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Stearoyl-CoA desaturase : role in metabolic syndrome, atherosclerosis and inflammation MacDonald, Marcia Leigh 2009

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STEAROYL-COA DESATURASE: ROLE IN METABOLIC SYNDROME, ATHEROSCLEROSIS AND INFLAMMATION  by MARCIA LEIGH MACDONALD B.Sc., Queen's University, 1994  A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE STUDIES (Genetics)  THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver)  October 2009 © Marcia Leigh MacDonald, 2009  Abstract Combination of the risk factors obesity, insulin resistance, dyslipidemia, and hypertension, often described as the "metabolic syndrome," increases the risk of developing diabetes and cardiovascular disease. Stearoyl-coenzyme A desaturase (SCD) activity has been implicated in the metabolic syndrome; however, earlier studies on the beneficial metabolic effects of SCD1 deficiency have been confined to normolipidemic mice, and the role of SCD in the context of atherosclerosis has not been examined. The primary purpose of this thesis was to investigate the effect of decreased SCD activity on susceptibility to atherosclerosis in mice. Thus, the overarching hypothesis driving the work was that SCD activity is atherogenic.  As well, we  examined the effect of absence of SCD1 on features of the metabolic syndrome and chronic inflammation in a mouse model of familial hyperlipidemia.  An  additional objective was to determine the effect of SCD1 deficiency on dextran sulfate sodium (DSS)-induced acute colitis with DSS dosing adjusted to account for genotypic differences in fluid consumption. These studies help us understand SCD functions in lipid metabolism and evaluate its attractiveness as a therapeutic target for the metabolic syndrome or atherosclerosis. After the Scd1ab-J mutation was characterized, mice carrying this allele were crossed with the low density lipoprotein receptor (LDLR)-deficient mouse strain. Features of the metabolic syndrome and atherosclerotic lesion area were evaluated in mice challenged with a Western diet for twelve weeks. In studies of colonic inflammation, wild-type controls were treated with 3.5% DSS for 5 days to induce moderately severe colitis, while the concentration of DSS given to SCD1deficient mice was lowered to 2.5% to control for increased fluid consumption. Despite an antiatherogenic metabolic profile, SCD1 deficiency increased atherosclerosis in hyperlipidemic LDLR-deficient mice. Absence of SCD1 also led to chronic inflammation of the skin and increased plasma inflammatory markers, but did not accelerate inflammation in DSS-induced acute colitis when DSS intake was controlled. These findings reinforce the crucial role of chronic inflammation in promoting atherosclerosis, even in the presence of antiatherogenic metabolic  ii  characteristics, and suggest that inflammation must be closely monitored in studies of SCD inhibitors for treatment of the metabolic syndrome in humans.  iii  Table of Contents Abstract..............................................................................................ii Table of Contents...............................................................................iv List of Tables....................................................................................viii List of Figures....................................................................................ix List of Abbreviations...........................................................................xi Acknowledgements...........................................................................xiii Dedication........................................................................................xiv Co-authorship Statement...................................................................xv CHAPTER 1 Introduction.....................................................................1 1.1 De novo lipogenesis......................................................................................1 1.2 The stearoyl-CoA desaturase protein............................................................3 1.2.1 SCD activity ..........................................................................................3 1.2.2 SCD genes in mice and humans............................................................4 1.2.3 SCD index as a surrogate for SCD activity.............................................5 1.2.4 Mutations in SCD genes.........................................................................6 1.2.5 The skin phenotype of the SCD1-deficient asebia mouse......................7 1.3 The metabolic syndrome..............................................................................9 1.3.1 Role of human SCD in obesity and insulin resistance..........................10 1.3.2 Role of murine SCD in the metabolic syndrome..................................12 1.3.3 The role of SCD in mediating the lipogenic effects of LXR agonists.....14 1.4 Atherosclerotic cardiovascular disease.......................................................15 1.4.1 Fatty acids in atherosclerotic cardiovascular disease..........................15 1.4.2 Possible pro- and anti- atherogenic effects of SCD activity ................16 1.4.3 The LDLR-deficient mouse model of metabolic syndrome and atherosclerosis.............................................................................................18 1.5 Inflammation...............................................................................................19 1.5.1 Role of SCD activity in mediating the lipogenic innate immune response.......................................................................................................20 1.5.2 Proinflammatory effects of saturated fatty acids.................................20 1.5.3 Role of hepatic SCD in inflammatory liver disease..............................21 1.5.4 Inflammatory bowel disease................................................................21 1.5.4.1 The dextran sulfate sodium model of acute colitis.......................22 1.5.4.2 The effect of SCD1 deficiency in experimental colitis..................22 1.6 Thesis hypotheses and objectives..............................................................23 1.7 References..................................................................................................30  iv  CHAPTER 2 Comparative analysis of the vertebrate SCD gene families and characterization of the mouse Scd1ab-J allele...............................47 2.1 Introduction................................................................................................47 2.2 Methods......................................................................................................49 2.2.1 Genomic sequence analysis................................................................49 2.2.2 Phylogenetic analysis..........................................................................49 2.2.3 Gene expression data..........................................................................50 2.2.4 Regulatory element analysis...............................................................50 2.2.5 Animals................................................................................................50 2.2.6 Genotyping and mutational analysis of Scd1ab-J.................................51 2.3 Results........................................................................................................51 2.3.1 Multiple SCD gene duplications have occurred in the rodent lineage..51 2.3.2 SCD5 gene loss in the rodent lineage..................................................53 2.3.3 Phylogenetic analysis and expression patterns of vertebrate SCD genes............................................................................................................53 2.3.4 Regulatory sequence analysis.............................................................55 2.3.5 Characterization of the asebia-J mutation............................................56 2.4 Discussion...................................................................................................57 2.5 References..................................................................................................66 CHAPTER 3 Absence of stearoyl-CoA desaturase-1 ameliorates features of the metabolic syndrome in LDLR-deficient mice..............................72 3.1 Introduction................................................................................................72 3.2 Methods......................................................................................................74 3.2.1 Animals and diet .................................................................................74 3.2.2 Adiposity measurements using magnetic resonance imaging and relaxometry..................................................................................................74 3.2.3 Fat pad measurements and histology..................................................75 3.2.4 Lipid and lipoprotein analysis..............................................................75 3.2.5 Hepatocyte isolation and radiolabeling with [3H]acetate....................75 3.2.6 Physiological analysis..........................................................................76 3.2.7 Real-time PCR and immunoblotting.....................................................76 3.2.8 Statistical analysis...............................................................................77 3.3 Results........................................................................................................77 3.3.1 SCD1 deficiency reduces weight gain and adiposity in Ldlr-/- mice.....77 3.3.2 SCD1 deficiency reduces hepatic steatosis in Ldlr-/- mice..................78 3.3.3 SCD1 deficiency reduces plasma lipids and improves lipoprotein profiles in