fluid. Assays were performed in the presence or absence of glucose-6-phosphate (15 mM) to allow calculation of the "activity ratio" (-/+ glucose-6-phosphate). 2.2.10 Protein Determination Protein concentration was determined by the Bradford dye-binding method (Bradford, 1976), using bovine gamma globulin as a protein standard. The assay was generally linear from 0 to 20 pg. Experimental protein concentrations were estimated within the linear portion of the standard calibration. 2.2.11 Statistical Analysis Results are generally expressed as mean +/- S.E.M. The statistical significance of differences was determined using a two-tailed t-test and confidence levels are indicated in the figure legends. 41 3.0 RESULTS AND DISCUSSION 3.1 KINASE EXPRESSION IN RAT WHITE ADIPOSE TISSUE 3.1.1 Comparison of Fat Pads, Adipocytes, and 3T3-L1 Cells The choice of an experimental system is an important decision in studies of insulin action. The system used must display insulin-responsiveness, to allow assessment of the hormone effects. Care must also be taken to ensure that the system used gives an accurate picture of the physiological effects of insulin in the cell. For adipose tissue, there are several options to be considered. The closest approximation to an in vivo situation is the use of isolated fat pads, which can be kept metabolically active in Krebs-Henseleit buffer. The isolated fat pads demonstrate good insulin sensitivity when isolated from smaller rats (< 200g), but the insulin responsiveness is lost as the animals get larger (Vernon, 1980). In some cases, it is preferable to digest the fat pads to obtain isolated fat cells (adipocytes). Care must be taken during the collagenase digestion procedure to retain the insulin-responsiveness of the cells, as excessive digestion can result in the loss of the insulin receptors from the cell surface. Although isolated fat cells are one step further removed from adipose tissue in vivo, they offer the advantage of increased homogeneity and thus reduce the sample variability than can be encountered with fat pads. Another system used by many researchers is 3T3-L1 cells, in a tissue culture system. The tissue culture system offers many advantages. One advantage is that the researcher does not sacrifice animals to obtain the tissue. In addition, the cells are theoretically identical from one experiment to the next, reducing variability. Also, transfection experiments can be easily performed in these cells, allowing the researcher to 42 express proteins of interest. When using 3T3-L1 cells, however, the researcher must keep in mind that these are cultured cells, and care must be taken when the results are interpreted in terms of physiological responses. Properties of 3T3-L1 cells are further discussed in the next section. Both fat pads and adipocytes were investigated as potential systems in which to study the effects of PKC inhibitors on ACC activation. Multi-kinase analysis was performed to determine the expression of various kinases in both fat pads and adipocytes. The multi-kinase analysis is an application of Western blotting that can be used to screen tissue extracts for the presence of a variety of kinases. The extracts are run on SDS-PAGE to separate proteins by molecular weight, then transferred to nitrocellulose membranes. A multi-channel blotting system is then used to detect several kinases of different molecular weights simultaneously in each channel. This is a proprietary technique, performed at Kinexus Bioinformatics Corp. (Vancouver, BC). Using this technique, both fat pad and adipocyte extracts were screened for kinase expression. In particular, expression of the various PKC isozymes was assessed. Differences between fat pads and adipocytes were also examined. Of the PKC isozymes, gamma and theta were not expressed in any of the extracts examined. The theta isozyme is primarily expressed in skeletal muscle, and the lack of expression in adipose tissue was not particularly surprising (Osada et al, 1992). The gamma isozyme is expressed exclusively in the central nervous system, and its absence from adipose tissue was also expected (Hashimoto et al., 1988). PKC-delta, -zeta and -mu (PKD) were the most highly expressed isozymes in both fat pads and adipocytes (Figure 3.1). Expression of PKC-alpha, -beta, and -lambda 43 was quite low in adipocytes, essentially at the limits of detection of the multi-kinase analysis. This was also true of the lambda isozyme in fat pads, whereas expression of PKC-alpha and -beta was slightly higher than seen in adipocytes. The epsilon isozyme was expressed at reasonable levels, and expression was similarly higher in fat pads than in adipocytes. PKC-mu, which is highly expressed in both fat pads and adipocytes, appears as two bands in the kinase analysis (Figure 3.1). In fat pads, the kinase is distributed equally between the two bands, but in adipocytes the kinase is found almost exclusively in the upper band. The reason for the bandshift is not known, but may represent a phosphorylation event. This probably does not reflect kinase activation, as the distribution between the upper and lower bands is unchanged in adipocytes treated with Ro 31-8220, which has been shown to inhibit PKD activation in cells (Zugaza et al., 1996). PKD/PKC-mu differs from other PKC isozymes in that it possesses both a putative transmembrane domain and a pleckstin-homology domain. In addition, the catalytic domain and substrate preferences of this isozyme are unlike other PKC isozymes (Nishikawa et al., 1997; Valverde et al., 1994). Three kinases were detected in fat pads that were not present in adipocytes: p45, Cot and PKG. Since some tissue is lost during the preparation of isolated fat cells, the potential exists for the loss of particular kinases. In particular, the vascular tissue and pre-adipocytes are discarded following collagenase digestion of adipose tissue. Since p45 and Cot have been implicated in cell differentiation and transformation, it seems reasonable that they would not be expressed in fully differentiated fat cells. The loss of 44 Figure 3.1: Multi-Kinase Analysis a) PKC Isozymes alpha FP AD beta FP AD delta FP AD epsilon FP AD .... JH 1 j-ft"? RI: 1916 367 2189 300 11437 4134 4390 1078 zeta lambda mu FP AD FP AD FP AD ^^^^^^ i W ^ H P RI: 18172 12338 250 367 15786 6195 b) differences in kinase expression p45 FP AD Cot FP AD PKG FP AD RI: 4778 0 3978 0 4460 0 PAKa FP AD CamKIV FP AD RI: 2200 4750 Fat pad (FP) and adipocyte (AD) extracts were prepared as previously described, but with microcystine added to the buffer. For each extract 500 ug of total protein was analyzed for kinase expression by Kinexus, using a Western blotting technique. The numbers indicated below the protein bands represent the relative intensity (RI) of the bands. Analysis of PKC isozymes is shown in panel (a). PKC isozymes gamma and theta were not detected in either extract. Differences in kinase expression between fat pad and adipocyte extracts are shown in panel (b). Results of a single experiment are shown. 45 PKG, which is involved in nitric oxide signaling, may also indicate specific expression of this kinase in the vasculature or pre-adipocytes. Interestingly, two kinases were detected in adipocytes that did not appear to be expressed in fat pads. These were p21-activated protein kinase alpha (PAKalpha) and calcium-calmodulin-dependent protein kinase IV (CaMKIV). Detection of these kinases in adipocytes but not fat pads may suggest an induction of kinase expression following collagenase digestion. As the time elapsed between collagenase digestion and the termination of cell incubations was less than two hours, it seems unlikely that significant protein synthesis would have occurred. An alternative explanation is that these kinases are more highly expressed in mature fat cells, and were thus enriched in the adipocyte extracts. 