UBC Theses and Dissertations

UBC Theses Logo

UBC Theses and Dissertations

Mechanisms of regulation of acetyl-CoA carboxylase Quayle, Katherine Amanda 1990

Your browser doesn't seem to have a PDF viewer, please download the PDF to view this item.

Item Metadata

Download

Media
831-UBC_1990_A1 Q39.pdf [ 10.05MB ]
Metadata
JSON: 831-1.0098784.json
JSON-LD: 831-1.0098784-ld.json
RDF/XML (Pretty): 831-1.0098784-rdf.xml
RDF/JSON: 831-1.0098784-rdf.json
Turtle: 831-1.0098784-turtle.txt
N-Triples: 831-1.0098784-rdf-ntriples.txt
Original Record: 831-1.0098784-source.json
Full Text
831-1.0098784-fulltext.txt
Citation
831-1.0098784.ris

Full Text

MECHANISMS OF REGULATION OF ACETYL-CoA CARBOXYLASE by K A T H E R I N E A M A N D A Q U A Y L E B.Sc(Hons) Heriot-Watt University, 1984 M.Sc, University of British Columbia, 1986 A THESIS S U B M I T T E D IN 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 T H E D E G R E E O F D O C T O R O F P H I L O S O P H Y in T H E F A C U L T Y O F G R A D U A T E STUDIES B I O C H E M I S T R Y We accept this thesis as conforming to the required standard T H E U N I V E R S I T Y O F BRITISH C O L U M B I A S E P T E M B E R , 1990 © K A T H E R I N E Q U A Y L E , 1990 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at The University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the Head of my Department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of Biochemistry The University of British Columbia 2075 Wesbrook Place Vancouver, Canada V 6 T 1W5 Date: September 1,1990 ABSTRACT: One of the major physiological responses to insulin secretion is the activation of lipogenesis in target tissues (principally fat and liver). As acetyl-CoA carboxylase (ACC) is the rate limiting enzyme in fatty acid synthesis, the mechanisms involved in the short term regulation of this enzyme represent a pertinent model system for detenmning elements involved in amplification of the signals produced in response to stimulation of cells with lipogenic and counter regulatory hormones. The regulation of mammalian A C C by hormones is a complex phenomenon involving interplay between allosteric and covalent mechanisms. While the effects of adrenaline and glucagon are well characterised, the mechanism of regulation by insulin has still to be defined and formed the focus for the work presented in this thesis. To study the role of phosphorylation in the response of A C C to insulin, the site-specific phosphorylation of the enzyme observed following exposure of intact cells to insulin has been reproduced in vitro. These studies for the first time describe the conditions required to achieve distribution of [32P] in vitro among sites of acetyl-C o A carboxylase, very similar to that seen after hormone treatment of intact cells and employing endogenous polyamine-sensitive kinase(s). No corresponding increase in catalytic activity was detected following phosphorylation, in vitro, of insulin directed phosphorylation sites on purified rat liver acetyl-CoA carboxylase in these studies. Subsequently, A C C was phosphorylated by an exogenous protein kinase from maturation activated sea-star oocytes which led to high stoichiometric incorporation of 3 2 P into the unique site (I-site) phosphorylated in intact cells in ii response to insulin (0.3 mol phosphate / mol 240,000 kD subunit). Again no change in A C C activity was observed following I-site phosphorylation. The peptide containing the I-site was separated from other tryptic phosphopeptides by reverse phase H P L C and then sequenced. Phosphorylation of serine 1186 was detenriined to be the major phosphorylation site of A C C in response to insulin. The amino acid sequence corresponding to the peptide containing Ser 1186 is located in the putative "hinge" region of A C Q which is some 300 amino acids towards the C-tenrunal of the biotin binding site (Lys-784). Subsequent re-evaluation of the kinetic properties of acetyl-CoA carboxylase during purification has led to the identification of a fraction containing low M r inhibitor(s) and an apparently novel protein activator present in rat liver. Affinity purified rat liver acetyl-CoA carboxylase can be activated 2-3 fold at physiological citrate concentrations (0.1-0.5mM) by the addition of the heat and pro tease-sensitive cytosolic protein. The A C C activator has been extensively purified (though not yet to homogeneity) from a 100,000 g supernatant fractions from rat liver extract, by a combination of ammonium sulphate precipitation, ion-exchange chromatography and gel filtration. From these results we concluded that the activator is a protein and the native molecular weight in solution is estimated to be approximately 75 kDaltons. A popular hypothesis regarding the short term regulation of A C C involves a phosphorylation-dephosphorylation mechanism resulting in inhibition and activation respectively. A number of experiments have been carried out in order to test the hypothesis that the activator preparation may contain protein phosphatase activity directed towards A C C . The results strongly suggest that under the assay conditions iii described for the expression of activation of catalytic activity of A C C , there is little or no apparent dephosphorylation. Indeed, the most purified preparations of A C C activator do not contain any detectable phosphatase activity towards the model substrates histone III-S and casein. The activation of A C C occurs rapidly, in a time dependent manner (within 20 min at 37°C) and involves protein-protein interaction which is antagonized by avidin. The interactions between A C C , avidin and activator protein suggest that the activator not only induces conformational change at the active site of A C C but may also bind in such a way as to be displaced (perhaps directly) by avidin. From the data presented it is concluded that this acetyl-CoA carboxylase activator protein represents a novel factor which may be involved in the short term regulation of A C C activity. iv T A B L E O F C O N T E N T S A B S T R A C T ii T A B L E O F C O N T E N T S v LIST O F T A B L E S viii LIST O F F I G U R E S ix LIST O F A B B R E V I A T I O N S xi A C K N O W L E D G E M E N T S xii C H A P T E R 1 I N T R O D U C T I O N 1 1.1 Insulin 1 1.1.1 Physiological Role 1 1.1.2 Insulin Structure 2 1.1.3 Insulin Secretion 3 1.2 The Insulin Receptor 4 1.2.1 Structure and Cellular Processing 4 1.2.2 Tyrosine Kinase Activity 7 1.3 Signalling Mechanisms Implicated in the Mechanism of Insulin Action. ...8 1.3.1 Overview 8 1.3.2 Membrane Hyperpolarisation 8 1.3.3 Calcium and Calmodulin 9 1.3.4 Mediators 10 1.3.5 Protein Phosphorylation 12 1.4 Model Systems used for Studying Insulin Action 17 1.4.1 Overview 17 1.4.2 Whole Animal Studies and Models of Insulin Resistance and Diabetes 17 1.4.3 Clinical Studies 18 1.4.4 Intact Tissue Models 19 1.4.5 Cell Culture Models 19 1.5 Acetyl-CoA Carboxylase 21 1.5.1 Overview 21 1.5.2 Structure and Metabolic Role of Acetyl-CoA Carboxylase 21 1.5.3 Regulation of Acetyl-CoA Carboxylase 25 1.6 Thesis Investigation 34 C H A P T E R 2 E X P E R I M E N T A L P R O C E D U R E S 37 2.1 M A T E R I A L S 37 2.2 M E T H O D S 37 2.2.1 Determination of Acetyl-CoA Carboxylase Activity 37 2.2.2 Purification of Acetyl-CoA Carboxylase from Rat Liver 38 2.2.3 Phosphorylation of Acetyl-CoA Carboxylase 39 2.2.4 SDS-Polyacrylamide Gel Electrophoresis 40 2.2.5 Phospho-Peptide Analysis 40 v Table of Contents 2.2.6 Protein Determination 41 2.2.7 Preparation of Rat Liver High Speed Supernatant 41 2.2.8 Preparation of a Low Molecular Weight Inhibitor of Acetyl-CoA Carboxylase 42 2.2.9 Assay of Acetyl-CoA Carboxylase Inhibitor Efficacy 42 2.2.10 Partial Purification of A C C by F P L C (preparation of M Q - A C C ) 43 2.2.11 Assay for Acetyl-CoA Carboxylase Activator 43 2.2.12 Partial Purification of Acetyl-CoA Carboxylase Activator 44 2.2.13 Other Methodology 45 C H A P T E R 3 SITE-SPECIFIC P H O S P H O R Y L A T I O N AS A M E C H A N I S M F O R T H E S H O R T T E R M R E G U L A T I O N O F A C E T Y L - C o A C A R B O X Y L A S E 46 3.1 R E S U L T S A N D DISCUSSION 46 3.1.1 Purification of Rat Liver Acetyl-CoA Carboxylase 46 3.1.2 Site-Specific Phosphorylation of Acetyl-CoA Carboxylase 54 3.1.3 Phosphorylation of Affinity-Purified Acetyl-CoA Carboxylase by Endogenous Protein Kinase 55 3.1.4 Phosphorylation of Acetyl-CoA Carboxylase which Leads to Major Incorporation of [32P] into the A-Peptides or B-Peptides 61 3.1.5 Effects of Site-Selective Phosphorylation on the Activity of Acetyl-CoA Carboxylase 65 3.2 S U M M A R Y 70 C H A P T E R 4 IDENTEFICATION O F A L O W M O L E C U L A R W E I G H T R E G U L A T O R O F A C E T Y L - C o A C A R B O X Y L A S E 77 4.1 R E S U L T S A N D DISCUSSION 77 4.1.1 Low Molecular Weight Effectors of Acetyl-CoA Carboxylase 77 4.1.2 Identification of a Low Molecular Weight Inhibitor of Acetyl-CoA Carboxylase 80 4.1.3 Characterization of the Low Molecular Weight Inhibitor of Acetyl-CoA Carboxylase 84 4.1.4 Investigations of Levels of Low Molecular Weight Acetyl-CoA carboxylase Inhibitor In Vivo 96 4.2 S U M M A R Y 104 C H A P T E R 5. IDENTIFICATION A N D C H A R A C T E R I S A T I O N O F A C O M P O N E N T O F R A T L I V E R C Y T O S O L T H A T A C T I V A T E S A F F I N I T Y PURIFIED A C E T Y L - C o A C A R B O X Y L A S E 107 5.1 R E S U L T S A N D DISCUSSION 107 5.1.1 Activation of Acetyl-CoA Carboxylase 107 5.1.2 Evidence for an Activator of Acetyl-CoA Carboxylase Activity in Rat Liver Cytosol 108 vi Table of Contents 5.1.3 Partial Purification of Acetyl-CoA Carboxylase Activator from rat liver I l l 5.1.4 Estimation of Molecular Size of the Acetyl-CoA Carboxylase Activator 114 5.1.5 Heat and Protease Sensitivity of the Acetyl-CoA Carboxylase Activator Preparation 116 5.1.6 Investigations into the Mechanism of Activation of Acetyl-CoA Carboxylase by the Novel Protein Activator 119 5.2 S U M M A R Y 129 C H A P T E R 6. I-SITE P H O S P H O R Y L A T I O N O F A C E T Y L - C o A C A R B O X Y L A S E 132 6.1 R E S U L T S A N D DISCUSSION 132 6.1.11-site Phosphorylation of A C C by a Fat Pad Kinase 132 6.1.2 Phosphorylation of Acetyl-CoA Carboxylase with Myelin Basic Protein Kinase 136 6.1.3 Amino Acid Sequencing of the I-peptide of Acetyl-CoA Carboxylase 136 6.2 S U M M A R Y 139 C O N C L U S I O N S 140 vii LIST OF TABLES Table 1. Purification of Acetyl-CoA Carboxylase from Rat Liver Cytosol 53 Table 2. Incorporation of [32P] into Sites on Acetyl-CoA Carboxylase by Different Protein Kinase Activities 62 Table 3. Effects of Site-specific Phosphorylation on the Catalytic Activity of Acetyl-CoA Carboxylase 69 Table 4. Effects of Rapid Gel Filtration on Acetyl-CoA carboxylase Activity in Extracts Prepared from Control and Insulin Treated Rat Adipose Tissue 79 Table 5. Extraction of the Acetyl-CoA Carboxylase Inhibitor Fraction with Organic Solvents 85 Table 6. Acid Treatment of the Acetyl-CoA Carboxylase inhibitor Fraction 88 Table 7. Treatment of Acetyl-CoA Carboxylase Inhibitor fraction with Acid Charcoal 89 Table 8. Effects of Glucose/Hexokinase and 5' Nucleotidase on Acetyl-C o A Carboxylase Inhibitor Fraction 90 Table 9. Effects of Proteases on the Acetyl-CoA Carboxylase Inhibitor Fraction 92 Table 10. Detection of Acetyl-CoA Carboxylase Inhibitor During the Diurnal Cycle 101 Table 11. Effects of Insulin Stimulation of Liver in vivo on Inhibitor Activity in Rat Liver Extracts 103 Table 12. Heat Sensitivity of Acetyl-CoA carboxylase Activator 117 Table 13. Protease Sensitivity of Acetyl-CoA Carboxylase Activator 118 viii LIST O F FIGURES Figure 1. Schematic representation of the insulin receptor in the plasma membrane 5 Figure 2. The reaction mechanism for formation of malonyl-CoA by acetyl-CoA carboxylase 22 Figure 3. Working model of the organisation of the catalytic domains of acetyl-CoA carboxylase 24 Figure 4. Phosphopeptides of acetyl-CoA carboxylase 32 Figure 5. Proteolysis of Acetyl-CoA Carboxylase 47 Figure 6. Affinity purified rat liver Acetyl-CoA Carboxylase 50 Figure 7. Purified Acetyl-CoA Carboxylase isolated from rat liver and adipose tissue 51 Figure 8. [32P] Phospho-peptides generated by the phosphorylation of acetyl-CoA carboxylase by endogenous protein kinase activity 56 Figure 9. Effect of Spermine on the endogenous phosphorylation of acetyl-CoA carboxylase 59 Figure 10. [32P] Phosphopeptides generated by the phosphorylation of acetyl-CoA carboxylase with protein kinase activity of rat liver fraction eluted from DEAE-cellulose at high salt (500 m M KC1) 63 Figure 11. [32P] Phosphopeptides generated by the phosphorylation of acetyl-CoA carboxylase by endogenous and cyclic A M P dependent protein kinases 64 Figure 12. Responses to citrate of three differently phosphorylated forms of acetyl-CoA carboxylase 67 Figure 13. Phosphorylation sites within acetyl-CoA carboxylase 71 Figure 14. Schematic representation of [32P] labelled phosphopeptides of A C C generated by tryptic digestion and resolved by two-dimensional thin layer mapping indicating the relative mobility of the A,B,C and I-sites 72 Figure 15. Activation of acetyl-CoA carboxylase by filtration 82 Figure 16. Inhibition of affinity purified acetyl-CoA carboxylase by the low molecular weight inhibitor 83 Figure 17. Effects of Magnesium and ultrafiltration on Acetyl-CoA carboxylase activity in high speed supernatants prepared from livers of starved rats 95 Figure 18. Specific activity of acetyl-CoA carboxylase during the diurnal cycle 98 ix List of Figures Figure 19. The citrate response of acetyl-CoA carboxylase during the diurnal cycle 100 Figure 20. Changes in the citrate sensitivity of acetyl-CoA carboxylase during purification 110 Figure 21. Steps utilized for the partial purification from rat liver of an activator of acetyl-CoA carboxylase 112 Figure 22. Gel filtration of rat liver acetyl-CoA carboxylase activator 114 Figure 23. Polyacrylamide gel electrophoresis of fractions generated during the partial purification of A C C activator 115 Figure 24. Time course of response of A C C to the activator enriched from rat liver cytosol 120 Figure 25. Co-migration of A C C activator with endogenous A C C during ion exchange chromatography 122 Figure 26. Absence of ACC-directed phosphatase activity in the A C C activator preparation 125 Figure 27. Quantification of phosphatase activity in the A C C activator preparation using [32P]-labelled casein and histone as substrates 126 Figure 28. Estimation of K m of Acetyl-CoA carboxylase for Acetyl-CoA 128 Figure 29. Fractionation of adipose tissue extracts by ion exchange chromatography 134 Figure 30. I-site phosphorylation of acetyl-CoA carboxylase 135 Figure 31. Amino acid sequence of the I-peptide following phosphorylation of A C C with sea star M B P kinase 138 x L I S T O F A B B R E V I A T I O N S . A C C Acetyl-CoA carboxylase IGF-I Insulin like growth factor-I IGF-II Insulin like growth factor-II E G F Epidermal growth factor P D G F Platelet derived growth factor Mr Apparent molecular mass A T P Adenosine triphopshate A D P Adenosine diphosphate A M P Adenosine monophosphate G T P Guanosine triphosphate PI Phosphatidyl inositol kD Kilo Daltons S D S - P A G E Sodium dodecyl sulphate-polyacrylamide gel electrophoresis M A P Microtubule associated protein H P L C High pressure liquid chromatography F P L C Fast protein liquid chromatography M Q Mono-Q C o A Coenzyme A Ca dep. P K Calcium dependent protein kinase C K I Casein kinase I C K n Casein kinase II P K C Protein kinase C C A K Cyclic AMP-dependent protein kinase A M P - P K Adenosine monophosphate dependent protein kinase Ser Serine Lys Lysine Thr Threonine MOPS Morpholinopropane sulphonic acid TRIS Tris(hydroxymethyl)aminomethane E D T A Ethylene diamine tetra acetic acid E G T A Ethylene glycol-bis-(9-aminoethyl ether) N,N,N',N'-tetra acetic acid PMSF Phenyl methyl-sulphonyl fluoride D E A E Diethylamino ethyl H M G 3-hydroxy-3 methylglutaryl-CoA xi A C K N O W L E D G E M E N T S First and foremost I would like to take this opportunity to thank Dr. Roger Brownsey for inviting me to Canada to study in his laboratory and so complete the requirements for graduating with a PhD degree from the University of British Columbia. I have appreciated his continual support and enthusiasm throughout this whole process. Secondly, I would like to acknowledge the others who have been involved in generating some of the results presented in this thesis: Dr. Steven Pelech, Dr. Jasbinder Sanghera and Dr. Reudi Aebersold, who were involved with the peptide sequencing, and Renata Oballa, a summer student in the lab who helped with some of the enzyme assays. My appreciation for the moral support and verbal abuse is extended to those members of the lab including John Lew and Bob Winz who contributed as they saw fit, on a day to day basis, depending on their mood. Others who have contributed to the "Canadian Experience" include; Pieter Cullis, for the endless supplies of beer, wild parties, ski trips, computer time etc., etc. and for entertainment and a good time, Tom Redelmeier, Helen Loughrey, Tom Madden, Linda Tai, Richard Harrigan, Neil Kitson, Michel LeFleur and Helene Cote. Last but least (Oh no, that's not right), my thanks to Richard Harrigan for his time spent editing and formating this thesis into a presentable form. This work was supported financially by grants from the Canadian Medical Research Council and B.C. Health Care. xii 1 1.1 INSULIN. 1.1.1 PHYSIOLOGICAL ROLE Imulin is a 5.8 kilodalton polypeptide hormone which plays a critical role in the regulation of intermediary metabolism (for a review, see Norman and Litwack, 1987). The major effects of insulin in target tissues (which in quantitative terms are primarily liver, muscle and adipose tissue) involves the stimulation of anabolism of carbohydrate, protein and fat, all of which result in promotion of glucose utilisation with subsequent regulation of blood sugar levels. In parallel, apparent rates of catabolic pathways are also coordinately regulated. Maintenance of a relatively constant blood sugar concentration is essential for life in higher vertebrates. Hyperglycaemia results in severe wasting of metabolic energy, osmotic diuresis and metabolic acidosis as well as long term effects on protein integrity involving non-enzymatic glycosylation. A reduction of blood glucose levels below the normal range on the other hand causes brain malfunction, convulsions and ultimately death. The pancreas plays a central role in the maintenance of a stable blood glucose concentration and responds to a variety of signals by secretion of insulin or glucagon as well as other polypeptides including somatostatin and amylin. Classically, glucose is itself considered a major factor in promoting insulin secretion, although the integrity of many factors is undoubtedly important (see below). Malfunction of insulin action can occur at a multitude of levels. For example within the pancreas itself, synthesis, storage and secretion of insulin may be affected (as in type I diabetes), as a result of factors which lead to an autcnimmune reaction directed against 0 -cells. At the level of target tissues insulin receptor number or binding capacity or indeed receptor function (e.g. as expressed by tyrosine kinase activity) may also be able to account for altered 2 insulin response. Finally disruption of the regulation of the metabolic pathways involved in a normal insulin response in target tissues either at the level of cell signalling or terminal metabolic enzymes may also be abnormal. The consequences of altered insulin availability or activity can be wide-ranging, resulting in pathogenesis of a variety of metabolic disorders including obesity, ageing and diabetes mellitus. Clearly a comprehensive understanding of the structure, function and mechanism of action of insulin is of central importance. 1.1.2 INSULIN STRUCTURE. Insulin was first identified in 1921 by the Canadian scientists Banting, Best and McCleod working in Toronto, who demonstrated that diabetic symptoms in dogs could be relieved by intravenous injection of pancreatic extracts which resulted in a lowering of elevated blood sugar levels. Insulin itself was subsequently isolated from these extracts and since its identification, has become one of the most extensively studied proteins to date. Insulin was the first protein to be completely sequenced at the amino acid level (Frederick Sanger, 1958) and in 1969 the three dimensional structure was determined by the pioneering work of Dorothy Crowfoot-Hodgkin, who, using X-ray crystallographic techniques, demonstrated the insulin molecule to be a compact structure with a non-polar core, stabilised by salt bridges, hydrogen bonds and two interchain disulphide bonds. In 1979 the insulin gene was cloned and sequenced (Rutter and Goodman) and it has become apparent subsequently from crystallographic studies of Blundell, Dodson and others that insulin belongs to a family of growth factors including relaxin, the insulin like growth factors IGF I and II, other somatomedins and nerve growth factor. In 1980, human insulin became the first recombinant protein to become available for clinical use. 3 1.1.3 INSULIN SECRETION. Insulin is synthesized, stored in and secreted from the p -cells of the pancreas. It is synthesized as a precursor, preproinsulin which is modified in the lumen of the endoplasmic reticulum to proinsulin. Proinsulin is transported to the Golgi and then to secretory granules, where proteolysis occurs resulting in the formation of the mature insulin molecule, composed of two polypeptide chains. The A-chain (21 amino acid residues) contains a single mtra-chain disulphide bridge and is joined by two disulphide bridges to the B-chain (30 amino acid residues). The rate of secretion of insulin is dependent on the extracellular glucose concentration which must be maintained between strict limits (80-110 mg/100 mL). The rate of insulin secretion is also influenced by amino acids and other hormones e.g. glucagon and the adrenergic hormones adrenaline and noradrenaline and by the parasympathetic nervous system. Quantitatively the major target tissues of insulin are the liver, muscle and adipose tissue although the majority of other vertebrate cells have receptors and may require insulin especially for longer-term maintenance (e.g. as shown by the requirement of cells for insulin to ensure long-term viability in culture). The cells of these major insulin target tissues have 20,000-100,000 insulin receptors of which only approximately 10% need be occupied by an insulin molecule to elicit a full physiological response. In liver, the major metabolic responses to insulin include stimulation of synthesis of glycogen, fatty acids and glycerolipids and suppression of gluconeogenesis. Uptake of glucose by muscle and adipose tissue and uptake of amino acids by muscle is accompanied by stimulation of protein synthesis and the synthesis of glycogen and triglyceride as well as suppression of adipose tissue lipolysis (for reviews, see Denton, 1987, Denton and Tavare, 1988,). The biological action of insulin is initiated by the binding of an insulin molecule to its specific cell surface receptor which results in rapid reversible changes in the catalytic activity of many key 4 enzymes involved in these metabolic processes. 12 THE INSULIN RECEPTOR. 1.2.1 STRUCTURE AND CELLULAR PROCESSING. The complete predicted arnino-acid sequence of the human insulin receptor (based on cDNA sequencing) was reported independently by two groups in 1985 (Ullrich et al. and Ebina et al.). The insulin receptor is an integral membrane glycoprotein (apparent molecular mass (Mr) of 350-400,000 KDaltons) containing 1370 amino acids and composed of two a-subunits (Mr 130,000) and two p-subunits (Mr 90,000). The receptor a and p subunits are held together by strong disulphide bridges to form a-p dimers and further, weaker disulphide bonds also hold thea-0 dimers together to form the native hetero-tetrameric structure. Both subunits are glycosylated, suggesting that parts of both are exposed on the extracellular surface of cells however studies involving cross-linking of radioactive insulin derivatives indicate that insulin binds predominantly to the a-subunit. The ^-subunit of the insulin receptor contains an ATP binding site and exhibits intrinsic tyrosine kinase activity which catalyses autophosphorylation of several tyrosine residues within the cytoplasmic domain (Kasuga et al., 1982a, 1982b, Roth and Cassell, 1983, Van Obberghen et al., 1983) and which is also able to phosphorylate tyrosine residues of exogenous substrates (Kasuga et al., 1983, Stadtmauer and Rosen, 1983). The organisation of the receptor in the membrane is depicted schematically in Figure 1. 5 FIGURE 1. SCHEMATIC REPRESENTATION OF THE INSULIN RECEPTOR IN THE PLASMA MEMBRANE. ln«ulin 135 KD Extracellular Ligand-binding Domain NH NH 2 2 NH 0 -S-S--s-s-Cy* Rich Domain NH -S-S-COOH COOH m mm* 95 KD Cytoplasmic Tyrosin* Kinas* Domain COOH COOH 6 In the late seventies, it became apparent from both morphological and biochemical evidence that termination of the signal generated by binding of insulin to the receptor at the plasma membrane was not occurring simply as a result of dissociation of the two components at the cell surface, but that the receptor/ligand complex was internalised within minutes of insulin binding (Bergeron et al., 1985). Receptor mediated endocytosis is now recognised as an important cellular mechanism involved in the transport of many ligands and receptors to their appropriate intracellular destinations (for review see Goldstein et al., 1985). Further, some authors argue that internalisation of insulin and/or the receptor may play a functional role in mediating at least some of the actions of insulin (Posner et al., 1980, Hedo and Simpson, 1984, Posner et al., 1988). After ligand binding, The insulin receptor is found to aggregate in clathrin coated pits which are distributed over the plasma membrane and subsequently internalised as coated vesicles by an energy dependent process. The vesicles are rapidly uncoated and fuse with endosomes, which comprise the compartment for uncoupling and sorting of receptor-ligand complexes. The acid interior of the endosomes facilitates the uncoupling of insulin from its receptor and sorting occurs allowing recycling of the insulin receptor back to the plasma membrane and delivery of insulin to the lysosomes for degradation (Stahl and Schwartz, 1986). This process occurs on a time scale of minutes, with products of insulin degradation seen in the medium within 4-5 min of insulin stimulation and free receptors recycled within 5-10 min. The proposed function of this process is two-fold, firstly to rapidly terminate the insulin signal by transfer of the ligand to the lysosomes for degradation, coupled with efficient recycling of the receptor itself back to the plasma membrane ready for further insulin stimulation and secondly, as a mechanism for facilitating signal transduction within the cell cytoplasm and intracellular organelles. Further investigations however are required in order to substantiate this latter hypothesis. 7 1.2.2 TYROSINE KINASE ACTIVITY The insulin receptor belongs to a (rapidly growing) family of protein tyrosine kinases which includes receptors for E G F , P D G F , and IGF-1, as well as the oncogenes which express protein tyrosine kinase activity. The oncogene v-ros shows the highest degree of homology with the insulin receptor although significant homology also exists with other src gene family members. One of the earliest events detected after insulin binds at the extracellular surface of cells is autophosphorylation of multiple tyrosine residues within the £-subunit of the receptor. This phosphorylation reaches steady state within 20s after the addition of insulin (White et al., 1985a) and serves to activate phosphotransferase activity allowing tyrosine phosphorylation of exogenous substrates. It is generally accepted that the autophosphorylation event is integral to insulin signalling as blocking expression of this activity either by site-directed mutagenesis (Ebina et al., 1987, Chou et al., 1987) or by the use of monoclonal antibodies (Morgan and Roth, 1987) directed against the appropriate domain results in abolition of the insulin response. Many other observations have been made which corroborate these findings including clinical studies which show that insulin stimulated tyrosine kinase activity is reduced by about 50% in patients with non-insulin-dependent diabetes mellitus (Fink and Freidenberg, 1988). However there are contrary findings which suggest that some caution may yet be required in defining the role of the insulin receptor kinase. For example the work of Goldfine and and co-workers have studied receptor kinetics with monoclonal antibodies, MA-5 and MA-20 which stimulate glucose transport but do not activate the tyrosine kinase of the/9 subunit (reviewed by Espinal, 1988). 8 1.3 SIGNALLING MECHANISMS IMPLICATED IN THE MECHANISM OF INSULIN ACTION. 1.3.1 OVERVIEW Although insulin was discovered as early as 1921, it is only comparatively recently that techniques have been available to allow studies of the pure biochemical nature of the signalling mechanism(s) involved in insulin action. A number of hypotheses have been postulated to account for the myriad of intracellular changes that occur within cells in response to insulin and yet to date there is still no consensus of opinion that goes unrefuted. As a consequence it is now generally conceded that multiple mechanisms are likely to be involved in signal transduction and that the integration of these mechanisms is very important for the coordination of an insulin response. In an attempt to give a flavour for the diversity of ideas and hypotheses that have been generated over the the years, some of the more popular themes are outlined below although it must be understood that this discussion is not intended to be an exhaustive review of all the observations which have been made over the years which contribute to our current understanding of insulin action and to a vast literature. 1.3.2 MEMBRANE HYPERPOLARISA TION One of the earliest detectable events that occurs subsequent to insulin binding to the insulin receptor and which is often neglected in discussions of mechanisms of hormonal signalling, is hyperpolarisation of the plasma membrane (reviewed by Zierler, 1988). The electrical potential across non-excitory cells ranges from -40 to -90mV. Insulin induced hyperpolarisation was first documented as occurring in skeletal muscle in 1957 (Zierler) and has subsequently been reported to occur in adipose tissue and adipocytes, (Cheng et al., 1981, Davis et al., 1981) in addition to a number of other tissues, where the response to insulin is to 9 stimulate glucose transport. The hyperpolarisation effect is of the order of 5-10 mV and can be measured within 500 msec after exposure to insulin. Hyperpolarisation is believed to be caused by a decrease in the permeability coefficients of both sodium and potassium ions, with a major effect on the permeability of the plasma membrane to sodium ions. Some evidence to support hyperpolarisation as the primary event in signal transduction includes the observations that inhibiting the depolarisation is correlated with a decrease in insulin stimulated glucose uptake and that electrically induced hyperpolarisation can cause increases in glucose transport in muscle. Although these observations give some insight into early events occurring after insulin binding, the mechanism by which this triggers other intracellular effects which occur after insulin treatment of cells is not clear. 