Ldlr-/- mice...................................................................................78 3.3.4 SCD1 deficiency reduces plasma apolipoproteins in Ldlr-/- mice........79 3.3.5 SCD1 deficiency reduces fatty acid synthesis in Ldlr-/- mice...............79 3.3.6 SCD1 deficiency reduces insulin resistance in Ldlr-/- mice..................80 3.3.7 SCD1 mediates the plasma lipid response to LXR agonist treatment in Ldlr-/- mice....................................................................................................80 3.4 Discussion...................................................................................................81 v  3.5 References..................................................................................................94 CHAPTER 4 Despite antiatherogenic metabolic characteristics, SCD1deficient mice have increased inflammation and atherosclerosis.......101 4.1 Introduction..............................................................................................101 4.2 Methods....................................................................................................102 4.3 Results......................................................................................................102 4.3.1 SCD1 deficiency increases atherosclerosis in Ldlr-/- mice.................102 4.3.2 SCD1 deficiency promotes inflammation in Ldlr-/- mice....................104 4.3.3 SCD1 deficiency alters HDL-associated proteins in Ldlr-/- mice........105 4.3.4 SCD1 deficiency does not alter macrophage function.......................105 4.3.5 Macrophage SCD1 deficiency does not alter atherosclerosis in Ldlr-/mice............................................................................................................106 4.4 Discussion.................................................................................................107 4.5 References................................................................................................116 CHAPTER 5 Absence of stearoyl-CoA desaturase-1 does not promote DSS-induced acute colitis.................................................................119 5.1 Introduction..............................................................................................119 5.2 Methods....................................................................................................121 5.2.1 Animals and diet................................................................................121 5.2.2 Induction of colitis.............................................................................121 5.2.3 General assessment of colitis............................................................122 5.2.4 Histological assessment of colitis......................................................122 5.2.5 Statistical analysis.............................................................................123 5.3 Results......................................................................................................123 5.3.1 SCD1 deficiency increases water consumption in C57BL/6 mice.....123 5.3.2 SCD1 deficiency does not accelerate DSS-induced acute colitis.......124 5.4 Discussion.................................................................................................125 5.5 References................................................................................................134 CHAPTER 6 Conclusions, perspectives, and future directions............138 6.1 Summary of findings.................................................................................138 6.2 Mammalian SCD gene families.................................................................139 6.3 Role of SCD in ameliorating the metabolic syndrome in hyperlipidemic mice ........................................................................................................................140 6.3.1 Relationship between fatty liver and insulin resistance.....................141 6.3.2 Tissue-specific function of SCD..........................................................142 6.3.3 Relationship between insulin resistance and inflammation...............144 6.3.4 Fate of excess fatty acids..................................................................144 6.4 Role of SCD in susceptibility to atherosclerosis........................................146 6.4.1 Function of SCD in immune cells.......................................................147 6.4.2 Mechanism of inflammation in SCD1 deficiency................................148 6.5 Pharmacological inhibition of SCD............................................................150  vi  6.6 References................................................................................................153 APPENDIX A Sequence information..................................................160 APPENDIX B Animal care certificates ...............................................162 APPENDIX C Supplemental methods................................................166 C.1 Animals and diet.......................................................................................166 C.2 Histological analysis.................................................................................166 C.3 Immunohistochemical studies..................................................................168 C.4 Quantitative RT-PCR.................................................................................168 C.5 Measurements of inflammatory molecules. ...........................................168 C.6 Apolipoprotein analysis.............................................................................169 C.7 Paraoxonase (PON1) activity....................................................................169 C.8 Bone marrow transplantation...................................................................169 C.9 Macrophage functional studies ...............................................................170 C.10 Statistical analysis..................................................................................170 C.11 References..............................................................................................171 APPENDIX D Supplemental tables & figures.....................................172  vii  List of Tables Table 2.1: Comparison of coding sequences of human and mouse SCD genes.. .61 Table 3.1: Plasma lipid and apolipoprotein levels in Ldlr-/- mice lacking SCD1.. .85 Table 5.1: Histological assessment of DSS-induced colon damage in SCD1deficient mice.....................................................................................................129 Table A.1: Genomic sequences used for analysis of vertebrate SCD gene families. ............................................................................................................................160 Table A.2: Coding sequences used for phylogenetic analysis of vertebrate SCD gene families......................................................................................................161 Table D.1: Conversions between conventional units and SI units. ....................172 Table D.2: Serum lipid levels in Ldlr-/- mice transplanted with bone-marrow derived cells lacking SCD1..................................................................................173  viii  List of Figures Figure 1.1: SCD in the de novo lipogenesis pathway............................................25 Figure 1.2: SCD in CE and TG synthesis................................................................26 Figure 1.3: SCD in lipoprotein metabolism............................................................27 Figure 1.4: Effect of SCD1 deficiency on tissue lipids...........................................28 Figure 1.5: Experimental design...........................................................................29 Figure 2.1: Schematic diagram of synteny and genomic organization of the SCD genes in human and mouse. ...............................................................................62 Figure 2.2: Phylogenetic relationships and sites of expression of SCD genes. ....63 Figure 2.3: VISTA plots of the MLAGAN alignment of human SCD genomic sequence with co-orthologous sequences from mouse and rat............................64 Figure 2.4: Analysis of the Scd1ab-J mutation. ...................................................65 Figure 3.1: Total body and fat pad weights of Ldlr-/- mice lacking stearoyl-CoA desaturase (SCD1). ..............................................................................................86 Figure 3.2: Adiposity in Ldlr-/- mice lacking SCD1. ..............................................87 Figure 3.3: Hepatic lipids in Ldlr-/- mice lacking SCD1. ........................................88 Figure 3.4: Plasma lipids and lipoprotein profiles in Ldlr-/- mice lacking SCD1. . 