3.1.2 Comparison of Primary and 3T3-L1 Adipocytes As many of the PKC studies have been performed in 3T3-L1 adipocytes, kinase expression in these cells was compared to kinase expression in primary adipocytes. Table 3.1 lists the major differences observed in kinase expression. There were many differences in kinase expression between primary and 3T3-L1 adipocytes. The kinases detected only in primary adipocytes exhibited very low expression levels, and their absence from 3T3-L1 cells may simply indicate that expression is below the detection limits of this technique. Expression of the kinases that were detected only in 3T3-L1 adipocytes was generally well above the detection limits, and these probably represent real differences in kinase expression between these two cell types. 46 Table 3.1: Comparison of Kinase Expression in Primary and 3T3-L1 Adipocytes Expressed only in Primary Adipocytes Expressed only in 3T3-L1 adipocytes Higher level of expression in 3T3-L1 Adipocytes PKCalpha, PKClambda, ROKalpha, Syk p45, Cdk9, Mekl, Cot, IKKalpha, Lck, BMX, Pyk2, ZAP70, CaMKK, Nek2 PKBalpha, Cdk4, Cdk5, Cdk7, PKCbeta, SAPK p54, Mos, Mek2, Mek6, PAKalpha, PKR, GSK3beta, p90S6K, Rskl, Rsk2, CK2alpha, GRK2, Hpk Kinase expression was determined by multi-kinase analysis, performed by Kinexus. A higher level of expression was defined as an increase of >10 times. Rskl expression was higher by a factor of 100 and is indicated in bold type. For a large number of the kinases detected in the multi-kinase analysis, expression was increased by a factor of ten or more in 3T3-L1 adipocytes. One kinase, Rskl, was expressed at a level one hundred times greater than in primary adipocytes. As many of the overexpressed kinases are involved in cell-cycle regulation, the increased expression may be an indication of the replicative nature of this cell line. It should also be noted that expression of PKC-beta, PKB-alpha, and GSK3-beta were increased in 3T3-L1 adipocytes. As these kinases are implicated in some of the metabolic effects of insulin signaling, the increased expression could influence the results of experiments done in this cell type. 47 3.2 PHOSPHORYLATION OF PURIFIED ACC BY PKC ISOFORMS 3.2.1 Screening PKC Isoforms The ability of PKC to phosphorylate ACC has been demonstrated, both in intact cells and in vitro (Haystead and Hardie, 1988; Vaartjes et al., 1987; Hardie et al, 1986). Stimulation of PKC in cells, by treatment with phorbol esters, activated ACC in hepatocytes, but not in adipocytes (Haystead and Hardie, 1988; Vaartjes et al., 1987). Treatment of adipocytes with phorbol esters did stimulate phosphorylation of ACC, on the same peptide that is phosphorylated in response to insulin treatment (Haystead and Hardie, 1988). ACC is also phoshorylated by purified PKC in vitro, though the effect of this phosphorylation on enzyme activity is controversial. Hardie et al. (1986) claim that PKC phosphorylates the same site as PKA and inhibits ACC. However, others found that the phosphorylation activated ACC approximately two-fold (Vaartjes et al., 1987), although the site of phosphorylation was not defined. The explanation for these very different results is still unclear, but may have been affected by the purity of the PKC used. The kinase used may have contained different mixtures of PKC isozymes, or may have been contaminated with additional ACC kinases. At the time of these earlier studies no effort was made to differentiate between PKC isozymes, as the complexity of the PKC family was not yet evident. Thus, by looking at the individual PKC isoforms, it may be possible to more accurately determine their roles in the phosphorylation of ACC and any resultant effects on ACC activity. Members of the classical (a, pi, Pn, y), novel (8, 8, n) and atypical i) groups of PKC isoforms were tested for the ability to phosphorylate rat liver ACC in vitro. Liver ACC was used for these experiments so that phosphorylation of both ACC-1 and ACC-2 48 could be assessed. The ability of each PKC isozyme to phosphorylate ACC was determined by the relative P incorporation into each ACC isoform (Figure 3.2). The ratio of ACC-1 to ACC-2 in the liver is three to one, which should be kept in mind when interpreting the results. Some variability was observed in the degree of basal ACC phosphorylation. For ACC phosphorylation by each of the PKC isozymes, a corresponding basal phosphorylation is shown for comparison. The basal phosphorylation likely results from the co-purification of an endogenous kinase and is commonly observed in ACC phosphorylation experiments. Variability in the extent of basal phosphorylation is often observed when different preparations of ACC are compared, but the stoichiometry of basal phosphorylation is quite low. The results are expressed relative to the level of ACC basal phosphorylation, which is assigned a value of one. Of the classical PKC isozymes, PKC-alpha phosphorylated ACC most efficiently, yielding two-fold phosphorylation. PKC-betal did not appear to phosphorylate ACC, whereas PKC-betall and PKC-gamma each resulted in a 1.5-fold phosphorylation. None of the classical isozymes demonstrated any specificity for one of the ACC isoforms over another. A similar picture emerged with the novel PKC isozymes, in that neither ACC isoform appears to be specifically phosphorylated. Overall, ACC was a poor substrate for the novel PKC isozymes, with phosphorylation similar to basal ACC phosphorylation levels. PKC-delta resulted in a slight increase, with 1.5-fold phosphorylation. 49 Figure 3.2: ACC Phosphorylation by PKC Isoforms a) Classical PKC Isoforms basal alpha betal basal betall gamma . 15 30 60 120 15 30 60 120 15 30 60 120 15 30 60 120 15 30 60 120 15 30 60 120 ACC Phosphorylation by Classical PKC Isoforms b) Novel PKC Isoforms basal delta epsilon 15 30 60 120 15 30 60 120 15 30 60 120 basal eta 15 30 60 120 15 30 60 120 ACC Phosphorylation by Novel PKC Isoforms I A C C - 1 I A C C - 2 Time (min) 50 c) Atypical PKC Isoforms A X FhcBptarylaticn by Apical PKCisofcrm as "firre(rrir| Affinity-purified ACC was incubated with purified preparations of the indicated PKC isoforms in the presence of [y- P]-ATP for 15 to 120 minutes. ACC-1 (lower band) and ACC-2 (upper band) were then resolved by SDS-PAGE and visualized by Coomassie staining. The 32P-labelled bands were visualized and quantitated by phosphoimaging (upper panels). Members of the classical (a), novel (b), and atypical (c) groups of PKC isoforms were used. Results were expressed relative to basal ACC phosphorylation (lower panels). ACC phosphorylation experiments were performed twice, and representative results are shown. 51 Only the atypical PKC isoforms demonstrated a clear time-dependent increase in ACC phosphorylation and ACC isoform specificity, with a preference for phosphorylation of ACC-1. PKC-zeta phosphorylated ACC more efficiently than did PKC-iota. Of all the PKC isoforms tested, PKC-zeta was the most efficient ACC kinase, demonstrating a 3-fold increase in phosphorylation of ACC-1 relative to basal phosphorylation. Even in this case, the rate and extent of phosphorylation was small when compared to phosphorylation seen with AMPK or PKA (Figure 3.3). Although the phosphorylation of ACC by PKC-zeta was relatively low, it was comparable to that reported for nucleolin, a reported substrate protein (Zhou et al, 1997). Relatively little is known about activation of the atypical PKC isoforms, making it difficult to interpret poor phosphorylation of a putative substrate protein. The low level of phosphorylation observed with the classical and novel PKC isozymes is quite interesting, in light of previous reports that ACC is phosphorylated by PKC. The phosphate incorporation observed in the earlier reports was between 0.54 and 0.75 moles per mole of ACC subunit and the kinase responsible was shown to be calcium- and phospolipid-dependent. The calcium-dependency, in particular, suggests that one of the classical PKC isozymes was responsible for ACC phosphorylation. The kinase used for these previous studies was purified from bovine brain and likely contained a number of PKC isozymes (Hardie et al., 1986). In the studies of ACC phosphorylation reported here, in contrast, each kinase used was a highly purified recombinant human PKC isozyme. Clearly there is some difference between these kinase preparations with respect to the ability to phosphorylate rat liver ACC, as the highly purified classical PKC isozymes phosphorylated ACC inefficiently. 