1.3.3 CALCIUM AND CALMODULIN Calcium ions have been shown to play an important role in the action of a number of hormones and neural transmitters and the concentration of free ionised calcium in cell cytoplasm increases rapidly in response to stimulation of the phosphau^ylinositol (PI) cycle (Berridge, 1984). In light of this, many studies have been undertaken to address the possibility of calcium having an integral role in insulin signalling. Several observations have been made which give some indication that calcium and calmodulin (one of a family of calcium binding proteins which mediates the intracellular free calcium concentration) do indeed have a role in insulin action. Studies by Bonne et al., (1978) have demonstrated that extracellular calcium is required for maximal glucose uptake and (Desai et a l , 1978) for insulin binding. However there is a very large calcium gradient maintained across the plasma membrane of practically all cells and therefore it is not surprising that events involving interactions at the plasma membrane are sensitive to fluctuations in the calcium gradient which occur when extracellular 10 calcium concentrations are adjusted. In short, many cell functions are likely to be altered by varying extracellular calcium and a specific effect on insulin action is difficult to discern. There is considerable evidence for insulin effects on calcium flux within cells (M cCormack and Denton, 1984). However, changes in intracellular concentration of free ionized calcium can not be detected (Thomas et al., 1985). Better evidence for a role for calcium in signalling is the fact that an insulin dependent association of calmodulin with the plasma membrane has been demonstrated in adipocytes and that calcium and calmodulin can affect the extent of phosphorylation of the insulin receptor in response to insulin stimulation (Plewhe et al., 1983). Additionally calmodulin may become phosphorylated in response to insulin in adipocytes (Colca et a l , 1987) although this is disputed (Blackshear and Haupt, 1989). Studies in vitro using highly purified insulin receptors indicate that calmodulin is phosphorylated exclusively on tyrosine residues and that this phosphorylation alters the binding of calcium to calmodulin (Sacks et al., 1989). Evidence against a pertinent role for calcium in insulin action includes studies using the calcium sensitive probe quin 2, which is sensitive to changes in calcium concentrations in the nanomaolar to micromolar range. Insulin stimulation of cells loaded with quin 2 does not appear to induce any changes in intracellular free calcium levels, neither does insulin appear to antagonise effects of adrenergic agonists which do promote increases in intracellular calcium concentrations. 1.3.4 MEDIATORS Rail and Sutherland (1958), who determined the role of cyclic A M P in hormone signalling and signal amplification, were the first to describe the concept of a second messenger mechanism for signal transduction within the cell. In 1972, Lamer proposed a similar mechanism for insulin action, suggesting that insulin 11 promotes the release of low molecular weight intracellular mediators from the plasma membrane into the cytoplasm, which are then free to interact with and/or indirectly regulate the catalytic activity of the key rate limiting enzymes of the metabolic pathways affected during the insulin response. This model was based on several observations of enzymes which appear to be modulated by putative insulin mediators, including pyruvate dehydrogenase, glycogen synthase, cyclic-AMP dependent protein kinase and adenylate cyclase. It was argued that the regulation of these key enzymes would consequently lead to changes in the major overall metabolic pathways; lipogenesis, lipolysis and glycogen synthesis (for reviews, see Jarret, 1988, Schwartz et al., 1988). As a consequence of these observations, several laboratories have undertaken the task of identifying the second messenger(s) involved in insulin action. Owing to the difficulty of isolating and characterising low molecular weight effectors (and apparently difficulties in reproducing results in different laboratories) this has remained a controversial topic. More recently however these ideas have once again risen into the limelight, as Saltiel and Cuatrecasus (1986) described the partial purification and characterisation of an insulin mediator containing phosphatidyl inositol, glucosamine and other carbohydrates. This "PI glycan" is thought to be anchored in the plasma membrane by the phospholipid moiety, which is sensitive to cleavage by an insulin dependent phospholipase C (Farese et al., 1986, Goldman and Rybicki, 1986) the action of which would liberate diacylglycerol and inositol phosphate glycan (IPG). IPG is thus solubilised and available for interaction with cytoplasmic components in the cell. Subsequently, further evidence for the mechanism of action and characterisation of the mediator has failed to appear in the literature and perhaps this reflects once again, the difficulties involved with identifying low molecular weight effectors in complex cell systems. This remains a particularly intriguing area considering the established anchoring of proteins through phosphatidyl inositol glycan linkages (for reviews, see Low, 1987, 12 Ferguson, 1988) and the ability of insulin to promote release of Pl-glycan anchored proteins. 1.3.5 PROTEIN PHOSPHORYLATION Increased phosphorylation of the insulin receptor is observed within seconds of exposure of intact cells to insulin. (Kasuga et al., 1982a and b). Phosphorylation of the insulin receptor occurs both on tyrosine and serine residues, although tyrosine phosphorylation occurs much more rapidly and is the result of intramolecular autophosphorylation (Shia et al., 1983, Sweet et al., 1986). Autophosphorylation activates the tyrosine kinase activity of the insulin receptor towards exogenous substrates by more than an order of magnitude (Rosen et al., 1983). The insulin receptor protein kinase shows essentially absolute specificity for tyrosine residues as phosphate acceptor in a protein or peptide substrate (Wallcer et al., 1987). Several proteins thought to act as intracellular substrates for the insulin receptor tyrosine kinase activity have now been described in the literature (for review see Zick, 1988). Identification of substrates with a definable cellular function however has not been straightforward. One approach has been to look for tyrosine phosphorylation of proteins in intact tissue in response to insulin. A protein of Mr 185,000 (ppl85) was isolated from FaO cells using antiphosphotyrosine antibodies (White et al., 1985b) and was proposed to be a putative endogenous substrate for the insulin receptor tyrosine kinase. Subsequently phosphorylation of proteins of similar molecular mass, in response to activation of the insulin receptor tyrosine kinase have been observed in a number of studies (Gibbs et al., 1986, Kadowaki et al., 1987, Shemer et al., 1988, Momomura et al., 1988). Despite accumulating evidence supporting the initial proposal that ppl85 is an important physiological substrate for the insulin receptor tyrosine kinase, there is no evidence to support this hypothesis. Recently studies 13 have been undertaken to investigate phosphorylation of ppl85 in a cell-free system (Tashiro-Hashimoto et al., 1989), which will allow examination of the biological activity of this protein. A 120 kD glycoprotein ppl20 has been identified in rat liver plasma membrane preparations (Rees-Jones and Taylor, 1985). Phosphorylation has been demonstrated both in cell-free systems and in intact cells (Perrotti et al., 1986). This protein has been proposed as a possible specific substrate for the insulin and IGF-1 receptor tyrosine kinases (Kadowaki et al., 1987) and was recently identified as a plasma membrane ecto-ATPase (pp 120/HA4) (Margolis et al., 1990). Other proteins which have been isolated by immunoprecipitation using anti-phosphotyrosine antibodies include two 160 kD proteins. One of these is reportedly a glycoprotein (Yu et al., 1987) and the other a cytosolic protein (Madoff et al., 1988). Both have been proposed as physiological substrates for the insulin receptor tyrosine kinase, however this has still to be demonstrated. Several reports have proposed that calmodulin is a substrate for the insulin receptor tyrosine kinase in vivo in intact cells (Colca et al., 1987) and by plasma membrane preparations enriched for the insulin receptor in cell-free systems (Graves et al., 1986, Sacks and McDonald, 1988, Sacks et al., 1989). This has, however, been disputed (Blackshear and Haupt, 1989), in studies which have indicated that calmodulin phosphorylation is negligible and that insulin does not stimulate calmodulin phosphorylation under conditions which lead to increased phosphorylation of other proteins. Other proteins which have been identified as substrates for the insulin receptor tyrosine kinase include a 15 kD cytosolic protein (ppl5) which accumulates when 3T3-L1 adipocytes are treated with phenylarsineoxide (demonstrated to block glucose transport). This protein is thought to have an intermediary transient role in insulin-stimulated glucose uptake (Bernier et al. 1988) In addition to studies with intact cells several proteins and peptides have been screened as substrates for preparations of purified insulin receptors. These in 14 vitro insulin receptor substrates include tubulin, microtubule associated proteins, the progesterone receptor, GTP-binding proteins, casein and histones, angiotensin, a synthetic peptide containing the tyrosine phosphorylation site of pp60Src and many more (reviewed by Zick, 1988). Despite the intensive input in this field, the physiological substrates for the insulin receptor tyrosine kinase remain largely unidentified and so no clear evidence for signalling immediately beyond the receptor has so far emerged from these studies. Insulin has also been demonstrated to activate cyclic AMP independent serine-threonine protein kinases in a large number of different cell types and in turn this leads to increases in the phosphorylation state of several proteins integral to the anabolic pathways which are activated during the insulin response (Cobb and Rosen, 1983, Tabarini et al., 1985, Cobb, 1986, Nemenoff et al., 1986, Brownsey et al., 1984, Brownsey et al, 1988). In light of these observations, an attractive hypothesis for insulin induced signal transduction and amplification is phosphorylation of the receptor and activation of a phosphorylation cascade. This is analogous to that described for hormones which raise intracellular cyclic AMP concentrations and thus activate cyclic AMP-dependent protein kinase (which subsequently phosphorylates a number of proteins including other protein kinases thus regulating their activity and propagating the signal). Proteins which are phosphorylated in response to insulin include ACC, ribosomal protein S6, ATP citrate lyase and other proteins of apparent subunit Mr of 46,000 and 22,000 as estimated by SDS-PAGE. The role of phosphorylation of the latter two examples in response to hormones is poorly understood as their functions are unknown. Studies with ATP citrate lyase have demonstrated that phosphorylation occurs in response to both insulin and to hormones that increase cyclic AMP concentrations (Pierce et al., 1982). The phosphorylation in both cases is stoichiometric yet HPLC analysis of tryptic peptides coupled with peptide sequencing reveals that both hormones promote phosphorylation of the same serine residue. No striking 15 changes in catalytic activity have been found after phosphorylation of ATP-citrate lyase (Houston and Nimmo, 1985) and thus no regulatory role has been assigned to this covalent modification. On the other hand the phosphorylation of both S6 and A C C is associated with changes in catalytic activity (reviewed by Denton, 1986). Phosphorylation of these proteins occurs entirely on serine residues thus there is no direct link between these phosphorylation events and the activated tyrosine kinase activity expressed by the insulin receptor itself. The search for substrates for the insulin receptor tyrosine kinase has been carried out vigorously in many laboratories and although evidence for tyrosine phosphorylation of several proteins has now been described, very few with a known physiological role have been identified to date. One exciting possibility is one of the msulin-stimulated protein serine kinases designated the MAP2 kinase which is not only activated in response to insulin but also phosphorylated on tyrosine and threonine residues (Ray and Sturgill, 1988). It appears that tyrosine phosphorylation of the MAP2 kinase activates serine kinase activity of this protein towards a number of substrates, the most interesting being the ribosomal S6 kinase II (Sturgill et al., 1988) which in turn is activated in response to insulin and phosphorylates S6 (Lastick and M cConkey, 1981, Martin-Perez and Thomas, 1983, Gabriella et al., 1984) a ribosomal protein involved with the regulation of protein synthesis (Terao and Ogata, 1979, Traugh, 1981). Conclusive evidence for a central role for tyrosine phosphorylation as the link between the insulin receptor and activation of intracellular serine protein kinases however remains to be established. Furthermore, the number of discrete protein serine kinases which may be activated by insulin has not been fully elucidated. These objectives will require the further isolation and characterisation of insulin stimulated serine kinases Protein kinase C has been implicated by several groups (Graves and MacDonald, 1985, Walaas and Horn, 1986, Witters et al., 1986) as a key element in insulin signalling although the evidence to support this hypothesis however is 16 rather indirect. Protein kinase C is activated following stimulation of the PI cycle and this involves activation of phospholipase C in the plasma membrane (probably via a receptor linked/associated G-protein) and subsequent generation of diacylglycerol and inositol 1,4,5 tris-phosphate which facilitate calcium release from intracellular stores. Protein kinase C requires diacylglycerol, calcium and phospholipid, (preferably phosphatidyl serine) for activation (Nishizuka, 1984) and the increased concentration of diacylglycerol is most likely the trigger to activation. It has been convincingly demonstrated that insulin does indeed stimulate phospholipid metabolism in insulin sensitive tissues (Farese et a l , 1985, Pennington, 1985), however there is no concurrent accumulation of intermediates of the PI cycle or changes in intracellular free calcium concentrations. Other evidence presented to implicate a role for protein kinase C , has been based upon studies using phorbol esters which activate protein kinase C and induce insulin like effects on glucose transport and protein synthesis as well as causing phosphorylation of some of the proteins that are phosphorylated in response to stimulation by insulin (Kirsch et al., 1985, Trevillyan et al., 1985). There is however no direct evidence for assuming a correlation between the mechanism of action of phorbol esters and that of msulin, in fact in adipocytes insulin has been shown to be as effective after prolonged treatment of the cells with phorbol esters, which results in chronic down regulation of protein kinase C (Blackshear et al., 1985, Blackshear, 1986). Further, the effects of insulin directly on protein kinase C activity have not been uniformly confirmed (i.e. in a number of studies insulin was not found to activate or cause translocation) (Rodriguez-Pena and Rozengurt, 1984, Blackshear et al., 1985). A regulatory role for protein kinase C (e.g. by feedback on the insulin receptor) or a transient role however is difficult to rule out. 17 1.4 M O D E L SYSTEMS USED F O R STUDYING INSULIN A C T I O N . 1.4.1 OVERVIEW As the metabolic effects of insulin are so diverse, there are many cell components which are affected as part of the insulin response and which must be considered if a valid hypothesis for the mechanism of insulin action is to be established. The major metabolic pathways which are activated in insulin sensitive tissue are glycogen synthesis, lipogenesis and protein synthesis. The key proteins for which evidence indicates regulation in response to insulin in these anabolic pathways are the glucose transporter itself, glycogen synthase, pyruvate dehydrogenase, A C C and the ribosomal complex probably at least in part, via the S6 protein. In addition to these effects, the catabolic pathways are switched off implicating regulation of many other proteins which facilitate the insulin response. In order to simplify the problem, model systems are selected which will allow characterisation of the regulatory mechanisms within a single metabolic pathway, by a combination of in vivo and in vitro techniques and definition of conditions which control for as many of the variables as possible. There are four main categories of model systems that are currently employed for studying insulin action; i) whole animal models, ii) clinical studies iii) intact tissue models, iv) cell culture models. The advantages and limitations of these methods are outlined below. 1.4.2 WHOLE ANIMAL STUDIES AND MODELS OF INSULIN  RESISTANCE AND DIABETES Whole animal models have been used to address the physiological and biochemical impairments of insulin action which result in a variety of disease states. The two agents most widely used to induce diabetic symptoms in animal models are streptozotocin and alloxan which chemically induce a disease state by selective cytotoxicity of the p -cells of the pancreas (Chang and Dianni, 1976, 18 Kobayashi and Olefsky, 1979). Insulin production is reduced to approximately half the normal level after treatment with streptozotocin. This method has been used effectively in a number of laboratories and is generally accepted as a representative model of the insulin-dependent diabetic state. There are however some disadvantages to this technique, namely that all animals do not develop disease symptoms at the same rate indicating that some animals are at least partially resistant to the toxin and may maintain some functional p -cells to allow adequate production of insulin. It may therefore be difficult to determine if all animals in the sample are responding similarly to subsequent treatments. In addition to these insulin-dependent diabetic animal models, there are several techniques used for rendering animals obese and insulin resistant (Le Marchand et al., 1978, Le Marchand et al, 1979). One method for example involves injection of young mice with gold thioglucose. Following this treatment, the animals become hyperphagic, obese and insulin resistant. Alternatively there are genetically selected animal models available which include the insulin resistant C57B16J obese ob/ob mouse, the insulin hyper-responsive obese Zucker fa/fa rat and the non-obese diabetic strain (NOD) of mice. With the advent of transgenic technology, it is likely that many new animal models will become available in the future. 1.4.3 CLINICAL STUDIES A n understanding of the pathophysiology of diabetes can only be gained by applying to clinical situations the basic biochemical knowledge attained from studying model systems. In this regard a variety of observations have been made in studying patients suffering from non-insulin-dependent diabetes mellitus (NIDDM) and obesity since both conditions are characterised by insulin resistance (Kolterman et al., 1980, Kolterman et al., 1981, Kashiwagi et al., 1983, Lonnroth et al., 1983). Analysis of the dose response curves for insulin action together with 19 glucose disposal rates and measurements of insulin binding in these patients, can serve to indicate physiological significance of the binding and post-binding receptor defects in insulin action (Fink and Freidenberg, 1988). Recent developments in this field have led to new diagnostic procedures and a better understanding of the impact of insulin ao^ministration for patients with Type II diabetes mellitus. 1.4.4 INTACT TISSUE MODELS. The four major target tissues of insulin in mammals are liver, muscle, white adipose tissue and brown adipose tissue. Muscle and white fat are particularly convenient in that these tissues can be removed intact and incubated in appropriate oxygenated buffer systems that maintain the cells in a viable state for several hours. As insulin effects are manifested within tens of minutes after exposure to the hormone, this system can be used to study a number of biochemical effects of insulin action. One of the major advantages of this technique is that tissue integrity is maintained with minimal cell disruption. Intact tissue models have been used successfully to study glucose uptake, lipogenesis and glycogen synthesis in addition to the regulation of specific enzyme activities involved in these pathways. This technique has provided an important link between in vitro and in vivo systems. For liver and brown adipose tissue (as well as white adipose tissue) extensive studies have been possible with isolated cells released from intact tissue by digestion with collagenase. This results in preparations of homogeneous cells with no stromovascular cells but the receptors may be damaged and the yield diminished. 1.4.5 CELL CULTURE MODELS The 3T3-L1 cell line represented the first cultured cell line to be used for studying insulin action (Green and Kehinde, 1974). These primary fibroblasts can 20 be induced to differentiate into adipocyte-like cells and exhibit insulin responsiveness similar to that seen with primary adipocytes. As culture techniques have developed, insulin action has subsequently been studied in a wide variety of cell types maintained in culture, including endothelial cells, microvascular cells, cells derived from nervous tissue and hepatocytes (for reviews see Pederson et al., 1988, Bar and Sadra, 1988, Heidenreich, 1988). As the liver plays a central role in maintaining glucose homeostasis and insulin is the major anabolic stimulus for this organ, cell culture has proved very useful for studying the mechanisms involved in the insulin response in this organ. Techniques which have been used very successfully include intact hepatocytes maintained in primary culture to demonstrate insulin/receptor complex internalisation (Fehlmann et al, 1982), regulation of insulin binding and receptor tyrosine kinase activity (Lerea and Livingston, 1988). Hepatocytes have also been used to investigate regulation of key enzymes in glycogen synthesis and in lipogenesis (Ichihara et al., 1982), in addition to the effects of insulin on protein synthesis (Wettenhall et al, 1982). Several hepatoma cell lines have been exploited for the study of insulin action, including H4-II-E-C3 cells which are derived from the Reuber H35 rat hepatoma (Pitot et al., 1964) Fao cells (Deschatrette and Weiss, 1974) and HepG2 cells (Knowles et al., 1980). These cell lines possess a large number of insulin receptors and are extremely sensitive to the hormone, yet maintain many functions similar to normal hepatocytes. Cell culture has therefore provided an alternative approach to the use of intact tissue, which for organs like the liver has considerable limitations in that it is impossible to maintain the liver, as a whole, in vitro other than for brief periods of perfusion, ideally with whole blood which necessitates the additional cost of several donor animals for each perfusion (Topping and Mayes, 1972). 21 1.5 ACETYL-CoA CARBOXYLASE. 1.5.1 OVERVIEW. The regulation of acetyl-CoA carboxylase (ACC), the rate limiting enzyme of fatty acid synthesis, has been studied in many laboratories since among many intriguing facets it offers a model for establishing the components involved in hormonal signal transduction. The data presented in this thesis and the subsequent discussion of current ideas about the mechanism of insulin action is based on studies of A C C activity in both liver and adipose tissue. 1.5.2 STRUCTURE AND METABOLIC ROLE OF ACETYL-COA  CARBOXYLASE. Acetyl-CoA Carboxylase (E.C. 6.4.1.2.) catalyses the reaction which commits acetyl- carbon towards the synthesis of long chain fatty acids and is subject to complex short-term control involving allosteric regulation as well as covalent control via phosphorylation at multiple serine residues (for recent reviews see Kim et al., 1989, Hardie, 1989, Brownsey and Denton, 1987, Numa and Tanabe, 1984). The reaction catalysed by A C C involves the Mg-ATP dependent carboxylation of acetyl-CoA to malonyl-CoA The generally accepted reaction mechanism is described by a two-site "ping-pong" model (Fig. 2.). 22 FIGURE 2. THE REACTION MECHANISM FOR FORMATION OF MALONYL-CoA BY ACETYL-CoA CARBOXYLASE. ATP A V HCO A V 3 ADP A V Pi A Acetyl CoA A Malonyl A CoA nz—Biotin Enz—Biotin —CO 2 Enz—Biotin 23 This reaction is recognised to play a highly significant role in the regulation of fatty acid synthesis, for several reasons: i) malonyl-CoA has no apparent metabolic fate other than as a substrate for fatty acid synthetase, which catalyses the formation of palmitoyl-CoA; ii) rates of fatty acid synthesis are proportional to the cellular concentration of malonyl-CoA; iii) the reaction is displaced far from equilibrium implicating this step as rate-limiting; iv) the enzyme is acutely regulated in response to hormones and in addition is also subject to longer-term regulation through turn-over involving protein synthesis and degradation. The predicted subunit molecular weight of ACC, based upon cDNA sequencing of the rat mammary enzyme (Lopez-Casillas et al., 1988) is calculated to be 265,220 and the enzyme consists of 2,345 amino acid residues. Sequence data for the chicken liver enzyme has also been reported (Takai, 1988) and the two sequences share a high degree of homology. The gene for A C C has been localised to human chromosome 17 (Milatovich et al., 1988). Under the range of "normal" physiological conditions, the half life of A C C is 36-48 hr (Numa and Yamashito, 1974). This enzyme is regulated at the transcriptional level by various nutritional and developmental signals. Recent studies by Kim and co-workers have shown that multiple forms of A C C mRNA occur in rat liver and mammary tissue and that unique message levels are increased under lipogenic conditions (Lopez-Casillas and Kim, 1989). A working model for the catalytic domains of the enzyme has been proposed based on homologies with propionyl CoA carboxylase and carbamoyl phosphate synthetase (Lopez-Cassilas et al., 1988) and is shown in Figure 3. Potential regulatory regions containing serines which can be reversibly phosphorylated are found in the N-terminus and the hypothetical "hinge" region which is located between the two catalytic domains and is adjacent to the domain containing the biotin binding site. 24 F I G U R E 3. W O R K I N G M O D E L O F T H E ORGANISATION O F T H E C A T A L Y T I C DOMAINS O F A C E T Y L - C o A C A R B O X Y L A S E . The symbols (^) represent reversible phosphorylation sites of A C C and the numbers indicate the corresponding serine residue location within the primary sequence based on the complete ammo acid sequence of rat mammary gland A C C . 2 3 2 5 2 9 3 4 7 7 7 9 9 5 N H . I I I 1 • • » • i i i T i B i o t i n B ind ing S i t * 1 2 0 0 1 2 1 5 I I • • COOH N - T « r m i n a l B i o t i n C a r b o x y l a s e H ing« Reg ion C a r b o x y l T r a n s f e r a s e C - T e r m i n a l 25 1.5.3 REGULATION OF ACETYL CoA CARBOXYLASE 1.5.3.1 Allosteric Regulation.  1.5.3.1.1 Citrate A C C is allosterically regulated by the tricarboxylic acid citrate (and to a lesser extent isocitrate). Exposure of purified A C C to citrate promotes aggregation of A C C from inactive dimers into active filamentous polymers (for reviews see Lane et al., 1974, Volpe and Vagelos, 1976, Kim, 1983). The polymerised form of A C C appears to reach a maximum of approximately twenty dimers corresponding to a sedimentation coefficient of 30-60 S. Other studies have shown that stable polymerised forms of A C C of intermediate size may also exist (Lee and Kim, 1978, Ahmad et al., 1978, Swenson and Porter, 1985). These intermediate species appear to have submaximal enzyme activity in vitro (Lent et al., 1978) and the exact relationship between activity and polymeric state is not clear. Studies involving digitonin permeabilisation of liver cells indicated that the rate of elution of A C C was slower in the presence of citrate and agents that promote polymerisation (Meredith and Lane, 1978,) however the rate did not change in response to insulin treatment (Witters, 1985), suggesting any effects of insulin on polymerisation do not persist under these conditions and may not occur in vivo. More recently, studies involving rapid separation of A C C by size exclusion chromatography using the F P L C system have demonstrated that exposure of rat epididymal adipose tissue to insulin caused an increase in M r of A C C indicative of polymerisation (Borthwick et al., 1987). As there is no evidence supporting the hypothesis that the intracellular concentration of citrate increases in response to insulin (Denton and Halperin, 1968, Saggerson and Greenbaum, 1970, Brownsey et al., 1977), it is possible the polymerisation results from interactions of A C C with 26 other cytosolic effectors or from covalent modifications which alter the sensitivity of A C C to citrate, allowing polymerisation to occur at physiological citrate concentrations (0.1-0.5mM). Given the evidence described above it seems unlikely that effects of insulin on A C C activity are brought about simply as a result of interaction with the allosteric activator, citrate. 1.5.3.1.2 Fatty Acvl-CoA Esters. In addition to allosteric regulation by citrate, A C C interacts in a similar fashion with micromolar concentrations of long chain acyl-CoA esters which promote depolymerisation of the enzyme and thus render it inactive (Numa and Tanabe, 1984). It is very difficult to estimate the intracellular concentration of the free form of long chain acyl-CoA esters as the majority of the cytosolic acyl-CoA esters are complexed with proteins including fatty acid binding proteins and perhaps partitioned into subcellular membrane fractions. A n alternative approach to address the possibility of acyl-CoA being involved in the regulation of A C C in response to insulin has been described by Brownsey et al (1988). Inhibition of A C C with additions of palmitoyl-CoA were effectively reversed by subsequent treatment with Lipidex (Lipidex is a lipophilic Sephadex which has been used to delipidate serum albumin and fatty acid binding proteins). Treatment of extracts prepared from control and insulin treated tissue with Lipidex however does not abolish the insulin induced activation of A C C . This strongly suggests that the effects of insulin are not due to differences in residual allosteric interaction of long chain acyl-CoA esters with A C C . 1.5.3.2 Covalent Modification. The earliest indication that A C C was a potential candidate for reversible phosphorylation came about by the discovery that the purified enzyme contains two moles of inorganic phosphate (Inoue and Lowenstein., 1972). Subsequently in 27 1973, Ki-Han Kim and co-workers reported that A C C became inactivated after incubation of a crude homogenate with Mg-ATP and that the reduction of catalytic activity of A C C after this treatment may be explained by phosphorylation. Covalent modification as a mechanism for short term regulation of A C C became an area of great interest and it was demonstrated that A C C was also phosphorylated within intact fat cells (Brownsey et al., 1977) and liver cells (Pekala et al., 1978, Witters et al., 1979), indeed in fat cells A C C is one of the major cell phosphoproteins. Studies with mammary enzyme have shown that A C C is phosphorylated at multiple sites (Hardie and Cohen, 1978a, 1978b, Hardie and Guy, 1980) and within intact cells the degree of phosphorylation is increased in response to insulin as well as to hormones that raise the intracellular concentration of cyclic A M P (Witters, 1981, Brownsey and Denton, 1982, Witters et al., 1983, Sim and Hardie, 1988). Two-dimensional analysis of tryptic phosphopeptides generated from A C C immunoprecipitated from intact tissue of control and hormone treated samples resulted in different phosphopeptide patterns. In initial studies these different phosphopeptides were designated by letter or number codes. Thus phosphorylation of " C or "control" sites occurs in the absence of hormones, phosphorylation induced by glucagon occurs at control and "A-sites" and that induced by insulin at control and the "I-site". From these observations, it was proposed that site-specific phosphorylation of A C C on different serine residues within the molecule could regulate the catalytic activity of the enzyme (Brownsey and Denton, 1982). A considerable effort has been directed towards defining the role of phosphorylation of A C C in the responses of this enzyme to hormones. 