89 Figure 3.5: Plasma apolipoproteins in Ldlr-/- mice lacking SCD1. ........................90 Figure 3.6: Fatty acid synthesis and hepatic gene expression in Ldlr-/- mice lacking SCD1. .......................................................................................................91 Figure 3.7: Glucose tolerance and insulin resistance in Ldlr-/- mice lacking SCD1. ..............................................................................................................................92 Figure 3.8: Plasma lipid response to LXR agonist treatment in Ldlr-/- mice lacking SCD1. ..................................................................................................................93 Figure 4.1: Lesion area in Ldlr-/- mice lacking SCD1. .........................................110 Figure 4.2: Lesion morphology Ldlr-/- mice lacking SCD1. .................................111 Figure 4.3: Skin of Ldlr-/- mice lacking SCD1. ....................................................112 Figure 4.4: Inflammation in Ldlr-/- mice lacking SCD1. ......................................113 Figure 4.5: HDL phenotype in Ldlr-/- mice lacking SCD1. ..................................114 Figure 4.6: Macrophages from mice lacking SCD1. ...........................................115 Figure 5.1: Water consumption in C57BL/6 mice lacking SCD1. ........................130  ix  Figure 5.2: DSS-induced acute colitis in C57BL/6 mice. .....................................131 Figure 5.3: DSS-induced colon damage in SCD1-deficient mice. .......................132 Figure 5.4: DSS-induced acute colitis in SCD1-deficient mice. ..........................133 Figure 6.1: Effect of SCD1 deficiency on susceptibility to atherosclerosis..........152 Figure D.1: Lesion area in Ldlr-/- mice lacking SCD1. ........................................174 Figure D.2: Lesion morphology in Ldlr-/- mice lacking SCD1. .............................175 Figure D.3: Skin of Ldlr-/- mice lacking SCD1. ....................................................176 Figure D.4: Inflammation in Ldlr-/- mice lacking SCD1. .....................................177  x  List of Abbreviations 16:0 16:1n7 18:0 18:1n9 ab ACAT ACC ACS ANOVA AOX apo ASO AUC CE CIH ChREBP CoA CPT CVD DMEM DNL DSS ER EST FACE FAS FC FH FPLC GNF GPAT H&E HDL HTG ICAM-1 IDF IL LPC LDL LDLR Ldlr-/LPS LXR MUFA NCBI NCEP PGC  palmitic acid palmitoleic acid stearic acid oleic acid asebia acyl-CoA:cholesterol acyltransferase acetyl-CoA carboxylase acetyl-CoA synthetase analysis of variance acyl-CoA oxidase apolipoprotein antisense oligonucleotide area under the curve cholesteryl esters chronic intermittent hypoxia carbohydrate response element binding protein coenzyme A carnitine palmitoyltransferase cardiovascular disease Dulbecco's modified Eagle's medium de novo lipogenesis dextran sulfate sodium endoplasmic reticulum expressed sequence tag long chain fatty acyl elongase fatty acid synthase free cholesterol familial hypercholesterolemia fast protein liquid chromatography Genomics Institute of the Novartis Research Foundation glycerol-3-phosphate acyltransferase hematoxylin and eosin high density lipoprotein hypertriglyceridemia intercellular adhesion molecule-1 International Diabetes Federation interleukin lysophatidylcholine low density lipoprotein LDL receptor homozygous for the Ldlrtm1Her allele lipopolysaccharide liver X receptor monounsaturated fatty acid National Center for Biotechnology Information National Cholesterol Education Program PPAR-γ coactivator xi  PKC PNS PON1 PPAR PUFA RANTES SAA SCD Scd1-/SFA SI SINE SNP SREBP TC TG TNFα VLDL WTD  protein kinase C peripheral nervous system paraoxonase-1 peroxisome proliferator-activated receptor polyunsaturated fatty acid Regulated upon activation, normal T-cell expressed, and secreted (CCL5) serum amyloid A stearoyl-CoA desaturase homozygous for either of the Scd1ab-J or Scd1ab-2J alleles saturated fatty acid Système International short interspersed element single nucleotide polymorphism sterol regulatory element binding protein total cholesterol triglycerides tumour necrosis factor-α very low density lipoprotein Western diet  xii  Acknowledgements First and foremost, I thank my thesis supervisor, Michael Hayden, for his scientific guidance and for providing numerous opportunities to expand my knowledge and experience as a researcher.  Thank you also to my thesis  committee members: Bruce McManus, for friendly support and for introducing me to the intricacies of atherosclerosis; Elizabeth Simpson, for her mouse expertise; and Brian Rodrigues for his openness and scientific curiosity. Thank you all for believing in me. Thank you to past and present members of the Hayden lab, with whom I've shared many stimulating discussions, particularly Jennifer Collins, Stef Butland, Terry Pape, Jeff Helm, Roshni Singaraja, Paul Orban, Liam Brunham, Rona Graham, Martin Kang, Mahmoud Pouladi, Jeff Carroll, Fiona Young, Janine Kruit, Henk Visscher, and Kuljeet Vaid. A special thank you to my former benchmate, Yvonne Bombard, for expanding my knowledge of the ethical, legal, and social implications of genetics, and to Simon Warby, a constant source of debate and fun throughout my time in the Hayden lab. Finally, I thank my family: my parents and sister, for their encouragement and support; and Tucker, for his patience and understanding.  xiii  Dedication  To my parents, Maureen and Lloyd, and my sister, Dana, who give me constant love and encouragement  xiv  Co-authorship Statement This thesis includes work performed in part via collaboration with other researchers. Those involved in the research are listed in the publication citations in each of the chapters. Their individual contributions to the research are outlined below. Chapter 2 I designed and performed the experiments described in Figures 1.1 to 1.3, analyzed the data, and prepared the manuscript. Terry Pape performed the mapping of the asebia-J deletion in Figure 1.4, under my direction. Chapter 3 I designed the experiments and performed the animal work, including body weight measurements, glucose and insulin tolerance testing, and LXR treatment. I also performed the hepatic lipid analyses, some plasma lipid analyses, and all gene expression analyses. Nagat Bissada and Piers Ruddle assisted with blood and tissue collection. Andrew C. Yung performed the MRI analysis described in Figure 1.2. Brian W. C. Wong performed the imaging of ORO-stained liver sections. Catherine Fievet and Emmanuelle Vallez evaluated plasma lipids, apolipoproteins, and lipoprotein profiles. Laura Hargreaves isolated the primary hepatocytes for assessment of fatty acid synthesis, performed by Russell Watts. Roshni Singaraja performed the ABCA1 immunoblotting. I analyzed the data and prepared the manuscript. Chapter 4 I designed the experiments and performed the lesion area quantitation, gene expression analysis, measurements of inflammatory molecules, and culture of thioglycollate-elicited peritoneal macrophages. Nagat Bissada assisted with blood and tissue collection. Brian W. C. Wong assisted with imaging. Bruce M. McManus performed the semi-quantitative assessment of lesion severity. Catherine Fievet performed the apolipoprotein analyses. Anatol Kontush and Hala Hussein evaluated serum paraoxonase activity. Miranda van Eck and Reeni B. Hildebrand performed the bone marrow reconstitution experiment. I analyzed the data and prepared the manuscript. Chapter 5 I designed the experiments and performed the animal work, including evaluation of water consumption, DSS treatment, and general assessment of colitis. Nagat Bissada assisted with tissue collection. Bruce A. Vallance performed semiquantitative assessment of colon damage. I analyzed the data and prepared the manuscript.  xv  CHAPTER 1 Introduction 1.1 De novo lipogenesis Fatty acids are essential components of all organisms and are used in the formation of bipolar lipids that make up cellular membranes. They also provide a concentrated energy source and modulate cellular signalling and metabolism; however, even at low concentrations, non-esterified fatty acids are cytotoxic (1), and require esterification as triglycerides (TG) to provide an energy reserve during times of nutrient deprivation. Mammals use complex lipid delivery systems for transport of hydrophobic TG through the aqueous medium of blood plasma for storage or utilization in target tissues.  The majority of the body's fatty acid requirements are provided by  absorption of dietary TG, secreted into the circulation by enterocytes of the gut in the form of TG-rich lipoproteins called chylomicrons.  In the post-absorptive  phase, however, most of the TG-rich lipoproteins in the plasma consist of very low density lipoproteins (VLDL) (2), which are synthesized by hepatocytes of the liver from both exogenously (dietary) and endogenously derived TG.  Some of the  esterified fatty acids transported in TG-rich lipoproteins are released into the plasma by lipolysis in extrahepatic tissues expressing lipoprotein lipase (LPL). Fatty acids released by LPL are used by muscle tissue as an energy source or are re-esterified to TG in adipose tissue, forming the primary energy storage depot in the body.  The remainder of fatty acids released by LPL remain in the blood  plasma as part of the non-esterified fatty acid pool, becoming available as a substrate for hepatic re-esterification. Esterifed fatty acids from the diet that do not undergo lipolysis in extrahepatic tissues instead enter the liver as chylomicron remnants. While fatty acids are primarily derived from the diet, a variety of tissues also have the enzymatic machinery for synthesizing fatty acids from excess carbohydrates and other substrates that produce acetyl-CoA during their catabolism.  This type of biosynthesis of lipids is termed de novo lipogenesis  (DNL; Figure 1.1 on page 25). The liver is considered to be the major site of fatty acid synthesis in mammals, and possesses a striking ability to regulate  1  lipogenesis in response to diet (3). DNL also occurs in human adipose tissue (4), although the lipogenic capacity of human adipose tissue is lower than that of rodent adipose (5) and does not appear to be actively involved in DNL induced by high-carbohydrate diets (6). Contrary to a long-held assumption, there is now some evidence that DNL can also occur in skeletal muscle (7) and that muscle DNL can be increased by glucose (8). The physiological significance of muscle DNL is not clear, but it has been hypothesized that it could function to dispose of excess circulating glucose as muscle TG for subsequent fatty acid oxidation and thermogenesis (9). Lipogenic enzymes are also present in other tissues, such as skin, brain and pancreatic islets, although in these tissues, newly synthesized fatty acids may primarily function to form specific lipid structures or to regulate signalling and metabolism, rather than to store energy. For instance, fatty acids synthesized in sebocytes are used in sebum production (10), while fatty acids in the hypothalamus activate peroxisome proliferator-activated receptor (PPAR)-α and stimulate food intake (11). Fatty acids are synthesized by the sequential addition of two-carbon units, provided by malonyl-coenzyme A (malonyl-CoA), to an initial starting molecule, acetyl-CoA (12).  