52 It is possible that the kinase preparation used in the previous studies was contaminated with another kinase, which was responsible for ACC phosphorylation. The impurity of the kinase preparation seems likely, since the phosphorylation was shown by different groups to result in either inhibition or activation of ACC, suggesting that the sites of phosphorylation were not identical. Alternately, it is possible that a mixture of kinases leads to synergistic or additive ACC phosphorylation. 3.2.2 ACC Phosphorylation by PKCzeta To further assess the role of PKC-zeta in ACC phosphorylation, increasing quantities of the kinase were used for in vitro phosphorylation, and the total phosphate incorporation into ACC-1 and ACC-2 was determined. Two different preparations of PKC-zeta were used for ACC phosphorylation experiments. The kinase obtained from Upstate Biotechnology (Lake Placid, NY) demonstrated both more efficient phosphorylation of ACC and a preference for ACC-1, resulting in up to 5-fold phosphorylation. The kinase obtained from Calbiochem (San Diego, CA), in constrast, showed no obvious isoform preference and was less efficient at ACC phosphorylation, generating a 2-fold phosphorylation. Both kinase preparations were human recombinant enzyme purified from bacculovirus-infected insect cells. The reason for the differences in kinase activity is currently unclear. The ACC phosphorylation results shown were performed with the kinase from Upstate Biotechnology. 53 Figure 3.3: ACC Phosphorylation with Increasing Amounts of PKC-zeta a) 25ng lOOng PKA Basal PKC^ Basal PKC<; 15 30 60 120 15 30 60 120 15 30 60 120 15 30 60 120 15 30 60 120 b) Phosphate Incorporation into ACC by PKA 15 P K A 30 60 Time (min) I ACC-1 IACC-2 c) Phosphate Incorporation into ACC by PKCzeta c 0.04 J 0.035 jo 0.03 O Q025 § Q02 0 Q015 E 0.01 57 0.006 1 0 E I • 1 1 • • • • • 1 I ACC-2 15 30 60 120 15 30 60 120 25ng 100ng Tirre(rrin) Affinity-purified rat liver ACC was incubated with PKA, PKC, or no kinase (auto) in the presence of [y-32P]-ATP for 15 to 120 minutes (as indicated). The phosphorylated proteins were resolved by SDS-PAGE and visualized by Coomassie staining, followed by phosphoimaging (a). The bands corresponding to ACC isoforms (upper and lower bands are ACC-2 and ACC-1, respectively) were then cut from the gel, digested and P was determined. The phosphate incorporation into each ACC isoform by PKA (b) and PKC 9^ (c) was calculated for P incorporation and mol ACC applied to each gel lane (5 ug, as determined by ACC activity). Results shown are representative of two experiments. 54 ACC phosphorylation by PKA is shown as a control. PKA preferentially phosphorylated ACC-2, as expected, and the total phosphate incorporation reached a maximum of 0.3 moles per mole of ACC-2 subunit at 120 minutes (Figure 3.3). With PKC-zeta, the maximum phosphate incorporation was 0.024 moles of phosphate per mole of ACC-1 subunit. This is less than 10% of the 0.3 moles of phosphate per mole of ACC-2 subunit incorporation obtained with PKA. Increasing the amount of PKC-zeta by a factor of four resulted in only a small increase in ACC phosphorylation, from 0.014 to 0.024 moles/mole. To see any effects of the phosphorylation on ACC activity, an incorporation of at least 0.2 moles/mole would be desirable. The relatively small increase in phosphorylation observed with increasing amounts of PKC-zeta could indicate that ACC is a very poor (and likely irrelevant) substrate, or that there is some limiting factor in the assay, preventing full kinase activity. It is difficult to know what factor might be limiting in the PKC-zeta assay, as little is known about the mechanism of activation of this kinase. Unlike other members of the PKC family, this atypical PKC is not activated by calcium or diacylglycerol. Phosphatidylserine, a known cofactor for the atypical kinases, was included in the assay at 100 ug/ml. The assay conditions used were comparable to those used by others (Kotani et al, 1998; Standaert et al., 1997), and resulted in reasonable kinase activity when the epsilon peptide was used as a substrate. Other reported activators of PKC-zeta are phosphatidylinositol-3,4,5-trisphosphate and long chain acyl CoA esters (Nakanishi et al., 1993; Yaney et al., 2000). The ability of these compounds to stimulate atypical PKC activity was not examined in these studies. 55 The poor phosphorylation of ACC by the purified kinase may be a result of incomplete activation of PKC-zeta in vitro. Alternately, ACC may simply be a poor substrate for the kinase. Due to the insufficient understanding of the mechanism of PKC activation, a different approach was undertaken, to ensure that the kinase used in the assay was fully activated. 3.2.3 Immunoprecipitation of PKCzeta from Adipose Tissue To further investigate the possible role of atypical PKC isozymes in the insulin-stimulated phosphorylation and activation of ACC, PKC-zeta and PKC-lambda were immunoprecipitated from insulin-treated fat pads and used to phosphorylate ACC. Insulin treatment of the intact fat pads was expected to produce highly active kinase. The antibody used for immunoprecipitation (nPKC<^ (C-20) from Santa Cruz Biotechnology) recognizes both atypical PKC isoforms. No ACC phosphorylation was observed when the immunoprecipitated PKC was used in an in vitro kinase assay (Figure 3.4a). A positive control using purified recombinant PKC-zeta shows preferential phosphorylation of ACC-1, indicating that the assay is operative. No differences were observed between the control and insulin-treated samples. The recovery of PKC-zeta from the adipose tissue extracts was assessed by immunoblotting (Figure 3.4c). PKC-zeta was present in the irnmunoprecipitates, with no apparent difference in recovery between the control and insulin-treated samples. Some kinase was left unbound. It is unclear whether this is due to insufficient antibody used, or due to some property of the kinase. The lack of ACC phosphorylation by the 56 Figure 3 . 4 : ACC Phosphorylation by Immunoprecipitated PKC-zeta/-lambda a) Insulin(min) P K C C IP 0 10 20 Background 0 10 20 P K C ; P K A ^V-n w % m w b) Epsilon Peptide Phosphorylation by Immunoprecipitated PKCzeta 0 10 20 Insulin Treatment (min) 0 IP 0 10 20 Unbound 10 20 + PKC antibody (nPKC(^ (C-20)) was used to immunoprecipitate PKC-zeta from rat adipose tissue treated with insulin for 0, 10, or 20 minutes, as indicated. The complex was recovered by binding to protein A-agarose beads and centrifugation. Immunoprecipitated kinase was incubated with affinity-purified rat liver ACC (a) or with the epsilon peptide substrate (b). Phosphorylation of ACC was assessed by SDS-PAGE followed by phosphoimaging. Peptide phosphorylation was assessed by Cerenkov counting of labeled peptide bound to P81 phosphocellulose paper. The immunoprecipitated complexes were also analyzed for kinase content by immunoblotting (c). Results shown are representative of five experiments. 