1.5.3.2.1 Intact Cell Studies. Following exposure to glucagon, catecholamines or insulin, modest increases in the overall phosphorylation of A C C is observed within intact fat and 28 liver cells. The increases in phosphorylation of A C C occur within 5-10 min of hormonal stimulation in fat and liver cells and rises by 10-40% following insulin treatment and by 18-60% with adrenaline or glucagon (Brownsey et al., 1977, Brownsey et al., 1979, Witters et al., 1979, Brownsey, 1981, Witters, 1982, Holland et al., 1985). The activation of A C C in response to insulin and the inhibition in response to glucagon or adrenaline are associated with increased phosphorylation of different sites (for reviews see Brownsey and Denton, 1987, Hardie, 1989, Kim et al., 1989). Studies of Denton and Brownsey showed that phosphorylation of control (or C-sites) is observed in the absence of hormones and hormone treatment had no apparent effect on the extent of phosphorylation of these sites. Incorporation into the A-sites is increased approximately two fold by epinephrine and incorporation into the I-site is increased approximately five fold by insulin. Phosphopeptide analysis by reverse phase chromatography on an HPLC system (Witters et al., 1983, Holland and Hardie, 1985) or two-dimensional mapping of tryptic digests of [32P]-labelled A C C have given similar but not identical results. The effects of insulin on peptide phosphorylation were smaller if A C C was isolated by avidin-Sepharose affinity chromatography prior to HPLC analysis (Witters, 1983, Holland and Hardie, 1985) than by immunoprecipitation prior to two-dimensional analysis (Brownsey and Denton, 1982). This may reflect differences in recovery achieved by the two methods, perhaps due to selectivity of avidin-Sepharose for different phosphorylated forms of the enzyme. Thus the recovery by immunoprecipitation was better than 90%, but employing avidin-Sepharose less than 30%. More extensive analysis of sites phosphorylated on A C Q including amino acid sequencing of phosphopeptides is described in detail below. 1.5.3.2.2 Studies with Protein Kinases The effects on the phosphorylation of A C C of increasing intra-cellular cyclic AMP concentrations within intact cells appears to be reproduced in vitro by 29 incubation of purified A C C with the catalytic subunit of cyclic AMP-dependent protein kinase. A number of studies have demonstrated parallel phosphorylation and inactivation of A C C (Hardie and Guy, 1980, Witters et al., 1983, Holland et al., 1984, Holland and Hardie, 1985) upon incubation with cyclic AMP-dependent protein kinase and the sites phosphorylated in vitro with purified preparations appeared to be very similar to the sites phosphorylated within intact cells exposed to adrenaline or glucagon (Brownsey and Hardie, 1980, Witters et al., 1983, Holland et al., 1984). In addition to the effects of cyclic AMP-dependent protein kinase, initial studies indicated at least two different cyclic AMP-independent protein kinases were able to phosphorylate and bring about inhibition of A C C (Hardie and Cohen, 1978, Lent et al., 1978, Lent and Kim, 1982). Phosphorylation by a number of other protein kinase preparations including casein kinase I, casein kinase II (Witters et al., 1983, Munday and Hardie, 1984, Tipper et al., 1983) and calcium dependent protein kinases (Hardie et al., 1986) appears not to be associated with any apparent alteration in catalytic properties of ACC. The enzyme has also been reported to be a poor substrate for protein kinase C, glycogen synthase kinase 3 and for phosphorylase kinase (Witters, 1985). More recently, phosphorylation of A C C by the AMP-dependent protein kinase which regulates several key enzymes of lipid metabolism (Hardie et al., 1989) has been extensively characterised (Davies et al., 1989,1990). A comprehensive analysis of phosphorylation of A C C has involved amino acid sequencing by Hardie and co-workers (Munday et al., 1988, Haystead and Hardie, 1988, Haystead et al., 1988). The latest peptide sequencing studies appear to have confirmed early suspicions (Lent and Kim, 1983) that effects of cyclic AMP-dependent protein kinase on A C C are indirect. Thus it appears that AMP-directed protein kinase is responsible for phosphorylation of Ser 79 of A C C in response to glucagon (Sim and Hardie, 1988). In one report (Witters et al., 1988) insulin was found to induce dephosphorylation of A C C in a hepatoma cell line, 30 although one may speculate that this is likely to involve the reversal of the effects of cyclic A M P dependent and 5-AMP-dependent protein kinases. More recently, further data has been presented to support the hypothesis that activation of rat liver A C C in response to insulin occurs as a result of dephosphorylation of the enzyme (Mabrouk et al., 1990). However the insulin induced activation of A C C in these experiments is not abolished by exposure to phosphatase 2 and therefore a more complex response to insulin appears likely. Interestingly these results are attained from A C C purified by avidin-Sepharose affinity chromatography and indicate that the activation of A C C is retained through this purification step. This would conflict with previous data of Witters et al., (1983). A n intriguing possibility proposed by Sommercorn and Krebs (1987) is that insulin may induce a transient activation of casein kinase II resulting in phosphorylation of A C C followed by dephosphorylation of the enzyme due to site-site interactions which would induce increased accessibility to protein phosphatases following the action of C K II. Nevertheless, in normal fat and liver cells, insulin added as the sole hormone does not apparently induce site-specific dephosphorylation (Brownsey and Denton, 1987, Brownsey, 1981, Witters 1982, Brownsey and Denton, 1982). 1.5.3.2.3 Studies with Protein Phosphatases. Several different protein phosphatase preparations have been described which are able to activate A C C (Hardie and Cohen, 1979, Krakower and Kim, 1981, Wada and Tanabe, 1983, Thampy and Wakil, 1985). Intracellular protein phosphatase activity has been thoroughly characterised by Cohen and co-workers (Ingebritsen and Cohen, 1983, Cohen, 1988). Apparently four protein phosphatases account for the majority of intracellular serine/threonine phosphatase activity and these are designated phosphatases 1, 2 A 2B and 2C. It is likely that the A C C protein phosphatase preparations described previously contain one or more of these phosphatase activities. Protein phosphatase 1 has been 31 shown to dephosphorylate A C C in vitro (Hardie and Guy, 1980) however neither inhibitor 1 or 2 (cytosolic proteins found to complex with phosphatase 1 and regulate its activity) blocked the effect of phosphatase activity in crude fractions prepared from rat liver extracts (Ingebritsen et al., 1983). This suggests phosphatase 1 is not causing dephosphorylation under these conditions. Phosphatase 2A activity is reportedly stimulated by M g 2 + and M n 2 + (Witters and Bacon, 1985, Thampy and Wakil, 1985) and dephosphorylates sites on A C C which are phosphorylated in response to casein kinases I and n. The A C C phosphatase activities in extracts of adipose and mammary tissue are activated by M g 2 + and C a 2 + (Brownsey et al., 1979, McNeillie et al., 1981) and co-migrate with the phosphatases 2A and 2C in liver (Ingebritsen et al., 1983). There do not appear to be any ACC-specific protein phosphatases and the role of dephosphorylation in regulation of A C C in response to insulin remains controversial. 1.5.3.2.4 Characterisation of site-specific phosphorylation of A C C by  identification of specific phosphorylated serine residues. A considerable body of work (principally by Hardie and co-workers) has led to a rather complete documentation of the various phosphorylation sites on A C C . A compilation of the data available from studies with chymotryptic and tryptic peptides separated by either reverse phase H P L C chromatography or two-dimensional thin layer analysis and including the amino acid sequence data of the phosphopeptides is presented in Figure 4. Ser 95 is not identified on the two-dimensional map and sequences for the I and B peptides are not known. It is not clear if the double spot seen in the A * position is a result of phosphorylation of either Ser 77 or Ser 79, or if partial oxidation of the peptide occurs during the analysis. 32 FIGURE 4. PHOSPHOPEPTIDES OF ACETYL-CoA CARBOXYLASE. Panel a) shows the amino acid sequences for tryptic phosphopeptides of ACC resolved by reverse phase HPLC. Serine residues which are phosphorylated in vitro by a variety of protein kinases are indicated. The numbers refer to the amino acid location within the primary sequence of ACC based on the complete amino acid sequence of rat mammary ACC. Panel b) shows phosphopeptides of ACC resolved by two-dimensional thin layer mapping on cellulose. The numbers indicate the location of serine residues within the primary amino acid sequence based on the deduced amino acid sequence of the rat mammary enzyme. A: sites phosphorylated in intact cells in response to hormones that elevate the concentration of intracellular cyclic AMP. B: sites phosphorylated in vitro by cytosolic rat liver protein kinase activity. C: sites phosphorylated in intact cells in control and hormone treated samples. I: site phosphorylated in intact cells in response to insulin. 23 29 28 34 F I I G S V S E D N S E O E I S N L V K 77 79 8S S M S G L H L V K O G R D R K K I D S Q R 1200 1215 Hinge r-I M S F A S N L N H Y G M T H V A S V S D V L L D N A F T P P C Q R egion Amino Acid Protein Kinase Residues Activity 23 ? 25 Calmodulin/Ca dep. PK 8 Major 29 CK II Phosphorylat ion 77 PKC/CAK Sites 79 AMP-PK 95 PKC 1200 AMP-PK/CAK 1215 AMP-PK N Terminal Elec t rophores is Relat ive to DNP-Lys ine 33 It is obvious that A C C is a good substrate for many different protein kinases in vitro. The recent reports from Hardie's group (Davies et al., 1990, Haystead et al., 1990) support the hypothesis that phosphorylation of a single serine residue (Ser 79) accounts for the acute inhibition of A C C seen in response to hormones that elevate intracellular cyclic-AMP concentrations and that phosphorylation of other residues have little or no direct effect on catalytic activity. The function of phosphorylation of these other residues therefore may be more subtle and will require further studies. 34 1.6 THESIS INVESTIGATION The work discussed in this thesis was initiated to test the general hypothesis that reversible site-specific phosphorylation of serine residues within the regulatory regions of A C C could explain (at least in part) the short term regulation of catalytic activity seen in response to hormonal stimulus. The primary objective was to generate a phosphorylated form of A C C in which the distribution of phosphate among the different serine residues was similar to that found in intact cells stimulated with insulin. Ideally a purified preparation of protein kinase able to phosphorylate the I-site could be obtained. This would allow an investigation of the effects of phosphorylation on the catalytic activity of the enzyme under a variety of conditions designed to substantiate the validity of the hypothesis within physiological limits. Folllowing a description of methods (Chapter 2), the major results are described in Chapters 3-6. The work presented in Chapter 3 describes the preparation of differently phosphorylated forms of ACC. Subsequently the data showing the effects of site-specific phosphorylation of A C C on catalytic activity is presented. From these studies we confirmed that phosphorylation of A peptides led to a decline in A C C activity. However, despite achieving incorporation of inorganic phosphate into other serine residues known to be phosphorylated in vivo in response to insulin, the covalent modification per se can not account for the rapid increase in catalytic activity of A C C which has been measured in response to stimulation of cells with insulin. These results indicate that phosphorylation of the I-peptide alone is insufficient to induce rapid activation of ACC, and that other factors may well be involved in mediating the effects of insulin on this enzyme. In light of the fact that no direct activation of A C C was observed following phosphorylation similar to that induced by insulin, the studies described in Chapter 4 were carried out. In these studies a strategy was adopted in order to address the possibility that other effectors may play an integral role in the short term 35 regulation of A C C . The approach was based upon observations published in the literature in 1983 by Saltiel and in 1986 by Hardies' group in Dundee which indicated that a low molecular weight activator caused the increased catalytic activity of A C C seen in response to stimulation of cells with insulin. Despite considerable perseverance, these results could not be reproduced; indeed we made quite contrary findings in studies with rat liver A C C . A C C activity in a crude extract of rat liver was increased dramatically at physiological citrate concentrations by the removal of cytosolic components which pass through a 10 KDalton cut-off membrane. The low molecular weight fraction was considered to contain an inhibitor and some of the characteristics of this inhibitor are discussed. A n attempt to investigate the physiological role of this component is also described. So far we have been unable to conclusively determine the chemical nature of this inhibitor (or mixture of inhibitors). However, some interesting properties of A C C were revealed in these studies. The findings described in Chapter 4 led to further re-evaluation of data that had accumulated over the years, regarding the catalytic properties of A C C and resulted in the conclusion that affinity purified enzyme may be activated in vitro by the addition of a cytosolic protein which has been further characterised. In Chapter 5 I describe the purification scheme that was so far developed for enriching this A C C activator protein and also discuss some of the properties of the protein that have been established. So far, no direct data has been attained to show a physiological role for this protein in the short term regulation of A C C in response to hormones. However, in light of the fact that phosphorylation of A C C alone does not account for the insulin-induced increase in catalytic activity, it would seem likely that other factors must be involved and that this protein may well play an important role in regulation of fatty acid synthesis. Finally, despite the intensive studies that were undertaken in Chapter 3 (and considering that no groups so far have succeeded in sequencing the insulin-directed phosphorylation site), I-site phosphorylation is readdressed in the last Chapter of the thesis. As a result of collaborative studies with another research group at the University of B.C. (directed by Dr. Steven Pelech, of the Biomedical Research Centre) it became evident that A C C could be efficiently phosphorylated 36 with protein serine kinase activities enhanced in sea star oocytes upon maturation. I was not personally involved in the preparation of the sea star extracts or purification of protein kinases from these extracts and therefore these techniques are not given in detail here. This collaboration has therefore led to a fortuitous extension of our phosphopeptide analysis. Phosphorylation of A C C with a protein kinase isolated from sea star oocytes is also shown to result in incorporation of [32P] primarily into the I-site. Using this approach we have been able to obtain sufficient I-peptide to undertake amino acid sequencing. Preliminary assignment of the sequence of this phosphopeptide and its location in the primary amino acid sequence of the A C C molecule is discussed. 37 C H A P T E R 2 E X P E R I M E N T A L P R O C E D U R E S 2 . 1 MATERIALS. Male Wistar rats (180-200g) were allowed free access to water and Purina rat chow up to the time of killing (9-10am). Most laboratory chemicals and solvents were obtained from B D H Chemicals Canada Ltd. (Vancouver, B.C. Canada). Most biochemicals were from Sigma Chemical Company (St. Louis, M O , USA) including the proteinase inhibitors (pepstatin, leupeptin and benzamidine), insulin, ox heart protein kinase catalytic subunit, rabbit muscle protein kinase inhibitor (0.17 unit/^g protein) and avidin. Radioisotopes including [7- 3 2P]-ATP and [14C]-sodium bicarbonate and A C S scintillation fluid were from Amersham International (Oakville, Ontario, Canada). Samples of A T P were dried under a stream of nitrogen to remove ethanol prior to use, although more recently aqueous preparations have avoided this requirement. Reagents for polyacrylamide gel electrophoresis were from Biorad Laboratories (Canada) Ltd., Mississauga, Ontario, Canada. 22 M E T H O D S . 2.2.1 DETERMINATION OF ACETYL-CoA CARBOXYLASE ACTIVITY. Assays were essentially as described previously (Halestrap and Denton, 1973) following the incorporation of [1 4C] from bicarbonate into acid stable product. Briefly, following pre-incubation of acetyl-CoA carboxylase under the conditions indicated, assays were initiated by addition of 50/il (1-2 mU) of enzyme preparation to 0.45 m L assay medium (20 m M MOPS, p H 7.2) containing M g S 0 4 (10 mM), E D T A (0.5 mM), A T P (5 mM), 2-mercaptoethanol (5 mM), defatted serum albumin (10 mg/mL), acetyl-CoA (150 nM) and potassium [1 4C]-1 38 bicarbonate (15 m M and 600 dpm/nmol). Reactions were terminated after 2 min at 37°C by the addition of 200/iL HC1 (5 M). A sample (0.6 mL) was evaporated to dryness and [1 4C]-malonyl-CoA measured by liquid scintillation counting. 2.2.2 PURIFICATION OFACETYL-CoA CARBOXYLASE FROM RAT LIVER. Generally, six male Wistar rats (200-240g) were stunned, killed by decapitation and the livers were removed immediately and placed on ice. The livers were then rinsed with distilled water and homogenised gently in three volumes of extraction buffer (10 m M TRIS-HC1, 20 m M MOPS; p H 7.4, 250 m M sucrose, 2 m M E D T A 5 m M citrate, 5 m M 2-mercaptoethanol, 0.5 m M PMSF, 1 mg/mL bacitracin, 2 / ig/mL pepstatin, 2 Mg/mL leupeptin, 2.5 m M benzamidine, 0.02% sodium azide) using a Potter Elvejhem for 30 seconds at 4°C. A l l subsequent operations were carried out at 4°C, except where indicated. The homogenisation procedure is based on methods shown to produce mitochondria with good respiratory quotients, indicative that organelle integrity is retained. This suggests that lysosomes, a major source of proteases, may also remain intact. The homogenate was centrifuged at 3,000 g for 10 min. The supernatant was collected and filtered through eight layers of cheesecloth to remove the floating fat layer which appears at the surface of the tubes after centrifugation, then centrifuged at 25,000 g for 25 min to pellet the mitochondrial fraction. The supernatant was decanted and filtered as described above. This fraction was then centrifuged for 60 min at 125,000 g to pellet the microsomal fraction. The supernatant was collected and designated the cytosolic fraction. Rat liver cytosol was incubated (30 min at 30°C) in the presence of citrate (10 mM) and defatted serum albumin (3 mg/mL) to ensure the full polymerisation of acetyl-CoA carboxylase. Samples (15 mL) were layered onto sucrose cushions comprising a lower layer of 3 m L (60% w/v) and an upper layer of 2 mL (25% w/v). Acetyl-CoA carboxylase was concentrated into the sucrose cushions by centrifugation, for 90 min at 150,000 g at 39 25°C. The recovered sucrose cushions were then applied to a DEAE-cellulose column (20 x 1.6 cm) equilibrated to p H 7.5 with buffer containing MOPS (25 mM), TRIS-HC1 (50 mM), E D T A (1 mM) and 2-mercaptoethanol (5 mM) plus proteinase inhibitors (2 Mg/mL pepstatin, 2 Mg/mL leupeptin and 2.5 m M benzamidine). After washing with the same low-salt buffer to remove unbound protein, A C C was eluted in buffer containing KC1 (0.5 M). The eluted A C C was then immediately applied to an avidin-Sepharose column. Details of this final step are as reported previously (Song and Kim, 1981, Tipper et al., 1983). The D E A E -cellulose fraction containing A C C was applied to the column (10ml / hr) and unbound protein was washed off. A C C was eluted with biotin (10 mg / 50 mL) and those fractions containing A C C activity were pooled and stabilised by the addition of citrate (5 mM). This fraction was concentrated by dialysis against flaked polyethylene glycol ("Aquacide"), and the concentration of KC1 reduced to below 100 m M by dilution, before storage in small aliquots at - 8 0 ° C . 2.2.3 PHOSPHORYLATION OF ACETYL-CoA CARBOXYLASE. Purified A C C (2-3 ng) was pre-incubated with MgCL, (5 mM) and an appropriate protein kinase fraction for 2 min at 37°C in a sealed microcentrifuge tube in a final volume of 50 /xl of 50 m M TRIS buffer (pH 7.4) containing 2 m M E D T A 1 m M E G T A ) . The reaction was initiated by the addition of [-y-32P]-ATP (50/iM and 1,000 dpm/pmol) and allowed to proceed for up to 30 min at 37°C. Reactions were subsequently terminated either by the addition of an equal volume of SDS-sample digestion buffer prior to analysis by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) or by the addition of 10 m M E D T A and placed on ice until subsequent analysis of the catalytic activity of A C C . In the latter instance, phosphorylation was carried out using non-radioactive A T P . 40 2.2.4 SDS-POLYACRYLAMIDE GEL ELECTROPHORESIS. Separation of proteins was achieved by S D S - P A G E in 0.6 cm lanes on 5 or 7% (w/v) slab gels as according to the procedure of Laemmli (1970). Up to 20 ng of protein per sample was dissolved in digestion buffer p H 6.8, containing TRIS-HC1 (65 mM), SDS (100 mg/mL), sucrose (200 mg/mL), Bromophenol Blue (0.2 mg/mL) and 2-mercaptoethanol (100 mM) by heating at 95°C for 5 min. After electrophoresis, protein bands were visualised by staining with Coomassie Blue or silver stain and the gels sandwiched between cellophane, clamped to a glass plate and left to dry in the air stream of a fume hood overnight. 2.2.5 PHOSPHO-PEPTIDE ANALYSIS. The techniques employed here are essentially as described by Brownsey and Denton (1982). 10-30/xg of purified A C C was pre-incubated with MgCL, and the appropriate protein kinase fraction for 2 min at 30°C in sealed microfuge tubes. The phosphorylation was initiated by the addition of [7-32P]-labelled A T P (50/iM, and specific radioactivity 1,000 dpm/pmol) and incubated for up to 30 min at 30°C. Reactions were terminated with the addition of a ten-fold volume excess of "quench" buffer containing beta-glycerophosphate (80 mM, p H 7.2), E D T A (10 mM), sodium fluoride (50 mM). Ant i -ACC antiserum (Brownsey et al., 1977) was then added and mixtures incubated for 30 min at 30°C and finally for a further 20 min (with occasional vortexing) after the addition of protein A Mixed A C C -protein A precipitates were collected by centrifugation (1 min at 12,000 g, in Eppendorf centrifuge tubes) and washed extensively (5x with quench buffer followed by 2x with ammonium bicarbonate buffer, 50 mM, p H 8.2). Washed pellets were suspended in 200 A»L of the same ammonium bicarbonate buffer and after the addition of TPCK-treated trypsin (1% by weight) were incubated for 6 hr at 37°C with occasional vortex mixing and with addition of a second equal volume 41 of trypsin after 3 hr. Solubilised peptides were recovered after the removal of insoluble material (largely protein A residue) by centrifugation. Initial supernatants and washes were pooled and evaporated to dryness and to ensure removal of inorganic salt the washing/drying cycle was repeated three times. The residues were dissolved in a minimum volume and subjected to thin layer two-dimensional mapping on cellulose plates (Kodak). Peptide mapping involved high voltage electrophoresis at p H 3.6 (3 hr at 400 V), using a solvent system containing pyridine:glacial acetic acid:water (1:9:190, by volume) followed by ascending chromatography in the second dimension using a butan-l-ol:pyridine:glacial acetic acid:water (15:10:3:12 by volume) solvent system. Dinitrophenyl lysine was spotted with the sample onto the thin layer plates as a marker. After thorough air-drying of plates, [32P]-labelled peptides were visualised by autoradiography at -80°C, using cassettes containing Dupont Cronex intensifying screens. 2.2.6 PROTEIN DETERMINATION. Protein concentration was determined by the dye-binding method (Bradford, 1976). Protein samples containing 10-50 fig protein in 0.1 m L were pipetted into a test-tube, 5 m L of Bradford reagent was added and mixed by vortexing. The absorbance at 595 nm was measured 10-60 min after addition of the colour reagent, in 3 m L cuvettes against a reagent blank containing 0.1 m L of the appropriate buffer and 5 mL of Bradford reagent. Standard curves were prepared on each occasion, using bovine serum albumin. 2.2.7 PREPARATION OF RAT LTVER HIGHSPEED SUPERNATANT. Male Wistar rats were killed by asphyxiation in C 0 2 , and the livers removed and placed on ice. The livers were then homogenized in three volumes of buffer p H 7.4 containing TRIS-HC1 (50 mM), E D T A (2 mM), E G T A (1 mM), sucrose 42 (250 mM), pepstatin (2 ng/mL), leupeptin (2 Mg/mL), benzamidine (2.5 mM), PMSF (0.5 mM) and 2-mercaptoethanol (5 mM). A l l subsequent procedures were carried out at 4°C. The homogenate was centrifuged at 3,000 g for 10 min, the supernatant collected and filtered through 8 layers of cotton gauze to remove the floating fat layer and subsequently centrifuged at 27,000 g for 20 min to pellet the dense membrane fraction. The supernatant was collected and filtered as above and the high speed supernatant prepared by ultracentrifugation at 365,000 g for 10 min, using a Beckman TL-100 benchtop ultracentrifuge. The supernatant was filtered through an 0.22 /xm filter, the resulting fraction being referred to as "Extract". 2.2.8 PREPARATION OF A LOW MOLECULAR WEIGHT INHIBITOR OF  ACETYL-CoA CARBOXYLASE. The high speed supernatant, described above (2.2.7), was diluted with extraction buffer containing 1.0 M KC1 to give a final salt concentration of 0.25 M . This sample was filtered by centrifugation through membranes with a size exclusion of 10 kD. The centrifugal ultrafiltration in centricon tubes (supplied by Amicon) was carried out for 1 hr at 4,500 g (1.0 m L per centricon). The filtrate represents the inhibitor fraction "Inh". The retentate was washed with extraction buffer in the centricon (2x1 mL) to remove salt and the volume adjusted to 750 /xL per centricon. This fraction represents the supernatant fraction from which the inhibitor had been removed "Filtered Extract". 2.2.9 ASSAY OF ACETYL-CoA CARBOXYLASE INHIBITOR EFFICACY. The efficacy of the A C C inhibitor was measured by determining the activity of A C C in samples of filtered supernatant after incubation with the inhibitor over a range of citrate concentrations. Typically 30 / x L of filtered supernatant was pre-incubated with the inhibitor fraction for 20 min at 37°C over a range of citrate 43 concentrations from 0-10 mM. The ACC activity data was fitted to the equation: v = V o + (Vm a x-V o)C -------------where VQ = ACC activity at zero citrate and C = citrate concentration. 2.2.10 PARTIAL PURIFICATION OF ACC BYFPLC (PREPARATION OF  MO-ACC). Rat liver cytosol was prepared as described previously for the purification of ACC. A sample (0.5 mL) of the cytosolic fraction was applied to a mono-Q ion exchange column equilibrated with 50 mM TRIS buffer; pH 7.5, containing EDTA (2 mM), EGTA (1 mM), benzann'dine (2.5 mM), pepstatin (2/xg/mL), leupeptin (2 /ig/mL), 2-mercaptoethanol (5 mM) and 0.02% sodium azide. The unbound protein was washed off with the same buffer and bound protein was eluted with a salt gradient (0-0.5 M KC1). Fractions containing ACC activity (usually in the range 50-250 mM) were pooled and used in assays to investigate the citrate response of ACC prepared by this procedure. 2.2.7/ ASSAY FOR ACETYL-CoA CARBOXYLASE ACTIVATOR 1-2 ng of affinity purified ACC was pre-incubated with defatted bovine serum albumin 5 mg/mL and up to 50 ng of cytosolic protein over a range of citrate concentrations (0-10 mM) for 20 min at 37°C in 50 mM TRIS buffer pH 7.4 containing EDTA (2 mM), EGTA (1 mM), 2-mercaptoethanol (5 mM). The assay was performed as described above for ACC. One unit of activator activity was arbitrarily defined as the amount of activator required to give a 2-fold increase in the catalytic activity of 1 ng of affinity purified ACC, measured at 0.2 mM citrate. 44 2.2.12 PARTIAL PURIFICATION OF ACETYL-CoA CARBOXYLASE  ACTIVATOR Two-four male Wistar rats (180-200g) were killed by C O 2 asphyxiation, the livers were removed and immediately covered with ice. Al l further manipulations were carried out at 4°C. The livers were homogenised with a loose fitting Potter Elvejhem homogenizer in extraction buffer p H 7.4 containing TRIS 50 mM, 2 m M E D T A 1 m M E G T A 250 m M sucrose, 0.5 m M PMSF, 2 / x g / m L pepstatin, 2 / x g / m L leupeptin, 2.5 m M benzamidine, 5 m M 2-mercaptoethanol). The homogenate was centrifuged to remove cell debris (3,000 g, 10 min), "mitochondrial fraction" (25,000 g, 25 min) and microsomal membranes (100,000 g, 60 min) The resulting 100,000 g supernatant was filtered through 8 layers of cotton gauze to remove the floating fat layer. Protein was precipitated with addition of solid ammonium sulphate (40% saturation). After stirring on ice (30 min), the protein pellet was recovered by centrifugation (25,000 g, 30 min), resuspended in 10 m L of extraction buffer and filtered through a 0.22 /im filter. This filtered fraction was applied to Sepharose-avidin to remove A C C and the unbound protein was collected (avidin-void). The avidin-void was then applied, at a flow rate of 20 ml/hr, to a DEAE-cellulose ion-exchange column (1.6 cm x 30 cm) equilibrated with column buffer (extraction buffer without sucrose or PMSF, p H 7.5) at 20 ml/hr. The unbound protein was washed off with column buffer and bound protein was eluted with the same buffer containing a salt gradient (0-500 m M KC1). Fractions containing A C C activator were pooled and subsequently desalted and concentrated by addition of ammonium sulphate (40% saturation). After collection of the protein by centrifugation the pellet was resuspended in column buffer and then applied to a mono-Q ion-exchange column, using the Pharmacia Fast Protein Liquid Chromatography (FPLC) system. Unbound protein was washed off and bound protein resolved with a salt gradient (0-500 m M KC1). The 45 solutions used were the same for mono-Q as for DEAE-cellulose, described above. Fractions containing A C C activator were pooled and subsequently concentrated and desalted by centrifugal ultrafiltration in centricon tubes (10 kDalton cut-off, supplied by Amicon). This fraction (0.5 mL) was applied to a Sephacryl-200 gel filtration column (1 cm x 32 cm) equilibrated with buffer, p H 7.4, containing 50 m M MOPS, KC1 (50 mM), E D T A (2 mM), E G T A (1 mM), 2-mercaptoethanol (5 mM) and (0.02% sodium azide w/v), at a flow rate of 2 mL/hr. The peak fractions of "enriched" A C C activator activity were pooled and stored in small aliquots at -80°C. 2.113 OTHER METHODOLOGY Statistical analysis involved standard or paired t-tests as indicated and values are reported as means and standard errors of the means. The activity of acetyl-CoA carboxylase is reported in units, where one unit catalyses the formation of one n mol of malonyl-CoA per min under the defined conditions at 37°C. 46 C H A P T E R 3 SITE-SPECIFIC P H O S P H O R Y L A T I O N AS A M E C H A N I S M F O R T H E S H O R T T E R M R E G U L A T I O N O F A C E T Y L - C o A C A R B O X Y L A S E . 