Acetyl-CoA carboxylase (ACC) is the rate-limiting step in this  process; it catalyzes the carboxylation of acetyl-CoA to generate malonyl-CoA (13). In animals, the only other protein required for synthesis of long-chain fatty acids is fatty acid synthase (FAS).  FAS is a multifunctional cytoplasmic enzyme  that catalyzes the entire pathway of synthesis of 16-carbon fatty acids, ending with the production of palmitic acid (16:0), a saturated fatty acid (SFA) (14). The end product of mammalian lipogenesis is usually a monounsaturated fatty acid (MUFA), oleic acid (18:1n9); compared with SFA, oleic acid is more readily incorporated into TG (15) and mobilized from fat stores (16). Elongation of 16carbon SFA and MUFA by two carbons is catalyzed by a specific long chain fatty acyl elongase enzyme (17), while desaturation of palmitic acid (16:0) and stearic acid (18:0) is performed by a specific enzyme with Δ9 desaturase activity, called stearoyl-CoA desaturase (SCD) (18).  Polyunsaturated fatty acids (PUFA) are  produced from oleic acid and palmitoleic acid (16:1n7) by further elongation and Δ6/Δ5 desaturation reactions (19, 20). Substrates for each of these desaturase and elongase enzymes can be derived from both products of DNL and fatty acids absorbed from the diet. 2  1.2 The stearoyl-CoA desaturase protein 1.2.1 SCD activity The desaturation of newly synthesized fatty acids can be considered to be the last stage of DNL, acting in parallel with FAS activity to prevent excess accumulation of saturated fatty acids (21).  SCD is an iron-containing enzyme  responsible for introducing a cis-double bond in the ∆9 position of CoA esters of fatty acid substrates. This oxidative reaction occurs in the endoplasmic reticulum (ER) and involves cytochrome b5, NADH-cytochrome b5 reductase, NADH, and molecular oxygen (22). Eight histidines contained in three regions of the enzyme are catalytically essential for SCD activity (23). Although several substrates are known, including vaccenic acid (11-18:1n7, trans or cis), the preferred substrates of SCD are palmitic acid (16:0) and stearic acid (18:0), generating the MUFA, palmitoleic acid (16:1n7) and oleic acid (18:1n9),  respectively  (18).  These  MUFA  are  key  components  of  TG,  diacylglyerols, phospholipids, cholesteryl esters (CE), and wax esters (24). Oleic acid is the major fatty acid found in TG and CE (Figure 1.2 on page 26) (25).  It the preferred substrate for diacylglycerol acyltransferase, the enzyme  that catalyzes the terminal step in TG synthesis from diacylglycerol and fatty acyl-CoA substrates (26). This preferred usage of oleic acid in TG synthesis is hypothesized to result from the close proximity of SCD and diacylglycerol acyltransferase in the ER (27). Similarly, oleoyl-CoA is the preferred fatty acid substrate of another ER enzyme, acyl-CoA:cholesterol acyltransferase (ACAT), which produces CE from long-chain fatty acyl-CoA and cholesterol (25). Accordingly, SCD-mediated synthesis of TG and CE plays an important role in the production of VLDL particles and storage of neutral lipids in adipose and other tissues (24, 28, 29). While much of the research into SCD activity has focussed on its effect on synthesis of TG and CE, it also modulates the fatty acid composition of several other lipids.  For instance, SCD activity increases the ratio of MUFA to SFA in  phospholipids, which can affect membrane fluidity and efflux of cellular cholesterol (30). Apart from being components of lipids, MUFA have also been implicated in mediation of signal transduction and differentiation of neurons and other cells (31), and can even control food intake in the brain (32).  3  1.2.2 SCD genes in mice and humans A number of mammalian SCD genes have been reported, including human SCD on 10q24 (33) and a cluster of four genes on mouse chromosome 19 that are orthologous to human SCD.  The four mouse genes, Scd1 (34), Scd2 (35),  Scd3 (36), and Scd4 (37), appear to have resulted from local duplications arising after divergence of the primate and rodent genomes, and are thus considered to be co-orthologous to human SCD. Human SCD comprises 6 exons, contained in a span of only 18 kb. This gene can produce two transcripts, 3.9 kb and 5.2 kb long, depending on the alternative usage of polyadenylation sites (33). Human SCD and other SCD genes have unusually long 3'UTRs (ranging from ~3.4 to 3.9 kb (38)) that are derived from a single exon.  The function of the alternative  polyadenylation sites and long 3'UTRs are not known, although they have been hypothesized to regulate the stability and/or translatability of the transcripts (39). An additional SCD gene, SCD5 (also known as SCD2 and ACOD4), which predates separation of the primate and rodent lineages, has been more recently identified (40).  Human SCD5 is on 4q21, and in this case no orthologous  sequence has been reported in the rodent lineage. Similar to SCD, two transcripts have been reported for human SCD5 (3.9 and 3.0 kb) (41). Like other lipogenic genes, mammalian SCD genes are highly regulated, and the diverse functions of SCD activity in lipid homeostasis may be facilitated by distinct tissue-specific roles.  While the expression of human SCD5 is brain-  specific (40), the predominant site of expression for SCD is adipose (42), with some lower level expression in liver (41) and brain (33). Rodent-specific duplications led to both subfunctionalization and neofunctionalization, such that mouse Scd1, Scd2, Scd3, and Scd4 have diverged to be expressed specifically in adipose and liver, brain, skin, and heart, respectively (36, 37). Of the SCD genes, regulation of expression has been examined the most extensively for mouse Scd1.  Expression of mouse Scd1 is increased in liver  and/or adipose by both dietary and hormonal factors, such as glucose (43), fructose (43), SFA (44), cholesterol (45), and insulin (46). Similar to their effects on other lipogenic genes, carbohydrates activate hepatic Scd1 transcription via the transcription factors liver X receptor (LXR) (47), sterol regulatory element binding protein 1c (SREBP-1c) (43), and carbohydrate response element binding  4  protein (ChREBP) (48). Furthermore, drugs that agonize LXR (47) also increase Scd1 transcription. Conversely, mouse Scd1 expression is decreased by PUFA (49), leptin (28), and TNFα (50).  PUFA repress expression of Scd1 by promoting SREBP-1c  transcript instability (51) and decreasing SREBP-1c protein maturation (45). Transcriptional and translational regulation have not been studied as extensively for human SCD genes, although like mouse Scd1, human SCD is repressed by polyunsaturated fatty acids via SREB-1 (52). SCD proteins are embedded in the ER via four transmembrane domains, with the N- and C- termini oriented toward the cytoplasm (53). The protein sequences of mammalian SCD isoforms range in length from 330 (40) to 359 aa (36), resulting in proteins with a molecular weight of 37.5 (40) to 41.5 kDa (36). Degradation of the mouse SCD1 protein is controlled by the ubiquitinproteasome-dependent pathway (54) and a plasminogen-like protein (55).  1.2.3 SCD index as a surrogate for SCD activity Direct measures of human SCD expression and activity are difficult to obtain in humans, and therefore many studies have relied on surrogate measures of desaturase activity. These surrogate measures estimate SCD activity using an SCD (or Δ9D) index, a ratio of 18:1n9/18:0 or 16:1n7/16:0, in some preparation of lipids. In rodents, the SCD index is usually calculated from triglycerides in liver (56) or adipose (56); however, in humans the SCD index is most frequently calculated from lipids in fasting plasma, while occasionally subcutaneous adipose (57) or skeletal muscle (58) are evaluated. Plasma lipid fractions may include TG (59), CE (60), phospholipids (61), or non-esterified fatty acids (62), although frequently a more easily obtained measurement of the SCD index in total plasma lipids is reported (63). Caution must be used when drawing conclusions about SCD function from the SCD index values in humans (64). For instance, 18:1 is always higher in TG than in other lipid fractions.  Consequently, observations of elevated SCD indices in  total plasma lipids can often be explained by increased levels of plasma TG rather than by actual SCD activity (64). Another concern is that the SCD index can be very dependent on recent food intake.  In particular, the SFA composition of  plasma lipid esters and adipose tissue has been demonstrated to mirror the fatty  5  acid pattern of diet (65), and relatively large within-person variability in plasma CE and phospholipid oleic acid has been reported (66). An added difficulty is there is no consensus around which ratio, 18:1n9/18:0 or 16:1n7/16:0, best represents SCD activity or which lipid fraction to analyze. Stable-isotope-tracer data indicates that conversion of dietary 18:0 to 18:1 is several fold greater than conversion of dietary 16:0 to 16:1 in humans (67), suggesting that human SCD may prefer stearic acid as a substrate. Nevertheless, even the rate of desaturation of 18:0 is much lower in human liver microsomes (6.1 to 14.2 %) (68, 69) than in rats (33 to 55 %) (70) or mice (83 %) (71). Setting aside these concerns, one cannot discount the intriguing data that comes from studies of SCD index in humans. Recent reports of an increased 16:1n7/16:0 ratio in serum CE or VLDL-TG with increased dietary saturated fat (72) or carbohydrate intake (21), respectively, likely demonstrate actual increases in SCD activity.  