57 immunoprecipitated kinase is intriguing since the purified kinase clearly phosphorylates ACC-1. The presence of PKC activity in the immunoprecipitates was confirmed by phosphorylation of the epsilon peptide substrate. There was no increase in the activity of the kinase that was precipitated from insulin-treated tissue as compared to the control. Instead, a slight decrease in kinase activity was observed with increasing length of insulin treatment (Figure 3.4b). The epitope recognized by the antibody used for kinase immunoprecipiation is found at the carboxy-terminus of the kinase and lies within the catalytic domain. It is therefore possible that binding of the antibody-proteinA-agarose complex to the kinase impedes its association with some substrate proteins. ACC, in particular, is a very large protein, and may not be able to associate with the immunoprecipitated kinase complex. Antibody binding does not completely abolish the kinase activity, however, as the peptide substrate is efficiently phosphorylated. Interestingly, insulin treatment of the fat pads did not appear to activate atypical PKC isozymes, as demonstrated by phosphorylation of the epsilon peptide by the immunoprecipitated kinase. Some authors report the translocation of PKC-zeta to the plasma membrane following insulin stimulation. It is therefore possible that the immunoprecipitation experiment is preferentially targeting the less active, cytosolic PKC-zetaZ-lambda. No effort was made in these experiments to enrich for membrane associated kinase. Since ACC-1 is considered to be largely cytosolic, regulation by a membrane-associated kinase seems unlikely. 58 3.2.4 PKCzeta Membrane Translocation In order to detect a possible membrane translocation, the relative amount of PKC-zeta/-lambda in plasma membrane and cytosolic fractions was compared for insulin treated and untreated fat pads. An increase in the proportion of PKC-zeta found in the plasma membrane fraction compared to the cytosolic fraction would be expected in the insulin-treated tissue if translocation occurred. . To compare PKC associated with plasma membranes to cytosolic enzyme, the extracts were first cleared of cellular debris and organelles by centrifugation at slow speed (1000 x g for 10 minutes). Subsequent centrifugation at 100 000 x g, for 10 minutes, was used to pellet the less dense membranes, including the plasma membrane. The supernatent was retained as the cytosolic fraction. PKC was clearly present in all cellular fractions analyzed and there was no apparent difference in the proportion of kinase that was membrane-associated in the insulin treated fat pads compared to control tissue (Figure 3.5). There does not, therefore, appear to be any net translocation of PKC-zeta to the plasma membrane following insulin stimulation of adipose tissue. Whether PKC-zeta undergoes translocation to the plasma membrane following insulin stimulation is still debated. When enzyme activity is used as a measure of PKC-zeta content in different cellular fractions, an insulin-stimulated translocation from the cytosol to the plasma membrane is generally observed (Mosthaf et al, 1996; Standaert et al, 1999b). When translocation is assessed by immunoblotting, however, the results are less clear, with some authors reporting that translocation occurs (Ishizuka et al., 1998) 59 and others reporting that it does not (Bandyopadhyay et al., 1997; Frevert and Kahn, 1996). It seems possible that a preferential activation of the membrane-associated kinase rather than a translocation, occurs following insulin stimulation of cells. Alternately, the translocation may be transient, and thus detection would depend on the time expired between insulin stimulation of cells and tissue homogenization. No assessment was made of the kinase activity in the different cellular fractions. Figure 3.5: PKC-zeta Does Not Translocate to the Plasma Membrane Following Insulin Stimulation Insulin-Treated lk 12k 100k P S P S P S Control lk 12k 100k P S P S P S Insulin-treated and untreated rat fat pads were homogenized and subjected to sequential centrifugation steps to generate cellular fractions. Following centrifugation, the pellet was resuspended to the starting volume with homogenization buffer. Equal volumes of each of the fractions were run on SDS-PAGE and transferred to PVDF membrane for immunoblotting. Purified recombinant PKCzeta was used as a positive control (+). The sequential centrifugation steps were 1000 x g (lk), 12000 x g (12k), and 100000 x g (100k). The pellet (P) and supernatent (S) from each centrifugation were analyzed. The plasma membrane and cytosolic fractions are the 100k pellet and supernatant, respectively. Results of a single experiment are shown. 60 3.3 EFFECTS OF PKC INHIBITORS IN INTACT TISSUES AND CELLS 3.3.1 Choice of PKC Inhibitors Due to the difficulties encountered with studies of PKC-zeta in vitro, another experimental approach was undertaken. To determine the effects of PKC isoforms on ACC activation in a cell-based system, selective PKC inhibitors were chosen. Since the role of PKC-zeta, in particular, was to be investigated, two inhibitors were chosen such that only one of them would be effective against this atypical PKC isoenzyme. Based on the information provided by the supplier (Table 3.2), the compounds Ro 31-8220 and Go 6983 were chosen. Go 6983 has been shown to inhibit PKC-zeta (IC50 = 60 nM). Thus, if this isozyme is involved in the insulin-stimulated activation of ACC, treatment of cells with Go 6983 should prevent or reduce the activation. Table 3.2: Reported IC50 (uM) Values for PKC Isozyme Inhibition PKC Isozyme Compound Ro 31-8220 Go 6983 a 0.005 0.007 P - 0.007 PI 0.024 -pn 0.014 -Y 0.027 0.006 6 - 0.01 s 0.024 -c - 0.06 - 20 *1 - -61 3.3.2 Effect of PKC Inhibitors on Purified ACC The inhibitors were first tested for any direct.effects on ACC activity. Ro 31-8220 has been reported to inhibit PKC by competition for the ATP-binding site (Davis et al., 1992). Go 6983 is presumed to act via a similar mechanism. As ACC also contains an ATP-binding site, the inhibitors might have a direct effect on carboxylase activity. Purified liver ACC, containing both ACC-1 and ACC-2 was used to assess the effects of the inhibitors. Neither inhibitor had any discernible effect on ACC activity at the experimental concentration (5 uM Ro 31-8220; 2 uM Go 6983) (Figure 3.6). Figure 3.6: PKC Inhibitors Do Not Directly Affect ACC Activity Effect of PKC Inhibitors of Purified Rat Liver ACC 0.8 [citrate] (mM) Affinity-purified rat liver ACC was pre-incubated for 20 minutes with Ro 31-8220 (5 pM), Go 6983 (2 pM), or without inhibitor, at various citrate concentrations (as indicated). ACC activity was then determined by C0 2 fixation from 1 4 C-HC0 3 into malonyl-CoA. Results of a single experiment are shown. 62 3.3.3 Effects of PKC Inhibitors in Fat Pads and Adipocytes Preliminary experiments were performed in both fat pads (epidiymal and perirenal) and in adipocytes. Insulin signaling was effective in both systems, and ACC activation was detectable. The effectiveness of the PKC inhibitors in these tissues was assessed by exploiting a side effect of Ro 31-8220. This compound has been shown to inhibit glycogen synthase kinase 3 (GSK-3) effectively (IC50 = 2.8 nM) (Hers et al, 1999). This results in a net activation of glycogen synthase by preventing inhibitory phosphorylation by GSK-3 (Hers et al, 1999; Standaert et al, 1999a). Thus when cells are treated with Ro 31-8220, glycogen synthase is activated, even in the absence of insulin. The effect is additive with activation by insulin. In extracts of insulin-treated and inhibitor-treated fat pads, insulin induced activation of glycogen synthase, with the activity ratio increasing from 0.14 to 0.23 (Figure 7a). Treatment of the fat pads with Ro 31-8220 alone did not result in activation of glycogen synthase, although Ro 31-8220 and insulin together did result in a slight increase in the activity ratio to 0.32 compared to 0.23 with insulin alone. Incubation with Go 6983 had no apparent effect on glycogen synthase activity. The effect of Ro 31-8220 observed in fat pads is much less than has been reported using adipocytes or L6 myotubes (Standaert et al, 1999a). Based on the lack of effect of this compound on glycogen synthase activity in the absence of insulin, it seems likely that the inhibitor is not reaching the interior of the cells. With fat pads, this may be due to the large pieces of tissue, where the majority of the cells are found in the interior and are thus inaccessible. 