3.1 R E S U L T S AND DISCUSSION 3.1.1 PURIFICATION OF RAT LIVER ACETYL CoA CARBOXYLASE. Established purification procedures have mostly employed the preparation of high speed supernatant fractions from adipose tissue, liver or mammary gland followed by precipitation of enzyme activity with ammonium sulphate, polyethylene glycol or a combination of both treatments. The resuspended A C C is then subjected to affinity chromatography over the matrix Sepharose-avidin with specific elution by buffer containing biotin. These techniques have given good yields (50% or more) of acetyl-CoA carboxylase from mammary gland or adipose tissue but relatively poor yields (10-20%) from rat liver. Yields from rat liver have been poor despite the use of a range of proteinase inhibitors and rapid procedures. In our studies we have found that the resuspension of A C C following precipitation by either ammonium sulphate or polyethylene glycol was difficult and substantial losses were incurred prior to affinity chromatography. The recovery could be improved slightly by extended dialysis (which presumably allowed slow removal of precipitant) but the extended dialysis probably allowed the opportunity for partial proteolysis even with the addition of a cocktail of proteinase inhibitors. A shift in the 240,000 band in this fraction appeared following resuspension and extended dialysis. This band contains both A C C and fatty acid synthetase, which co-migrate on S D S - P A G E under these conditions. The loss of the 240,000 band and appearance of a band of Mr 220,000 may be the result of limited proteolysis of A C C with the loss of a fragment of approximately Mr 20,000 leaving the subunit at apparent Mr 220,000 (see Fig. 5.). 47 F I G U R E 5. PROTEOLYSIS OF A C E T Y L - C o A C A R B O X Y L A S E . Approximately 20-30 ug of protein from each sample were subjected to SDS-P A G E on a 5% polyacrylamide gel. Fractions applied were (A) 125,000 g supernatant freshly prepared from rat liver, (B) fraction A enriched by sucrose density centrifugation, (C) fraction B precipitated by polyethylene glycol (3-6% cut, by volume) after dialysis (8hr, 4°C). _250 _225 • n I" ! 48 The procedure described in the Methods section (Chapter 2.) has largely overcome these problems and the final A C C preparation displays a major protein band with subunit Mr approximately 240,000 as judged by SDS-polyacrylamide gel electrophoresis relative to the migration of the twin heavy chains of erythrocyte spectrin. This is the only band detectable using Coomassie Blue staining. Upon staining with silver stain (see Fig. 6.) a minor band at slightly higher Mr of approximately 250,000 and some minor contaminants at lower Mr are observed but these represent less than 5% of total protein by relative staining. We have been concerned that the major band may represent a proselyting fragment (generated during the isolation) from the 250,000 kD species despite all of the precautions taken and the rapid procedures which are completed within 12 hr. However, this seems unlikely for several reasons. Firstly, the major band of purified rat liver A C C co-migrates with A C C isolated by rapid immunoprecipitation from fat or liver supernatants and indeed co-migrates on S D S - P A G E with the major high Mr protein band present in preparations of purified adipose tissue A C C (Fig. 7.). This same Mr band is apparent even in fractions directly precipitated by acid immediately after homogenisation and detected following western blotting and detection with antibody binding. Secondly, the kinetic properties displayed by this purified A C C include low specific activity (1.5-2.5 units/mg protein) and high for the substrate acetyl-CoA (70-80 t i M ) which are unlike the properties of the proteolytically modified enzyme described earlier (Song and Kim, 1981) which include values of 10-15 units/mg protein and KJJJ of 7-10 j*M respectively. The minor high molecular weight band has not yet been identified, but from the results of other studies being carried out in our laboratory, it appears to contain biotin. Following limited proteolysis the two proteins display peptide "fingerprints" which have many similarities. This suggests that these two polypeptides are very similar. Indeed an isozyme of A C C has been described 49 which is uniquely expressed in rat cardiac and skeletal muscle and co-expressed with the major form of A C C in rat liver, mammary gland and brown adipose tissue (Thampy, 1989, Bianchi et al., 1990). Taken together, therefore, this evidence indicates that the Mr 240,000 subunit is indeed the major native form of rat liver A C C and is most unlikely to have been produced by the action of proteases. 50 F I G U R E 6. AFFINITY PURIFIED RAT LIVER A C E T Y L - C o A C A R B O X Y L A S E . Approximately 5 ug of the purified A C C preparation was subjected to S D S - P A G E on a 5% polyacrylamide gel. The gel was fixed and stained with silver stain. Values (xlO"3) indicate the subunit Mr and position of migration of the twin bands of the molecular weight marker spectrin. 51 F I G U R E 7. PURIFIED ACETYL-CoA C A R B O X Y L A S E ISOLATED F R O M RAT LIVER AND ADIPOSE TISSUE. Approximately 5 ug of protein from each preparation was subjected to SDS-PAGE on a 5% polyacrylamide gel. The gel was fixed and stained with Coomassie Blue protein stain. Lane A is protein from rat liver; lane B is from adipose tissue. _ 2 5 0 - 2 2 5 52 The overall purification procedure adopted is outlined in Table 1 which summarises the results from five separate preparations each beginning with four-six rat livers. The least effective single step is DEAE-cellulose chromatography, but this serves the important function of allowing removal of citrate and sucrose and exchange into the correct high-salt buffer for subsequent affinity chromatography. This is achieved with speed and with little dilution of the sample. 53 T A B L E 1. PURIFICATION O F A C E T Y L - C o A C A R B O X Y L A S E F R O M RAT LIVER C Y T O S O L The values represent the mean values for five different preparations, beginning with 4-6 rat livers. F R A C T I O N P R O T E I N ACnvrrY SPECIFIC Y I E L D (mg) (mU) A C T I V I T Y (%) (mU/mg) 100,000 g C Y T O S O L 5522 15458 2.8 100 S U C R O S E C U S H I O N 2093 12650 6.1 82 D E A E - C E L L U L O S E (500 m M KC1) 1128 9872 8.8 64 S E P H A R O S E A V I D I N E L U A T E 2.7 5874 2200 38 54 3.1.2 SITE-SPECIFIC PHOSPHORYLATION OF ACETYL-CoA  CARBOXYLASE. An increase in A C C activity can be detected in cell free extracts after insulin stimulation of cells (Halestrap and Denton, 1973, Lee, et al., 1973). Brownsey and Denton (1982) demonstrated that insulin also increased the phosphorylation state of A C C and that this phosphorylation occurred at sites distinct from those phosphorylated by cyclic AMP-dependent protein kinase. From these observations, a mechanism involving site-specific phosphorylation by insulin activated serine kinase activity was proposed for the short term regulation of A C C . By employing different conditions and protein kinase preparations I therefore set out to generate different phosphorylated forms of purified rat liver A C C in which [32P] is incorporated into the various sites on the protein. In this way it was hoped to test the hypothesis that site-specific phosphorylation affected the activity by assaying the catalytic activity of A C C in its various phosphorylated forms. Following phosphorylation under different conditions, the distribution of [32P] between the several sites on rat liver A C C has been determined by tryptic digestion of [32P]-labelled A C C and subsequent two-dimensional peptide mapping as described in the methods. These techniques have been used to separate at least four different phosphopeptides that can be generated in vitro which are also recognised following the phosphorylation of A C C within intact fat cells The four phosphopeptides separated are designated as C-sites ("control"), A and A*-sites ("adrenaline") and the I-site ("insulin") (see Introduction for full description). In addition to these four peptides, another group of peptides designated B-sites have been identified in vitro. The C-sites, which appear in two loci near the electrophoretic origin (but towards the chromatographic solvent front) represent "control" phosphorylation observed in A C C from cells incubated in the absence of 55 hormones. Peptides designated as groups A and A* exhibit increased phosphorylation following treatment of cells with hormones which increase cellular cyclic A M P concentrations, including adrenaline or glucagon. A n additional phosphopeptide is observed (designated I-peptide) after exposure of cells to insulin (Brownsey and Denton, 1987). The B-sites have not been observed in previous studies with intact cells and therefore the physiological significance of this phosphorylation is not clear. Recent data from Borthwick et al. (in press) however, describe the partial purification of a protein kinase which reportedly phosphorylates A C C on the I-site. The peptide maps generated from A C C phosphorylated by this protein kinase appear remarkably similar to those showing B-site phosphorylation that are described here. Phosphorylation of the B-sites of rat liver A C C is a major in vitro event. Indeed, the I-sites and B-sites may be identical, however this can only be confirmed by further comparison of the peptides involving H P L C analysis and ultimately the sequence of the I-site and B-sites. 3.1.3 PHOSPHORYLATION OF AFFINITY-PURIFIED ACETYL-CoA  CARBOXYLASE BY ENDOGENOUS PROTEIN KINASE. Purified rat liver A C C was incubated in the presence of [7- 3 2P]-ATP but in the absence of any other protein fraction and then subjected to trypsin digestion and phosphopeptide analysis as described above. The resulting two-dimensional maps from two different preparations of A C C are illustrated in Figure 8. From a series of four separate preparations the incorporation of [32P] into the major phosphopeptides was as follows : C-group (64 ± 4 % of total), I-peptide (25 ±2%) , B-group (not detectable) and A-group detectable in 3 of 4 preparations (14 ±6%) . 56 F I G U R E 8. r z P ] PHOSPHO-PEPTIDES G E N E R A T E D BY T H E P H O S P H O R Y L A T I O N O F ACETYL-CoA C A R B O X Y L A S E BY E N D O G E N O U S P R O T E I N KINASE ACTIVITY. Affinity purified acetyl-CoA carboxylase was incubated in the presence of [7-32P]-A T P but with no other protein fraction added, for 1 hr at 3 7 ° C [nP]-ACC was immunoprecipitated, digested with trypsin and the released peptides separated by two-dimensional mapping. The figure illustrates autoradiographs of phospho-peptides from two preparations of A C C purified from rat liver. Arrows indicate the migration of the I-peptide. Bars represent the distance of migration of DNP-lysine standard in electrophoresis (horizontal bar) and of the solvent front in chromatography (vertical bar). 57 It should be stressed that this endogenous A C C kinase activity is apparent despite purification involving sucrose density centrifugation, DEAE-cellulose chromatography and also extensive washing in the presence of 500 m M KC1 while A C C is bound to Sepharose-avidin. Intriguingly, this endogenous phosphorylation represents the only condition studied employing the rat liver fractions in which phosphorylation of the I-peptide has been consistently observed. The total incorporation of [32P] into acetyl-CoA carboxylase under these conditions reaches 0.1-0.2 mol phosphate per mol subunit Mr 240,000 in 30 min at 37°C. The incorporation of [32P] into the I-peptide reaches a very similar proportion, although lower stoichiometry to that observed on A C C isolated from intact fat cells exposed to insulin and represents the most "insulm-like" phosphorylated form of A C C we have so far achieved with A C C protein and protein kinases from rat liver. The failure to observe incorporation of [32P] into the I-peptide in the presence of other fractions from rat liver suggests that a negative interaction may be expressed either at the level of the different sites on A C C itself (such that the phosphorylation of one site may restrict the phosphorylation of another site) or, perhaps more likely that the protein kinase activity itself is either unstable or inhibited in these crude fractions because of other protein components that are present. It is not possible to draw any clear deductions about the explanation for the low apparent I-peptide phosphorylation in fractions other than highly purified A C C . A n important limitation of this observation is that since I-peptide phosphorylation is not detected using crude kinase fractions, I have been unable to make any estimate of the recovery of the protein kinase able to phosphorylate the I-peptide. The inclusion of spermine in the phosphorylation reaction buffer, promoted a 2-3 fold stimulation of incorporation of [32P] into A C C by protein kinase activities present in the affinity-purified preparations of A C C . This effect was 58 apparent with the polyamine added at concentrations in the range 0.1-1.0 mM and no further enhancement was observed by increasing the spermine concentration to 2.0 mM (see Fig. 9.). 59 FIGURE 9. EFFECT OF SPERMINE ON THE ENDOGENOUS PHOSPHORYLATION OF ACETYL-CoA CARBOXYLASE. Approximately 2-3 ng of affinity-purified rat liver A C C was phosphorylated by the addition of pPJ-labelled Mg-ATP (50 /xM) for 1 hr at 37°C with no further additions or in the presence cl spermine: 0.5 mM; 1.0 mM; 2.0 mM. 0* mm mi 250 225 S P E R M I N E / m m o l o 0.5 1.0 2.0 60 Similar effects were observed with spermidine or putrescine although the effects were smaller than with spermine. Analysis of tryptic phosphopeptides shows that inclusion of spermine leads to increased incorporation of [32P] into C-and I-peptides, reflecting increased activity of the cyclic A M P independent endogenous A C C kinase. This effect of spermine may be indicative of contamination of the purified A C C preparation by casein kinase II, which is activated by polyamines (Hathaway and Traugh, 1982) and has been reported to cause C-site phosphorylation of A C C (Haystead et al., 1988). No increase in the phosphorylation of A A * or B-peptides was observed. At present it is not possible to determine with any certainty if the effects of the added polyamine is brought about by activation of protein kinase activity directly or if the effect is due to primary binding to A C C itself which induces an alteration in the accessibility of the phosphorylation sites. Nevertheless the observation does provide a potentially valuable characterisation of the observed endogenous phosphorylation. A n important advantage is that the enhanced endogenous phosphorylation observed in the presence of spermine has provided a method to generate increased stoichiometric incorporation of phosphate into A C C , although incorporation into the I-site may still not be sufficient to draw irrefutable conclusions from the results of the effects of I-site phosphorylation on the activity of A C C . Finally, it may be pointed out that the phosphorylation of the I-peptide has not so far been observed in the absence of the simultaneous (or perhaps prior) phosphorylation of control or C-sites either in these experiments or in previous studies of acetyl-CoA carboxylase isolated from intact cells exposed to different hormone treatments (Brownsey and Denton, 1982). Indeed the C-sites have been shown to be phosphorylated under all conditions so far tested, corroborating the observations of Hardie and co-workers (1988) that these sites are acted upon by a number of different protein kinases. 61 3.1.4 PHOSPHORYLATION OF ACETYL-CoA CARBOXYLASE WHICH  LEADS TO MAJOR INCORPORATION OF f2P] INTO THE A-PEPTIDES  OR B-PEPTIDES. Incubation of purified A C C with a cytosolic protein kinase activity enriched from rat liver extracts by DEAE-cellulose chromatography, leads to extensive incorporation of [32P] into B-sites with remaining incorporation of [32P] into C-sites. Little or no incorporation of [32P] into A \ A * peptides or the I-peptide could be detected. The effects of the different kinase preparations on the phosphorylation of the various sites on A C C and examples of two-dimensional maps are shown in Table 2. and in Figures 10 and 11. 62 T A B L E 2. INCORPORATION O F [ 3 2 P] INTO SITES O N A C E T Y L - C o A C A R B O X Y L A S E BY DIFFERENT PROTEIN KINASE ACTIVITIES. P R O T E I N K I N A S E [32P] I N C O R P O R A T I O N (% O F T O T A L ) C-SITES I-SITES A-SITES B-SITES E N D O G E N O U S A C T I V I T Y 6 4 ± 4 2 5 ± 2 1 4 ± 6 D E A E - C E L L U L O S E 27 ± 2 (500 m M KC1) C Y C L I C A M P 35 ± 5 D E P E N D E N T P R O T E I N K I N A S E 7 1 ± 8 6 7 ± 9 Peptide maps generated from four separate incubations of A C C with each protein kinase, were analysed by densitometric scanning. The incorporation of [ P] into each site was estimated as the mean percentage of the total ± the standard error from the mean. 63 F I G U R E 10. r*P] PHOSPHOPEPTIDES G E N E R A T E D BY T H E P H O S P H O R Y L A T I O N O F A C E T Y L - C o A C A R B O X Y L A S E WITH P R O T E I N KINASE ACTIVITY O F RAT LIVER FRACTION E L U T E D F R O M D E A E -C E L L U L O S E A T H I G H SALT (0.5M K C L ) . Details are as described in the legend to Figure 8. Phosphorylation was for 15 min at 37°C. Arrows indicate the migration of B-peptides. The phosphopeptide maps were obtained with column fractions from two separate preparations. 64 FIGURE 11. [^P] PHOSPHOPEPTIDES GENERATED BY THE PHOSPHORYLATION OF ACETYL-CoA CARBOXYLASE BY ENDOGENOUS AND CYCLIC AMP DEPENDENT PROTEIN KINASES. Details are as described in the legend to Figure 8. Phosphorylation reactions were carried out in the absence (upper panel) or in the presence (lower panel) of the catalytic subunit of cyclic AMP dependent protein kinase for 30 min at 37°C. Arrows indicate the migration of A-peptides. 65 The results indicated that under these conditions the incorporation of [32P] into C-sites accounted for 27 ± 2 % of the total with 73 ± 8 % of total entering B-sites. As indicated in the discussion above, the phosphorylation of the B-sites appears to reduce endogenous incorporation of into I- and A-peptides observed with the purified A C C preparation incubated alone. Indeed even the apparent incorporation into C-sites was reduced in some cases. Under these conditions the incorporation of [32P] into A C C was equivalent to 0.5-0.8 mol [P] per mol subunit. For comparative purposes we have also carried out phosphorylation of purified rat liver A C C with the free catalytic subunit of cyclic A M P dependent protein kinase (see Fig. 11.). Under these conditions, as expected, incorporation of J3 2?] occurred mainly into A-peptides and the extent of phosphorylation reached 0.5 mol [P] per subunit of A C C within 30 min at 37°C. During these incubations 67 ± 9 % was found in [32P] entered the A-peptides with 33 ± 5 % of total in the C-sites. This represents a distribution which is very similar to that observed for the fat cell A C C isolated from cells incubated in the presence of adrenaline (Brownsey et al., 1979, Holland et al., 1984). 3.1.5 EFFECTS OF SITE-SELECTIVE PHOSPHORYLATION ON THE  ACTIVITY OF ACETYL-CoA CARBOXYLASE. In previous studies the effects of incubating fat or liver cells with hormones including insulin, glucagon or adrenaline have illustrated important changes in the kinetic properties of A C C (for review see Brownsey and Denton, 1987). One property in particular which reflects the effect of hormones is the response of A C C to citrate addition during pre-incubation prior to assay. We have therefore investigated the response to citrate of the purified rat liver A C C following phosphorylation in three different forms described in detail above. The three different phosphorylated forms of A C C studied are: (i) purified A C C phosphorylated in the presence of the catalytic subunit of cyclic A M P dependent 66 protein kinase (30% C-sites, 70% A-peptides): (ii) phosphorylation by addition of cytosolic rat liver protein kinase activity enriched by DEAE-cellulose chromatography (eluted as the high-KCl fraction from DEAE-cellulose) (30% C-sites, 70% B-peptides): and (iii) purified acetyl-CoA carboxylase incubated in the presence of 1.0 m M spermine but with no other added protein fraction (60% C-sites, 25% I-peptide and 15% A-peptides). In all cases the incubations were carried out so as to generate total incorporation of [32P] into A C C of at least 0.2-0.5 mol [P] per mol subunit Mr 240,000. The incorporation in each experiment was determined by parallel incubations in which incubations were carried out with unlabeled A T P for determination of A C C activity or were carried out with [nf-32P]-A T P and samples separated by S D S - P A G E in order to determine [32P] in the 240,000 subunit. In addition to the determination of total subunit [32P], gel slices were also extracted with trypsin to confirm the phosphopeptide distribution under the three different phosphorylation conditions. The results of these experiments are shown in Figure 12. which summarises the values obtained with three different preparations of A C C . It can be seen that the incorporation of 0.3-0.5 mol [P] into the A-peptides (per mol A C C subunit) leads to an inhibition of A C C which is apparent at zero, low (1 mM) and high (10 mM) concentrations of added citrate. 67 F I G U R E 12. RESPONSES T O CITRATE O F T H R E E D I F F E R E N T L Y P H O S P H O R Y L A T E D F O R M S O F A C E T Y L - C o A C A R B O X Y L A S E . Purified rat liver A C C was incubated in the absence (open bars) or in the presence (closed bars) of A T P (100nM) together with cyclic A M P dependent protein kinase catalytic subunit (A-sites), or the protein fraction eluted from DEAE-cellulose at 500 m M KC1 (B-sites) or addition of spermine at 1 m M (C+I-sites). In each case the designation of the groups indicates the major phosphorylation sites which become [^P] labelled under the different conditions. After protein kinase treatment for 30 min at 37°C acetyl-CoA carboxylase activity was determined as described in methods. The histograms represent mean data from three experiments, with the bars representing S.E.M. 2000--1500 1000 500-- „ 0 1 A B C + l 0 mM A B C + l 1 mM CITRATE A 8 C + l 1 0 m M 68 The extent of phosphorylation observed is similar to that observed in a number of previous experiments, which have been described in the introduction. It can be seen that the similar incorporation of phosphate into the alternate sites on A C C , either into a combination of C-plus B-peptides or into the combination of C-plus I-peptides brings about very little apparent change in the kinetic responses of A C C to citrate. Incorporation into C and I-sites in the presence of spermine is accompanied by a small inhibition of A C C activity (Table 3), it is not clear if this inhibitory effect is directly attributable to the phosphorylation of A C C or to the presence of spermine in the assay. A C C is expected to become more negatively charged as the extent of phosphorylation is increased and spermine (being a very basic compound) will potentially bind in greater quantities to highly phosphorylated A C C . In the light of these observations we have also investigated the responses of A C C to other potentially important ligands including long-chain acyl-CoA esters but I have not been able to detect any activation of A C C even with the additional range of assay and pretreatment conditions. It must therefore be concluded that the alterations in phosphorylation of the B or I-sites of A C C by the incubations described above are not accompanied by parallel changes in enzyme activity, although I was able to confirm inhibition under conditions which lead to phosphorylation of the sites contained within the A-peptides. 69 T A B L E 3. E F F E C T S O F SITE-SPECIFIC P H O S P H O R Y L A T I O N O N T H E C A T A L Y T I C ACTIVITY O F A C E T Y L - C o A C A R B O X Y L A S E . SITES O F C I T R A T E (mM) P H O S P H O R Y L A T I O N 0 1 1+albumin 10 10+pal-CoA N O N E 22.3 ±2.2 57 .7±4.6 74.5 ±7.5 100 52.7 ±9.7 A-SJTES 16.5 ±4.3 44.0 ±5.3 72.7 ±3.2 87.5 ±4.2 28 .0±11.8 B-SITES 17.7 ±2.7 60.7 ±5.7 67.3 ±7.9 92 ±4 .0 42.5 ±6.5 C+I-SITES N D 14.0±9.0 43.0 ± 7.0 85.5 ± 15.0 N D C+I-SITES 9.0 ±1.0 34.0 ±4.0 55.2 ±4.0 72 .0±10 .1 29.3 ±7.8 ( +SPERMINE) Affinity purified rat liver A C C (2-3 ng) was incubated for 30 min at 37°C either without Mg-ATP (no phosphorylation) or with Mg-ATP with the addition of the catalytic subunit of cyclic AMP-dependent protein kinase (A-sites), the D E A E -cellulose (500 m M KC1) fraction (B-sites), no added protein kinase (C+I-sites) or no added protein kinase but in the presence of spermine (C+I-sites + spermine). The samples were subsequently incubated for a further 20 min at 37°C at different citrate concentrations, supplemented with albumin (5 mg/mL) or in the presence of palmitoyl-CoA (pal-CoA 2 mM). The activity of non-phosphorylated A C C assayed at 10 m M citrate is arbitrarily assigned maximal activity (100%). Values are then expressed as a percentage of Vmax. Each value represents the mean value from three experiments ± the standard deviation from the mean. N D = not determined. 70 32 SUMMARY Detailed analysis of the phosphorylation-sites of A C C has been described by Hardie and co-workers and is summarised in a review paper (Hardie, 1989). These studies have largely employed HPLC techniques to achieve separation of phospho-peptides generated by proselyting cleavage and the separated peptides then subjected to amino acid sequence. The results indicate there are at least eight serine residues which can be phosphorylated in vitro in the A C C molecule (Munday et al., 1988, Haystead and Hardie, 1988, Haystead et al., 1988, Davies et al. 1990). By comparison with the deduced amino acid sequence of ACC, six of these sites occur in the amino terminal domain (Ser 23, Ser 25, Ser 29, Ser 77, Ser 79 and Ser 95) and two in the putative hinge region (Ser 1200, Ser 1215). In Figure 13 (reproduced from Fig. 4., Introduction), I have summarised the tryptic peptides which are phosphorylated and the protein kinases which are responsible for the phosphorylation of the individual serine residues. Correlating the data from the observations of Hardie and co-workers with our own observations and data based on a large number of whole cell studies using the 2-dimensional peptide separation technique (see Methods), has allowed a tentative designation of phosphorylation of particular serine residues within the groups of sites resolved by 2-dimensional analysis which are seen consistently. There are four major groups of phosphopeptides resolved by thin layer analysis (control or C-sites, A\A*-sites, B-sites and the I-site). These groups of phospho-peptides are illustrated schematically in Figure 14. (reproduced from Fig. 4., Introduction). 71 FIGURE 13. PHOSPHORYLATION SITES WITHIN ACETYL-CoA CARBOXYLASE. Peptide sequence of rat mammary gland ACC indicating the serine residues which are known to be phosphorylated and the protein kinases for which they are substrates in vitro. N Terminal Region Hinge Region 1200 1215 [ M S F A S N L N H Y G M T H V A S V S D V L L D N A F T P P C Q R Amino Acid Residues Protein Kinase Activity 23 25 29 77 79 95 1200 1215 Calmodulin/Ca dep. PK 8 Major Phosphorylation Sites PKC/CAK AMP-PK PKC CKII AMP-PK/CAK AMP-PK 72 FIGURE 14. SCHEMATIC REPRESENTATION OF [32P] LABELLED PHOSPHOPEPTIDES OF ACC GENERATED BY TRYPTIC DIGESTION AND RESOLVED BY TWO-DIMENSIONAL THIN LAYER MAPPING INDICATING THE RELATIVE MOBILITY OF THE AJB,C AND I-SITES. Electrophoresis Relative to DNP-Lysine 73 The C-sites represent a phosphopeptide generated from the amino terminal domain of ACC. This peptide contains three reversibly phosphorylated serines at positions 23, 25 and 29 and a fourth serine at position 34 which, being surrounded by acidic amino acids may potentially act as a substrate for casein kinase II (see Fig. 13.). In intact tissue, this peptide is phosphorylated regardless of hormone treatment and the extent of phosphorylation is constant under all short term experimental conditions tested (Brownsey and Denton, 1982). The phosphorylation has no apparent effect on catalytic activity of ACC. Thin layer separation resolves this peptide into four distinct spots with different mobilities in the electric field. This is presumably indicative of phosphorylation of multiple serine residues as the peptide would be expected to migrate further towards the anode as it becomes more highly phosphorylated and has more negative charge. These observations indicate the 2-dimensional mapping procedure is more discruninating than the HPLC method, as this peptide is generally resolved as a single peak by reverse phase chromatography (Haystead et al., 1988). Ser 25 has been demonstrated to be a substrate for the calmodulin-dependent protein kinase in vitro and Ser 29 a substrate for casein kinase n. The protein kinase responsible for phosphorylation of Ser 23 is as yet unknown. All three sites are phosphorylated in control cells and an increase in incorporation into Ser 29 in response to insulin is consistent with a transient activation of casein kinase II which has been demonstrated in 3T-3 cells in response to insulin (Sommercorn et al., 1987). It is worth noting however that the increase in phosphorylation seen in the control sites (Ser 23, Ser 25, Ser 29) is very small (20% increase) compared to that into the I-site (5-fold increase). So far, the phosphorylation of Ser 34 has not been detected from sequencing studies. Acetyl-CoA carboxylase is also a substrate for protein kinase C in vitro. Protein kinase C is able to phosphorylate two serine residues Ser 77 and Ser 95. It is unlikely however that the insulin mimetic effects of phorbol esters on fatty acid 74 synthesis (Vaartjes and de Haas, 1985) are via protein kinase C phosphorylation of A C C , as these Ser residues are not phosphorylated in vivo (Hardie, 1989). However, stimulation of A C C activity in rat hepatocytes in response to phorbol esters has beeen demonstrated (Vaartjes et al., 1987). So far, we have not been able to indicate the possible mobility of the peptide containing Ser 95 on our own 2-D mapping system. The A and A * peptides are distinct, judging from their separation by two-dimensional thin layer mapping.. The A peptide contains serine 1200 and is located in the hinge region and the A * peptide contains serines 77 and 79 and is located near the N-terminal of A C C (see Fig. 13). These peptides are phosphorylated in response to hormones that raise intracellular concentrations of cyclic A M P (Brownsey and Denton, 1982). The catalytic activity of A C C is inhibited by the phosphorylation which occurs under these conditions (Lee and Kim, 1978, Brownsey et al., 1979). As a result of these observations many studies have been undertaken to investigate the effects of phosphorylation on the catalytic activity of purified A C C by the catalytic subunit of cyclic AMP-dependent protein kinase. Phosphorylation of rat mammary gland A C C with cyclic AMP-dependent protein kinase indicated that phosphorylation resulted in inactivation which was reversed by dephosphorylation (Hardie and Guy, 1980). Similar results were subsequently obtained for the rat fat pad enzyme and rat liver enzyme (Brownsey et al., 1981, Tipper and Witters, 1982). Holland et al. (1985) subsequently proposed that the effects of adrenaline are due to the direct phosphorylation of A C C by cyclic AMP-dependent protein kinase. More recently Carling and Hardie (1986) reported the partial purification of a cyclic AMP-independent protein kinase (acetyl-CoA carboxylase kinase-3) which phosphorylates A C C and causes inactivation of catalytic activity. This protein kinase is stimulated by phosphorylation and by micromolar concentrations of A M P (Carling et al., 1987). Subsequent studies have led to the identification of this protein kinase as the key 75 activity involved in the inactivation of A C C in response to hormones which raise intracellular cyclic A M P levels. In fact it appears that A C C is not a physiological substrate for cyclic AMP-dependent protein kinase, despite being a substrate in vitro (Haystead et al., 1990). Peptide analysis has revealed that ser 77 and ser 1200 are phosphorylated in vitro by cyclic AMP-dependent protein kinase and that ser 79 and ser 1200 are phosphorylated by AMP-dependent protein kinase (Haystead et al., 1990). In addition, a third residue, ser 1215 is also phosphorylated by the A T P -dependent protein kinase, though more slowly. Manipulation of conditions to allow selective phosphorylation of these serine residues indicates that the inactivation of catalytic activity of A C C by the AMP-dependent protein kinase is mediated entirely by phosphorylation of ser 79. The smaller effects of cyclic A M P -dependent protein kinase are due to phosphorylation of ser 77 (Davies et al., 1990). The AMP-activated protein kinase has been purified and characterised (Carling et al., 1989) and is found to be identical to the major H M G - C o A reductase kinase detectable in rat liver. This kinase appears to be a component of a protein kinase cascade which regulates several key enzymes of lipid metabolism including A C C , H M G - C o A reductase and hormone-sensitive lipase (Hardie, 1989). It is interesting to note that Lent and Kim (1983b) proposed that A C C was not phosphorylated directly by cyclic AMP-dependent protein kinase and indicated that the inactivation of A C C was due to phosphorylation by a cyclic A M P -independent protein kinase which was in turn activated by phosphorylation by cyclic AMP-dependent protein kinase. In contrast to Hardie's AMP-activated protein kinase, this kinase requires C o A (Lent and Kim, 1982, 1983a and 1983b). As the purification procedures described for these two protein kinase activities are similar, it is possible they are different forms of the same protein kinase. As no other peptide sequence data is yet available, it has not been possible to determine exactly which amino acid residues are phosphorylated in the I-peptide or the B-peptides or where in the primary amino acid sequence of the 76 A C C molecule these peptides are located. In regard to this, preliminary sequence data which has recently been obtained will be discussed in detail in Chapter 5. The data presented in this chapter demonstrates that, using selected in vitro conditions I have achieved phosphopeptide patterns very similar to those seen for A C C isolated from insulin treated fat pads. These phosphorylation events however do not appear to have a significant effect on catalytic activity. In conclusion, covalent modification alone is insufficient for the increased catalytic activity of A C C which is seen as a short term response to insulin stimulation of liver and adipose tissue in vivo. Other observations which support this conclusion include studies which demonstrate that effects of insulin on A C C activity are lost after purification by Sepharose-avidin affinity chromatography (Witters et al., 1983, Haystead and Hardie, 1986,). It is worth noting however that the recoveries of A C C from the affinity column in these experiments were less than 40% and may not be representative of the A C C sample applied. Secondly, dephosphorylation of the I-site with protein phosphatase I does not reverse the effects of insulin on catalytic activity of A C C in cell-free extracts of hormone treated adipocytes (Haystead and Hardie, 1986). It would seem likely therefore that other factors must be involved in the short term regulation of A C C in response to insulin action. 