There is also some evidence that SCD indices in  adipose tissue, including both 16:1n7/16:0 and 18:1n9/18:0 ratios, correlate with human SCD mRNA levels in subcutaneous adipose tissue biopsies (57).  These  studies provide some validation for the use of the SCD index as an estimate of SCD activity in humans, at least when calculated from 16:1n7/16:0 in serum CE and either 16:1n7/16:0 or 18:1n9/18:0 in adipose tissue. A recent study has investigated cross-sectional correlations between SCD indices in human adipose TG and serum lipid fractions, including phospholipids and non-esterified fatty acids, in 301 healthy 60-year-old men (73). The 16:1n7/16:0 ratio was significantly correlated between both serum fractions and adipose TG, whereas the 18:1n9/18:0 ratio was significantly correlated between serum phospholipids and non-esterified fatty acids, and between non-esterified fatty acids and adipose TG, but not between serum phospholipids and adipose TG (73). Overall, the SCD index in adipose TG was best reflected in the 16:1n7/16:0 ratio in serum non-esterified fatty acids (73).  In order to fully understand the  meaning of the human SCD index values collected in epidemiological studies, additional work is now needed to validate the use of SCD indices in other plasma lipid fractions, such as TG and CE, as an estimate of SCD activity in liver.  1.2.4 Mutations in SCD genes The only known mutation in a human SCD gene is the disruption of SCD5 by a balanced pericentric inversion, inv(4)(p13q21), identified in a father and son with 6  orofacial clefting (40); in the absence of additional associations, however, the role of SCD5 in the orofacial clefting phenotype is unclear. Fortunately, mutations in mouse SCD genes have provided ample opportunity to study the phenotypic effect of reduced SCD activity.  Seven mouse Scd1  mutant alleles have now been described, including four spontaneous mutations, BALB/c-Scd1ab (74), ABJ/Le-Scd1ab-J (75), DBA/1LacJ-Scd1ab-2J/J (76), and KunmingScd1Xyk(77), one chemically-induced mutation, C57BL6/J-Scd1flk (78), and two targeted mutations, 129S6-Scd1tm1Ntam (79), and  B6129S1F2-Scd1tm1Wst (80).  While a targeted mutation of Scd2 has been produced (81), no spontaneous mutations have been detected in any mouse SCD gene other than Scd1, highlighting the importance of this gene to lipogenesis in the mouse. Of the null mutations in mouse Scd1, the asebia series of alleles (ab, ab-J, and ab-2J) has been studied the most extensively. The name “asebia” comes from a specific skin phenotype that includes the absence of normal sebaceous gland lipids and hypoplastic sebacous glands (82).  1.2.5 The skin phenotype of the SCD1-deficient asebia mouse Homozygosity for each of the mouse Scd1 null alleles in the asebia series is associated with extreme sebaceous gland hypoplasia, alopecia, scaly skin, and a huntched posture (82); these phenotypes are also observed in all other mice carrying disruptions of the gene (77, 79, 80). The large number of spontaneous Scd1  mutations  identified  can  likely  be  explained  by  this  conspicuous  macroscopic skin phenotype. Asebia mice have a sparse coat with bald areas prominent on the upper backs, although ventral fur is also sparse (83). The epidermis of asebia mice is hyperplastic: 3 to 6 keratinocytes versus 1 to 2 keratinocytes in wild-type mice (83, 84). The skin displays increased edema, disordered collagen bundles, and keratinocyte ballooning (84).  There is a marked increase in trans-epidermal  water loss, indicating that the epidermis of asebia mice does not provide a normal barrier (76). The increased trans-epidermal water loss (76) is most likely responsible for the increased water consumption observed in asebia mice (76). In  the  C57BL6/J  genetic  background,  SCD1-deficient  mice  also  exhibit  spontaneous skin ulceration, and increased susceptibility to gram-positive bacterial infection (78).  7  SCD1-deficient asebia mice display abnormal sebaceous cytodifferentiation, resulting in fewer lipid droplets in sebacous cells (85) and a reduction in sebaceous gland lipids, including wax diesters and monoesters and sterol esters (86). This sebaceous gland hypoplasia produces a hair shaft that is unable to shed its sheath, and instead grows in reverse toward the subcutis (76). When the hair fibre perforates the follicle base, this results in a foreign body response to fragments of hair fibre in the dermis, followed by follicle loss, dermal scarring, and epidermal hyperplasia (76). SCD1-deficient mice evidently have mild chronic dermal inflammation, indicated by increased vascularity (84), increased skin histamine (2.1-fold greater than controls) (83) and increased numbers of mast cells (3.1-fold higher in asebia mice than controls) (83, 87) and macrophages (88), but only rare lymphocytes or neutrophils in the dermis (84, 88).  The macrophage infiltrate contains lipid  crystals, which were originally thought to form the basis for the dermal inflammation (88). Increased mRNA encoding ICAM-1 has also been reported in asebia dermis (83), and subcutanous cyclosporine A can inhibit ICAM-1 expression and reduce mast cell numbers in the skin, restoring the wild-type skin phenotype (83). The skin of SCD1-deficient mice is constricted around the eyes, giving a pronounced 'squinting' appearance (83).  The eyelids are deficient in meibum  (79), the lipid film secreted by a specialized sebaceous gland called the meibomian gland that spreads over the ocular surface and protects it from dehydration (89).  With insufficient levels of meibum, the eyelids of SCD1-  deficient mice are more susceptible to bacterial infections (79). The asebia mouse has been proposed as a model for the pathogenesis of some forms of human scarring alopecias (90), including cutaneous lupus erythematosus, alopecia mucinosa, lichen planopilaris, and pseudopelade, which characteristically begin with sebaceous gland ablation (76).  The phenotype of  SCD1-deficient mice has also been suggested to mirror a very rare skin disorder, ichthyosis follicularis with atrichia and photophobia syndrome (OMIM 308205) (78), which involves atrophic sebaceous glands, alopecia and recurrent skin infections (91). The asebia model is less similar to psoriasis, although both have epidermal hyperproliferation and dermal inflammation, but from different causes (92, 93). The hydration  abnormality  in asebia mice  is also somewhat analogous 8  eczematous skin disorders (86), which are characterized by decreased hydration of the stratum corneum and show a predilection for sebaceous-gland-depleted extremities (94, 95).  The asebia model also suggests that alteration of SCD  activity in the eyelid can be implicated in human eye diseases (79). It is now known that chronic blepharitis and dry eye syndrome, which constitutes one of the most common and frustrating eye disease conditions in humans, are due to lipid abnormalities in meibum (96), but a role for SCD activity in these conditions has not been investigated.  1.3 The metabolic syndrome After much early work on the skin phenotype of asebia mice, now known to be deficient in SCD activity, most recent investigations into the physiological role of SCD activity have focused on metabolic phenotypes. More specifically, SCD has become a key enzyme implicated in the “metabolic syndrome”, a cluster of metabolic  characteristics  that  includes  visceral  dyslipidemia and hypertension (reviewed in  obesity,  (97, 98)).  hyperglycemia, This cluster of  characteristics is estimated to be present in between 20% and 30% of the adult population in most countries (98), and the prevalence is expected to increase with projected increases in obesity (99). More precise estimates of prevalence and comparisons between populations have been difficult to attain, due to evolving diagnostic criteria over the last decade (98). The two major definitions of metabolic syndrome are currently those recommended by the International Diabetes Federation (IDF) (100) and the National Cholesterol Education Program (NCEP) (101), recently updated by the American Hearth Association/National Heart, Lung and Blood Institute (102). Under both sets of criteria, metabolic syndrome is diagnosed when three of the following five features are present: increased waist circumference (population specific), increased plasma TG (≥ 150 mg/dL1), decreased plasma high density lipoprotein (HDL) cholesterol (< 40 mg/dL in men or < 50 m/dL in women), impaired fasting plasma glucose (≥ 100 mg/dL; includes insulin resistance and diabetes), and increased blood pressure (≥ 130/85 mm Hg). The main difference between the two definitions is that the IDF has a requirement for increased waist 1Lipid and glucose values are reported using conventional units of measurement for ease  of interpretation by North American readers. Table D.1 on page 172 shows conventional and SI units and conversion factors for these substances.  9  circumference, plus two of the other features, whereas the NCEP does not. Nevertheless, regardless of the clinical criteria used to identify affected persons, the risk-factor clustering that characterizes the metabolic syndrome is widely recognized to have a dramatic effect on morbidity and mortality worldwide. That is, it increases the risk of developing diabetes by 5-fold (103) and the risk of developing atherosclerotic cardiovascular disease (CVD) by 2-fold (104). Metabolic syndrome is often associated with other medical conditions, including cholesterol gallstones (105), sleep apnea (106), and fatty liver, especially with coincident obesity (107). The link between metabolic syndrome and fatty liver is strengthened by observations that the degree of steatosis in non-alcoholic fatty liver disease is proportional to the degree of obesity (108), and insulin resistance is almost universally observed in non-alcoholic fatty liver disease (109).  