63 When the same experiments were performed in adipocytes, the effect of Ro 31-8220 was readily observed. The activity ratio for glycogen synthase activity was 0.26 when adipocytes were incubated with Ro 31 -8220 in the absence of insulin, and increased further to 0.43 in the presence of insulin (Figure 7b). In the absence of this compound, insulin increased the glycogen synthase activity ratio from 0.09 to 0.23. The effect of insulin on glycogen synthase activation was more apparent in adipocytes than in fat pads. The effect of Ro 31-8220 was apparent in unstimulated adipocytes, and the effects of insulin and the inhibitor were additive. The other inhibitor used, Go 6983, had no effect on glycogen synthase activity. The effectiveness of Ro 31-8220 in adipocytes but not in fat pads further indicated that the inhibitor was limited in its ability to penetrate cells. Thus the use of individual cells likely permitted better access of the inhibitor to intracellular proteins. For Go 6983, there was no comparable way to verify its effectiveness in adipocytes. Based on the similar structures of the two compounds (Figure 3.8) and similar properties, it was assumed that Go 6983 was also capable of penetrating adipocytes. 64 Figure 3.7: Effects of PKC Inhibitors on Glycogen Synthase Activity In Extracts of Rat Fat Pads or Adipocytes a) Fat Pad Extracts 0.4 T -£< 0.35 > DMSO RO31-8220 G&S983 Treatment Fat pads (a) or isolated adipocytes (b) were pre-incubated for 30 minutes in the presence of the PKC inhibitors Ro 31-8220 or Go 6983 or with DMSO (control). Insulin was then added, where indicated, and the incubations were continued for 30 minutes. The tissues were then homogenized and centrifuged at 12000 x g for 15 minutes, and 25 pi of the supernatent was used to assay glycogen synthase activity by 14C-UDP-glucose incorporation into glycogen in the presence and absence of glucose-6-phosphate. Glycogen synthase activity is expressed as the activity ratio (- glucose-6-phosphate/+glucose-6-phosphate). Values are expressed as mean +/- standard error (n=3 for fat pads, n=15 for adipocytes). Insulin response is significant where indicated by * and Ro 31-8220 treated sample is significantly higher than control where indicated by ** (PO.007) by t-test. 65 Figure 3.8: Structures of the Calbiochem Compounds Ro 31-8220 and Go 6983 Ro 31-8220 G o 6983 Structures of the PKC inhibitors Ro 31-8220 (3-[l-[(Amidinothio)propyl-lH-indol-3-yl]-3-(l-methyl-lH-indol-3-yl)maleimide) and Go 6983 (2-[l-(3-Dimethylaminopropyl)-5-methoxyindol-3-yl]-3-(lH-indol-3-yl)maleimide), as indicated by Calbiochem. Structural differences between the two compounds are indicated in bold type. 66 3.3.4 Effect of PKC Inhibitors on ACC Activity in Adipocytes The effect of the PKC inhibitors on ACC activity was assessed in adipocytes. Given the isoenzyme specificities indicated by the supplier, Go 6983 was expected to inhibit PKC-zeta (IC50 = 60 nM) and PKC-delta. Ro 31-8220 was not expected to inhibit the atypical PKC isozymes, but was expected to inhibit the classical PKC isozymes (a, (31, (311, and y) as well as the novel isozyme PKC-epsilon. The goal was to use these compounds in adipocytes to inhibit PKC isozymes and determine the effect, if any, on ACC activation in response to insulin. If PKC-zeta is the kinase which phosphorylates ACC following insulin treatment, and this is involved in ACC activation, then inhibiting the kinase should prevent activation of ACC. Thus, treatment of adipocytes with Go 6983 was expected to prevent insulin stimulation of ACC activity, whereas treatment with Ro 31-8220 was not expected to have any effect. In the adipocyte incubations, 2% BSA was included to stabilize the cells during incubations. Significant amounts of BSA were therefore carried over into the adipocyte extracts, meaning that accurate determination of intracellular protein concentration was not possible. In order to correct for differences in protein concentration between samples, fully activated glycogen synthase activity was used as a measure of cytosolic protein content and recovery. For experiments with fat pads, where BSA was not included in the incubations, a good correlation was found between cellular protein concentration and maximal glycogen synthase activity (not shown). 67 Figure 3.9: Effect of PKC Inhibitors on ACC Activity in Rat Adipocytes a) + 20 mM citrate c) + 20 mM citrate E o si >h-<2 O a> o > **« <D a. 283 230 240 220 C 2D 3 180 Sia> 140 — 123 — iooir^ sol— 2D 40 60 lnsdinTresfarert(irin) • •-CTR.(DVK)) GB6BB3 E o si 2B0 230 240 = u. 220 s o ! " 2 3 0 <S=.1S0 O « e 180 O > — <3 ™ 0) 120 °i 1001 ^ 83 -•--CTR-(TJM9q[| - • — Fto3V8220 0 23 40 60 Insulin Treefrrert (mn) b) no preincubation d) no preincubation .E 260 E o 260 si >l-*= O o „ < o o > 24) 220 0) 3180 c 183 ~140 120 100 fc-r 80?— 4 ' ' •••••OR.PK) - * - - G b 6 9 B 3 20 63 InsuBn Treefrnat (mn) —•—R>31-8220 0 20 43 60 lrEUinTieefrmt(rrin) Adipocytes were pre-incubated with 5 pM Ro 31-8220, 2 pM Go 6983, or with 0.1% DMSO (CTRL) for 30 minutes. Insulin was then added, when indicated, for 0 (n=14), 8 (n=3), 15 (n=5), 30 (n=3), or 60 (n=6) minutes (in separate experiments). Adipocytes were homogenized and centrifuged at 12 000 x g for 15 minutes. ACC activity in 40 pi of supernatent was determined by CO2 fixation from 14C-bicarbonate into malonyl-CoA. Assays were performed with a 20 minute pre-incubation in the presence of 20 mM citrate or with no pre-incubation and no citrate. All assays were performed in duplicate. Each time point represents the average of at least three individual experiments. Error bars indicate the standard error. ACC activity is significantly higher than control where indicated by ** (PO.04). 68 ACC activity was determined under two separate assay conditions: with or without a 20 minute preincubation with the allosteric activator citrate. Maximal ACC activity was determined by pre-incubating the enzyme with 20 mM citrate, which induced polymerization and activation of ACC. In the past, ACC activity has been expressed as an activity ratio, where the initial ACC activity (assayed without preincubation) is expressed as a proportion of total activity (assayed with a pre-incubation in the presence of 20 mM citrate). It is apparent from the above graphs, however, that some ACC activation is observed even when assayed in the presence of 20 mM citrate, suggesting that there is some additional factor in the activation of ACC (Figure 3.9). The degree of ACC activation assessed at 20 mM citrate is somewhat smaller under these assay conditions (approximately 30-50%) than in the absence of citrate (2-fold or greater). This demonstrates that citrate does not fully activate ACC. The further activation of ACC activity in insulin-treated tissues may be attributable to the phosphorylation state of the enzyme. For example, the inhibitory sites phosphorylated by PKA or AMPK may become dephosphorylated. In addition, as previously noted, ACC is phosphorylated on a distinct site, within the 'I-peptide', following insulin stimulation and this may be involved in the activation of the enzyme. Phosphorylation of this site does not appear to directly activate ACC, but may instead be indirectly involved in activation. It is possible that phosphorylation of the I-site is involved in ACC activation by facilitating polymerization of the enzyme. The polymerized form of ACC is highly active. Another scenario is that this phosphorylation event provides a recognition site for some other protein, which regulates ACC activity. A likely candidate is the regulator 69 protein, which was previously identified in rat adipose tissue. The regulator protein associates strongly with ACC