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The Role of Vitamin B12 Deficiency on Hepatic Metabolism of Lipids

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The Role of Vitamin B12 Deficiency on Hepatic Metabolism of Lipids A Thesis Submitted for the Degree of PhD at the University of Warwick Permanent WRAP URL: http://wrap.warwick.ac.uk/144209 Copyright and reuse: This thesis is made available online and is protected by original copyright. Please scroll down to view the document itself. Please refer to the repository record for this item for information to help you to cite it. Our policy information is available from the repository home page. For more information, please contact the WRAP Team at: [email protected] warwick.ac.uk/lib-publications The role of vitamin B12 deficiency on hepatic metabolism of lipids By Joseph Boachie A thesis submitted to the Faculty of Medicine of the University of Warwick in partial fulfilment for the degree of Doctor of Philosophy in Medical Sciences Clinical Sciences Research Lab Division of Metabolic and Vascular Health Warwick Medical School University of Warwick England, UK September 2019 i Table of Contents Table of contents …………………………………………………………………………...ii List of figures ……………………………………………………………………………....x List of tables……………………………………………………………………………….xii Acknowledgement …………………………………………………………………….….xiii Declaration………………………………………………………………………………...xiv Abstract………………………………………………………………………………….....xv List of abbreviations……………………………………………………………………….xvi 1. Chapter One – Introduction…………………………………………………………....1 1.1 Metabolic syndrome ...…………………………………………..…………………...2 1.1.1 Background…………………………..………………………………………....2 1.1.2 Epidemiology…………………………….……………………………………..2 1.1.3 Pathophysiology of metabolic syndrome………………….……………………3 1.1.3.1 Insulin resistance (IR)………………………………………………...3 1.1.3.2 Obesity and increased waist circumference…………………………..4 1.1.3.3 Dyslipidaemia………………………………………………………...5 1.1.3.4 Hypertension………………………………………………………….5 1.1.4 Consequences of metabolic syndrome………………………………………….6 1.2 Diabetes mellitus (DM)……………………………………………………………....7 1.2.1 Background…………………………………………………………………......7 1.2.2 Types of Diabetes…………………………………………………………....….7 1.2.2.1 Type 1 diabetes mellitus (T1DM)…………………………………….7 1.2.2.2 Type 2 diabetes mellitus (T2DM)…………………………………….7 1.2.2.3 Gestational diabetes mellitus (GDM)………………………………....8 1.2.2.4 Specific type. ,………………………………………………………...8 1.2.3 Epidemiology of DM…………………………………………………………....8 1.2.4 Pathophysiology of DM ………………………………………………………..9 1.2.4.1 Type 2 Diabetes Mellitus (T2DM) …………………………………...9 ii 1.2.4.2 Gestational Diabetes Mellitus (GDM) ………………………………10 1.2.4.3 Type 1 Diabetes Mellitus (T1DM) ………………………………….11 1.2.5 Consequences of Diabetes Mellitus (DM)……………………………………..12 1.3 Cardiovascular disease (CVD)……………………………………………………...13 1.3.1 Epidemiology of CVD………………………………………………………....13 1.3.2 Pathophysiology of CVD……………………………………………………....14 1.3.3 Consequences of CVD…………………………………………………………16 1.4 Environmental factors and metabolic risk…………………………………………..16 1.5 Vitamin B12: Role in metabolic diseases………………………….………………..19 1.5.1 Vitamin B12 deficiency………………………………………………………..19 1.5.1.1 Background…………………………………………………………..19 1.5.1.2 Epidemiology………………………………………………………...20 1.5.1.3 Diagnostic markers and risk factors………………………………….20 1.5.2 B12: Biochemical structure and synthesis……………………………………..22 1.5.3 Sources, bioavailability, transport and hepatic uptake of B12………………....23 1.5.3.1 Hepatic metabolism of lipids………………………………………...24 1.5.4 Vitamin B12 deficiency is associated with obesity……………………………27 1.5.5 Vitamin B12 is associated with metabolic disorders ………………………….28 1.5.5.1 Vitamin B12 and insulin resistance………………………………….28 1.5.5.2 Vitamin B12 and dyslipidaemia……………………………………..29 1.5.5.3 Vitamin B12 and cardiovascular diseases…………………………...30 1.5.6 Molecular mechanisms regulating B12 deficiency and lipogenesis…………..32 1.5.7 Epigenetic mechanisms underlying B12 deficiency in lipid metabolism……..33 1.5.7.1 Regulation through methylation of DNA…………………………....34 1.5.7.2 Regulation through microRNAs (miRNAs)………………………....35 1.5.7.3 Regulation through modifications of histone………………………..36 1.5.8 Impact of one carbon micronutrient supplementation on hepatic lipid metabolism…………………………………………………………………………………..36 1.6 Conclusion and prospects………………………………………..………………….38 1.7 Hypothesis and Aims………………………………………………………………..39 iii 2. Chapter Two – Materials and Methods ……………………………………………..40 2.1 List of Materials and Reagents……………………………………………………...41 2.1.1 Reagents / materials for culture of Hep G2 cell line………………………….….41 2.1.2 Reagents for protein characterization studies………………………………..41 2.1.3 Reagents for RNA, cDNA and gene expression studies……………………..43 2.1.4 Genes for RT- qPCR…………………………………………………………44 2.1.4.1 Primers for Human genes…………………………………………….44 2.1.4.2 Primers for mouse genes …………………………………………….45 2.1.5 Reagents and materials for radioactive flux assay…………………………...45 2.1.6 Reagents and materials for seahorse flux assay……………………………...46 2.1.7.1 Media formulations…………………………………………………………..46 2.1.7.2 Buffers ……………………………………………………………………….47 2.1.8 Solutions………………………………………………………………….