1.3.1 Role of human SCD in obesity and insulin resistance As described above, the SCD index is frequently used as a surrogate measure of SCD activity in human studies. Several but not all (110, 111) observational studies in humans have shown an association between increased indices of SCD activity and components of the metabolic syndrome, including insulin resistance (57, 59, 112), obesity (29, 58-60, 113), and hypertension (114). It is important to note, however, that all studies that report a correlation between the SCD index and plasma TG (63, 115-118) have evaluated the SCD index in total plasma lipids rather than in individual lipid fractions. Therefore, given the known abundance of MUFA in TG (64), one must conclude that in these studies the 18:1n9/18:0 and 16:1n7/16:0 ratios are primarily influenced by plasma TG levels rather than by SCD activity. Human data evaluating expression of SCD genes is limited.  Elevated SCD  mRNA has been observed in skeletal muscle of obese humans (29), and SCD mRNA in subcutaneous adipose tissue of moderately overweight men is reduced with acute weight loss, particularly in response to a low-carbohydrate diet (119). In the latter study, adipose SCD mRNA also correlates with plasma TG levels (119).  Another study that involved humans with varying insulin sensitivity  identified a positive correlation between adipose SCD mRNA and mRNAs encoding two inflammatory markers produced by macrophages in adipose tissue, MCP-1 and CD68 (120).  In this study, however, there was no significant 10  relationship between adipose or muscle SCD mRNA and either body mass index or insulin sensitivity (120).  There have also been other studies that found no  association between adipose (110) or muscle (120) SCD expression and insulin sensitivity. These results indicate that transcriptional regulation of SCD in human adipose and muscle may play a role in body weight regulation but not insulin sensitivity.  If human SCD is involved in insulin sensitivity, it may be acting  primarily in the liver. Alternatively, adipose and muscle SCD may be involved in insulin sensitivity, but may be regulated primarily at the level of protein turnover rather than transcription (54, 55). More detailed investigations that employ both SCD index and human SCD mRNA and protein expression levels will be required to sort out the importance of tissue-specific SCD activity in human metabolic syndrome. While a number of analyses of SCD sequence variation have come out of agricultural research designed to alter the fatty acid composition of food products from animals (121-123), there are only a small number of studies of human SCD sequence variants. The first published study of polymorphisms in human SCD, which involved 608 diabetic subjects and 600 controls from the UK, found no significant association between type 2 diabetes and six variants in SCD (124). These single nucleotide polymorphisms (SNPs) included one non-synonymous variant, p.M224L, one variant in the 3'UTR, one variant in intron 4, and 3 variants in the promoter.  One SNP had a borderline association with diabetes when  evidence for linkage to 10q was taken into account in a subset of families (p = 0.059), but this relationship was not confirmed in a replication study involving 350 young-onset diabetic patients and 747 controls (124).  A more recent study  of 1143 elderly Swedish men identified 4 rare genetic variations in SCD, rs10883463, rs7849, rs2167444, and rs508384, not tested in the previous UK study, that were significantly associated with both insulin sensitivity and waist circumference (125). Interestingly, none of these 4 SNPs were associated with the 16:1n7/16:0 SCD index in serum CE from 489 individuals, while 2 intronic SNPs (rs3071 and rs3793767) that were not significantly associated with features of the metabolic syndrome did demonstrate a minor but significant association with SCD index (125). Replication of these associations is now needed in another large cohort that includes similar data from euglycemic-hyperinsulinemic clamping and fatty acid composition.  11  1.3.2 Role of murine SCD in the metabolic syndrome A much clearer picture of the role of SCD activity in the metabolic syndrome has emerged from studies in rodents. Unlike studies involving human subjects, studies of rodents are able to include liver tissue in analyses of fatty acids, SCD activity, and expression of SCD genes. For instance, an experiment involving rats has demonstrated increased 18:1n9/18:0 and 16:1n7/16:0 SCD indices in total liver lipids in a rat model of type 2 diabetes (126).  SCD activity in liver  microsomes, using 18:0 as a substrate, correlates with liver Scd1 mRNA, and both of these measurements are increased with diabetes (126). Most rodent studies that explore the role of SCD activity in features of the metabolic syndrome involve animals with deficiencies in SCD1 protein, either mediated by disruptions in the mouse Scd1 gene (24, 28, 44, 63, 80), or gene silencing using antisense oligonucleotides (ASO) (127) or small interfering RNA (128). Mice with a complete absence of SCD1 exhibit reduced plasma TG, increased insulin sensitivity, an increased metabolic rate, and resistance to diet-induced and genetic obesity (28, 129). An important caveat is that all previous studies of SCD1-deficient mice, including those involving diet-induced and genetic obesity, used animals with a very different lipoprotein profile from humans: high levels of antiatherosclerotic HDL and relatively low levels of proatherogenic LDL and TGrich VLDL. The impact of SCD1 deficiency on metabolic syndrome in animals that posess a human-like lipoprotein profile with high LDL and VLDL, and relatively low HDL, as found in genetically-modified hyperlipidemic mice, is not yet known. The most profound metabolic phenotype in SCD1-deficient mice is reduced liver TG, highlighting the important role of SCD1 activity in hepatic TG synthesis (Figure 1.4 on page 28). Liver TG are reduced by 40-65% in SCD1-deficient mice (24, 130) and synthesis of TG (130) and CE (24) is also reduced.  The highest  relative reductions in liver TG were observed in chow-fed SCD1-deficient genetic models of obesity (28) and lipodystrophy (131). Diets high in oleic acid do not restore the wild-type phenotype, indicating that SCD1-mediated synthesis of endogenous MUFA is required for normal hepatic TG synthesis (130). In addition to reducing synthesis of endogenous MUFAs for storage as TG, SCD1 deficiency has an effect at earlier stages of the DNL pathway.  Chow-fed SCD1-deficient  mice have been observed to have reduced expression of hepatic ACC and FAS  12  (129, 132), as well as reduced hepatic ACC activity (132) and reduced whole body DNL (127). The role of mouse SCD1 in regulating plasma TG has been evaluated in several studies but a reduction in plasma TG has not been consistently observed in SCD1-deficient mice.  For example, some studies have shown plasma TG  reduced by over 50% with SCD1 deficiency (24, 44, 63), but two studies have demonstrated no significant differences (43, 133). It is not known whether the phenotypic differences can be attributed to the variations in age, sex, diet, fasting protocol, or genetic background of mice in the different studies. The second aspect of dyslipidemia that defines the metabolic syndrome is reduced plasma HDL cholesterol (100, 102). In most studies of normolipidemic mice, SCD1 deficiency does not significantly alter levels of plasma HDLcholesterol (63, 134-136).  Nevertheless, there is evidence for model-specific  effects on HDL cholesterol, indicated by increased HDL cholesterol with SCD1 deficiency in the presence of an LXR agonist (136), but decreased HDL cholesterol with SCD1 deficiency in the presence of genetic obesity (135) or highcarbohydrate/low-fat diet (136). While SCD1-deficient mice are protected from weight gain in studies of highfat feeding or genetic models of obesity (28, 129), SCD1 deficiency does not alter body weight in lean mice.  Two studies of chow-fed lean mice have even  demonstrated that female but not male mice have increased body weight with SCD1 deficiency (28, 129), which might be attributed to an increase in lean mass (129). The mechanism for increased lean mass in chow-fed female mice with SCD1 deficiency is not understood. SCD1-deficient mice on a 129S6 or C57BL/6J genetic background are protected from diet-induced hepatic insulin resistance, evaluated by glucose and insulin tolerance testing (129), as well as euglycemic-hyperinsulinemic clamping (137). Increased insulin sensitivity is accompanied by increased hepatic insulin signaling and decreased glucose production due to decreased gluconeogenesis and decreased glycogenolysis (137).  The improvement in hepatic insulin  sensitivity with ASO-mediated acute reduction in SCD1 levels is evident even in the absence of reduction in liver TG or body weight, suggesting a direct effect of SCD activity on hepatic insulin action (137). Overexpression of SCD1 (138) decreases insulin signaling and glucose uptake in muscle cells, suggesting that absence of SCD1 may contribute to increased 13  insulin sensitivity in skeletal muscle in addition to the liver. This is supported by evidence that SCD1 deficiency elevates insulin-signaling components and downregulates protein-tyrosine phosphatase 1B in muscle (139). Since all earlier studies on the beneficial metabolic effects of SCD1 deficiency have been confined to normolipidemic mice, one of the objectives of this research is to investigate the impact of SCD1 deficiency on metabolic syndrome in genetically-modified hyperlipidemic mice that posess a human-like lipoprotein profile.  