and activates the carboxylase up to four-fold ( Borthwick et ah, 1990; Heesom et al., 1998). These possibilities are not necessarily mutually exclusive, as the final effect on ACC activation may be a result of a combination of phosphorylation state, polymerization and association with the regulator protein. Treatment of adipocytes with Ro 31-8220 resulted in a more rapid activation of ACC in response to insulin, with half-maximal activation observed at approximately 10 minutes. In the untreated adipocytes, half-maximal activation occurred at approximately 20 minutes. Go 6983 did not appear to affect ACC activation, as the maximal activation and the time course of activation observed were essentially identical to the control. Although treatment with Ro 31-8220 caused more rapid ACC activation by insulin, it did not affect either the maximal or basal ACC activity. Thus treatment with the compound alone did not affect ACC activity, but rather affected the response of the enzyme to insulin. This suggests that treatment with this compound is relieving an inhibition of ACC which normally results in a delay in ACC response to insulin. The reason for the lag in ACC response is unknown, but has been described by other researchers in both hepatocytes (Bijleveld et al., 1989) and hepatoma cells (Witters and Kemp, 1992). The ability of Ro 31-8220 to reduce the lag time for ACC activation suggests that the slow response of ACC to insulin is the result of an inhibition of the enzyme, rather than a simple delay in activation. It is interesting to note that other metabolic enzymes are activated quite rapidly following insulin stimulation of cells. Glycogen synthase activation was detected at 8 minutes, the earliest time point studied in these experiments. Protein kinase B is 70 similarly activated within 2-8 minutes in response to insulin (Moule et al, 1995; Cross et al, 1997). Studies using fat pads have demonstrated the insulin-induced activation of ACC at 15 minutes, with full activation of the enzyme requiring insulin treatment of 60 minutes (Lee et al, 1973). This is consistent with the time-course of ACC activation in adipocytes, as presented here. Studies done in vivo, however, have indicated ACC activation as early as 10 minutes after insulin injection (Stansbie et al, 1976). If enzyme activation is dependent on some intercellular signaling, it seems likely that an exaggerated delay would be seen in isolated tissues and cells. The exact length of the delay in ACC activation, therefore, may be shorter in vivo than in adipocytes. It is possible that the delay in ACC activation represents a mechanism of prioritizing the replenishment of energy stores. Given that energy stored as glycogen is quickly accessible and rapidly depleted, it seems reasonable that the cell would first replenish this energy store. The synthesis of fatty acids, on the other hand, is generally thought to be a long-term storage option. Thus it may be reasonable to expect that activation of ACC, which catalyzes the first committed step in fatty acid synthesis, would be repressed until the other energy stores are replenished. Although it is clear that Ro 31-8220 is acting on some target to produce a more rapid activation of ACC by insulin, the target itself is unclear. The compound was chosen as an isozyme-selective PKC inhibitor, but it appears that it is also an effective inhibitor of GSK-3. It seems possible, then, that the inhibitory properties of Ro 31-8220 have been incompletely defined. To address the effects of both Ro 31-8220 and Go 6983 in adipocytes, it was necessary to more accurately determine their kinase-specificity. 71 3.4 FURTHER CHARACTERIZATION OF PKC INHIBITORS 3.4.1 Inhibition of PKC Isoforms by Ro 31-8220 and Go 6983 We have been unable to find a comprehensive report that describes the effects of the PKC inhibitors on all the PKC isozymes. Due to gaps in the literature concerning the inhibition of various PKC isozymes by the inhibitors Ro 31-8220 and Go 6983, several isozymes were screened to determine IC50 values. The values obtained for inhibition of the various PKC isozymes are quite remarkable, for several reasons. First, there does not appear to be a great deal of specificity for individual isozymes, using these inhibitors. This is in contrast to the information provided by the supplier, which indicated that these compounds would effectively distinguish between isozymes. Secondly, the IC50 values are much higher than indicated by the supplier, generally by an order of magnitude. The exception to this trend was the inhibition of the pi and pil isozymes by Ro 31-8220, which occurred with IC50 values comparable to those indicated. One striking observation was that PKC-zeta was largely unaffected by even 5 uM Go 6983 (Figure 3.10). Neither of the atypical PKC isozymes were very efficiently inhibited by either Ro 31-8220 or Go6983. For PKC-zeta, only a slight inhibition was observed at the highest inhibitor concentrations tested, indicating that the IC50 values were well above the experimental concentrations used in the adipocyte studies. 72 Figure 3.10: Inhibition of PKC Isoforms by Ro 31-8220 and Go 6983 % PKC Activity (Relative to Control) a) PKC 125 1 | | i i 1 1 1 1 1 1 i c o - M h - - i ^ r I B -alpha b) PK 1251 1 1 1 1 1 1 | 1 1 i ico |3^^ = * c - z z -75 1 C-betal c) PKC-125 i i i i | i 1 , i i i 75 3^ •betall % PKC Activity (Relative to Control) 50 25 o l 1 1 1 1 1 1 1 1 1 1 9 8 •7 6 5 50 25 j o 1 M 1 I 1 1 1 1 ' 1 9 8 7 6 5 50 j - -25 o l 1 1 1 1 1 I I 1 1 1 9 8 llHtitfill 7 6 5 % PKC Activity (Relative to Control) -»-R>31^220 -•-G&6GB3 -»-Q>69B3 -»-R>3WI220 -»-Q>6BB3 % PKC Activity (Relative to Control) d) PKC 1251 | 1 | ] 1 1 1 1 1 1 75 "-gamma e) PKC 1251 i i i 1 i i 1 i i i 1O0 k = - | 5 r 75 >delta f) PKC 1251 i i i i i • i , , . , loo = = * - - § s -75 -epsilon % PKC Activity (Relative to Control) 50 25 o l 1 1 1 1 1 I 1 1 1 1 9 8 7 6 5 50 25 o l 1 I 1 1 1 1 1 1 1 1 9 S H 7 6 5 50 25 o l 1 1 1 1 1 1 1 1 1 1 1 9 8 7 6 5 % PKC Activity (Relative to Control) -•-RX31-8220 - * - Q > « 6 3 -•-F631-S220 -*-0>ea63 -*-RX31-8220 -^Q>6983 % PKC Activity (Relative to Control) g) PB 1251 | i | 1 1 1 1 1 1 I 1 100 | ^ g - - — 75 J B LC-eta h) PKC 1251 1 1 1 | | , | , , , , ico ! f c = p a c = = * 75 -^zeta i) PKC 1251 | j 1 1 1 1 | | j I I ioo»^=-5^- = «. 75 -iota % PKC Activity (Relative to Control) 50 25 o l 1 1 1 1 1 1 1 1 1 1 1 9 8 7 6 5 50 25 o 1 M 1 1 1 1 1 1 1 9 8 7 6 5 50 25 o 1 1 1 1 1 1 1 1 1 1 1 1 9 8 7 6 5 % PKC Activity (Relative to Control) -»-R>31-8220 -»-G>€983 -•-R>31-a220 -»-G>6E83 -»-R>31-8220 -1-Q>6SB3 % PKC Activity (Relative to Control) -log [inhibitor] PKC isoforms from the classical (a-d), novel (e-g), and atypical (h-i) subgroups were incubated with increasing concentrations of Ro 31-8220 or Go 6983 in the presence of [y-32P]-ATP and either histone HI (a-d) or epsilon peptide (e-i). 3 2 P incorporation was determined by Cerenkov counting and the results were expressed as a percentage of initial kinase activity (determined in the absence of inhibitors). Results representative of two experiments are shown. 