77 C H A P T E R 4. IDENTIFICATION O F A L O W M O L E C U L A R W E I G H T R E G U L A T O R O F AC ETYL-CoA C A R B O X Y L A S E . 4.1 R E S U L T S AND DISCUSSION 4.1.1 LOW MOLECULAR WEIGHT EFFECTORS OF ACETYL-CoA  CARBOXYLASE. One mechanism that has been proposed to account for insulin action, involves the generation and subsequent binding of low molecular weight effectors with key regulatory enzymes (Larner, 1972). In line with this mechanism, a putative mediator has been described which activates A C C (Saltiel et al., 1983). The activator was generated from liver plasma membranes after incubation with insulin and found to be water soluble. Subsequently Saltiel and co-workers (1986a, 1986b) have published data demonstrating that a low molecular weight mediator can be released from the plasma membrane in adipose tissue by a phosphatidylinositol-specific phospholipase C. The Pi-specific phospholipase is activated by insulin binding to its receptor resulting in release of phosphatidylinositol glycan (PIG) and diacylglycerol. Although PIG can modulate the activity of cyclic A M P phosphodiesterase and adenylate cyclase, it does not appear to activate purified A C C or A C C in extracts prepared from control or insulin treated adipocytes (Hardie, 1989). In 1986, Haystead and Hardie presented evidence indicating that activation of A C C induced by insulin stimulation of adipose tissue is lost after rapid gel filtration under conditions of high ionic strength. They proposed that activation of A C C is mediated by a tightly bound low molecular weight effector. Having established that I-site phosphorylation alone is insufficient for activation of catalytic activity (see Chapter 2), studies were initiated to test the hypothesis that 78 insulin-induced activation of A C C is brought about by the generation and binding of a low molecular weight activator. This is, of course, parallel to many studies of "insulin mediators" discussed over many years (see below and Introduction). A n important objective was to generate a fraction containing the low molecular weight activator in order to examine the effects of this fraction on the various phosphorylated forms of A C C which could be generated following the previous studies of site-specific phosphorylation. This approach would then be used to establish if I-site phosphorylation of A C C is involved in the activation mechanism in a cooperative fashion with another mechanism, perhaps allosteric. A l l attempts to repeat the observations documented by Haystead and Hardie were unsuccessful. In my hands there was no effect of rapid gel filtration on insulin stimulated A C C activity prepared from fat pad extracts. The results from three experiments are shown in Table 4. Insulin stimulated A C C activity 1.44 fold as measured at physiological citrate concentrations, with no effect on Vmax. After rapid gel filtration, the effect of insulin stimulation on specific activity was of similar magnitude to the effects obtained for the initial fractions as measured at 0.5 m M citrate. My results were confirmed in an independent laboratory (Dr. R. Denton, private communication). Furthermore, no additional reports to support the initial observations of Hardie et al. have been presented in the literature. In the course of extending my own studies, I have attempted to carry out experiments involving gel filtration of supernatant fractions from rat liver as well as from adipose tissue. In the experiments with the liver extracts a contrary and striking activation of A C C was observed by removal of a low molecular weight inhibitor fraction, and is described in the next section. 79 TABLE 4. EFFECTS OF RAPID GEL FILTRATION ON ACETYL-CoA CARBOXYLASE ACTIVITY IN EXTRACTS PREPARED FROM CONTROL AND INSULIN TREATED RAT ADIPOSE TISSUE. INSULIN SPECIFIC A C T I V I T Y (mU/mg) T R E A T M E N T 0.5 m M CIT 10 m M CIT N O R M A L E X T R A C T + 5.73+ .69* 8.3 + 4.0 3.97 ±.37 7.3 ± .22 G E L - F I L T E R E D E X T R A C T + 5.13±.31*« 8.03±.41 3.60±.22 8.57±.34 Supernatant fractions were prepared from control and insulin treated fat pads. Aliquots of normal extracts and gel filtered extracts (50 1^) were pre-incubated with 0.5 m M or 10 m M citrate for 20 min at 37°C prior to assay. Values represent the means ±the standard deviation about the mean of three experiments. Statistically significant effects of insulin are indicated by *P<.02 and **P,<.01. 80 4.1.2 IDENTIFICATION OF A LOW MOLECULAR WEIGHT INHIBITOR OF ACETYL-CoA CARBOXYLASE. Various inhibitors of A C C have been described in the literature and alternative mechanisms by which the catalytic activity of A C C may be regulated in vivo have been discussed (Abdel-Halim et al., 1985). Saltiel et al. (1982, 1983) describe the preparation of an ethanol soluble inhibitor fraction generated from liver plasma membranes. Other inhibitors of A C C which are found in rat liver supernatant fractions have a variety of characteristics. They include protein kinases (Brownsey and Hardie, 1980, Shiao et al., 1981, Lent and Kim, 1982, Munday and Hardie, 1984, Munday et al. 1988), a regulatory protein which may interact directly with A C C (Abdel-Halim and Porter, 1980, Abdel-Halim and Yousufzain, 1981) and a low molecular weight, heat stable factor (Aultman and Mapes, 1982). My own studies with A C C have led to the identification of a low molecular weight inhibitor of the en2yme. Figure 15. shows the effect of ultrafiltration of a rat liver high speed supernatant on the activity of A C C . A normal response of A C C activity to increasing citrate concentration (Hardie and Guy, 1980) is observed for the untreated high speed supernatant, with activity at zero citrate less than 10% of Vmax and half maximal activation occurring with citrate added in the range 1-2 mM. Removal of the low molecular weight fraction caused a dramatic shift in the citrate response curve, reducing Kcit to 0.2-0.5 m M and increasing Vmax by 25-50%. Thus at physiological citrate concentrations there is a 2-3 fold increase in catalytic activity of A C C upon removal of the low molecular weight fraction. This activation can be largely reversed by recombination of the low molecular weight fraction with the activated A C C preparation, indicating that these effects are unlikely to be a result of covalent modification. The inhibitor fraction can act directly on purified rat liver 81 A C C , effectively lowering catalytic activity at physiological citrate concentrations in a dose-dependent fashion (see Fig. 16.). The removal of the inhibitor (as measured by the activation of A C C ) upon ultrafiltration requires the presence of moderate salt concentrations, in the range 150-300 mM. Higher concentrations of KC1 added to the extract inhibit A C C activity (Lane et al., 1974), while below 50 m M the efficiency of removal of the inhibitor is reduced considerably. This suggests that the interaction between inhibitor and A C C is, at least in part, ionic. Addition of salt of up to 300 m M to these extracts had no effect on A C C activity. 82 FIGURE 15. ACTIVATION OF ACETYL-CoA CARBOXYLASE BY FILTRATION. Rat liver 100,000 g supernatant was filtered through an ultrafiltration membrane with a 10 kDalton cut-off (centricon) in the presence of 250 mM KC1. The catalytic activity of A C C was assayed after pre-incubation (20 min, 37°C) with various concentrations of citrate, in aliquots (50ul) of the untreated supernatant (O ), the filtered supernatant ( A ) and the filtered supernatant recombined with the low molecular weight filtrate (•). CITRATE (mM) 83 F I G U R E 16. INHIBITION O F AFFINITY PURIFIED A C E T Y L - C o A C A R B O X Y L A S E BY T H E L O W M O L E C U L A R W E I G H T INHIBITOR. A C C was assayed at 0.2 m M citrate in the presence of increasing amounts of the low Mr inhibitor after incubation for 20 min at 3 7 ° C . The inhibitor is expressed in arbitrary units. INHIBITOR (UNITS) 84 4.1.3 CHARACTERIZATION OF THE LOW MOLECULAR WEIGHT  INHIBITOR OFACETYL-CoA CARBOXYLASE. A variety of tests were performed on the inhibitor fraction in an attempt to establish its chemical nature. These tests are described in detail below. In all of the experiments, the inhibitor fraction was prepared and assayed as described in the methods. Briefly, freshly prepared rat liver supernatant (100,000 x g) were subjected to ultrafiltration in th4e presence of salt. A C C activity was then determined before and after ultrafiltration and also after readdition of the low Mr inhibitor fraction at the similar concentrations as that present prior to ultrafiltration. The values represent experiments performed in duplicate. 4.1.3.1 Lipid Extraction of Low Molecular Weight Acetyl-CoA Carboxylase  Inhibitor. The activity of A C C in cell free extracts can be altered in vitro by a variety of low molecular weight ligands. These include long chain acyl-CoA esters (Ogiwara et al., 1978) and phospholipids (Blytt and Kim, 1982). In order to address the possibility that the inhibitor might be a lipid, the inhibitor fraction was extracted with chloroform:methanol (Folch extraction) and subsequently assayed for the ability to inhibit ACC. Table 5 shows that the re-addition of the inhibitor fraction after lipid extraction brought about A C C inhibition just as effectively as the untreated inhibitor fraction. This confirms previous experimental data indicating that lipid extractable components from rat fat pads do not exert an inhibitory influence on A C C in these supernatant fractions (Brownsey et al., 1988). It had been shown previously that treatment with Lipidex (a high affinity lipid binding matrix) had no effect on ACC activity. Furthermore, in the experiments described here, the effects of inhibitor are apparent in the presence of a large excess of defatted serum albumin, which should provide extensive lipid "buffering". 85 TABLE 5. EXTRACTION OF THE ACETYL-CoA CARBOXYLASE INHIBITOR FRACTION WITH ORGANIC SOLVENTS. F R A C T I O N Kcit A C C A C T I V I T Y Vmax (mM) (% Vmax at (mU/mg) 0.5 m M cit) Extract 2.3 19.8 2.79 Filtered Extract 0.21 65.4 4.6 Filtered Extract+Inh 1.09 36.7 4.0 Filtered Extract+InhL 1.31 19.9 5.4 The inhibitor fraction was extracted with chloroform methanol according to the procedure of Folch (1951). Chloroform-methanol (2:1 v/v) was added to sample to give a 20 fold excess of solvent. This mixture was shaken with 0.2 volumes of 0.04% CaCL^ to aid separation into two phases. The aqueous layer was removed by drying under nitrogen and resuspended in extraction buffer (InhL). Values are the average of two separate experiments. 86 4.1.3.2 Acid Stability of Low Molecular Weight Acetyl-CoA Carboxylase Inhibitor. Many of the low molecular weight mediators which have been implicated in hormone action have been described as acid stable (Kiechle et al., 1980, Lamer et al., 1982). Table 6 shows the effects of acid treatment of the inhibitor fraction. The inhibition of A C C is unaffected by this treatment, indicating the inhibitor obtained by ultrafiltration of liver supernatants is also acid stable. 4.1.3.3 Investigation of the Possibility that the Low Molecular Weight Inhibitor of  Acetyl-CoA Carboxylase may be a Nucleotide The adenine nucleotides A T P and A M P which have been reported to inhibit A C C activity (Yeh et al., 1980) and guanine nucleotides which reportedly activate A C C activity (Witters et al., 1981, Buechler and Gibson, 1984) have been implicated in the regulation of A C C in response to hormones. Several tests were therefore carried out in order to investigate if the inhibition of A C C seen in the present studies was due to a nucleotide component of the low molecular weight fraction. These tests involved incubation with acid charcoal, incubation with glucose and hexokinase and incubation with 5' nucleotidase. Acid charcoal binds a variety of compounds including a range which are organic acids, perhaps classically long-chain fatty acids. It has been used successfully for the binding of nucleotides and was thus a suitable choice for our studies. Acid charcoal is known to bind up to at least m M amounts of nucleotides in the volume ratios used here, so that it is unlikely any free nucleotides could remain after this treatment. The results of the initial experiment involving incubation of the inhibitor fraction with acid charcoal are presented in Table 7. The data indicate that the inhibitor fraction is rendered ineffective by this treatment. The inhibitory component in the low molecular weight fraction therefore binds to acid charcoal and may well be an organic acid and perhaps a 87 nucleotide. In order to establish if the inhibitor might be A T P , the fraction was treated with glucose and hexokinase, sufficient to drive the conversion of 10 m M A T P in the sample to A D P within 2 min at 30°C. This treatment will catalyse the conversion of glucose into glucose 6-phosphate with the concomitant conversion of A T P into A D P . This treatment had no effect on the inhibitor (Table 8.). Furthermore, treatment of the inhibitor fraction with 5'nucleotidase specific for the breakdown of A M P (sufficient to drive the conversion of 10 m M 5 - A M P to product within 2 min at 30°C), also had no effect on the inhibitor (Table 8.). In conclusion therefore, despite the inhibitor binding to charcoal, it is unlikely that inhibition of A C C is due to the presence of adenine nucleotides which have long been recognised to cause inhibition of A C C . It should be stressed however that these experiments do not exclude the possibility that other non-adenine nucleotides may be responsible for these effects. Parenthetically, it is worth noting that the inhibitor fraction is effective in the absence of divalent metal ions, again suggesting that nucleotides are not involved (nucleotide di- or triphosphates are normally present within cells as a M g 2 * complex). 88 TABLE 6. ACID TREATMENT OF THE ACETYL-CoA CARBOXYLASE INHIBITOR FRACTION. F R A C T I O N Kcit A C C A C T I V I T Y Vmax (mM) (% Vmax at (mU/mg) 0.5 m M cit) Extract 2.4 18.2 7.7 Filtered Extract 0.05 80.9 11.5 Filtered Extract+Inh 1.8 25 9.2 Filtered Extract+ Inh. 3.4 13 7.5 A A n aliquot of the inhibitor fraction was dried under nitrogen and then incubated at room temperature for 5 hr in 02. mL of a mixture containing sodium acetate (25 mM, p H 3.5) and sodium nitrate (0.33 M). At the end of the incubation the p H was adjusted to 7.4 and the volume adjusted to that of the starting volume (InhA). 89 TABLE 7. TREATMENT OF ACETYL-COA CARBOXYLASE INHIBITOR FRACTION WITH ACID CHARCOAL. F R A C T I O N Kcit A C C A C T I V I T Y Vmax (mM) (% Vmax at (mU/mg) 0.5 m M cit) Extract 2.9 20.2 4.1 Filtered Extract 0.08 80.8 7.0 Filtered Extract+ Inh 1.4 41.7 9.1 Filtered Extract+Inh c 0.2 77.6 9.1 Inhibitor fraction was incubated for 30 min with acid charcoal. The charcoal was removed by centrifugation in an Eppendorf centrifuge (12,000 g for 5 min) and the p H of the supernatant adjusted to 7.4 prior to assay for inhibitor activity (Inhc). 90 T A B L E 8. E F F E C T S O F G L U C O S E / H E X O K I N A S E AND 5' N U C L E O T I D A S E O N A C E T Y L - C o A C A R B O X Y L A S E INHIBITOR FRACTION. F R A C T I O N Kcit A C C A C T I V I T Y Vmax (mM) (% Vmax at (mU/mg) 0.5 m M cit) Extract 2.4 18.2 7.7 Filtered Extract 0.43 56.7 12.0 Filtered Extract+Inn 1.77 29.1 7.9 Filtered Extract+InhHK 1.06 36.6 7.1 Filtered Extract+InhN 1.22 17.6 7.15 Inhibitor fraction was treated with glucose (1 mM) and hexokinase (InhH K) or 5' nucleotidase (InhN) for 30 min at 3 7 ° C , prior to assay for A C C inhibition. 91 4.1.3.4 Effects of Proteases on the Function of the Low Molecular Weight Inhibitor Especially in earlier studies of insulin second messengers, putative mediator fractions have been described as containing peptides (Seals and Czech, 1980, Sakamoto et al., 1982) although it is not clear that all mediator activities have been shown to be sensitive to enzymatic proteolysis (Lamer, 1983, Jarrett et al., 1983). In order to establish if the A C C inhibitor described in this thesis might be a small polypeptide, the inhibitor fraction was treated with trypsin, V8 protease, and carboxypeptidase Y . The fraction was stable to trypsin, but sensitive to V8 and carboxypeptidase Y as seen in Table 9. While exposure to V8 led to a smaller loss of inhibitor activity, the inhibitor appears to be most sensitive to carboxypeptidase Y . These experiments are not totally unambiguous, since the changes in Kcit and activity at 0.5 m M citrate are not obviously reflected in changes in Vmax. Nevertheless, as the inhibitory effect is reduced, the inhibitor may be a small polypeptide. To ensure that carry over of residual proteinase from the treatment of the inhibitor fraction was not responsible for the effects seen, samples were heat-treated prior to addition of A C C . Further, control additions of proteinases inactivated in this way produced no detectable changes in A C C activity. 92 T A B L E 9. E F F E C T S O F PROTEASES O N T H E A C E T Y L - C o A C A R B O X Y L A S E INHIBITOR FRACTION. F R A C T I O N Kcit A C C A C T I V I T Y Vmax (mM) (% Vmax at (mU/mg) 0.5 m M cit) Extract 1.72 25.0 9.4 Filtered Extract 0.3 66.6 11.9 Filtered Extract+Inh 1.0 39.3 8.65 Filtered Extract+Inhyg 0.7 43.6 8.7 Filtered Extract+Inh c p Y 0.15 79.2 7.7 Filtered Extract +11110^ 0.96 38.0 8.8 A C C inhibitor fraction obtained by centricon filtration was treated with indicated protease for 1 hr at 37°C. The proteases were present at 1 A*g/ml during the digestion and were inactivated by heating for 10 min at 95°C prior to re-addition of inhibitor fraction for assay of A C C activity. The inhibitor fraction is designated Inh V 8 , InhppY, or Inn- after treatment with V8 protease or carboxypeptidase Y , respectively. 93 4.1.3.5 Further Characterization of the Low Molecular Weight Acetyl-CoA  Carboxylase Inhibitor. In addition to the data described above I have attempted to establish other properties of the low molecular weight A C C inhibitor. It is possible that the inhibitor may be produced as an artefact of the tissue homogenisation technique used to prepare the rat liver extracts. In order to address this possibility, a rapid preparation was generated by freeze clamping tissue immediately after dissection from the animal and subsequently extracting the tissue with perchloric acid. The acid soluble fraction produced by this procedure was found to contain the A C C inhibitor. This would suggest that the inhibitor is not merely being formed as a result of the breakdown or release of metabolites in the rat liver supernatant fractions and may well represent an important factor involved in the regulation of A C C . A firm estimate of size has yet to be determined, however preliminary estimates by size exclusion chromatography suggest it is probably between 1500 and 5000 Daltons as it is retained by Sephadex G-25 (fractionation range 1000-5000 Daltons), but excluded by Sephadex G-15 (fractionation range 0-1500 Daltons). A C C is inhibited in vitro by malonyl-CoA the thioester bond of which is sensitive to alkali conditions. Subjecting the inhibitor fraction to alkaline conditions (pH 10) for 10 minutes had no effect on the activity of the inhibitor, although these conditions do ensure hydrolysis of malonyl C o - A It is therefore unlikely that the inhibition is a result of an interaction of malonyl-CoA N-ethyl maleimide (NEM) is used to modify -SH groups. There was no effect of N E M treatment on the inhibitor indicating no involvement of an -SH group in the mechanism of action. Further experiments were carried out in order to gain some insight into the effects of phosphorylation and dephosphorylation of A C C on the actions of the 94 endogenous inhibitor. These studies involved using rats which had been starved for 8 hr prior to use. In starved rats A C C is highly phosphorylated and has low specific activity (McNeillie and Zammit, 1982, Munday and Hardie, 1986a, 1986b). Assays for inhibitor activity under these conditions indicate that even after starvation the endogenous inhibitor was present and could be removed with consequent activation of A C C (see Fig. 17.). This observation implies that the inhibitor can bind to A C C even in a highly phosphorylated form. The specific activity of A C C in extracts prepared from rats starved for 8 hours, measured at 10 m M citrate (Vmax) is indeed lower (approximately 4 mU/mg) than in extracts from rats that have not been starved prior to killing (approximately 8 mU/mg). Furthermore, Figure 17 indicates that treatment of high speed supernatant (prepared from starved rats) with M g 2 + to induce dephosphorylation of A C C (Brownsey et al., 1979) resulted in activation of A C C (by 30%) but with no change in Kcit. The inhibitor was then removed from this "dephospho" fraction and a typical citrate response was seen, with the Kcit shifted to the 0.2-0.5 m M range. This data suggests that the activation associated with removal of the inhibitor can not be accounted for simply by dephosphorylation of A C C . 95 F I G U R E 17. E F F E C T S O F M A G N E S I U M AND U L T R A F I L T R A T I O N O N A C E T Y L - C o A C A R B O X Y L A S E ACTIVITY IN H I G H S P E E D SUPERNATANTS P R E P A R E D F R O M LIVERS O F STARVED RATS. Rats were fed ad lib from 8 p.m. to 8 a.m. when chow was removed. Rats killed at 4 p.m. represent the starved rats described here. The superaatants were Erepared and filtered as described previously in Methods. A C C was assayed in the ign speed supernatant ( • ), high speed supernatant treated with magnesium sulphate (6 mM) for 20 rnin at 37 °C (O) and the M g 2 * treated supernatant after filtration ( # ). A C C activity was measured in each of tehse three differently treated supernatant fractions over a range of citrate concentrations (0-10 mM). CITRATE (mM) 96 4.1.4 INVESTIGATIONS OF LEVELS OF LOW MOLECULAR WEIGHT  ACETYL-CoA CARBOXYLASE INHIBITOR IN VIVO. In a normal environment, rats are diurnal creatures that sleep during the day and feed during the night. In an attempt to assess the physiological role of the inhibitor in the regulation of A C C activity in response to hormonal fluctuations in the rat, a diurnal study was undertaken involving measurement of the effects of removing the inhibitor from high speed supernatant fractions at different time points during a 24 hr period. The rats were allowed free access to food and water during the night from 8 pm to 8 am after which time the food was removed. Measurements were made at six hour intervals. The data obtained from this set of experiments revealed changes in the kinetic properties of A C C which are described below. 4.1.4.1 Effect of the Diurnal Cvcle on the Specific Activity of Acetyl-CoA  Carboxylase. The specific activity of A C C in high speed supernatants prepared at 6 hr intervals over a 24 hour cycle is shown in Figure 18. These data indicate there are diurnal fluctuations in A C C activity. Immediately after feeding at the beginning of the day, the specific activity is 8.3 mU/mg, more than twice the specific activity measured at the 8 pm time point, 3.5 mU/mg, when the animals have gone without food for 12 hr. The half life of the enzyme is reportedly in the order of 36-48 hr (Numa and Yamashita, 1974) therefore over a twelve hour time interval loss of specific activity due to degradation of the enzyme can account for a maximum of 20% of total activity. Studies involving longer term fasting periods have demonstrated that over time the enzyme becomes increasingly phosphorylated and that highly phosphorylated A C C has a significantly lower specific activity than the dephospho form (Munday and Hardie, 1986). In addition to these observations, 97 recent reports by AJIred and Roman-Lopez, (1988, 1989) provide data to support the hypothesis that A C C exists in both soluble and membrane associated forms within the cell. The evidence presented indicates that A C C can associate with the outer mitochondrial membrane and that this results in a 20 fold decrease in specific activity. This pool of A C C associated with the mitochondrial membrane is proposed to act as a reservoir of A C C which can be mobilised and hence activated, depending on the physiological status of the animal (Allred et al., 1989). So far, these effects have only been documented in response to long term conditioning of dietary state and thus it is not clear if the effects on specific activity seen within a normal diurnal cycle are accounted for by either of these mechanisms. The diurnal fluctuations in A C C activity could not be explained by alterations in the apparent binding of low molecular weight inhibitor over this time period, since the diurnal changes in A C C activity were not abolished simply by the ultrafiltration treatment (Fig. 18.). The diurnal cycle of A C C activity could therefore be a result of either "chronic" fluctuations in the phosphorylation state of the enzyme, mobilisation of the enzyme from the mitochondrial pool or some other metabolic mechanism. In the latter case however, the mobilisation of A C C would have to be accompanied by an equivalent amount of inhibitor in order to be consistent with the results presented here. 98 FIGURE 1 8 . SPECIFIC ACTIVITY OF ACETYL-CoA CARBOXYLASE DURING THE DIURNAL CYCLE. Rats were fed ad lib from 8 p.m. to 8 a.m. and starved for the next 12 hr. Liver extracts were prepared at time points throughout the diurnal cycle. A C C activity was assayed in the initial high-speed supernatant fractions (•) and again following ultrafiltration of these supernatants (O). 12 cn O-l 1 — i 1 1 F 1 1 i 1 1 1 J 0 2 4 6 8 10 12 14 16 18 20 22 24 TIME 99 4.1.4.2 Effect of the Diurnal Cycle on Citrate Sensitivity of ACC. The citrate sensitivity of A C C was measured throughout the diurnal cycle and is presented in Figure 19. These data reflect the results obtained above for specific activity measurements, in that at times during the diurnal cycle when specific activity is high, the sensitivity of the enzyme towards citrate is also high and as the specific activity drops over the course of the day, the sensitivity to citrate decreases. These effects on Kcit are detected with the untreated high speed supernatant (Fig. 19a), however again the results were accentuated by the ultrafiltration, since after removal of the inhibitor by Centricon treatment the sensitivity of A C C to citrate is seen to fluctuate 4-fold during the course of the day (Fig. 19b.). Studies with purified rat liver A C C have shown that as the molar amount of phosphate incorporated into the enzyme is increased, the specific activity decreases and the Kcit increases (Jamil and Madsen, 1987a, 1987b). It is therefore possible to speculate that the effects described above are consistent with these observations and that over a 24 hr time period the total phosphate content of A C C follows a diurnal cycle similar to that shown in Figure 19b for the Centricon treated extract. The inhibitor can be generated from a high speed supernatant fraction at all times throughout the diurnal cycle as it is possible to activate A C C in extracts prepared at all time points, by Centricon treatment (Table 10.). However, the inhibition of A C C in extracts prepared during the starving period is less (48%) than that seen for A C C in extracts prepared during the feeding period (66%) of the diurnal cycle. 100 F I G U R E 19. T H E CITRATE RESPONSE O F A C E T Y L - C o A C A R B O X Y L A S E DURING T H E DIURNAL C Y C L E . a) Rat liver supernatant fraction extracts were prepared at time points throughout the diurnal cycle and A C C activity was assayed over a range of citrate concentrations (0-10 mM), and Kcit values calculated. 5 4 •• 2 1 -j 1 i 1 i 1 1 i 1 1 1 1 f 0 2 4 6 8 10 12 14 16 18 20 22 24 TIME b) The fractions prepared for the experiment described in panel 19a were subjected to Centricon ultrafiltration and A C C activity assayed over the same range of citrate concentrations above. 1.000 r - 1 O 0.000 - 1 1 1 1 1 1 1 1 1 1 1 1 0 2 4 6 8 10 1 2 1 4 1 6 18 20 22 24 TIME 101 T A B L E 10. D E T E C T I O N O F ACETYL-CoA C A R B O X Y L A S E INHIBITOR DURING T H E DIURNAL C Y C L E . SPECIFIC A C T I V I T Y (mU/mg) (0.5 m M C I T R A T E ) T I M E Extract Filtered Extract % Inhibition 08.00 2.6 7.6 66 14.00 1.6 3.3 51 20.00 1.0 1.9 48 02.00 1.9 4.6 58 A C C activity measured in freshly prepared supernatant was assayed in fractions of rat liver prepared at 0.5 m M citrate, were determined in untreated fractions ("extract"), and again after ultrafiltration by Centricon treatment ("filtered extract") at various times throughout the diurnal cycle. 