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The Regulation of Fatty Acid Synthesis in the Mammary Gland of the Lactating Rat

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The Regulation of Fatty Acid Synthesis in the Mammary Gland of the Lactating Rat THE REGULATION OF FATTY ACID SYNTHESIS IN THE MAMMARY GLAND OF THE LACTATING RAT KAREN ANGELA OTTEY Ph.D THESIS, 1992 Submitted to the University of London for the degree of Doctor of Philosophy. London School of Pharmacy Brunswick Square London 1 ProQuest Number: U065910 All rights reserved INFORMATION TO ALL USERS The quality of this reproduction is dependent upon the quality of the copy submitted. In the unlikely event that the author did not send a com plete manuscript and there are missing pages, these will be noted. Also, if material had to be removed, a note will indicate the deletion. uest ProQuest U065910 Published by ProQuest LLC(2017). Copyright of the Dissertation is held by the Author. All rights reserved. This work is protected against unauthorized copying under Title 17, United States C ode Microform Edition © ProQuest LLC. ProQuest LLC. 789 East Eisenhower Parkway P.O. Box 1346 Ann Arbor, Ml 48106- 1346 Abstract of Thesis The mammary gland of the lactating rat uses 30 mmol of glucose a day. 70% of this is used for the synthesis of fatty acids which is therefore stringently regulated. This thesis describes investigations designed to elucidate some of the mechanisms of this regulation. Acetyl-CoA carboxylase (ACC) and pyruvate dehydrogenase (PDH) are key regulatory enzymes in the pathway from glucose to fatty acid. Pyruvate dehydrogenase catalyses the oxidative decarboxylation of pyruvate to acetyl-CoA and ACC catalyses the committed step in the synthesis of fatty acids from acetyl-CoA. In lactating rat mammary gland the activities of both enzymes are profoundly inhibited by starvation and rapidly re-activated (within 3 hours) upon refeeding. Unexpectedly during these dietary manipulations the rate of fatty acid synthesis can be more closely correlated with PDH than ACC even though the latter is considered to be the major rate determining enzyme. Perfusion of the mammary gland in situ was performed using known inhibitors of fatty acid synthesis in the perfusate. Palmitic acid inhibited PDH but not ACC; acetoacetate had no effect on either enzyme. In vitro experiments suggest differential effects of palmitate (16:0) and oleate (18:1), palmitate being a less potent inhibitor of fatty acid synthesis and ACC than oleate. ACC in lactating rat mammary gland is phosphorylated and inactivated in response to 24hr starvation. One possible candidate for such phosphorylation is cAMP-dependent protein kinase. This had been purified from lactating mammary gland and its unusual tissue specific properties are described and discussed in relation to the results of in vivo experiments showing that in mammary gland the activity of this kinase does not correlate with the rate of fatty acid synthesis or ACC activity. The partial purification of a kinase capable of phosphorylating and inhibiting ACC in a manner identical to that which occurs during starvation in vivo is described. Its characteristics, regulation and physiological significance are discussed. 2 ACKNOWLEDGEMENTS Many thanks to my supervisors Drs. Michael Munday and Roger Clegg for their help, advice and support throughout the course of the work completed for this thesis. I would also like to thank my friends Kevork, Sab and Mika for their help and humour for which I was especially grateful in the latter months! Special thanks to Dr David Calvert for performing the mammary gland perfusions, Mrs Kate Roy for help with acini preparations, Tricia Worby for the artwork and Mrs Gill Patterson for typing this thesis and her patience despite the many alterations. Finally I thank the SERC and the Hannah Research Institute for their financial support and the use of facilities, without which none of this would have been possible. 3 ABBREVIATIONS Apart from those listed below, the abbreviations used throughout this thesis follow the recommendations of the IUPAC-IUB Joint Commission on Biochemical Nomenclature, as detailed in the Biochemical Journal (225. pp 1-26, 1985). AABS p-(p-aminophenylazo) -benzene sulphonic acid. AAT arylamine acetyl transferase ACC Acetyl-CoA carboxylase APAD acetylpyridine-adenine dinucloetide AMP-PK AMP-activated protein kinase A-V arterio-venous BSA bovine serum albumin BZ benzamidine CAMP-PK cAMP-dependent protein kinase CNBr cyanogen bromide CS citrate synthase DTT dithiothreitol F-l,6-P2 fructose-1,6-bisphosphate F-2,6-P2 fructose-2,6-bisphosphate FSBA fluorosulphonyl-benzoyl adeonsine FPLC fast protein liquid chromatography G-l-P glucose-l-phosphate G-6-P glucose-6-phosphate HPLC high pressure liquid chromatography IBMX 3-isobutyl-1-methyl xanthine MG mammary gland PDH pyruvate dehydrogenase PEG poly(ethylene) glycol PFK-1 6-phosphofructo-l-kinase PFK-2 6-phosphofructo-2-kinase PMSF phenylmethylsulphonyl fluoride SBTI soya bean trypsin inhibitor SDS-PAGE sodium dodecyl sulphate polyacrylamide gel electrophoresis 4 TLCK Na-tosyl-L-phenylalanine chloromethyl ketone n Note: the dimensions of chromatography coluir^ are quoted as bed height x internal diameter. 