73 Table 3.3: Summary of Inhibition of PKC Isoforms by Ro 31-8220 and Go 6983 (IC50 values in uM) PKC Isoform Ro 31-8220 Go 6983 a 0.100 0.112 PI 0.021 0.372 (3H 0.028 0.251 Y 0.229 0.708 5 0.246 0.282 s 0.204 0.263 0.100 0.191 c >5 >5 I no inhibition no inhibition The lack of PKC-zeta inhibition by Ro 31 -8220 in adipocytes was surprising, given recent reports demonstrating inhibition in rat adipocytes with IC50 = 4 uM as well as inhibition of the partially purified kinase with IC50 = 1 uM (Standaert et al., 1997). Go 6983 inhbition of partially purified PKC-zeta has also been previously demonstrated, with IC50 = 60 nM (Gschwendt et al., 1996). It has been demonstrated that many of the commonly used PKC inhibitors are much more effective against membrane-derived than cytosolic PKC (Budsworth and Gescher, 1995). The effect was most striking with Ro 31-8220, which inhibited cytosolic and membrane-derived PKC with IC50 values of 48 and 2.2 nM, respectively. No effort was made in that study to distinguish the activities of individual PKC isozymes. It seems, therefore, that inhibition of PKC-zeta by Ro 31-8220 and Go 6983 is dependent on some factor that was not duplicated in the assay in vitro. Inhibition may require a particular kinase conformation that occurs following membrane association in vivo. Given the report that Ro 31-8220 inhibits partially purified PKC-zeta 74 (IC5o = 1 uM) as well as PKC-zeta in adipocytes (IC50 = 4 uM), we must assume that the kinase was inhibited to some extent in the adipocyte experiments presented here, despite the lack of inhibition of purified PKC-zeta. The poor phosphorylation of ACC by PKC-zeta in vitro suggested that it was not a good substrate for the kinase. In addition, inhibiting PKC-zeta in rat adipocytes did not prevent activation of ACC by insulin, but rather was shown to accelerate the activation of the carboxylase. This is strong evidence that PKC-zeta is not the insulin-stimulated kinase which is involved in phosphorylation of the I-site and activation of ACC in adipose tissue. In contrast, PKC-zeta may be involved in the inhibition of ACC which delays insulin-mediated activation of the carboxylase. To better define the potential role of PKC-zeta in the inhibition of ACC activity in adipocytes, more selective and effective inhibition of this isozyme would be required. At present, potent and isozyme-specific inhibitors are not available. It is interesting that only PKC-betal and -betall isozymes display differential sensitivity to the PKC inhibitors. Both of these classical isoforms are more sensitive to Ro 31-8220 than to Go 6983. If the effects of Ro 31-8220 on ACC activation are a result of inhibition of specific PKC isozymes, PKC-betal and -betall would be likely candidates based on the inhibition profiles. This would suggest that these isozymes are involved in the inhibition of ACC activity, which would be consistent with the previous report suggesting that classical PKC isozymes phosphorylated and inhibited the carboxylase (Hardie et ah, 1988). The purified PKC-betal and -betall isozymes, however, do not appear to phosphorylate ACC directly (Figure 3.2). If these isoforms are involved in ACC inhibition, it is likely an indirect effect. 75 It is intriguing to note that PKD (PKC-mu) activation has been demonstrated to occur via a PKC-dependent mechanism, and is inhbited by Ro 31-8220. PKD is highly expressed in rat white adipose tissue (Figure 3.1), which presents this kinase as a potential link between PKC and ACC. Future experiments may explore this possibility. Another approach that could be taken to assess the role of PKC isozymes in ACC activation is the use of dominant-negative mutants. These mutant forms have been described for most PKC isozymes, and can be used in transfection studies to assess kinase function. Although transfection studies are generally performed in a tissue culture system, they can also be performed in adipocytes, where electroporation is an efficient transfection method. Alternately, the studies could be done in 3T3-L1 adipocytes, although these cells display many differences in kinase expression when compared to fat pads and adipocytes. It should also be noted that there are some difficulties associated with using dominant-negative mutants to assess the function of the different PKC isozymes. There is some convincing evidence that the mutant form of one isozyme can have an inhibitory effect on other isozymes. In particular, PKC-zeta mutants have been shown to inhibit both PKC-alpha and PKC-epsilon (Garcia-Paramio et al, 1998). It has also been demonstrated that the overexpression of one PKC isozyme can alter expression of other isozymes (Ways et al, 1995). These results suggest that there may be some interdependence between the different PKC isozymes with respect to expression and function. Thus the results of the transfection experiments should also be interpreted with caution. 76 3.4.2 Effect of PKC Inhibitors on Known ACC-Inhibitory Kinases 3.4.2.1 Inhibition of AMPK and PKA Based on the previous reports that Ro 31-8220 exerts some non-specific effects in cells, it was possible that the effect on insulin-mediated ACC activation exerted by this compound was attributable to inhibition of some kinase other than PKC. The effects of Ro 31-8220 and Go 6983 on two ACC-inhibitory kinases were examined. AMPK has been shown to phosphorylate ACC on Ser79, which strongly inhibits carboxylase activity (Davies et ah, 1990). ACC-1 is phosphorylated much more efficiently than ACC-2 by this kinase. In rat adipose tissue, where ACC-1 is the only isoform expressed, AMPK is probably the most relevant regulatory kinase. PKA preferentially phosphorylates ACC-2 though it does not appear to have a direct effect on carboxylase activity. This kinase has been implicated in regulation of ACC-2 in heart and skeletal muscle, which suggests its involvement in the regulation of fatty acid oxidation in these tissues. PKA phosphorylates ACC-1 much more slowly than AMPK. Both AMPK and PKA are inhibited by Ro 31-8220, with IC 5 0 values of 513 and 759 nM, respectively (Figure 3.11). These kinases are much less sensitive to Go 6983, which inhibits AMPK and PKA with IC50 values of 5 uM and 2 uM, respectively. Since the experimental concentrations used in the extracellular buffer were 5 uM Ro 31-8220 and 2 uM Go 6983, it is unlikely that Go 6983 was inhibiting AMPK and PKA in adipocytes. The concentration of Ro 31-8220 used experimentally, however, was sufficiently high to cause inhibition of both kinases. Although the IC50 values for PKA and AMPK were two to three times higher than seen for the PKC isozymes, it is difficult 77 to be confident that these compounds are truly selective in inhibiting the PKC isozymes. Effects on PKA and/or AMPK cannot be ruled out in intact cells. Since the effect of Ro 31-8220 on ACC activity was observed in rat adipocytes, AMPK is probably the more relevant regulatory kinase. It has been previously demonstrated that AMPK preferentially phosphorylates and inhibits ACC-1, the only isoform present in rat adipose tissue. There is considerable evidence that AMPK is the relevant kinase for inhibition of ACC activity in adipocytes in response to isoproterenol treatment. It has also been proposed that the delay in ACC activation following insulin stimulation is a result of phosphorylation and inhibition by AMPK (Witters and Kemp, 1992). 78 Figure 3.11: Inhibition of AMPK and PKA by Ro 31 -8220 and Go 6983 Inhibition of AMPK by Ro-31-8220 and Go-6983 - 125 •Ro-31-8220 • Go-6983 -log[inhib] 9 8 7 6 5 -log[inhib] C ) IC5o Values (nM 0 Ro-31-8220 Go 6983 PKA 759 4898 AMPK 513 2089 Purified AMPK (a) or PKA (b), incubated with increasing concentrations of Ro 31-8220 or Go 6983, was used to phosphorylate a peptide substrate in the presence of [y-32P]-ATP. The SAMS peptide and kemptide were used as substrates for AMPK and PKA, respectively. The 3 P incorporation into peptide was determined by Cerenkov counting and the results were expressed as a percentage of total kinase activity (determined in the absence of inhibitors). The concentrations required for 50% kinase inhibition (IC50) were determined graphically and are summarized for comparison (c). Results representative of two experiments are shown. 