1.3.3 The role of SCD in mediating the lipogenic effects of LXR agonists LXR is a transcription factor that up-regulates mouse Scd1 and other genes involved in fatty acid, cholesterol, and glucose homeostasis (134). Synthetic LXR agonists, such as T0901317  and GW3965, have shown promise in preclinical  mouse studies for the treatment of insulin resistance (140, 141), obesity, dyslipidemia (142, 143), and atherosclerosis (144).  The increased HDL  cholesterol and reduced atherosclerosis associated with LXR agonist therapy is thought be due to increased ABCA1 (145, 146), a protein that controls cholesterol efflux from lipid-loaded cells (147). Drugs that activate both LXRα and LXRβ isoforms have the undesirable effect of increasing plasma and liver TG through transcriptional activation of lipogenic genes, including those that encode SREBP-1c, FAS, and SCD (148). In order to ameliorate this effect, there has been an intense effort to identify LXR agonists specific for the LXRβ isoform, which is less involved in induction of lipogenesis (149). A study using homozygous 129S-Scd1tm1Ntam mice fed a chow diet has shown that absence of SCD1 protects against hypertriglyceridemia and increases plasma HDL-cholesterol induced by a synthetic LXR agonist, T0901317 (134), which increases cholesterol efflux and reduces atherosclerosis in hyperlipidemic mice (150); however, LXR activation exacerbates hypertriglyceridemia in dyslipidemic mice (150), and the role of SCD1 in regulating the severe LXR-induced HTG observed in dyslipidemic animals has not yet been determined. Therefore, one of the goals of this research is to determine whether decreasing SCD activity reduces LXR-induced HTG in a mouse model of hyperlipidemia.  14  1.4 Atherosclerotic cardiovascular disease Atherosclerotic CVD is the most common cause of illness and death worldwide (151). The risk of developing atherosclerotic CVD is increased by dyslipidemia, particularly by elevated levels of plasma cholesterol (152). As noted above, the risk of developing CVD is also increased 2-fold in the presence of metabolic syndrome (102, 104). Clinical trials have now confirmed that plasma cholesterol levels and aspects of the metabolic syndrome are important in the pathogenesis of CVD, and that interventions targeting these risk factors can be beneficial (153159). HMG-CoA reductase inhibitors, known as statins, are the current standard of care for individuals with hypercholesterolemia and are the most widely prescribed drug class in the world (160).  Unfortunately, even after treatment  with statins and other drugs that affect plasma cholesterol, some 70% of cardiovascular events (based on the number of events in control groups) are not prevented (161). As a result, there remains a significant need for new therapeutic approaches. Efforts to address this need have increased our understanding of the molecular pathogenesis of CVD, leading to new targets for therapies that lower plasma cholesterol and ameliorate features of the metabolic syndrome (162, 163).  1.4.1 Fatty acids in atherosclerotic cardiovascular disease In light of the reports of improved features of the metabolic syndrome with SCD deficiency, it has been suggested that reduced SCD activity may also provide protection from atherosclerotic CVD (164). Though many studies have examined a relationship between fatty acid composition and CVD in large human cohorts, only one has reported SCD indices (165): The Uppsala Longitudinal Study of Adult Men followed a group of 2009 50-year old Swedish men for more than 30 years, and found an association between the 16:1n7:16:0 SCD index in serum CE and CVD (165).  Another report from the same cohort demonstrated an  association between the 16:1n7:16:0 SCD index in serum CE and serum Creactive protein (166), a marker of systemic inflammation that has been linked to CVD (167). The putative atheroprotective effect of SCD inhibition may seem paradoxical, given the long-held belief that high intakes of SFA, substrates of SCD, increase 15  atherosclerosis (168). Public health organizations have instead recommended a “Mediterranean” diet containing high amounts of 18:1 (169), which is reduced in liver(24, 130), adipose (79, 133), and muscle (133) in the absence of SCD1. A dietary shift from SFA to MUFA was originally recommended based on geographic and migration studies demonstrating a positive correlation between consumption of SFA and mortality from coronary heart disease (170) and based on evidence that substitution of dietary SFA with MUFA can lower LDL cholesterol levels (171); however, intervention studies that test the relative effects of dietary SFA and MUFA on human CVD are notably absent. Nevertheless, existing dietary guidelines seem to be inconsistent with results from African green monkeys and mice that suggest that dietary MUFAs enrich LDL particles with cholesteryl oleate (172) and lead to increased, rather than decreased, atherosclerosis (173), when compared to diets substituted with SFA and polyunsaturated fatty acids.  1.4.2 Possible pro- and anti- atherogenic effects of SCD activity In addition to its role in TG synthesis and insulin signalling, the function of SCD in cholesterol homeostasis has suggested a more direct atherogenic role for SCD activity (Figure 1.3 on page 27).  When free cholesterol (FC) builds up in cell  membranes, it becomes coupled to long-chain fatty acids in the form of CE for storage in lipid droplets or secretion from hepatocytes or enterocytes as components of apolipoprotein (apo) B-containing lipoproteins. CE have long been recognized for their association with atherosclerosis, and their accumulation within macrophages and smooth muscle cells to form foam cells is a characteristic feature of the early stages of atherosclerotic plaques (174). As oleic acid is the preferred substrate for ACAT, the rate-limiting enzyme in cholesterol  esterification  (25),  it  has  been  proposed  that  endogenously  synthesized MUFA arising from SCD activity in the ER may be more available to ER-resident ACAT than dietary MUFA (18). Accordingly, mice lacking SCD1 have decreased synthesis of CE in the liver despite normal ACAT activity (24). Also, diets supplemented with MUFA fail to restore liver CE to levels found in normal mice (24). TG and CE containing oleic acid are major constituents of liver-derived apoB-containing VLDL particles, and SCD1-deficient mice have markedly reduced rates of VLDL production, sufficient to reduce VLDL-TG synthesis in obese leptindeficient mice to levels comparable to wild-type controls (28).  The impact of 16  reduced SCD activity on plasma lipoproteins in animals that posess a human-like lipoprotein profile is not known, but it can be predicted that SCD deficiency will reduce levels of CE-containing lipoproteins secreted by the liver, including both VLDL and atherogenic LDL, the metabolic by-product of VLDL (175, 176). Additional support for the hypothesis that SCD activity is atherogenic is provided by the findings that increased levels of SCD and its MUFA products inhibit cellular cholesterol efflux mediated by ABCA1, a protein with known atheroprotective function (30, 177-179).  Studies in CHO cells overexpressing  SCD reveal that SCD increases the 18:1/18:0 ratio in plasma membrane phospholipids  and  decreases  membrane-ordered  regions  in  the  plasma  membrane, decreasing the availability of cholesterol for efflux to apoA-I. Additional studies in macrophages indicate that the mechanism of SCD-mediated ABCA1 inhibition involves increased protein turnover rather than altered transcript levels (180). In vivo, SCD1 deficiency can increase hepatic ABCA1 in mice fed a very-low fat diet (181). The role of SCD in atherosclerosis may be more complex than was initially proposed. Predictions of an atherogenic role for SCD have thus far not taken into account  a  possible  atheroprotective  esterification in lesions.  role  for  SCD-mediated  cholesterol  Similarly, the inhibition of macrophage ACAT1 had been  proposed as a strategy to reduce foam cell formation and treat atherosclerosis, due to the accepted hypothesis that accumulation of cholesterol esters in arterial macrophages is a deleterious process. When macrophage ACAT1 was selectively deleted in a hyperlipidemic mouse model, it increased lesions (182), however, confirming other results in vitro and in vivo that ACAT1-dependent cholesterol esterification in macrophages is a protective response to toxicity from excess FC in conditions in which cholesterol efflux pathways become saturated (183, 184). While a role for SCD in cholesterol esterification in macrophages has not yet been examined, mouse Scd1 is known to be expressed in primary macrophages (30).  Also, SCD activity can be induced in macrophages by agonists of LXR  receptors (30, 180), which modulate lipid metabolism at the transcriptional level, and by agonists of toll-like receptors (78), which modulate innate immunity. It can be hypothesized that the synthesis of CE in macrophages is dependent on expression  of  SCD  genes  by  extension  from  the  enhanced  cholesterol  esterification observed in a Chinese hamster ovary cell line transiently transfected with mouse Scd1 (24).  When lipid composition of various tissues, 17  such as eyelid and skin, have been examined in chow-fed SCD1-deficient mice, increased FC and reduced CE have been observed (74, 79, 86), similar to the extensive deposition of FC in the skin and brain observed in ACAT1-deficient mice (183). Thus it would not be unexpected to find that an increased FC/CE ratio in SCD1-deficient macrophages increases severity of atherosclerotic lesions in a manner similar to macrophage ACAT1 deficiency. To complicate matters further, ABCA1-mediated cholesterol efflux is initially induced in cholesterol-loaded primary macrophages, but is inhibited by ~80% in pre-toxic FC-loaded macrophages through a mechanism of increased ABCA1 protein degradation (185). Although SCD activity has been implicated in the metabolic syndrome, the role of SCD in the development of atherosclerosis has not been previously examined. While the association between metabolic syndrome and atherosclerotic CVD in humans indicates that SCD activity should have a proatherogenic effect on atherosclerosis,  the  findings  of  ACAT1-deficient  animals  suggest  an  antiatherogenic effect of cholesterol esterification that may be mediated in part by SCD. A primary objective of this research is to investigate the role of SCD in susceptibility to atherosclerosis.  