5 CHAPTER 1 INTRODUCTION 1.1. Biosynthesis of fatty acids in mammals 16 1.2 Lipid content of milk 17 1.3 Regulation of fatty acid synthesis 20 1.3.1 Hormonal regulation oflipogensis 21 1.4 Potential sites of regulation of 22 fatty acid synthesis 1.4.1 Glucose transport 23 1.4.2 Hexokinase and 25 phosphofructokinase-1 1.4.3 Pyruvate dehydrogenase 27 1.4.4 Acetyl-CoA carboxylase 32 1.5 cAMP-dependent protein kinase 47 1.6 Aims of the present work 50 CHAPTER 2 MATERIALS AND METHODS 2.1 Animals 51 2.2 Chemicals 51 2.2.1 Biochemicals 51 2.2.2 Radiochemicals 52 2.2.3 Pharmacological chemicals 52 2.2.4 Peptide substrates and inhibitors 52 2.2.5 Protease inhibitors 52 2.3 Preparation of affinity chromatrography media. 53 2.3.1 Avidin Sepharose 55 2.3.2 Histone Sepharose 55 2.4 Treatment of fatty acid free BSA prior to use in biochemical assays. 55 2.5. Preparation of albumin bound fatty acid complex. 56 2.6 Purification of acetyl-CoA carboxylase from rat lactating mammary gland. 56 2.6.1 Preparative 57 2.6.2 Analytical 59 2.7 Purification of arylamine acetyltransferase 60 6 2.7.1 Assay of arylamine 60 acetyltransferase Preparative separation of cAMP-PK holoenzymes type I 61 and II from rat liver. Analytical Separation of cAMP-PK holoenzymes type I 64 and II from lactating rat mammary gland. Assay for 5 'AMP-activated protein kinase (AMP-PK) 65 Assay of AMP-PK kinase 66 'Spun-column' chromatography 67 Assay of cAMP-PK-dependent protein kinase 68 (cAMP-PK) 2.13.1 Activity ratio of cAMP-PK 69 Assay of acetyl-CoA carboxylase (ACC) 69 2.14.1 Assay in crude homogenate 70 2.14.2 Assay of purified ACC 71 Pyruvate dehydrogenase assay 72 Citrate Synthase assay. 73 Measurement of fatty acid synthesis 74 2.17.1 In isolated mammary acini 74 2.17.2 In vivo 77 Assay of free fatty acids 77 Assay of DNA in crude homogenates 78 Assay of protein 79 Metabolite assays 79 2.21.1 Blood Metabolites 79 2.21.2 Tissue Metabolites 80 Perfusion of mammary tissue 80 Surgical preparation of anoxic mammary glands 81 Preparation of acini from mammary tissue of lactating rat. 81 2.24.1 Wet weight determination of acini 84 2.24.2 Incubation of acini with palmitate or oleate 84 Polyacrylamide gel electrophoresis (PAGE) 85 2.25.1 Denaturing PAGE 85 2.25.2 Non denaturing PAGE 85 Protein detection after PAGE 86 2.26.1 Coomassie blue dye stain 86 7 2.26.2 Silver stain 86 2.26.3 Autoradiography of radioactive 86 proteins 2.27 Analysis of adenine nucleotides by HPLC 86 2.27.1 Tissue preparation 86 2.27.2 Measurement of ATP 87 2.27.3 Separation of nucleotides by HPLC 87 2.27.4 Calculation of adenine nucleotide 87 concentrations 2.28 Covalent modification of AMP-PK by [14C] Fluorosulphonylbenzoyladenosine. ([14C]-FSBA). 89 2.29 Analysis of phosphorylation sites on [32P]-labelled 89 acetyl-CoA carboxylase 2.30 Assay of alkali-labile phosphate in ACC 90 CHAPTER 3 CAMP-DEPENDENT PROTEIN KINASE IN RAT LACTATING MAMMARY GLAND 3.1 Introduction: cAMP-PK activity in mammary 92 tissue of the lactating rat. 3.2 Dependence of cAMP-PK on cAMP 94 3.3 Post extraction activation and inactivation 96 of cAMP-PK 3.4 Determination of the conditions necessary 97 to prevent artefactual changes in the activity of cAMP-PK 3.5 Effect of fasting and subsequent 99 refeeding on the activity of cAMP-PK 3.6 Effect of isoprenaline on cAMP-PK activity 99 3.7 Distribution of isoenzyme type I and II of 104 cAMP-PK purified from lactating mammary gland 3.8 Studies on the catalytic subunit of cAMP-PK 109 purified from lactating rat mammary gland 3.9 Purification of the catalytic subunit 109 3.9.1. Separation on phosphocellulose 109 3.9.2 Separation on hydroxyapatite 112 3.9.3 Separation by gel filtration 112 8 3.10 Substrate specificity of cAMP-PK from lactating 117 rat mammary gland and rat heart. 3.11 Sensitivity of cAMP-PK catalytic subunit 119 from lactating rat mammary gland and rat heart to the specific inhibitor of cAMP-PK. 3.12 Summary and Conclusions. 121 CHAPTER 4 AMP-ACTIVATED PROTEIN KINASE (AMP-PK) IN RAT 123 LACTATING MAMMARY GLAND 4.1 Purification of AMP-PK 124 4.1.1 Preparation of tissue; PEG - 6000 124 protein precipitation. 4.1.2 Separation by anion exchange - DEAE 125 and Mono-Q. 4.1.3. Separation by affinity chromatography 139 blue agarose and histone Sepharose. 4.1.4 Separation on the basis of molecular 136 weight - gel filtration. 4.1.5 Purification table for AMP-PK.
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