102 4.1.4.3 Insulin Stimulation of Liver In Vivo. Experiments involving in vitro incubation of liver samples can be limited by loss of cell viability due to poor diffusion of oxygen. In order to measure the effect of insulin stimulation of the liver on inhibitor levels, rats were starved for 8 hr then injected intraperitoneally with glucose. This approach was chosen since the technique is more technically straightforward than either of the two major alternatives - liver perfusion or preparations of isolated hepatocytes. Furthermore, this method has been used successfully to stimulate fatty acid synthesis and activation of A C C in vivo in the liver (Stansbie et al., 1976). The livers were removed 30 min after the injection of glucose and A C C activity in high speed supernatant fractions measured before and after Centricon treatment. Table 11. shows there was no significant difference in levels of A C C inhibitors in high speed supernatants obtained from rat liver following in vivo glucose injection. The low molecular weight effector was detectable in both control and insulin stimulated extracts and Centricon treatment activated both samples approximately 2-fold at 0.5 m M citrate. 103 TABLE 11. EFFECTS OF INSULIN STIMULATION OF LIVER//i vivo ON INHIBITOR ACTIVITY IN RAT LIVER EXTRACTS. F R A C T I O N Kcit % Vmax ( + or - INSULIN) V0.5 m M citrate E X T R A C T (+) 2.14 24.4 E X T R A C T (-) 2.86 17.7 F I L T E R E D 0.78 40.2 E X T R A C T ( + ) F I L T E R E D 0.40 49.9 E X T R A C T (-) Rats were injected intraperitoneally with 0.9% saline ( 1.0 mL) or saline containing 200 mg of glucose. After 30 min livers were removednd high speed supernatant fractions prepared. Samples of extract and filtered extract from control and glucose stimulated rats were assayed for A C C activity over a range of citrate concentrations. A C C activity was determined in the initial, untreated supernatant fractions ("extract"), and also following Centricon ultrafiltration ("filtered extract"). Symbols (+ or -) represent the presence or absence of glucose by i.p. injection, and indicate elevated or basal circulating insulin in vivo. 104 4.2 S U M M A R Y The activity of acetyl-CoA carboxylase (ACC) increases within minutes in adipose tissue and liver in vivo in response to insulin (Stansbie et a l , 1976) and similar observations have been made using isolated tissue, or cell preparations (see Introduction). The mechanism by which the activation of A C C occurs in response to insulin however has yet to be established. A C C is regulated allosterically (especially by citrate and fatty acyl-CoA esters, Vagelos, 1971) and by site-specific phosphorylation (Brownsey and Denton, 1982). Activation of A C C in response to insulin is probably not accounted for by changes in concentrations of citrate or long-chain acyl-CoA as the levels of citrate do not change in response to hormonal stimulation (Goodridge et al., 1973a, 1973b, Brownsey et al. 1977) and the effects of insulin persist when acyl-CoA esters are removed (Brownsey et al., 1988). Further, in the studies reported in Chapter 3 of this thesis, I observed no clear activation of purified rat liver A C C upon site-selective phosphorylation, in vitro, of sites which appear to reproduce those observed in response to treatment of intact cells with insulin. These observations suggest other factors may be involved in the regulation of A C C . Consequently I have proceeded with studies to look for other effectors which may be involved in regulating A C C activity. The initial results of these studies led to the identification of a low molecular weight fraction responsible for the inhibition of A C C in fresh liver supernatants. The activity of A C C in high-speed supernatant fractions is increased 2-3 fold upon rapid filtration in the presence of KC1 (0.25 M) using membranes with an exclusion limit of approximately 10 kD. Typically Kcit is reduced from 1-1.5 m M to 0.1-0.5 and Vmax is increased by 50%. The activity at physiological citrate concentrations is increased from 20-30% of Vmax to 60-70% of Vmax. Dose-dependent re-inhibition of A C C is observed upon re-addition of the low molecular weight filtrate. Parallel gel filtration studies indicate a molecular weight of 1500-5000 Daltons for the inhibitor fraction, which is stable to heating (60°C for 30 105 min), to acid and trypsin treatment, though it shows some sensitivity to V8 protease and carboxypeptidase Y . The ability of the low molecular weight fraction to inhibit A C C is independent of ATP-Mg and is unaffected by treatment with glucose plus hexokinase, 5' nucleotidase, Lipidex or albumin. Our attempts to detect physiological changes in concentrations of the inhibitor were not conclusive. We were not able to demonstrate that the concentrations of the inhibitor changed with insulin treatment, nor during a diurnal cycle. Both of these conditions lead to substantial changes in rates of fatty acid biosynthesis and in activity of A C C . Interestingly, the removal of the inhibitor fraction appears to "release" latent A C C activity during the lipogenic phase of the diurnal cycle. From these observations it is difficult to assign a clear characterisation of the inhibitor and it seems difficult to deduce a simple model which can account for these results. There are several points worth considering. One important concern is that it is possible that this inhibitor of A C C is generated as an artefact during the preparation of the extracts and that it is of no real consequence in the regulation of A C C in situ. The freeze-clamp experiments however suggest it is not spuriously generated. It may on the other hand represent an important effector that can be released from the plasma membrane or some other cell locus in response to hormone stimulation and contaminates cytosolic extracts as a consequence of the homogenisation technique. Certainly its removal from cytosolic fractions reveals some properties of the enzyme which would otherwise be neglected and indeed offers an explanation for a paradoxical activation of A C C observed in the initial stages of purification. Taking the view that this inhibitor does indeed play a role in the regulation of A C C , I would like to speculate on the results presented above. It is likely that the total phosphorylation state of the enzyme does indeed change biphasically over the course of the diurnal cycle on a time scale similar to that seen for the changes reported here for specific activity, and that this fluctuation in phosphate content is 106 responsible for the fluctuations in specific activity. This effect does not detract from the hypothesis that short term regulation of A C C is facilitated by site-specific phosphorylation events via insulin dependent and cyclic A M P dependent protein kinase activity. It is well established that the antagonistic hormones glucagon (or hormones that elevate intra-cellular cyclic A M P concentrations) and insulin cause modest increases in the phosphorylation state of A C C in intact tissue (Brownsey et al., 1977, Brownsey and Denton, 1982). At least in the case of the inhibitory hormones, site-specific phosphorylation results in a decrease in catalytic activity (Hardie, 1989). In order to reproduce these phosphorylation events, purified preparations require 30-60 min incubation in vitro to achieve stoichiometric incorporation of phosphate and yet the acute effects of adrenaline and insulin on catalytic activity can be measured within minutes. It becomes reasonable to suppose there may be additional regulatory mechanisms, which could enhance the speed of these in vitro responses and also accommodate responses in different time frames. The inhibitor described here may represent an important effector which can be readily mobilised (e.g. from the plasma membrane) to facilitate the rapid response of the system to stimulation, for example by adrenaline during the "fight or flight" response. Further experiments are necessary to extend these studies and should include confirmation that diurnal changes in phosphorylation of A C C do indeed occur and investigations to determine if the inhibitor can be generated from a specific cell fraction, such as a membrane fractions. The studies presented above serve to remind us that the integration of many factors may be required to coordinate the regulation of this key metabolic enzyme in these specialized tissues and emphasizes the importance of pursuing this type of investigation for a more complete understanding of the regulation of fatty acid metabolism. 107 CHAPTER 5. IDENTIFICATION AND CHARACTERISATION OF A COMPONENT OF RAT LIVER CYTOSOL THAT ACTIVATES AFFINITY PURIFIED ACETYL-CoA CARBOXYLASE. 5.1 RESULTS AND DISCUSSION 5.1.1 ACTIVATION OF ACETYL-CoA CARBOXYLASE. The activation of ACC in response to insulin occurs within 5-10 min of hormonal stimulation but the mechanisms involved remain to be fully established. Although ACC is activated allosterically in vitro by citrate, it seems unlikely that such a mechanism is involved in insulin action as intracellular citrate concentrations do not change in response to insulin stimulation. Indeed it has proven difficult to relate ACC activity to the intracellular concentration of a number of low molecular weight effectors (citrate, long chain acyl-CoA esters, coenzyme A nucleotides, phospholipids) that have been proposed to regulate ACC in intact tissue (for review, see Brownsey and Denton, 1987). The effects of ligands that reportedly activate ACC, are to promote polymerisation of the dimeric form of the enzyme (molecular mass approximately 500,000) to the polymeric form (molecular mass approximately 5-10 xlO6). The effects of insulin on ACC activity have been shown to persist after purification by ammonium sulphate precipitation and subsequent dialysis and also following citrate precipitation (Brownsey and Denton, 1982). More recently Borthwick et al. (1987), have shown the insulin effect is still apparent after gel filtration on Superose columns using the FPLC system. Sepharose-avidin affinity chromatography of fractions containing ACC however results in a loss of the insulin effect. This indicates the highly purified enzyme does not retain the properties induced by insulin action (Witters et al., 1983, Holland et al., 1984). Due to the persistence of the effect of insulin, it seems 108 improbable that activation of A C C is caused by the interaction with a low molecular weight ligand. Since the effects of insulin are however lost during affinity chromatography, this may indicate that a non-dialysable ligand with a high affinity and potential to interact with A C C is facilitating the activation seen in response to insulin stimulation. The studies presented below provides some evidence to support this hypothesis. 5.1.2 EVIDENCE FOR AN ACTIVATOR OF ACETYL-CoA CARBOXYLASE  ACTIVITY IN RAT LIVER CYTOSOL. The activity of A C C which is highly purified by affinity chromatography is compared with that of a partially purified preparation of A C Q generated by fractionation of a 100,000 g supernatant on a mono-Q ion exchange column using the Pharmacia Fast Protein Liquid Chromatography (FPLC) System, designated MQ-ACC (see Fig.20a). In the absence of citrate, the activity of both enzyme preparations is 10-20% of Vmax. The affinity purified A C C shows a citrate response curve with a half maximal activation by citrate (Kcit) of 1.5-2.0 mM. MQ-ACC however exhibits a markedly different response to citrate with a Kcit of only 0.1-0.2 mM. The mono-Q preparation of A C C exhibits 3-fold higher activity at physiological citrate concentrations (0.1-0.5 mM). This observation could conceivably be explained by a number of effects, but we hypothesised that it may be due to the dissociation of an activator of A C C which elutes with unbound protein during Sepharose-avidin chromatography. This hypothesis was supported by results of experiments in which purified A C C was recombined with the fraction containing the unbound protein "avidin void". This recombination of two protein fractions effectively reversed the changes in the kinetic properties of A C C observed after Sepharose-avidin chromatography (Fig. 20b). The observation of the reversible deactivation and reactivation of ACC by removal and re-addition of 109 the avidin void fraction provided a means to assay for the activator which facilitated the characterisation of this effect. 110 FIGURE 20. CHANGES IN THE CITRATE SENSITIVITY OF ACETYL-CoA CARBOXYLASE DURING PURIFICATION. a) The response of ACC activity to citrate was determined following affinity gurification of ACC ( # ) and also in a less purified preparation of a cytosolic action which had been enriched by ion exchange chromatography on a mono-Q column using the FPLC system (o). CITRATE (mM) b) The response of ACC activity to citrate was determined following affinitv purification of ACC (•) and also for affinity purified ACC recombined with the "avidin void" fraction (A) CITRATE (rr .M) Ill 5.1.3 PARTIAL PURIFICATION OF ACETYL-CoA CARBOXYLASE A CTTVA TOR FROM RA T LIVER Partial purification of a protein activator of A C C was achieved by a combination of purification techniques depicted schematically in Figure 21. and described in detail in the Methods. The activator was precipitated by addition of ammonium sulphate to concentrations in the range of 20-40% saturation. Attempts to refine this step were unsuccessful as A C C activator was equally distributed between protein pellets generated by precipitation with ammonium sulphate at concentrations of 25, 30, 35 and 40% saturation. The next purification technique involved ion-exchange chromatography on DEAE-cellulose and the A C C activator eluted as a broad peak between 200-400 m M KC1. Subsequently as a sharp peak from the mono Q column between 320-360 m M KC1. Estimates of recoveries of A C C activator are not straightforward as the assay is based on activity of A C C . The assignment of "units" of A C C activator at present, therefore remains rather arbitrary. Experience gained during the purification of A C C indicates there are many possible variabilities that will occur from one preparation to another, some of which we do not fully understand. For example, the inherent phosphorylation state of the enzyme as it is eluted from the Sepharose-avidin affinity column may vary between preparations and this will potentially alter the citrate sensitivity of the enzyme. Obviously as this comprises the basis of the assay for detection of the activator protein, it becomes a major concern to achieve consistency in the quality of A C C which is to be used as a substrate. Such changes in phosphorylation may be explained by diurnal or even seasonal changes, not all of which will be controlled. Furthermore, considering the wide range of potential allosteric regulators of A C C it is difficult to predict the optimum conditions required to expose the activity of the A C C activator protein. Clearly, much further work will be required to optimise these conditions. 112 F I G U R E 21. STEPS UTILIZED FOR T H E PARTIAL PURIFICATION F R O M RAT LIVER O F A N ACTIVATOR O F A C E T Y L - C o A C A R B O X Y L A S E . RAT LIVER HOMOGENATE I CENTRIFUGATION STEPS 5K (10 min), 15K (20 min), 40K (60 min) I AMMONIUM SULPHATE PRECIPITATION 40%SATURATION J AVIDIN SEPHAROSE (VOID) I DEAE-CELLULOSE (200-400 mM KC1) 1 AMMONIUM SULPHATE PRECIPITATION (40% SATURATION) I MONO-Q (320-360 mM KC1) 1 SEPHACRYL-200 113 5.1.4 ESTIMATION OF MOLECULAR SIZE OF THE ACETYL-CoA  CARBOXYLASE A CTIVA TOR Gel filtration chromatography on Sephacryl-200 and on Superose 6B using the F P L C system, under non-denaturing conditions gives an estimate of molecular weight of about 75 kDaltons (Fig. 22). Samples of fractions produced at various stages throughout the purification were subjected to S D S - P A G E . A typical gel showing separation of these proteins shows four major bands, as visualised by Coomassie Blue staining, with molecular weights corresponding to 96, 72, 42 and 40 kD (Figure 23). We are unable to conclude if one (or more) of these major bands or if minor components represent the A C C activator. Further purification steps are necessary to determine the molecular weight of the activator under denaturing conditions, as we have no information to predict if the activator is a monomer or complex in its native form. 114 FIGURE 22. GEL FILTRATION OF RAT LTVER ACETYL-CoA CARBOXYLASE ACTIVATOR An aliquot (0.5 mL) of the partially purified A C C activator was applied to a Sephacryl-200 gel filtration column (32xlcm) and eluted at a flow rate of 2 mL / hr. Fractions (0.5 mL) were collected and assayed for protein (O) and with A C C for activator activity (A). The column was calibrated using 7 -globulin, M 150,000, bovine serum albumin, M r 69,000 and ovalbumin, M 43,000. The molecular weights of these standards are indicated (xlO"3) along tne top axis; the activity of the activator is shown along the right hand axis. 0.300 j? 0.250-z: o 1— < ca \— z UJ 0 -z. o o 2: • 0.050+ o QC °- 0.000 0.200--0.150-0.100--400 300 T200 -100 5 10 15 20 25 30 35 40 45 50 55 60 FRACTION NUMBER 115 F I G U R E 23. P O L Y A C R Y L A M I D E G E L E L E C T R O P H O R E S I S O F FRACTIONS G E N E R A T E D DURING T H E PARTIAL PURIFICATION O F A C C ACTIVATOR. Samples containing approximately 10-20 ug of protein were subjected to SDS-P A G E on a 7% polyacrylamide gel. The gel was fixed and stained with Coomassie Blue protein stain. Numbers on the vertical axis represent the position of migration as well as subunit M r of molecular weight standards. Experimental samples applied were obtained at different stages of purification of A C C activator as follows: (a) 100,000 g supernatant, (b) DEAE-cellulose fraction eluted at 200-400 m M KC1, (c) protein pelleted by ammonium sulphate at 40% saturation, (d) mono-Q fraction eluted at 320-360 m M KC1, (e) Active fraction following chromatography on Sephacryl-200. 116 5.1.5 HEAT AND PROTEASE SENSITIVITY OF THE ACETYL-CoA  CARBOXYLASE A CTIVA TOR PREPARA TION. In order to test that the A C C activator is indeed a protein, heat and proteinase sensitivity were investigated. The activator was rendered ineffective by heating at 90°C for 10 min, but was found to be stable with heating for up to 30 min at 60°C (Table 12.). In fact shorter heat treatments, between 5-15 min at 60°C caused a 50% increase in activator activity. Trypsin treatment of the activator preparation indicated the activator is trypsin stable, however digestion with carboxypeptidase Y resulted in an approximately 90% loss of activity (Table 13.). As with the earlier experiments involving use of proteinases, precautions were taken to ensure (a) that sufficient proteinase was added to ensure proteolysis and (b) that conditions were adjusted to minimise the carry-over of proteinase into subsequent treatment of A C C itself. In these respects, (a) the proteases were observed to have a marked effect on the overall protein profile of activator preparations (judged by SDS-PAGE), and (b) control additions of inactivated proteinases to A C C alone produced no detectable changes in A C C activity. These observations strongly suggest that the activator is a soluble protein. 117 T A B L E 12. H E A T SENSITIVITY O F A C E T Y L - C o A C A R B O X Y L A S E ACTIVATOR. 0.5 m M 10 m M Citrate Citrate A C C A L O N E 1.14 2.58 A C C + A C T I V A T O R 2.18 3.60 H E A T T R E A T M E N T O F A C T I V A T O R 60 °C: 90 °C: 2 min 3.94 3.68 5 min 3.50 3.92 10 min 3.44 4.10 20 min 2.22 3.52 30 min 1.94 3.50 10 min 1.24 2.72 Partially purified acetyl-CoA carboxylase activator (eluted from DEAE-cellulose and precipitated by 40% ammonium sulphate - see page 111) was pre-incubated for the times indicated at 60°C. These samples were cooled and combined with 2-3 M g of purified A C C and incubated for 20 min at 37°C prior to assay for A C C activity. 118 T A B L E 13. P R O T E A S E SENSITIVITY O F A C E T Y L - C o A C A R B O X Y L A S E ACTIVATOR. SPECIFIC A C T I V I T Y (mU/mg) A C C alone 0.92 A C C + Activator 3.66 P R O T E A S E T R E A T M E N T O F A C T I V A T O R Trypsin 3.52 Proteinase K 2.84 Carboxypeptidase Y 1.03 The A C C activator (eluted from DEAE-cellulose and precipitated by 40% ammonium sulphate - see page 111) was dialysed to remove protease inhibitors which are added to the buffers during the partial purification procedures and subsequently treated with proteases as indicated above for 1 hr at 37°C. The protease activity was inhibited by addition of appropriate inhibitors (soya-bean trypsin inhibitor and PMSF for inhibition of proteinase K and Carboxypeptidase Y). These samples were incubated with 2-3 ng of purified A C C and incubated for 20 min at 37°C prior to assay for A C C activity. 119 5.1.6INVESTIGA TIONS INTO THE MECHANISM OF A CTIVA TION OF  ACETYL-CoA CARBOXYLASE BY THE NOVEL PROTEIN ACTIVATOR. Having established that a potent activator of A C C can be enriched from rat liver cytosol, we have attempted to determine the mechanism by which the activation is occurring. Two known mechanisms for activation of the enzyme in vitro are incubation with citrate which facilitates polymerisation of the dimers with a concurrent increase in catalytic activity and dephosphorylation of the enzyme with phospho-serine phosphatases. The following experiments describe some of the characteristics of the activation of A C C by the protein effector. 5.1.6.1 Time Course of Activation. Figure 24., shows the time course of activation of A C C in response to the activator at 37°C as measured under physiological citrate concentrations. The effect of A C C activator is time dependent, giving half maximal activation within 5-7 min and reaching a plateau after approximately 15 min. This indicates that the activator can stimulate A C C activity over a time scale similar to that observed following addition of citrate to cell extracts and also similar to the response time for A C C activation in intact tissue treated with insulin. The rate of activation of A C C with the protein activator is at least as rapid as that due to addition of citrate alone, indicating that physiologically, the activator has potential to produce a rapid response. 120 F I G U R E 24. T I M E C O U R S E O F RESPONSE O F A C C T O T H E A C T I V A T O R E N R I C H E D F R O M RAT LIVER C Y T O S O L . A C C was incubated in the presence or absence (O) of A C C activator (eluted from DEAE-cellulose and precipitated by 40% ammonium sulphate-see page 111) and 02 m M citrate at 37°C. Aliquots were removed at 5 min intervals for up to 30 min and assayed for A C C activity as described in the methods. The data is the average of duplicate experiments using different preparations of activator. TIME (min) 121 5.1.6.2 Co-migration of the Activator protein with Acetyl-CoA Carboxylase is  Disrupted by Avidin. Activator activity was observed to co-migrate with A C C through several purification steps, however this co-migration could be disrupted by avidin treatment of the fraction in solution. Initially it was this dissociation following Sepharose-avidin chromatography which allowed us to detect the activator. A high speed supernatant prepared from rat liver was fractionated over DEAE-cellulose before and after incubation with avidin (30 min on ice, 1 mg/10 mL). The elution profile for A C C activity in a high speed supernatant is shown in Figure 25a. A C C activity elutes between 50-150 m M KC1. The citrate sensitivity of A C C in these fractions is high, of the order of 0.2 mM, indicating these fractions contain both A C C activity and activator activity. There was no detectable activation of exogenous A C C after incubation with those column fractions that did not contain endogenous A C C . If the high speed supernatant was treated with avidin prior to application to DEAE-cellulose, endogenous A C C activity was completely inhibited as expected. Assays of column fractions for activator activity however indicated that the activator now binds to the column eluting between 200-300 m M KC1. The elution profile of activator activity under these conditions is shown in Figure 25b. These results suggest there is a strong association of the activator protein with A C C , that can be competitively displaced by avidin which is known to bind at the biotin-containing domain. Consequently the strength of binding of A C C activator to DEAE-cellulose is altered by removal of A C C itself. 122 FIGURE 25. CO-MIGRATION OF ACC ACTIVATOR WITH ENDOGENOUS ACC DURING ION EXCHANGE CHROMATOGRAPHY. a) Rat liver cytosol was fractionated over DEAE-cellulose. The unbound protein fraction was washed off and bound protein eluted with a linear salt gradient (0-500 m M KC1). Fractions (2 mL) were assayed for A C C activity (in the presence of 10 m M citrate) (•) and for protein ( A ) . The salt gradient is shown by the dotted line. J , z c < rr i— z U J C J z o a z u I— o rr a. £ o < a o < 15 20 25 30 35 40 45 50 55 60 65 70 F R A C T I O N N U M B E R b) Elution profile of A C C activator on DEAE-cellulose. Rat liver cytosol was stirred with avidin (1 mg/ 10 mL) at 4°C for 30 min prior to application to DEAE-cellulose. Unbound protein was washed off the column and bound protein was eluted with a salt gradient (0-500 mM). Fractions (2 mL) were collected and assayed for protein ( A ) and A C C activator ( • ) . FRACTION N U M B E R 123 5.1.6.3 Potential of Activation of ACC as a Result of Phosphatase Activity in the  Activator Preparation. O n the basis of a number of studies (Witters et al., 1979, Jamil and Madsen, 1987, Witters et al., 1988, Mabrouk et al., 1990) it is postulated by others that the dephosphorylation of A C C is the most likely mechanism to explain the activation seen in response to stimulation of cells by insulin. Despite this, direct determination of A C C phosphorylation reveals that insulin induces site-specific increases in phosphorylation in fat and liver cells (Brownsey and Denton, 1982, Witters et al., 1983, Holland and Hardie, 1985). Furthermore it is possible that in the four main target tissues, muscle, liver, adipose tissue and lactating mammary gland, the regulatory mechanisms may not turn out to be identical. In addition to in vivo studies, many groups have demonstrated in vitro that treatment of A C C with phosphatases also leads to dephosphorylation and activation (Hardie and Guy, 1980, Krakower and Kim, 1981, Ingebritsen et al., 1983, Carling et al., 1987). The mechanism involved in the activation of A C C in response to insulin remains controversial. It is clearly important to test for phosphatase activity in the activator preparation and establish if dephosphorylation of A C C under these conditions can explain the activation. There are four protein phosphatases which account for the majority of the cytosolic protein phosphatase activity (protein phosphatase-1, protein phosphatase 2A protein phosphatase-2B and protein phosphatase-2C). The properties of these enzymes have been studied in detail by Cohen and co-workers (Cohen, 1988). Two of these phosphatases could potentially be active in the activator preparation (protein phosphatase-1 and protein phosphatase-2A). These protein phosphatases do not require metal ions for activity and are able to dephosphorylate a wide variety of substrates in vitro. Phosphatases 2B and 2C are both metal ion dependent and would be essentially inactive under the conditions employed for A C C 124 activation in the presence of chelators. Purified rat liver A C C was phosphorylated using the catalytic subunit of cyclic AMP-dependent protein kinase for 30 min at 37°C. The [32P]-labelled A C C was then used as a substrate to test for A C C directed phosphatase activity in the A C C activator fraction. A C C and activator fraction were incubated together under conditions described for activation and aliquots were removed at 5 min intervals for up to 20 min. These samples were then subjected to SDS-polyacrylamide gel electrophoresis and the stained and dried gels exposed to X-ray film. The resulting autoradiogram from a representative experiment (of three) is shown in Figure 26. The results indicate that under conditions which lead to A C C activation the activator fraction does not cause dephosphorylation of A C C and this was confirmed by densitometric scanning of autoradiographs which indicated that after maximal A C C activation, the degree of dephosphorylation was less than 10%. In order to substantiate this data, the experiment was repeated using phosphorylated casein and histone JJI-S as potential phosphatase substrates. Phosphatase activity was not detectable in either case (Fig. 27.). These results indicate that the activation of A C C in response to the activator is not occurring as a result of dephosphorylation of C or A sites and that under the conditions used for expression of activation of A C C it is unlikely there is significant phosphatase activity present. 125 FIGURE 26. A B S E N C E O F A C C - D I R E C T E D PHOSPHATASE ACTIVITY IN T H E A C C ACTIVATOR PREPARATION. Affinity purified rat liver A C C was phosphorylated for 30 min at 37°C with [32P]-labelled Mg-ATP and the catalytic subunit of cyclic AMP-dependent protein kinase. The reaction was stopped by the addition of E D T A (5 mM). This sample was subsequently incubated with A C C activator at 37°C. Identical aliquots (containing 2-3 M g of A C C ) were removed at the times indicated and added to SDS-sample buffer in preparation for SDS-PAGE. Samples were applied to a 7% polyacrylamide gel. The gel was fixed and stained with Coomassie Blue protein stain and exposed to X-ray film for two days at -80°C with an intensifying screen. The autoradiograph is shown above. Migration of the two chains of spectrin (Mr 250,000 and 225,000) is indicated. _ 250 . 225 T I M E / m i n 0 5 10 15 20 126 F I G U R E 27. QUANTIFICATION O F P H O S P H A T A S E ACTIVITY IN T H E A C C A C T I V A T O R PREPARATION USING [ 3 2 P ] - L A B E L L E D CASEIN A N D HISTONE AS SUBSTRATES. The substrates casein (#), or Histone ITJ-S ( O ) were phosphorylated with [-Y-32P]-A T P and the catalytic subunit of cyclic AMP-dependent protein kinase and then exposed to the A C C activator fraction for the indicated times. Details of the incubations were as described in Figure legend 26. Samples were removed at the times indicated and spotted onto phospho-cellulose paper (in duplicate). Papers were washed with three changes of 0.5% phosphoric acid (200 mL) and P2]P incorporation determined by Cherenkov counting. Background incorporation was estimated by spotting reaction mix from which protein acceptor had been omitted, or reaction mix quenched at zero time. Both procedures gave similar results, and have been subtracted from the values presented. 2 0 0 0 - -1 0 0 0 - -0 J 1 1 1 1 0 5 10 15 2 0 TIME ( m i n ) 127 5.1.6.4 Effect of ACC activator on the Km ofAcetvl-CoA Carboxylase for its  substrates. The reaction catalysed by A C C involves the ATP-dependent carboxylation of acetyl-CoA to form malonyl-CoA Although the effects of insulin are evident even when all substrates are non-limiting (5-fold in excess of Km) even so, more subtle effects may also be involved. A regulatory mechanism could therefore involve changes in the affinity of the enzyme for acetyl-CoA Mg-ATP or bicarbonate. Of these different substrates, acetyl-CoA probably displays the largest changes in concentration under normal physiological conditions, as the abundance of acetyl-CoA changes with the fuel availability inside the cell (e.g. a high ratio of free fatty acids to glucose favours acetyl-CoA). We therefore investigated the possibility that the additions of A C C activator may influence the apparent affinity of A C C for acetyl-CoA Figure 28. shows the effect of the activator on the activity of A C C for acetyl-CoA Kinetic analysis of this data by the method of Eadie Hofstee indicates that the addition of the cytosolic protein has no effect on the K m for acetyl-CoA at physiological citrate concentrations. 