79 3.4.2.2 AMPK Activation in Adipocytes Previous studies have indicated that AMPK is inhibited in response to insulin stimulation and that this inhibition coincided with the activation of ACC (Witters and Kemp, 1992). The authors suggested that the delay in ACC activation following insulin stimulation was a result of inhibition by AMPK, and that the inhibition of this kinase was required to permit ACC activation. It is therefore tempting to speculate that Ro 31-8220 was acting on AMPK in the cell and that inhibition of the kinase was responsible for the more rapid activation of ACC. To assess this possibility, AMPK activity was assessed at various times following insulin stimulation of rat adipocytes. AMPK activity was also assessed in adipocytes treated with either isoproterenol (an AMPK activator) or with the inhibitor Ro 31-8220. In addition, the basal activity of ACC was assessed over time. Based on the report by Witters and Kemp (1992), insulin stimulation of adipocytes was expected to result in an inhibition of AMPK activity, with maximal inhibition occurring at 10 minutes. The kinase inhibition was expected to coincide with the beginning of ACC activation at approximately 20 minutes. To correlate this trend with the time-course of ACC activation seen in the present studies, AMPK and ACC activity were determined at 15 minute intervals from 15 to 60 minutes (Figure 3.12). 80 Figure 3.12: Time Course of AMPK Activity Following Insulin Stimulation AMPK Activity in Rat Adipocytes - - & - -+ Insulin — • — — Insulin A R o - 3 1 - 8 2 2 0 • I sopro te reno l Rat adipocytes were incubated with or without insulin for various times, as indicated. Two additional samples were incubated either with Ro 31-8220 or with isoproterenol, as indicated in the figure legend. Adipocyte extracts were prepared by homogenization in the presence of microcystin, followed by centrifugation at 12000 x g for 15 minutes to remove cellular debris. A 40 pi aliquot of extract was incubated with the SAMS peptide, in the presence of [y-32P]-ATP. 3 2P incorporation was determined by Cerenkov counting. Results of a single experiment are shown. The activity of AMPK increased with time in the absence of insulin, suggesting that AMPK was being activated under basal conditions. Treatment of the adipocytes with insulin, however, appeared to suppress AMPK activation. Therefore, rather than an inhibition of AMPK following insulin stimulation, a lack of activation was observed. As expected, isoproterenol treatment resulted in a strong activation of AMPK within fifteen minutes. Interestingly, treatment with Ro 31-8220 for 30 minutes did not result in AMPK inhibition, but rather a slight activation of the kinase. It would appear, therefore, that while Ro 31-8220 inhibits purified AMPK in vitro, it does not have this effect on the kinase in primary adipocytes. 81 If AMPK is phosphorylating and inhibiting ACC activity in adipocytes, then basal ACC activity should be decreasing with time, corresponding to the increase in AMPK activity observed. To assess this, the results of the adipocyte experiments were compiled to create a time course of basal ACC activity (Figure 3.13). Figure 3.13: Baseline ACC Activity in Adipocytes a) + 20 mM citrate — _ 1 o w E O c Z. 0.9 — o >. ••-> "g 0.8 fj o O 1 0 7 O u < ~~ 0.6 •CTRL •Insulin 20 40 time (min) 60 Adipocytes were incubated with or without insulin for 15 (n=5), 30 (n=3), or 60 (n=6) minutes, as indicated. Extracts were prepared by homogenization and clarified by centrifugation at 12000 x g. A 40 pi aliquot of extract was used to determine ACC activity either following pre-incubation with 20 mM citrate (a) or without preincubation (b). ACC activity was determined by C0 2 fixation from 14C-bicarbonate into malonyl-CoA. Total glycogen synthase activity was used as a measure of protein concentration in the extracts, and was used to correct for variations in protein concentration between adipocyte extracts. 82 From 15 to 60 minutes, ACC activity in unstimulated adipocytes remained reasonably constant. The activity was higher when ACC was preincubated with 20 mM citrate, as expected. The total activity was much lower when the enzyme was assayed without preincubation, but the trends observed were the same. Basal ACC activity remained constant from 15 to 60 minutes. Insulin treatment resulted in an increase in ACC activity over time, as previously discussed. Since AMPK activity increased with time in unstimulated adipocytes, but ACC activity remained constant, it appears that this kinase was not inhibiting ACC under the experimental conditions. In addition, treatment of adipocytes with Ro 31-8220 did not inhibit AMPK, but rather resulted in a slight stimulation of kinase activity. These results are inconsistent with the hypothesis that Ro 31-8220 inhibition of AMPK in adipocytes results in ACC activation, both because Ro 31-8220 stimulates AMPK and because AMPK is not inhibiting ACC in adipocytes. The effect of Ro 31-8220 on insulin-stimulated ACC activation in adipocytes must therefore be mediated by some other target of the compound. This is intriguing, as it implies that there is some other kinase which can inhibit ACC activity in adipocytes. 83 4.0 CONCLUSIONS Acetyl-CoA carboxylase exerts important control on the rate of de novo fatty acid biosynthesis. This enzyme is inhibited in response to catecholamines/glucagon and is activated by insulin treatment of cells. The inhibition of the enzyme has been well characterized, and is largely mediated by the phoshporylation of Ser-79 by AMPK in rat white adipose tissue. The mechanism of ACC activation by insulin is not as well characterized, but is associated with the phosphorylation of a specific serine residue, the T-site'. The kinase responsible for this phosphorylation has not been identified, but is believed to lie downstream of PI3K. One of the downstream targets of PI3K in insulin signaling is the atypical subgroup of PKC isozymes, PKC-zeta and PKC-iota. Of nine PKC isozymes tested, PKC-zeta was the most efficient ACC kinase in vitro. The stoichiometry of phosphorylation with this kinase was low, reaching a maximum of 0.024 moles of phosphate per mole of ACC-1 subunit. Given the limited understanding of the mechanism of atypical PKC activation, it was unclear whether ACC was simply a poor substrate for this kinase or whether the kinase was not fully activated in the in vitro assay. When immunoprecipitated kinase was used to phosphorylate ACC, no phosphorylation was observed. Since the immunoprecipitate did contain active atypical PKC, the lack of phosphorylation was attributed to a prevention of association of the kinase with ACC by the antibody complex. The PKC inhibitors Ro 31-8220 and Go 6983 were used to inhibit PKC isozymes in rat adipocytes, and the effect on ACC activity was determined. Ro 31-8220 accelerated the activation of ACC by insulin, indicating that ACC activation is normally delayed and that the delay is mediated by some target of Ro 31-8220. There was no 84 effect on ACC activity in adipocytes treated with Go 6983. The PKC isozymes display similar sensitivity to Ro 31-8220 and Go 6983, with the exception of PKC-betal and -betall which are more sensitive to Ro 31-8220 by an order of magnitude. This might implicate these PKC isozymes in ACC inhibition, and consequently in the acceleration of ACC activation by insulin in adipocytes treated with Ro 31-8220. The involvement of these isozymes in ACC inhibition is likely to be indirect, based on the poor phosphorylation of ACC in vitro. One potential explanation for the involvement of these isozymes is through activation of PKD. Activation of PKD occurs via a PKC-dependent mechanism, and is inhibited by Ro 31-8220. This kinase has not been tested for its ability to phosphorylate ACC. This would be a logical extension of the work presented here. Ro 31-8220 also inhibits PKA and AMPK in vitro. The latter kinase has been implicated in the inhibition of ACC in adipose tissue, and has been suggested to result in the delay in ACC activation following insulin stimulation. Although Ro 31-8220 inhibited AMPK efficiently in vitro, treatment of adipocytes with the compound resulted in a slight activation of the kinase. AMPK does not, therefore, appear to be involved in the delay in ACC activation in adipocytes. 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