…..48 2.1.9 Miscellaneous materials and reagents………………………………….…….48 2.2 Methods ………………………………………………………………………….….49 2.2.1 Cell culture …………………………………………………………………….49 2.2.1.1 Hep G2 cell line ……………………………………………………..49 2.2.1.2 Thawing of Hep G2 cells…………………………………………….49 2.2.1.3 Passaging of Hep G2 cells…………………………………………...50 2.2.1.4 Freezing of Hep G2 cells ……………………………………………50 2.2.1.5 Seeding of Hep G2 cells for B12-lipid experiments ………………..50 2.2.1.6 Harvest of Hep G2 cells for B12-lipid experiments ………………...51 2.2.2 Gene expression assay………………………………………………………....51 2.2.2.1 Total RNA isolation …………………………………………………51 2.2.2.2 DNASe treatment of RNA ……………………………………….….53 iv 2.2.2.3 Reverse transcription (cDNA synthesis) ………………………….…53 2.2.2.4 Real-time quantitative polymerase chain reaction (RT-qPCR)………53 2.2.3 Estimation of total intracellular TG levels in Hep G2 cells…………………....55 2.2.4 Radioactive flux assessment of TG synthesis………………………………….56 2.2.5 Lipid content in Hep G2 cells – Oil Red O (ORO) staining…………………...57 2.2.6 Protein characterization study (Western blot analysis) ………………………..57 2.2.6.1 Hep G2 cells harvest for western blot analysis ………………….…..57 2.2.6.2 Protein quantification using Bradford reagent ………………..……..58 2.2.6.3 Western blot analysis……………………………………………..….58 2.2.7 Analysis of fatty acids in total lipids of hepatic cell pellets ……………….…..59 2.2.8 Mitochondrial dysfunction assessment using Seahorse assay……….……....…60 2.2.8.1 Cell culture for seahorse assay……………………………………………….60 2.2.8.2 Cell density optimization and seeding for seahorse assay …………………..60 2.2.8.3 Seahorse inhibitors …………..………………………………………….…...60 2.2.8.4 Seahorse media optimization ………………………………………………..61 2.2.8.5 Sample preparation and seahorse assay……………………………………...62 2.2.9 Animal model……………………………………………………………………..62 2.2.10 Statistical analysis……………………….………...………………………….…63 3. Chapter Three – Results……………………………………………………………....64 3.0 Uptake and storage of vitamin B12 is modulated by CD320 receptor and TCN2 transporter in hepatocytes in various extracellular B12 concentrations...……………..65 3.1 Introduction…………………………………………………………………………65 3.2 Methods……………………………………………………………………………..67 3.2.1 Cell culture ………………..…………………………………………………..67 3.2.2 Animal model……………………………………….……...………………….67 3.2.3 RNA isolation and quantitative real time-PCR (qRT-PCR) ……………...…..67 v 3.2.4 Western blot analysis………………………………………………………….67 3.2.5 Measurement of intracellular B12 levels………...……………………………68 3.2.6 Measurement of B12 concentrations in EMEM media and plasma…………..68 3.3 Results………………………………………………………………………………69 3.3.1 Hepatic uptake of B12………………………………………………………....69 3.3.1.1 B12 uptake in Hep G2 cell line……………………………………...69 3.3.1.2 B12 uptake in mice liver…………………………………………….70 3.3.2 Transcobalamin receptor CD320………………...............................................71 3.3.2.1 Hep G2 cell line …………………………………………………….71 3.3.2.2 Mice liver …………………………………………………………...73 3.3.3 B12 transporter transcobalamin II (TCN2)…………………………………....74 3.3.3.1 Hep G2 cells…………………………………………………………74 3.3.3.2 Mice liver……………………………………………………………76 3.4 Discussion…………………………………………………………………………..77 3.5 Conclusion …………………………………………………………………………79 4.0 Vitamin B12 deficiency alters lipid synthesis triggering accumulation in the liver.........................................................................................................................................80 4.1 Introduction ………………………………………………………………………....80 4.2 Methods …………………………………………………………………………......86 4.2.1 Cell culture. …………………………………………………………………...86 4.2.2 Oil Red O staining and elution assay………………………………………….86 4.2.3 RNA isolation, cDNA synthesis and gene expression ………………………..86 4.2.4 Total intracellular triglyceride estimation ………………………………….....86 4.2.5 Radioactive flux assay for TG biosynthesis …………………………………..86 4.2.6 Analysis of fatty acids in total lipids of hepatic cell pellets …………………..87 4.3 Results ……………………………………………………………………………...87 4.3.1 Lipogenesis …………………………………………………………………...87 4.3.1.1 Effect of B12 on lipid droplets formation in hepatocytes …………..87 4.3.1.2 Effect of B12 on total intracellular triglyceride (TG) levels ………..88 vi 4.3.1.3 Effect of B12 on genes regulating fatty acid synthesis ……………..89 4.3.1.4 Effect of B12 on genes regulating TG synthesis …………………....91 4.3.1.5 Effect of B12 on TG synthesis utilizing radiolabelled FA….……….93 4.3.1.6 Effect of B12 on genes regulating cholesterol synthesis …….……...94 4.3.2 Fatty acid (FA) profiling in hepatocyte cell line….…………………………...95 4.3.2.1 Total fatty acids ……………………………………………………..95 4.3.2.2 Grouped fatty acids …………………………………………………96
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