128 FIGURE 28. ESTIMATION OF KM OF ACETYL-CoA CARBOXYLASE FOR ACETYL-CoA Affinity purified ACC was assayed over a range of concentrations of acetyl-CoA following incubation in the presence ( • ) or absence (O) of activator at 0.2 mM citrate. The data is presented as an Eadie-Hofstee plot. 100 20-0 4— 0.00 0.50 1.00 1.50 2.00 V/S (mUmL - 1 /JLM ]) 129 5.2 S U M M A R Y Since A C C was discovered in 1959, the determination of multiple factors able to alter its activity has led to our appreciation that the regulation of this enzyme is indeed complex. The precise definition of the mechanism involved in the rapid activation of A C C in response to lipogenic hormones such as insulin remains elusive. Both allosteric mechanisms and covalent modification by protein kinases activated as part of a protein kinase cascade, have been implicated and yet convincing evidence to support these hypotheses has still to be attained. In addition, there are apparently differences in the properties of A C C isolated from different tissues (Bianchi et al., 1990) and from our knowledge to date, it appears that regulation of these isozymes may also differ. It is not obvious why different regulatory mechanisms need exist in different tissues. The complexity of the organ or tissue may determine the complexity of the regulatory mechanisms required. The liver for example is one of the more complex organs in that it acts as a major target for many hormones and growth factors and has diverse metabolic functions. The signal transduction pathways involved may overlap and therefore require additional factors in order to increase the stringency of the response to a particular stimulus. During lactation, A C C is highly active in mammary tissue. Most of the glucose taken up by this tissue in these conditions is used for fatty acid synthesis. Starvation (Robinson et al., 1987) or feeding a high fat diet (Agius et al., 1980, Munday and Williamson, 1987) results in inhibition of A C C . In addition to changes in rates of protein synthesis and degradation, the regulation of lactating rat mammary A C C appears to be accounted for by reversible phosphorylation (McNeillie and Zammit, 1982, Munday and Hardie, 1986a, 1986b). Starvation or fat feeding was found to increase the total phosphate content of A C C with concurrent reduction of catalytic activity. The increased phosphorylation may be due to low circulating insulin concentrations (Jones et al. 1984). It is interesting to 130 note that in mammary tissue there is no requirement for a very rapid activation of A C C (within a few minutes) as seen in adipose tissue and liver. Mammary A C C is required to be fully active during the lactation period and only appears to be regulated under conditions of nutritional imbalance, with changes extending over hours. This may account for the apparent differences in the effects of insulin on A C C in this tissue. More recently dephosphorylation in response to insulin has been proposed as the mechanism of activation of A C C in Fao Reuber hepatoma cells (Witters et al., 1988) and in rat liver (Mabrouk et al., 1990). However convincing evidence that dephosphorylation of A C C abolishes the effect of insulin is not shown; indeed, Mabrouk et al. present data to the contrary. In these latter studies A C C can be activated by dephosphorylation but this treatment had no effect on the activation induced by insulin, which was still apparent. It is obvious there are still different views among investigators in this field concerning the mechanisms involved in the regulation of A C C in response to insulin. The data presented in this thesis emphasises that our understanding of the properties and regulation of A C C is far from complete, but opens some new avenues which suggest experimental work for the future. From the studies in Chapter 3, it appears that I-site phosphorylation alone is not sufficient to account for insulin-induced activation of rat liver A C C . The results described in this Chapter (and in Chapter 4) support the hypothesis that other factors in the cytosol can modulate A C C activity and may be important in the short term regulation of the enzyme. The data shows that a protein enriched from rat liver cytosol can alter the sensitivity of purified A C C to the allosteric activator citrate. This apparent activation of A C C at physiological citrate concentrations occurs in the absence of Mg-ATP. Furthermore the activation does not involve dephosphorylation of the enzyme. Indeed, phosphatase activity could not be detected in assays containing a variety of substrates including A C C , histone III-S and casein under the conditions used for expression of activation of A C C . 131 The activation occurs rapidly (within 20 min at 37°C) and has no detectable effects on the affinity of A C C for acetyl-CoA The activator is sensitive to treatment with heat and proteases and has a molecular weight of approximately 75 kD as estimated by size exclusion chromatography. From these results it is tempting to speculate that this activator is a cytosolic protein that is required for j , or involved in, activation of A C C in response to insulin action. Further studies are obviously required before this will become apparent. These studies should include investigations of the effects of the activator on different phosphorylated forms of A C C and address the possibility of I-site phosphorylation being involved in activator binding. 132 C H A P T E R 6. I-SITE PHOSPHORYLATION O F A C E T Y L - C O A C A R B O X Y L A S E 6.1 R E S U L T S AND DISCUSSION 6.1.1 I-SITE PHOSPHORYLA TION OF A CC BY A FA T PAD KINASE. Previous attempts to isolate I-site protein kinase activity from rat liver cytosol were not entirely satisfactory (see Chapter 2.). A n alternative approach was explored involving rapid fractionation of cell free extracts prepared from insulin treated adipose tissue. The fat pad extracts were fractionated using a mono-Q column with the F P L C system, and this has given promising results, with resolution of three peaks of A C C protein kinase activity from the bound protein with a salt gradient (0-500 m M KC1) (Fig 29). A C C kinase in freshly prepared fractions was determined in Mono Q fractions by phosphorylation of A C C and subsequent two-dimensional analysis of tryptic peptides. The results indicated that the fraction eluting between 175-225 m M KC1 contained I-site kinase activity (Fig. 30a). Unfortunately, so far this activity is very unstable and seems unable to withstand any of the standard techniques applied for the stable storage of proteins. This has made further enrichment of the kinase very difficult as the assay involving two-dimensional analysis of A C C is lengthy and tedious. 133 The kinase activity or activities in this Mono Q peak fraction also phosphorylate myelin basic protein in an insulin-dependent fashion. This has allowed us to quantitate the protein kinase activity much more rapidly as the assay using this protein as a substrate is both simpler and faster. Obviously it will be necessary to establish that the myelin basic protein kinase is indeed the I-site A C C kinase. In the meantime this approach should allow us to further purify the kinase and find conditions in which the kinase fraction is stable. Further, unanticipated studies have provided an even stronger rationale for extending the studies of M B P kinase as described in the next section. 134 F I G U R E 29. FRACTIONATION O F ADIPOSE TISSUE E X T R A C T S BY ION E X C H A N G E C H R O M A T O G R A P H Y . Adipose tissue cytosol (3 mL) was applied to a mono-Q column using the F P L C system. Unbound protein was washed off and bound protein eluted with a salt gradient (0-500 m M KC1). Fractions (1 mL) were assayed for protein kinase activity using A C C (2-3 ng per assay) as a substrate (15 min, 37°C). The reaction was stopped by addition or an equal amount of SDS-sample buffer and samples were subjected to S D S - P A G E on a 7% polyacrylamide gel. The gel was fixed and stained with Coomassie Blue protein stain and exposed to X-ray film. The incorporation of I32?] into A C C was estimated by scanning densitometry and expressed in arbitrary units. The salt gradient is shown by the dotted line. F R A C T I O N 135 FIGURE 30. I-SITE PHOSPHORYLATION OF ACETYL-CoA CARBOXYLASE. a) Affinity purified ACC (10 /ig) was phosphorylated in the presence of [32P]-labelled Mg-ATP with I-site kinase enriched from insulin stimulated rat adipose tissue (30 min, 37°C). The sample was subjected to two-dimensional tryptic peptide analysis and autoradiography. The bars indicate the migration of DNP-lysine (horizontal bars and the solvent front (vertical bar). The arrow indicates incorporation into the I-site. b) . Affinity purified ACC was phosphorylated by a protein kinase preparation isolated from extracts of maturation stimulated sea star oocyte. The autoradiograph resulting from subsequent two-dimensional tryptic peptide analysis is shown. Details are as for panel a). Note: the [32P] towards the anode (left)is resudual [32P]-ATP, not phosphopeptide. 136 6.1.2 PHOSPHORYLATION OF ACETYL-CoA CARBOXYLASE WITH MYELIN BASIC PROTEIN KINASE. A maturation-activated myelin basic protein kinase has been isolated from sea star oocytes by the group of Dr. Steven Pelech (BRC, U B C ) (Sanghera et al., 1990). Subsequently, in collaborative studies, purified rat liver A C C has been used as a substrate in studies characterising the properties of this protein kinase (Pelech et al., submitted). Two-dimensional peptide analysis of the phosphopeptides generated by tryptic digestion of A C C phosphorylated with this protein kinase is shown in Figure 30b. Under these conditions, A C C is phosphorylated with a stoichiometry of approximately 0.3 mol Pi/mol 240,000 kD subunit-Densitometric scanning of the thin layer plates indicates that approximately 80% of the p P ] is incorporated into the I-site (n=2). This protein kinase preparation has enabled us to generate enough pPJ-labelled I-peptide for peptide sequence analysis. Therefore, quite unexpectedly we have been able to contemplate the analysis of the insulin directed phosphorylation site of A C C (I-peptide) which has for so long been elusive. 6.1.3 AMINO ACID SEQUENCING OF THE I-PEPTIDE OF ACETYL-CoA  CARBOXYLASE. Affinity purified A C C was phosphorylated with a purified preparation of myelin basic protein kinase for 3 hr at 37°C in order to attain significant incorporation of p P ] into the I-site. Phosphopeptides generated by trypsin digestion with 1% of the sample by weight were separated by reverse phase H P L C using a C-18 Waters H P L C column equilibrated with buffer containing 0.1% T F A . Peptides were resolved by elution with an acetonitrile gradient (0-70%). The major phosphopeptide eluted as a single peak at 30% acetonitrile. This fraction was subjected to amino acid analysis using the peptide sequenator facility in the 137 laboratory of Dr. Reudi Aebersold (BRC, UBC). Furthermore, following H P L C , this phosphopeptide was re-chromatographed by two-dimensional thin-layer mapping, and its identity as the I-peptide was confirmed. A n umambiguous sequence was detected corresponding to D N T ( X ) W E F . The sequence data obtained indicates that this peptide corresponds to a tryptic peptide located in the hinge region of the protein. The most likely complete sequence, based on the rat mammary and chicken liver sequences for A C C is shown in Figure 31 and contains both a serine residue (Ser 1186/1179) and threonine residues (Thr 1185/1178 and 1174/1167) for rat and chicken respectively. A full search of the two published sequences of A C C indicate that this sequence is not repeated elsewhere in the protein. The myelin basic protein kinase preparation used in these experiments phosphorylates primarily the serine residues of A C C , with less than 5% of [32P] incorporated as phosphothreonine. This suggests that Ser 1186 is the most likely residue to be phosphorylated within this peptide of the rat enzyme (Pelech et al., submitted). Further sudies will be carried out to corrfirm this identification. This peptide appears to be distinct from those previously described by Hardie and co-workers (see Introduction and Chapter 3.) and on the basis of two-dimensional peptide mapping appears to represent the major site on A C C that is phosphorylated within intact fat cells, in response to insulin. Further studies are currently underway to extend these studies in order to confirm the amino-acid sequence with the oocyte kinase corresponds to that with insulin-stimulated fat cell protein serine kinases. 138 FIGURE 31. AMINO ACID SEQUENCE OF THE I-PEPTIDE FOLLOWING PHOSPHORYLATION OF ACC WITH SEA STAR MBP KINASE. Affinity purified rat liver ACC was phosphorylated with a purified preparation of myelin basic protein kinase and [32P]-labelled Mg-ATP and subsequently subjected to digestion with trypsin. Tryptic phosphopeptides were resolved by reverse phase HPLC and the peptide peak containing the majority of counts was sequenced. The amino acid sequence of this tryptic peptide is given below, based upon the rat mammary sequence and chicken liver sequence for the whole protein. The number indicates the amino acid residue location within the primary amino acid sequence. Rat Mammary ACC: 1186 D N T C V V E F Q F M L P T S H P N R Chicken Liver ACC: 1179 D N T Q V V E F Q F M L P T S H P N R 139 6.2 SUMMARY The identification of the site within the primary amino acid sequence of A C C that is phosphorylated in response to insulin has proven difficult to ascertain. Problems have been encountered due to technical difficulties in preparing protein kinase fractions which are stable and subsequently, in generating enough material for sequencing. A protein kinase isolated from sea star oocytes has enabled us to overcome these problems and preliminary sequence data is presented above. Immature oocytes can be induced to undergo maturation by the addition of 1-methyladenine. After germinal vesicle breakdown is detected, cytosolic fractions prepared from these oocytes are found to contain several different protein kinases that phosphorylate a variety of substrates (Pelech et al., 1987). A C C was found to be a substrate for one of these activated protein kinases and further investigation (Pelech et al., submitted) led to the conclusion that this protein kinase is the myelin basic protein kinase, previously purified and characterized by Sanghera et al. (1990). It is intriguing a peak of the insulin stimulated A C C kinase in adipose tissue co-migrates on Mono Q chromatography with a myelin basic protein kinase and it is tempting to speculate that these two protein kinases are related. These observations add to the rapidly growing number of observations that show protein kinase cascades are involved in the regulation of cellular metabolism by growth factors and hormones at all stages of development and that these protein kinases may be conserved. 140 CONCLUSIONS If we are to understand the complexities of the mechanisms involved in the regulation of cell metabolism, it is important to be able to define all the parameters involved. It is becoming increasingly obvious that insulin stimulation of cells induces changes in a multitude of cellular components and that the changes are not necessarily the same in all cell types. There are many approaches to studying hormonal signalling. So far these have focussed either on studies of ligand binding to the appropriate receptor (and identifying subsequent effects occurring at the cell membrane) or on characterising the regulatory mechanisms involved in the control of key enzymes in the metabolic pathways which are affected as part of the insulin response. The ultimate objective of understanding the factors involved in linking these two classes of events will provide considerable further challenges. The data presented in this thesis describe studies that were undertaken using the approach of working from the properties of a target metabolic enzyme, in order to understand how ACC may be regulated in response to hormones. ACC plays a critical role in the determination of the rate of fatty acid synthesis, one of the major metabolic pathways stimulated in response to insulin in liver and adipose tissue. The recognition of reversible protein phosphorylation as an important regulatory mechanism in metabolism and other cellular processes has evolved over the last twenty years. Acetyl-CoA carboxylase is reversibly phosphorylated on at least eight serine residues, in vitro, by a variety of different protein kinases. The enzyme is also phosphorylated in vivo in response to insulin and to hormones which induce an increase in the intracellular concentration of cyclic AMP. Covalent modifications can induce changes in the tertiary and quaternary structure of proteins which may affect the catalytic activity of the enzyme by a variety of mechanisms, including disruption of substrate binding sites, 141 disruption of protein-protein interactions, facilitating binding of inhibitors, relocation of the enzyme in terms of subcellular distribution etc. It is therefore important to establish if the phosphorylation of A C C occurring in response to insulin is able to directly affect the catalytic activity of the enzyme. We have therefore studied in detail the effects on A C C catalytic activity of phosphorylation of different combinations of serine residues within the enzyme, and find that this covalent modification does not lead to activation of A C C under the range of conditions tested. This has been true for phosphorylation of A C C by liver, adipose tissue and sea star kinases, which give extensive phosphorylation of the insulin-directed phosphorylation site (I-site). Having confirmed that phosphorylation per se is insufficient for the activation of catalytic activity, we have proceeded to characterize cytosolic fractions which were identified as ACC effectors. These effectors appear to be members of two quite separate classes, namely a low molecular weight heat and acid stable inhibitor and a non-dialysable 75 kD protein activator. Over the past 20 years, a number of low molecular weight effectors have been reported, that are apparently detected in a variety of cell types in response to insulin treatment, These effectors have been reported to regulate the activity of many of the key enzymes involved in the insulin response and also to affect flux through the metabolic pathways. Experiments were carried out to measure levels of this inhibitor in vivo in different nutritional states (starved and fed) throughout the diurnal cycle and in response to acute insulin stimulation by glucose injection. It was not possible to detect an effect of insulin on inhibitor levels or effectiveness in these studies, although differences in the characteristics of the inhibition were observed during the diurnal cycle. The inhibitor was more effective with the dephosphorylated enzyme in extracts prepared from fed rats than that from starved rats. A C C was activated in a time dependent fashion by a 75 kD cytosolic protein 142 in a manner similar to that seen in response to treatment of intact cells with insulin. Further characterisation of this protein revealed that the interaction with A C C alters the sensitivity of the enzyme to the allosteric regulator citrate. The protein binds tenaciously to A C C and under the conditions tested, is only displaced from the enzyme by avidin, a particularly stringent condition. This suggests that the activator interacts with A C C strongly and quite probably near the active site. Importantly, the identification of this activator protein offers an explanation for the extensive apparent loss of A C C activity which is evident in most preparations employing Sepharose-avidin affinity chromatography. The studies described here indicate that many factors may be involved in the regulation of A C C in response to insulin. Further studies are required to investigate the role of I-site phosphorylation in the short term regulation of A C C and the effects of site-specific phosphorylation on the binding of the activator. These studies will lead to a more comprehensive understanding of the regulation of intracellular metabolism in response to insulin end help to identify important factors involved in this complex mechanism. 143 R E F E R E N C E S Abdel-Halim M.N. and Porter J.W. (1980): A new mechanism of regulation of rat liver acetyl-CoA carboxylase. J. Biol. Chem., 255:441-444. Abdel-Halim M.N. and Yousufzai (1981): Purification and properties of rat liver acetyl-CoA carboxylase protein inhibitor. Int. J. Biochem., 13:1171-1176. Abdel-Halim M . N . and Farah S.I. (1985): Review. Short-term regulation of acetyl-C o A carboxylase: is the key enzyme in long-chain tatty acid synthesis regulated by an existing physiological mechanism. Comp. Biochem. Physiol., 81:9-19. Agius L. , Rolls B J . , Rowe E J . and Williamson D . H . (1980): Impaired lipogenesis in mammary glands of lactating rats fed on a cafeteria diet. Biochem. J., 186:1005-1008. Ahmad F., Ahmad P.M., Pieretti L . and Waters G . (1978): Purification and subunit structure of rat mammary gland acetyl-Coenzyme A carboxylase. J. Biol. Chem., 253:1733-1737. Allred J.B. and Roehrig K.L . (1973):, Inhibition of rat liver acetyl-Coenzyme A carboxylase by N 6 ,Or -dibutyryl cyclic adenosine 3':5'-monophosphatein vitro. J. Biol. Chem. 248:4131-4133. Allred J.B. and Roman-Lopez C R . (1988): Enzymatically inactive forms of acetyl-C o A carboxylase in rat liver mitochondria., Biochem. J. , 251:881-885. Allred J.B., Roman-Lopez C.R., Jurin R.R. and McCune S.A. (1989): Mitochondrial storage forms of acetyl C o A carboxylase: mobilization/activation accounts for increased activity of the enzyme in liver of genetically obese Zucker rats: J . Nutrition, 119:478-483. Aultman K.S. and Mapes J.P. (1982): Inhibitors of rat liver acetyl-coA carboxylase. Fed. Proc.,41(4):1191. Bar R.S. and Sadra A (1988): Insulin receptors in vascular epithelium. In; Receptor Biochemistry and Methodology, Vol 12A Ed. Kahn C R . and Harrison L .C . , Liss, New York. pp. 267-279. Bernier M . , Laird D . M . and Lane M . D . (1988): Identification and role of a 15-kilodalton cellular target of the insulin receptor tyrosine kinase. In; Insulin Action and Diabetes. Ed. Goren H.J. et al., Raven Press, New York. pp. 117-128. Bergeron J.J.M., Cruz J., Khan M . N . and Posner B.I. (1985): Uptake of insulin and other ligands into receptor-rich endocytic components of target cells; the endosomal apparatus. Ann. Rev. Physiol., 47:383-403. -Berridge M . (1984): Inositol trisphosphate and diacylglycerol as second messengers. Biochem. J., 220:345-360. 144 Beynon A C , Vaartjes W J . and Geelen M J . H . (1979): Opposite effects of insulin and glucagon in acute hormonal control of hepatic lipogenesis. Diabetes 28:828-835. Bianchi A. , Evans J.L., Iverson A J . , Nordlund A C , Watts T .D . and Witters L . A (1990): Identification of an isozymic form of acetyl-CoA carboxylase. J. Biol. Chem., 265:1502-1509. Blackshear P.J., Witters L . A , Girard P.R., Kuo J.F. and Quamo S.N. (1985): Growth factor-stimulated protein phosphorylation in 3T3-L1 cells: Evidence for protein kinase C-dependent and -independent pathways. J. Biol. Chem., 260:13304-13315. Blackshear P J . (1986): Protein phosphorylation in cultured cells : Interactions among insulin, growth factors and phorbol esters. In; Mechanisms of Insulin Action, Vol. 7. Ed. Stralfors P. et al., Elsevier, Amsterdam, pp. 211-227. Blackshear P J . and Haupt D . M . (1989): Evidence against insulin-stimulated phosphorylation of calmodulin in 3T3-L1 adipocytes. J. Biol. Chem., 264:3854-3858. Blytt H J . and Kim K . H . (1982): Phosphoinositide inhibition of acetyl- C o A carboxylase from rat liver. Arch. Biochem. Biophys., 213:523-529. Bonne D., Belhadj O. and Cohen P. (1978): Calcium as modulator of the hormonal-receptors-biological-response coupling system. Eur. J . Biochem., 86:261-266. Borthwick A C , Edgell N J and Denton R . M . (1987): Use of rapid gel-permeation chromatography to explore the inter-relationships between polymerization, phosphorylation and activity of acetyl-CoA carboxylase. Effects of insulin and phosphorylation by cyclic A M P -dependent protein kinase., Biochem. J., 241:773-782. Bradford M . M . (1976): A rapid and sensitive method for quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem., 72:248-254 Brownsey R.W., Hughes W . A , Denton R . M . and Mayer R J . (1977): Demonstration of the phosphorylation of acetyl-CoA carboxylase within intact rat epididymal fat cells. Biochem. J. , 168:441-445. Brownsey R.W., Bridges B. and Denton R . M . (1977): Effects of fluoroacetate and (-)-hydroxycitrate on fatty acid synthesis in rat epididymal adipose tissue. Biochem. Soc. Trans., 5:1286-1288. Brownsey R.W., Hughes W . A and Denton R . M . (1979): Adrenaline and the regulation of acetyl-CoA carboxylase in rat epididymal adipose tissue. Inactivation of the enzyme is associated with phosphorylation and can be reversed by dephosphorylation. Biochem. J., 184:23-32. Brownsey R.W. and Hardie D . G . (1980): Regulation of acetyl-CoA carboxylase: identity of sites phosphorylated in intact cells treated with adrenaline and i n vitro by cyclic AMP-dependent protein kinase. FEBS Lett., 120:67-70. 145 Brownsey R.W., Belsham G J . and Denton R . M . (1981): Evidence for phosphorylation of acetyl-CoA carboxylase by a membrane-associated cyclic AMP-independent protein kinase. Relationship to the activation of acetyl-CoA carboxylase by insulin. F E B S Lett., 124:145-150. Brownsey R.W. and Denton R . M . (1982): Evidence that insulin activates fat-cell acetyl-CoA carboxylase by increased phosphorylation of a specific site. Biochem. J., 202:77-86 Brownsey R.W., Edgell N.J., Hopkirk T J . and Denton R . M . (1984): Studies on insulin-stimulated phosphorylation of acetyl-CoA carboxylase, A T P -citrate lyase and other proteins in rat epididymal adipose tissue. Biochem. J., 218:733-743. Brownsey R.W. and Denton R . M . (1987): Acetyl-Coenzyme A Carboxylase. In; The Enzymes Vol .XVffl . Ed. Boyer P. and Krebs E . G . , Academic Press, New York. pp. 123-146. Brownsey R.W., Dong G.W., Lam V . and McGreer W. (1988): Studies on protein phosphorylation using subcellular fractions from insulin-treated white adipose tissue of rats. Biochem. Cell Biol., 66:296-308. Brownsey R.W. Quayle K . A and Mui A L . F . (1988): Studies on the regulation of acetyl-CoA carboxylase by insulin. In: Insulin Action and Diabetes. Ed. Goren H.J. Raven Press, New York. pp. 173-184. Buechler K . F . and Gibson D . M . (1984): Guanosine triphosphate and colchicine affect the activity and the polymeric state of acetyl-coA carboxylase. Arch. Biochem. Biophys., 233:698-707. Carling D . and Hardie D . G . (1986): Isolation of a cyclic AMP-independent protein kinase from rat liver and its effects on the enzymic activity of acetyl-C o A carboxylase. Biochem. Soc. Trans., 14:1067-1077. Carling D., Zammit V . A and Hardie D . G (1987): A common bicyclic protein kinase cascade inactivates the regulatory enzymes of fatty acid and cholesterol biosynthesis. FEBS Lett., 223:217-222. CarHng D., Clarke P.R., Zammit V . A and Hardie D . G . (1989): Purification and characterisation of the AMP-activated protein kinase. Eur. J. Biochem., 186:129-136. Cheng K., Groake J., Ostimehin B., Haspel H . C . and Sonenberg M . (1981): Effects of insulin, catecholamines and cyclic nucleotides on rat adipocyte membrane potential. J. Biol. Chem., 256:649-655. Chou C K , , Dull T.J., Russell D.S., Gherzi R., Lebwohl D., Ullrich A and Rosen O . M . (1987): Human insulin receptors mutated at the ATP-binding site lack protein tyrosine kinase activity and fail to mediate postreceptor effects of insulin. J. Biol. Chem., 262:1842-1847. Cobb M . H . and Rosen O . M . (1983): Description of a protein kinase derived from insulin-treated 3T3-L1 cells that catalyses the phosphorylation of ribosomal protein S6 and casein. J. Biol. Chem., 258:12472-12481. 146 Cobb M . H . (1986): A n insulin-stimulated ribosomal protein S6 kinase in 3T3-L1 cells. J. Biol. Chem., 261:12994-12999. Cohen P. (1988): In; Molecular Aspects of Cellular Regulation, Vol . 5. Ed. Cohen P. and Klee C , Elsevier, Amsterdam, pp. 145-193. Colca J.R., DeWald D.B., Pearson J.D., Palazuk B.J., Laurino J.P. and McDonald J .M. (1987): Insulin stimulates the phosphorylation of calmodulin in intact adipocytes. J. Biol. Chem., 262:11399-11402. Davies S.P., Carling D . and Hardie D . G . (1989): Tissue distribution of the A M P -activated protein kinase, and lack of activation by cyclic-AMP-dependent protein kinase, studied using a specific and sensitive peptide assay., Eur. J. Biochem., 186:123-128. Davies S.P., Sim A T . R . and Hardie D . G (1990): Location and function of three sites phosphorylated on rat acetyl-coA carboxylase by the A M P -activated protein kinase. Eur. J . Biochem., 187:183-190. Davis (1981): The effect of insulin on plasma membrane and mitochondrial membrane potentials in isolated fat -cells. Biochem. J., 196:133-147. Denton R . M . and Halperin M . L . (1968): The control of fatty acid and triglyceride synthesis in rat epididymal adipose tissue. Biochem. J., 110:27-38. Denton R . M . (1986): Early Events in Insulin Action. In; Advances in Cyclic Nucleotide and Protein Phosphorylation Research, Vol . 20. Ed . Greengard P. and Robison G . A , Raven Press, New York. pp. 293-341. Denton R . M . and Tavare J .M. (1988): Insulin action: mechanisms involved in the rapid effects of insulin on lipid metabolism. In; The Diabetes Annual/4. Ed. Albert K . G . M . M . and Krall L.P. Elsevier Science Publishers, pp 546-563. Desai K.S., Zinman B., Steiner G . and Hollenberg C H . (1978): Effect of calcium on [125I]-insulin binding to rat adipocytes. Can. J. Biochem., 56:843-848. Deschatrette J. and Weiss M . C (1974): Characterization of differentiated and dedifferentiated clones from a rat hepatoma. Biochimie, 56:1603-1611. Ebina Y. , Araki E . , Taira M . , Shimanda F., Mori M . , Craik C , Siddle K. and Pierce S. (1987): Proc. Natl. Acad. Sci. U S A , 84:704-708. Ebina Y. , Ellis L., Jarnagin K., Edery M. , Graf L. , Clauser E. , Ou J., Masiarz F., Kan Y.W., Goldfine I.D., Roth R.A. and Rutter W J . (1985): The human insulin receptor cDNA: The structural basis for hormone-activated transmembrane signalling. Cell, 40:747-758. Espinal J. (1988): What is the role of the insulin receptor tyrosine kinase? TIBS 13 10:367-368. 147 Farese R.V., Davis J.S., Barnes D.E., Standaert M.L., Babischkin J.S., Hock R., Rosie N.K, and Poller R J . (1985): The de novo phospholipid effect of insulin is associated with increases in diacylglycerol, but not inositol phosphates or cytosolic C a 2 + . Biochem. J., 231:269-278. Farese R.V., Kuo J.Y., Babischkin J.S. and Davis J.S. (1986): Insulin provokes a transient activation of phospholipase C in the rat epidiymal fat pad. J. Biol. Chem., 261:8589-8592. Fehlmann M., Carpentier J.L., LeCam A,, Thamm P., Saunders D., Brandenburg D., Orci L. and Freychet P. (1982) Biochemical and morphological evidence that the insulin receptor is internalized with insulin in hepatocytes. J. Cell. Biol., 93:82-87. Ferguson M . A J . (1988): Cell-surface anchoring of proteins via glycosyl-phosphatidylinositol structures. Ann. Rev. Biochem., 57:285-320. Fink R.I. and Freidenberg G. (1988): Clinical application of insulin receptor studies: Diabetes and states of glucose intolerance. In; Receptor Biochemistry and Methodology, Vol. 12B. Ed. Kahn C R . and Harrison L.C., Liss, New York. pp. 75-105. Folch J . , Ascoli I., Lees M., Meath J . A and LeBaron F.N. (1951): Preparation of lipide extracts from brain tissue. J. Biol. Chem., 191:833. Gabriella B., Wettenhall R.E.H., Kemp B.E., Quinn M . and Bizonova L. (1984): Phosphorylation of ribosomal protein S6 and a peptide analogue of S6 by a protease-activated kinase isolated from rat liver. FEBS Lett, 175:219-224. Gibbs E.M., Allard WJ. and Lienhard G.E. (1986): The glucose transporter in 3T3-L1 adipocytes is phosphorylated in response to phorbol ester but not in response to insulin. J. Biol. Chem., 261:16597-16603. Goldman J. and Rybicki B. A (1986)^Diabetes, 35:320_._ - : ^ Goldstein J.L., Brown M.S., Anderson R.G.W., Russell D.W. and Schneider WJ. (1985): Receptor-mediated endocytosis: Concepts emerging from the L D L receptor system. Ann. Rev. Cell. Biol., 1:1-39. Goodridge A G . (1973a): Regulation of fatty acid synthesis in the liver of prenatal and early postnatal chicks. J. Biol. Chem., 248:1939-1945. Goodridge A G . (1973b): Regulation of fatty acid synthesis in isolated hepatocytes. J. Biol. Chem., 248:4318-4326. Green H. and Kehinde O. (1974) Sublines of mouse 3T3 cells that accumulate lipid. Cell 1:113-116 Graves C.B. and MacDonald J.M. (1985): Insulin and phorbol ester-stimulate phosphorylation of a 40-KDa protein in adipocyte plasma membranes. J. Biol. Chem., 260:11286-11292. 148 Graves C.B., Gale R.D., Laurino J.P. and McDonald J.M. (1986): The insulin receptor and calmodulin: Calmodulin enhances insulin-mediated receptor kinase activity and insulin stimulates phosphorylation of calmodulin. J. Biol. Chem., 263:10429-10438. Halestrap A P . and Denton R . M . (1973): Insulin and the regulation of adipose tissue acetyl-coenzyme A carboxylase. Biochem. J. 132:509-517. Halestrap A P . and Denton R . M . (1974): Hormonal regulation of adipose-tissue acetyl-Coenzyme A carboxylase by changes in the polymeric state of the enzyme. Biochem. J., 142:365-377. Hardie D . G . and Cohen P. (1978a): The regulation of fatty acid biosynthesis. F E B S Lett., 91:1-7. Hardie D . G . and Cohen P. (1978b): Purification and physiochemical properties of fatty acid synthetase and acetyl-CoA carboxylase from lactating rabbit mammary gland. Eur. J. Biochem., 92:25-34. Hardie D . G . and Cohen P. (1979): Dephosphorylation and activation of acetyl-C o A carboxylase from lactating rabbit mammary gland. F E B S Lett., 103:333-338. Hardie D . G . and Guy P.S. (1980): Reversible phosphorylation and inactivation of acetyl-CoA carboxylase from lactating rat mammary gland by cyclic AMP-dependent protein kinase. Eur. J. Biochem., 110:167-177. Hardie D .G. , Carling D., Ferrari S., Guy P.S., Aitken A (1986): Characterization of the phosphorylation of rat mammary ATP-citrate lyase and acetyl-C o A carboxylase by C a 2 + and calmodulin-dependent multiprotein kinase and C a 2 + and phospholipid-dependent protein kinase. Eur. J. Biochem. 157:553-561. Hardie D . G . (1989): Regulation of fatty acid synthesis via phosphorylation of acetyl-CoA carboxylase. [Review], Progress In Lipid Research 28:117-46. Hardie D .G. , Carling D. and Sim A T . (1989): The AMP-activated protein kinase: a multisubstrate regulator of lipid metabolism. TIBS, 14:19-23. Hathaway G . M . and Traugh J.A. (1982): Casein kinases-multipotential protein kinases. Curr. Top. Cell. Regul., 21:101-127. Haystead T . A and Hardie D . G . (1986): Evidence that activation of acetyl-CoA carboxylase by insulin in adipocytes is mediated by a low-Mr effector and not by increased phosphorylation. Biochem. J. 240:99-106. Haystead T . A , Campbell D . G . and Hardie D . G , (1988): Analysis of sites phosphorylated on acetyl-CoA carboxylase in response to insulin in isolated adipocytes. Comparison with sites phosphorylated by casein kinase-2 and the calmodulin-dependent multiprotein kinase. Eur. J. Biochem., 175:347-354. 149 Haystead T.A. , Moore F., Cohen P. and Hardie D . G . (1990): Roles of the A M P -activated and cyclic-AMP-dependent protein kinases in the adrenaline-induced inactivation of acetyl-CoA carboxylase in rat adipocytes. Eur. J. Biochem., 187:199-205. Heidenreich K . A (1988): Structure of insulin receptors in nervous tissue. In; Receptor Biochemistry and Methodology, Vol 12A Ed. Kahn C R . and Harrison L . C . , Liss, New York. pp. 281-292. Hedo J . A and Simpson L A (1984): Internalisation of insulin receptors in the isolated rat adipose cell. Demonstration of the vectorial disposition of the receptor subunits. J. biol. Chem., 259:11083-11089. Holland R., Witters L . A and Hardie D . G . (1984): Glucagon inhibits fatty acid synthesis in isolated hepatocytes via phosphorylation of acetyl-coA carboxylase by cyclic AMP-dependent protein kinase. Eur. J . Biochem., 140:325-333. Holland R., Hardie D .G. , Clegg R . A and Zammit V . A (1985): Evidence that glucagon-mediated inihbition of acetyl-CoA carboxylase in isolated adipocytes occurs as a result of increased phosphorylation of the enzyme by cyclic AMP-dependent protein kinase. Biochem. J., 226:139-149. Holland R. and Hardie D . G . (1985): Both insulin and epidermal growth factor stimulates fatty acid synthesis and increase phosphoryaltion of acetyl-CoA carboxylase and ATP-citrate lyase in isolated hepatocytes. FEBS Lett., 181:308-312. Houston B. and Nimmo H . G . (1985): Effects of phosphorylation on the kinetic properties of rat liver ATP-cirate lyase. Biochem. Biophys. Acta., 844:233-239. Ichihara A , Nakamura T. and Tanaka K. (1982): Use of hepatocytes in primary culture for biochemical studies on liver functions. Mol. Cell. Biochem., 43:145-160. Ingebritsen T.S. and Cohen P. (1983): Protein phosphatases: properties and role in cellular regulation. Science, 221:331-338. Ingebritsen T.S., Blair J., Guy P., Witters L . and Hardie D . G . (1983): The protein phosphatases involved in cellular regulation. Eur. J. Biochem., 132:275-281. Inoue H . and Lowenstein J . M . (1972): Acetyl coenzyme A carboxylase from rat liver. J. Biol. Chem., 247:4825-4832. Jamil H . , Madsen N.B. (1987): Phosphorylation state of acetyl-coenzyme A carboxylase. I. Linear inverse relationship to activity ratios at different citrate concentrations. J. Biol. Chem., 262:630-637. Jamil H . , Madsen N.B. (1987): Phosphorylation state of acetyl-coenzyme A carboxylase. II. Variation with nutritional condition. J. Biol. Chem., 262:638-642. 150 Jarett L. , Kiechle F.L. , Parker J.C. and Macauley S.L. (1983): The chemical mediators of insulin action: possible targets for postreceptor defects. Am. J. Med., 74 (Suppl. IA) 31-37. Jarett L J . (1988): Mediators of insulin action. In; Insulin Action and Diabetes. Ed. Goren H J . et al., Raven Press, New York, pp 105-111. Jones R . G . , Ilic V. and Williamson D . H . (1984): regulation of lactating-rat mammary-gland lipogenesis by insulin and glucagon in vivo. Biochem. J., 223:345-351. Kadowaki T , Koyasu S., Nishida E . , Tobe K,, Izumi T., Takaku F., Sakai H . , Yahara I. and Kasuga M . (1987): Tyrosine phosphorylation of common and specific sets of cellular proteins rapidly induced by insulin, insulin-like growth factor, and epidermal growth factor in an intact cell. J. Biol. Chem., 262: 7342-7350. Kashiwagi A , Verso M . A Andrews J., Vasquez B., Reaven G . and Foley J .E . (1983): In vitro insulin resisitance of human adipocytes isolated from subjects with non-insulin dependent diabetes mellitus. J. Clin. Invest, 72:1246-1254. Kasuga M . , Fujita-Yamaguchi Y. , Blithe D .L . and Kahn C R . (1983): Tyrosine specific protein kinase activity is associated with the purified insulin receptor. Proc. Natl. Acad. Sci. U S A 80:2137-2141. Kasuga M . , Karlsson F . A and Kahn C R . (1982a): Insulin stimulates the phosphorylation of the 95,000-dalton subunit of its own receptor. Science, 215:185-187. Kasuga M . , Zick Y. , Blithe D.L. , Crettaz M . and Kahn C R . (1982b): Insulin stimulates tyrosine phosphorylation of the receptor in a cell-free system. Nature, 298:667-669. Kiechle F .L . , Jarett L . , Popp D . A and Kotagal N. (1980): Isolation from rat adipocytes of a chemical mediator for insulin activation of pyruvate dehydrogenase. Diabetes, 29:852-855. Kim K . H . (1983): Regulation of acetyl-CoA carboxylase. Curr. Top. Cell. Regul., 22:143-176. Kim K . H . , Lopez-Casillas F., Bai D.H. , Luo X., Pape M . E . (1989): Role of reversible phosphorylation of acetyl-CoA carboxylase in long-chain fatty acid synthesis. [Review], F A S E B Journal 3:2250-2256. Kirsch D., Obermaier B. and Haring H . U . (1985): Phorbol esters enhance basal D-glucose transport but inhibit insulin stimulation of D-glucose transport and insulin binding in isolated rat adipocytes. Biochem. Biophys. Res. Commun., 128:824-833. Knowles B.B., Howe C C , Aden D.P. (1980): Human hepatocellular carcinoma cell lines secrete the major plasma proteins and hepatitus B surface antigen. Science, 209:497-499. 151 Kobayashi M . and Olefsky J .M. (1979): Effects of streptozotocin-Lnduced diabetes on insulin binding, glucose transport, and intracellular glucose metabolism in isolated rat adipocytes. Diabetes, 28:87-95. Kolterman O.G. , Insel J., Saekow M . and Olefsky J .M. (1980): Mechanisms of insulin resistance in human obesity. Evidence for receptor and post-receptor defects. J . Clin. Invest., 65:1273-1284. Kolterman O.G. , Gray R.S., Griffin J., Burstein P., Insel J., Scarlett J . A and Olefsky J .M. (1981): Receptor and post-receptor defects contribute to the insulin resisitance in non-insulin dependent diabetes mellitus. J . Clin. Invest., 68:957-969. Krakower G.R. and Kim K . H . (1981): Purification and properties of acetyl-CoA carboxylase. J. Biol. Chem., 256:2408-2413. I^emmli U .K. (1970): Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature, 227:680-685. Lane M.D. , Moss J. and Polakis S.E. (1974): Acetyl Coenzyme A Carboxylase. Curr. Top. Cell. Regul, 8:139-195. Larner J. (1972): Insulin and glycogen synthase. Diabetes, 21:428-438. Larner J., Cheng K., Schwartz C , Kikuchi K., Tamura S., Creacy S., Dubler R., Galasko G. , Pullin C. and Katz M . (1982): A proteolytic mechanism for the action of insulin via oligopeptide mediator formation. Fed. Proc, 41:2724-2729. Larner J . (1983): Mediators of postreceptor action of insulin. A m . J. Med., 74 (Suppl. IA) 38-51. Lastick S.M. and McConkey E . H . (1981): HeLa ribosomal protein S6. Insulin and dibutyrylcyclic A M P affect different phosphopeptides. J. Biol. - Chem., 256:583-585. - -Le Marchand-Brustel Y. , Freychet P. and Jeanrenaud B. (1978): Longitudinal study on the establishment of insulin resistance in hypothalmic obese mice. Endocrinology, 102:74-85. Le Marchand-Brustel Y . and Freychet P. (1979): Effect of fasting and streptozotocin diabetes on insulin binding and action in the isolated soleus muscle J. Clin. Invest., 64:1505-1515. Lee K . H . and Kim K . H . (1978): Effect of epinephrine on acetyl-CoA carboxylase in rat epididymal fat tissue. J. Biol. Chem., 253:8157-8161. Lee K . H . , Thrall T. and Kim K . H . (1973): Hormonal regulation of acetyl-CoA carboxylase-effect of insulin and epinephrine. Biochem. Biophys. Res. Comm., 54:1133-1140. Lent B . A , Lee K . H . and Kim K . H . (1978): Regulation of rat liver acetyl-CoA carboxylase. J. Biol. Chem., 253:8149-8156. 152 Lent B.A. and Kim K . H . (1982): Purification and properties of a kinase which phosphorylates and inactivates acetyl-CoA carboxylase. J. Biol. Chem., 257:1897-1901. Lent B . A and Kim. K . H . (1983a): Phosphorylation and activation of acetyl-Coenzyme A carboxylase kinase by the catalytic subunit of cyclic AMP-dependent protein kinase. Arch. Biochem. Biophys., 225:972-978. Lent B . A and Kim. K . H . (1983b): Requirement of acetyl-Coenzyme A carboxylase kinase for coenzyme A Arch. Biochem. Biophys., 225:964-971. Lerea K . M . and Livingston J.N. (1988): Insulin receptors in specialised tissues-hepatocytes. In; Receptor Biochemistry and Methodology, Vol . 12A Ed. Kahn C R . and Harrison L.C. , Liss, New York. pp. 205-219. Lonnroth P., DiGirolamo M . , Krotkiewski M . and Smith U . (1983): Insulin binding and responsiveness in fat cells from patients with reduced glucose tolerance and type II diabetes. Diabetes 32:748-754. Lopez-Casillas F., Bai D .H. , Luo X.C. , Kong I.S., Hermodson M . A and Kim K . H , (1988): Structure of the coding sequence and primary amino acid sequence of acetyl-coenzyme A. carboxylase. Proc. Natl. Acad. Sci. U S A , 85:5784-5788. Lopez-Casillas F. and Kim K . H . (1989): Heterogeneity at the 5' end of rat acetyl-coenzyme A carboxylase m R N A Lipogenic conditions enhance synthesis of a unique m R N A in liver. J. Biol. Chem., 264:7176-7184. Low M . G . (1987): Biochemistry of the glycosyl-phosphatidylinositol membrane protein anchors. Biochem. J., 244:1-13. Madoff D . H . , Martensen T . M . and Lane M . D . (1988): Insulin and insulin-like growth factor 1 stimulate the phosphorylation on tyrosine of a 160 kDa cytosolic protein in 3T3-L1 adipocytes. Biochem. J. , 252:7-15. Martin-Perez J. and Thomas G . (1983): Ordered phosphorylation of 40S ribosodmal protein S6 after serum stimulation of quiescent 3T3 cells. Proc. Natl. Acad. Sci. U S A , 80:926-930. Mabrouk G . M . , Helmy I.M., Thampy K . G . and Wakdl S J . (1990): Acute hormonal control of acetyl-CoA carboxylase. J. Biol. Chem., 265:6330-6338. Margolis R.N. , Schell M.J., Taylor S.I. and Hubbard A L . (1990): Hepatocyte plasma membrane ecto-ATPase (ppl20/H4) is a substrate for tyrosine kinase activity of the insulin receptor. Biochem. Biophys. Res. Comm., 166:562-566. McCormack J . G . and Denton R . M . (1984): Role of Ca 2 + ions in the regulation of intrramitochondrial metabolism in rat heart. Evidence from studies with isolated mitochondria that adrenaline activates the pyruvate dehydrogenase and 2-oxoglutarate dehydrogenase complexes by increasing the intramitochondrial concentration of Ca . Biochem. J., 218:235-247. 153 McNeillie E .M., Clegg R . A and Zammit V . A (1981): Regulation of acetyl-CoA carboxylase in rat mammary gland. Effects of incubation with C a 2 + , Mg 2* and ATP on enzyme activity in tissue extracts. Biochem. J., 200:639-644. McNeillie E.M. and Zammit V A (1982): Regulation of acetyl-CoA carboxylase in rat mammary gland. Effects of starvation and of insulin and prolactin deficiency on the fraction of the enzyme in the active form in vivo. Biochem. J., 204:273-280. Meredith M J . and Lane M.D. (1978): Acetyl-CoA carboxylase. J. Biol. Chem., 253:3381-3383. Milatovich A , Plattner R., Heerema N.A, Palmer C.G., Lopez-Casillas F., Kim K.H. (1988): Localization of the gene for acetyl-CoA carboxylase to human chromosome 17., Cytogen. Cell Gene., 48:190-192. Momomura K,, Tobe K., Seyama Y. Takaku F. and Kasuga M. (1988): Insulin-induced tyrosine-phosphorylation in intact rat adipocytes. Biochem. Biophys. Res. Commun., 155:1181-1186. Morgan D.O. and Roth R . A (1987): Acute insulin action requires insulin receptor kinase activity: Introduction of an inhibitory monoclonal antibody into mammalian cells blocks the rapid effects of insulin. Proc. Natl. Acad. Sci., USA, 84:41-45. Munday M.R. and Hardie D.G. (1984): Isolation of three cyclic-AMP-independent acetyl-CoA carboxylase kinases from lactating rat mammary gland and characterisation of their effects on enzyme activity. Eur. J. Biochem., 141:617-627. Munday M.R. and Hardie D.G. (1986): Insulin activation of acetyl-CoA carboxylase in isolated mammary acini from lactating rats fed on a high-fat diet, Biochem. Soc. Trans., 14:1075-1076. Munday M.R. and Hardie D.G. (1986a): The role of acetyl-CoA carboxylase phosphorylation in the control of mammary gland fatty acid synthesis during the starvation and re-feeding of lactating rats., Biochem. J., 237:85-91. Munday M.R., Haystead T A . . , Holland R., Carling D . A and Hardie D.G. (1986b): The role of phosphorylation/dephosphorylation of acetyl-CoA carboxylase in the regulation of mammalian fatty acid biosynthesis. Biochem. Soc. Trans. 14:559-562. Munday M.R. and Williamson D.H. (1986): Insulin activation of lipogenesis in isolated mammary acini from lactating rats fed a high-fat diet. Evidence that acetyl-CoA carboxylase is a site of action. Biochem. J., 242:905-911. Munday M.R., Campbell D.G., Carling D. and Hardie D.G.(1988): Identification by amino acid sequencing of three major regulatory phosphorylation sites on rat acetyl-CoA carboxylase. Eur. J. Biochem. 175:331-338. 154 Munday M.R., Carling D . and Hardie D . G . (1988): Negative interactions between phosphorylation of acetyl-CoA carboxylase by the cyclic A M P -dependent and AMP-activated protein kinases., FEBS Lett., 235:144-148. Nemenoff R . A , Gunsalus J.R. and Avruch J. (1986): A n insulin-stimulated (ribosomal S6) protein kinase from soluble extracts of H4 hepatoma cells. Arch. Biochem. Biophys., 254:196-203. Nishizuka Y . (1984): The role of protein kinase C in cell surface signal transduction and tumour promotion. Nature, 308:693-697. Norman A W . and Litwack G . (1987): Pancreatic hormones. In; Hormones. Academic Press Inc., pp 263-320. Numa S. and Tanabe T. (1984): Acetyl-coenzyme A carboxylase and its regulation. In; Fatty Acid Metabolism and its Regulation. Ed. Numa S., Elsevier, New York. pp. 1-27. Numa S. and Yamashito S. (1974): Regulation of lipogenesis in animal tissue. Curr. Top. Cell Regul., 8:197-246. Ogiwara H . , Tananbe T , Nikawa J. and Numa S. (1978): Inhibition of rat liver acetyl-CoA carboxylase by palmitoyl-CoA: formation of equimolar enzyme-inhibitor complex. Eur. J. Biochem., 89:33-41. Pederson O. (1988): Insulin receptors in human adipocytes. In; Receptor Biochemistry and Methodology, Vol 12B. Ed. Kahn G R . and Harrison L .C . , Liss, New York. pp. 63-73. Pelech S.L., Meijer L . and Krebs E . G . (1987): Characterisation of maturation-activated histone H I and ribosomal S6 kinases in sea star oocytes. Biochemistry, 26:7960-7968. Pelech S.L., Sanghera J.S., Padden H.B., Quayle K . A . and Brownsey R W . (1990): Identification of the major maturation-activated acetyl-CoA carboxylase kinase in sea star oocytes as p44 m p . Submitted. Biochem. J. Pennington S.R. and Martin B.R. (1985): Insulin-stimulated phosphoinositide metabolism in isolated fat cells. J. Biol. Chem., 260:11039-11045. Perotti N., Accili D., Rees-Jones R.W. and Taylor S.I. (1986): Diabetes, 35, Suppl. 1, 9 A Pekala P.H., Meredith M.J., Tarlow D . M . and Lane M.D. (1978): Multiple phosphorylation ofacetyl-CoA carboxylase in chick liver cells. J. Biol. Chem., 253:5267-5269. Pierce M.W., Palmer J.L., Keutmann H.T. Hall T .A. and Avruch J. (1982): The insulin-directed phosphorylation sites on ATP-citrate lyase. J. Biol. Chem., 257:10681-10686. Pitot H . C , Peraino C , Morse P . A and Potter V.R. (1964) Hepatomas in tissue culture compared with adapting liver in vivo. Natl. Cancer Inst. Monogr. 13:229-242. 155 Plehwe W.E. , Williams P.F., Caterson I.D. Harrison L . C . and Turtle J.R. (1983): Calcium-dependence of insulin receptor phosphorylation. Biochem. J., 214:361-366. Posner B.I., Kahn M . N . and Bergeron J .M. (1988): The role of endosomal kinase activity in insulin action. In; Insulin Action and Diabetes. Ed. Goren H J . et al., Raven Press, New York, pp 141-150. Posner B.I., Patel B., Verma A . K . and Bergeron J.J.J.M. (1980): Uptake of insulin by plasmalemma and Golgi subcellular fractions of rat liver. J . Biol. Chem., 255:735-741. Ray L .B . and Sturgill T.W. (1988): Characterization of insulin-stimulated microtubule-associated protein kinase. J, Biol. Chem., 263:12721-12727. Rail T.W. and Sutherland E.W. (1958): Formation of a cyclic adenine ribonucleotide by tissue particles. J. Biol. Chem., 232:1065-1076. Rees-Jones R.W. and Taylor S.I. (1985): A n endogenous substrate for the insulin receptor-associated tyrosine kinase. J. Biol. Chem., 260:4461-4467. Robinson A M . , Girard J.R. and Williamson D . H . (1978): Evidence for a role of insulin in the regulation of lipogenesis in lactating rat mammary gland. Biochem. J. 176:343-346. Rodriguez-Pena A and Rozengurt E . (1984): Disappearance of Ca2+-sensitive, phospholipid-dependent protein kinase activity in phorbol ester-treated 3T3 cells. Biochem. Biophys. Res. Comm., 120:1053-1059. Rosen O . M . , Herrera R., Olowe R., Petruzelli L . M . and Cobb M . H . (1983): Phosphorylation activates the insulin receptor tyrosine protein kinase. Proc. Natl. Acad. Sci., U S A , 80:3237-3240. Roth R . A and Cassell D J . (1983): Insulin receptor: Evidence that it is a protein kinase. Science, 219:299-301. Sacks D.B. and McDonald J .M. (1988): Insulin-stimulated phosphorylation of calmodulin by rat liver insulin receptor preparations. J. Biol. Chem., 263:2377-2383. Sacks D.B., Fujita-Yamaguchi Y. , Gale R.D. and McDonald J .M. (1989): Tyrosine-specific phosphorylation of calmodulin by the insulin receptor kinase purified from human placenta. Biochem. J., 263:803-812. Saggerson E . D . and Greenbaum A L . (1970): The regulation of triglyceride synthesis and fatty acid synthesis in rat epididymal adipose tissue. Biochem. J., 119:193. Sakamoto Y., Kuzuya T. and Sato J, (1982): Demonstration of a pyruvate dehydrogenase activator in insulin treated human placental membrane. Biomed. Res., 3:599-605. 156 Saltiel A R . , Siegel M.L, Jacobs S. and Cuatrecasas P. (1982): Putative mediators of insulin action: Regulation of pyruvate dehydrogenase and adenylate cyclase activities. Proc. Natl. Acad. Sci. U S A , 79:3513-3517. Saltiel A.R. , Doble A , Jacobs S. and Cuatrecasas P. (1983): Putative mediators of insulin action regulate hepatic acetyl-CoA carboxylase activity. Biochem. Biophys. Res. Comm., 110:789-795. Saltiel A R . and Cuatrecasas P. (1986a): Insulin stimulates the generation from hepatic plasma membranes of modulators derived from an inositol glycolipid. Proc. Natl. Acad. Sci. U S A , 83:5793-5797. Saltiel A R., Fox J . A , Sherline P. and Cuatracasas P. (1986b): Insulin-stimulated hydrolysis of a novel glycolipid generates modulators of c A M P phosphodiesterase. Science, 233:967-971. Sanghera SJ . , Padden H.B., Bader S . A and Pelech S.L, (1990): Purification and characterisation of a maturation-activated myelin basic protein kinase from sea star oocytes. J. Biol. Chem., 265:52-57. Schwartz C.F.W., Villar-Palasi C , Malchoff C D . and Lamer J. (1988): Isolation and characterisation of insulin mediators. In; Receptor Biochemistry and Methodology, Vol., 12B. Ed . Kahn C R . and Harrison L .C . , Liss, New York. pp. 155-170. Seals J.R. and Czech M.P. (1980): Evidence that insulin activates an intrinsic plasma membrane protease in generating a secondary chemical messenger. J. Biol. Chem., 255:6529-6531. Shia M.A. , Rubin J.B. and Pilch P.F. (1983): The insulin receptor protein kinase. Physio-chemical requirements for activity. J. Biol. Chem., 258:14450-14455. Shiao M.S., Drong R.F. and Porter J.W. (1981): The purification and properties of a protein kinase and the partial purification of a phospho-protein phosphatase that inactivate and activate acetyl-CoA carboxylase. Biochem. Biophys. Res. Comm., 98:80-87. Shemer J. , Adamo M . , Wilson G.L. , Heffez D., Zick Y . and LeRoith D. (1988): Insulin and insulin-like growth factor-I stimulate a common endogenous phospho-protein substrate ppl85 in intact neuroblastoma cells. J. Biol. Chem., 262:15476-15482. Sim A T . and Hardie D . G , (1988): The low activity of acetyl-CoA carboxylase in basal and glucagon-stimulated hepatocytes is due to phosphorylation by the AMP-activated protein kinase and not cyclic AMP-dependent protein kinase. F E B S Lett. 233:294-298. Skoglund G. , Hansson A and Ingelman-Sundberg M . (1985): Rapid effects of phorbol esters on isolated rat adipocytes. Relationship to the action of protein kinase C. Eur. J. Biochem. 148:407-412. Sommercorn J., Mulligan J . A , Lozeman F J . and Krebs E . G . (1987): Activation of casein kinase II in response to insulin and to epidermal growth factor. Proc. Natl. Acad. Sci. U S A , 84:8834-8838. 157 Song C. S. and Kim K . H . (1981): Re-evaluation of the properties of acetyl-coA carboxylase from rat liver. J. Biol. Chem., 256:7786-7788. Stadmauer L . A . and Rosen O . M (1983): Phosphoryaltion of exogenous substrates by the insulin receptor-associated protein kinase. J. Biol. Chem., 258:6682-6685. Stahl P. and Schwartz A L . (1986): Receptor-mediated endocytosis. J. Clin. Invest., 77:657-662. Stansbie D. , Brownsey R.W., Crettaz M . and Denton R . M . (1976): Acute effects in vivo of anti-insulin serum on rates of aftty acid synthesis and actiivties of acetyl-CoA carboxylase and pyruvate dehydrogenase in liver and epididymal adipose tissue of fed rats. Biochem. J., 160:413-416. Sturgill T.W., Ray L.B. , Erikson E . and Mailer J.L. (1988): Insulin-stimulated MAP-2 kinase phosphorylates and activates ribosomal protein S6 kinase II. Nature, 334:715-718. Sweet L.J . , Wilden P . A and Pessin J .E . (1986): Dithiothreitol activation of the insulin receptor/kinase does not involve subunit association of the native a-, b? insulin receptor subunit complex. Biochemistry, 25:7068-7074. Swenson T . L . and Porter J.W. (1985): Mechanism of glucagon inhibition of liver acetyl-coA carboxylase. J. Biol. Chem., 260:3791-3797. Tabarini D., Heinrich J. and Rosen O . M . (1985): Activation of S6 kinase activity in 3T3-L1 cells by insulin and phorbol ester. Proc. natl. Acad. Sci. U S A , 77:2542-2545. Takai T., Yokoyama C , Wada K., Tanabe T, (1988): Primary structure of chicken liver acetyl-CoA carboxylase deduced from c D N A sequence. J . Biol. Chem., 263:2651-2657. Tashiro-Hashimoto Y. , Tobe K., Koshio O., Izumi T., Takaku F., Akanuma Y . and Kasuga M . (1989): Tyrosine phosphorylation of ppl85 by insulin receptor kinase in a cell-free system. J. Biol. Chem., 264:6879-6885. Terao K. and Ogata K. (1979): Proteins of small subunits of rat liver ribosomes that interact with poly (v).II. Crosslinks between poly(v) and ribosomal proteins in 40S subunits induced by U V irradiation. J. Biochem. (Tokyo), 86;605-617. Thampy K . G . and Wakil S J . (1985): Activation of acetyl-CoA carboxylase: Purification and properties of a Mn 2 +-dependent phosphatase. J. Biol. Chem., 250:6318-6323. Thampy K . G . , Wakil S J . (1988): Regulation of acetyl-coenzyme A carboxylase. I. Purification and properties of two forms of acetyl-coenzyme A carboxylase from rat liver. J. Biol. Chem., 263:6447-6453. Thampy K . G . , Wakil S J . (1988): Regulation of acetyl-coenzyme A carboxylase. II. Effect of fasting and refeeding on the activity, phosphate content, and aggregation state of the enzyme., J. Biol. Chem., 263:6454-6458. 158 Thampy K . G . (1989): Formation of malonyl coenzyme A in rat heart. Identification and purification of an isozyme of acetyl-CoA carboxylase from rat heart., J. Biol. Chem., 264:17631-17634. Thomas A P . , Martin-Requero A and Williamson J.R. (1985): Interactions between insulin andaj-adrenergic agents in the regulation of glycogen metabolism in isolated hepatocytes. J. Biol. Chem., 260:5963-5973. Tipper J.P. and Witters L . A (1982): In vitro phosphorylation and inactivation of rat liver acetyl-CoA carboxylase purified by avidin affinity chromatography. Biochem. Biophys. Acta., 715:162-167. Tipper J.P., Bacon G.W. and Witters L . A (1983): Phosphorylation of acetyl-CoA carboxylase by casein kinase I and casein kinase TJ. Arch. Biochem. Biophys. 227:386-396 Topping D . L . and Mayes P . A (1972): The immediate effects of insulin and fructose on the metabolism of the perfused liver. Biochem. J., 126:295-311. Traugh J.A. (1981): Regulation of protein synthesis by phosphorylation. In; Biochemical Actions of Hormones. Ed. Litwack G . , Academic Pres, New York. pp. 167-208. Trevillyan J .M. , Perisic O., Traugh J . A and Byus C V . (1985): Insulin- and phorbol ester-stimulated phosphorylation of ribosomal protein S6. J. Biol. Chem. 260:3041-3044. Ullrich A , Bell J.R., Chen E.Y. , Herrera R., Petruzelli L . M . , Dull T J . , Gray A , Cousens L. , Liao Y . C , Tsubokawa M . , Mason A , Seeburg P.H., Grunfeld C , Rosen O . M . and Ramachandran J. (1985): Human insulin receptor and its relationship to the tyrosine family of oncogenes. Nature, 313:756-761. Vaartjes W.J., de Haas C . G . , Geelen M J . and Bijleveld C. (1987): Stimulation by a tumor-promoting phorbol ester of acetyl-CoA carboxylase activity in isolated rat hepatocytes. Biochem. Biophys. Res. Comm., 142:135-410. Vaartjes W J . and de Haas C . G . M . (1985): Acute effects of tumour-promoting phorbol esters on hepatic intermediary metabolism. Biochem. Biophys. res. Comm., 129:721-726. Vagelos P.R. (1971): Regulation of fatty acid biosynthesis. Curr. Top. Cell. Regul., 4:119-166. Van Obberghen E . , Rossi B., Kowalski A , Gazzano H . and Ponzio G . (1983): Receptor-mediated phosphorylation of the hepatic insulin receptor: Evidence that the M 95,000 receptor subunit is its own kinase. Proc. Natl. Acad. Sci. USA\ 80:945-949. Volpe J J . and Vagelos P.R. (1976): Mechanisms and regulation of biosynthesis of saturated fatty acids. Physiol. Rev., 56:339-417. 159 Wada K. and Tanabe T. (1983): Dephopshorylation and activation of chicken liver acetyl-Coenzyme A carboxylase. Eur. J. Biochem., 135:17-23. Walaas O. and Horn R.S. (1986): The effect of insulin on C a 2 + and phospholipid dependent protein kinase in muscle. In; Mechanisms of Insulin Action, Vol . 7. Ed. Stralfors J. et al., Elsevier, Amsterdam, pp. 239-248. Walker D.H. , Kuppuswamy, D., Visvanathan A and Pike L.J . (1987): Substrate specificity and kinetic mechanism of human placental insulin receptor/kinase. Biochemistry, 26:1428-1433. Wettenhall R . E . H . , Cohen P., Caudwell B. and Holland R. (1982): Differential phosphorylation of ribosomal protein S6 in isolated rat hepatocytes after incubation with insulin and glucagon. F E B S Lett. 148:207-213. White M.F. , Takayama S. and Kahn C R . (1985a): Differences in the sites of phosphorylation of the insulin receptor in vivo and in vitro. J. Biol. Chem., 260:9470-9478. White M.F. , Maron R. and Kahn C R . (1985b): Insulin rapidly stimulates tyrosine phosphorylation of a M -185,000 protein in intact cells. Nature, 318:182-186. Witters L . A , Kowaloff E . M . and Avruch J. (1979): Glucagon regulation of protein phosphorylation. J. Biol. Chem., 254:245-248. Witters L . A , Moriaty D . and Martin D.B. (1979): Regulation of hepatic acetyl coenzyme A carboxylase by insulin and glucagon. J. Biol. Chem., 254:6644-6649. Witters L . A , (1981): Insulin stimulates the phosphorylation of acetyl-CoA carboxylase. Biochem. Biophys. Res. Comm., 100:872-878. Witters L . A , Friedman S.A., Tipper J.P. and Bacon G.W. (1981): Regulation of acetyl-CoA carboxylase by guanine nucleotides. J. Biol. Chem., 256:8573-8578. Witters L . A , Tipper J.P. and Bacon G.W. (1983): Stimulation of site-specific phosphorylation of acetyl-CoA carboxylase by insulin and epinepherine. J. Biol. Chem., 258: 5643-5648. Witters L . A , (1985): Regulation of acetyl-coA carboxylase by insulin and other hormones. In; Molecular Basis of Insulin Action. Ed. Czech M.P., Plenum, New York. pp. 315-326. Witters and Bacon G.W. (1985): Protein phosphatases active on acetyl-CoA carboxylase phosphorylated by casein kinase I, casein kinase II and the cAMP-dependent protein kinase. Biochem. Biophys. Res. Comm., 130:1132-1138. Witters L .A. , Colliton J.W., McDermott J .M. , Fang Z . and Glynn B.P. (1986): Regulation of adipocyte metabolism by insulin and phorbol esters: potential role of protein kinase C. In; Mechanisms of Insulin Action, Vol. 7. Ed. Sralfors et al., Elsevier, Biomedical Press, Amsterdam, pp. 229-237. 160 Witters L .A . , Watts T.D. , Daniels D.L. and Evans J.L. (1988): Insulin stimulates the dephosphorylation and activation of acetyl-CoA carboxylase. Proc. Natl. Acad. Sci. U S A , 85:5473-5477. Yeh L . A , Lee K . H . and Kim K . H . (1980): Regulation of rat liver acetyl-CoA carboxylase; regulation of phosphorylation and inactivation of acetyl-C o A carboxylase by the adenylate energy charge. J. Biol. Chem., 55:2308-2314. Y u K.T. , Khalaf N. and Czech M.P. (1987): Insulin stimulates the tyrosine phosphorylation of a Mr = 160,000 glycoprotein in rat adipocyte plasma membranes. J. Biol. Chem., 262:7865-7873. Zick Y . (1988): Natural and synthetic substrates for the insulin-receptor kinase. In; Receptor Biochemistry and Methodology, Vol . 12A Ed. Khan C R . and Harrison L . C , Liss, New York. pp. 147-161. Zierler K . L . (1957): Increase in resting membrane potential of skeletal muscle produced by insulin. Science, 126:1067-1068. Zierler K . L . (1988): Electrical events in transduction of insulin action and insulin action on electrical events. In; Insulin Action and Diabetes. E d Goren H J . et al., Raven Press, New York. pp. 91-103. 

Cite

Citation Scheme:

        

Citations by CSL (citeproc-js)

Usage Statistics

Share

Embed

Customize your widget with the following options, then copy and paste the code below into the HTML of your page to embed this item in your website.
                        
                            <div id="ubcOpenCollectionsWidgetDisplay">
                            <script id="ubcOpenCollectionsWidget"
                            src="{[{embed.src}]}"
                            data-item="{[{embed.item}]}"
                            data-collection="{[{embed.collection}]}"
                            data-metadata="{[{embed.showMetadata}]}"
                            data-width="{[{embed.width}]}"
                            async >
                            </script>
                            </div>
                        
                    
IIIF logo Our image viewer uses the IIIF 2.0 standard. To load this item in other compatible viewers, use this url:
http://iiif.library.ubc.ca/presentation/dsp.831.1-0098784/manifest

Comment

Related Items