1.4.3 The LDLR-deficient mouse model of metabolic syndrome and atherosclerosis Familial hypercholesterolemia (FH; OMIM 143890), characterized by markedly elevated LDL-cholesterol levels and premature atherosclerosis, was the first genetic disease of lipid metabolism to be clinically and molecularly defined (186). The risk to FH patients of developing coronary disease is further increased by the presence of metabolic syndrome (187) or its individual components, including low HDL-cholesterol (188), high triglycerides(188), and insulin resistance (189). The FH phenotype is reflected in the LDLR-deficient hyperlipidemic mouse, which is frequently used as a model for the alterations in metabolic function that lead to atherosclerosis (190).  When fed a Western diet (21% milk fat, 0.2%  cholesterol), LDLR-deficient mice exhibit a lipoprotein phenotype similar to that seen in human patients with FH (190) and develop advanced atherosclerosis over 2 to 3 months (191, 192). In contrast to the most popular atherogenic model, apoE-deficient mice (193), LDLR-deficient mice develop diet-induced obesity and insulin resistance (191, 192).  The increase in body weight is predictive of 18  atherosclerotic lesion area (194), reflecting the link between metabolic syndrome and  CVD  in  humans.  In  addition,  such  high-fat  diets  increase  hypercholesterolemia (195), hepatic steatosis (193), and plasma markers of systemic inflammation (195), and these inflammatory markers are associated with increased lesion area independent of plasma lipoprotein levels (196). For these reasons, the LDLR-deficient mouse model was selected for studies of metabolic syndrome and atherosclerosis in this research.  1.5 Inflammation Inflammation is now recognized to play a major role in all stages of atherogenesis  (197),  and plasma markers of systemic  predictive for cardiovascular events in humans (198).  inflammation are Moreover, chronic  inflammatory diseases such as psoriasis (199), rheumatoid arthritis (200), periodontal disease (201) and systemic lupus erythematosus (202), associated with increased cardiovascular risk.  are  Standard atheropreventive drug  therapies such as aspirin and statins are known to have antiinflammatory properties and have been shown to be most beneficial in individuals with elevated inflammatory markers at baseline, even in those with relatively low serum cholesterol levels (203). The underlying mechanisms by which inflammation influences progression of atherosclerosis have been an area of intense research (197, 204).  These  mechanisms are now known to involve accumulation of modified LDL in the artery wall, activating endothelial cells to increase expression of adhesion molecules and chemokines (205).  This results in recruitment of monocytes to the  subendothelial space, where macrophage colony-stimulating factor produced by inflamed intima induces them to differentiate to macrophages (206).  Modified  LDL particles are internalized by macrophages and cholesterol accumulates in cytosolic droplets, turning the macrophages into foam cells, the prototypical cell in atherosclerosis. Activation of macrophage toll-like receptors stimulates NFκB activation, producing proinflammatory cytokines, such as interleukin (IL)-6 and tumour necrosis factor-α (TNFα) (207).  These cytokines have both local and  systemic effects, including hepatic expression of the acute phase proteins Creactive protein and serum amyloid A, which are both associated with increased cardiovascular risk (196, 208, 209). As the disease progresses, foam cells die,  19  producing an extracellular pool of debris and cholesterol that forms the necrotic core of mature lesions. In addition to the atherosclerotic CVD risk factors required for formal diagnosis, the metabolic syndrome is associated with a state of chronic low-grade inflammation, which also plays a role in promoting atherogenesis (210). The precise role of inflammation in metabolic syndrome remains to be determined, but  in  other  chronic  inflammatory  diseases,  including  atherosclerosis,  inflammatory skin disorders (psoriasis and eczema) and inflammatory bowel disease (Crohn disease and ulcerative colitis), cytokines recruit leukocytes to the site  of  their  secretion,  thereby  amplifying  perpetuating the initial tissue damage.  the  inflammatory  state  and  Genetic and environmental factors,  specific cytokine involvement, and inflammatory infiltrate differ with the tissue involved,  but  the  inflammatory  processes  are  common  to  all  chronic  inflammatory diseases (211).  1.5.1 Role of SCD activity in mediating the lipogenic innate immune response The innate immune system responds to infection and injury with changes in lipid and lipoprotein metabolism, including increased DNL, which functions to increase availability of lipid substrates for the activated immune system and tissue repair (212). Several pro-inflammatory cytokines, including TNFα, IL-6, and IL-1, increase hepatic DNL by acutely increasing intracellular concentrations of citrate (213, 214), an allosteric activator of ACC.  Surprisingly, given the  importance of lipogenesis to the innate immune response, a direct role for SCD activity in mediating the response to infection and inflammatory stimuli has not yet been examined. It might be predicted that increased SCD activity, resulting in increased TG synthesis and VLDL secretion, is part of the pro-lipogenic innate immune response.  Evidence supporting such a role includes the observations  that agonists of the toll-like receptor 2 induce SCD1 in peritoneal macrophages (78), and that hepatitis B virus X protein induces SCD1 in liver (215).  1.5.2 Proinflammatory effects of saturated fatty acids There is a much greater body of evidence that SCD activity may play a role in preventing induction of an inflammatory response in cells exposed to high levels of the SCD substrate, SFA.  For example, non-esterified palmitic acid activates 20  the protein kinase C (PKC)/nuclear factor-κB pathway, leading to impaired insulin signalling and induction of the pro-inflammatory cytokine interleukin (IL)-6  in  muscle cells (216). This effect is reversed by co-incubation with oleic acid, which acts by increasing TG synthesis and channelling palmitic acid away from diacylglycerol and consequent activation of PKC (216). A similar induction of IL-6 and another pro-inflammatory cytokine, TNFα, is observed when adipose (217), macrophage (218) and endothelial (219) cells are incubated with palmitic acid. There is also some support for an anti-inflammatory role for oleic acid in humans. In a randomized crossover trial, a diet containing 8% oleic acid led to a significant decreased in plasma IL-6 relative to a diet containing 8% stearic acid (220). Further studies are needed to determine whether SCD activity plays a role in mediating the inflammatory effects of fatty acids in vivo.  1.5.3 Role of hepatic SCD in inflammatory liver disease The SCD1-deficient mouse provides a useful model in which to explore inflammatory pathways as they relate  to lipid synthesis and metabolic  characteristics. However, the specific characteristics of each disease model must be taken into consideration when drawing extensions to human pathology. Two different studies have recently reported on the relationship between SCD1 deficiency and inflammatory liver disease (221, 222). SCD1 deficiency provides protection from concanavalin-A-induced nonalcoholic steatohepatitis (221), but SCD1 deficiency increases liver damage in a methionine-choline-deficient dietary model of nonalcoholic steatohepatitis (222). In the former model, steatohepatitis is induced by a T lymphocyte mitogen, while phosphatidylcholine synthesis and VLDL secretion are blocked in the latter model (223). These conflicting results seem to stem from the differing mechanisms of liver damage in the two models, suggesting that SCD activity may be involved in immune-mediated induction of fatty liver and liver inflammation under normal or high-fat dietary conditions, while SCD activity plays an important role in preventing lipotoxicity in the context of hepatic accumulation of free fatty acids.  1.5.4 Inflammatory bowel disease Inflammatory bowel disease, which includes Crohn disease and ulcerative colitis, is yet another chronic disease with a systemic inflammation component.  21  Plasma markers of systemic inflammation, including IL-6, serum amyloid A, and C-reactive protein, correlate with disease severity in patients with inflammatory bowel disease (224, 225), and numerous studies have reported the beneficial effect of antibodies targeted to inflammatory molecules, including  anti-TNFα  (226), anti-IL-12p40 (227), anti-IL6 receptor (228), and anti-α4β7 integrin (229) in patients with inflammatory bowel disease.  1.5.4.1 The dextran sulfate sodium model of acute colitis Dextran sulfate sodium (DSS)-induced colitis is a mouse model used by several groups to study the link between the immune system and colitis. In this model, plasma markers of inflammation increase in response to DSS treatment, and these markers are associated with disease severity (230). DSS is a sulfated heparin-like polysaccharide. Depending on the time course of oral administration in drinking water, it can induce both acute and chronic colitis, inhibiting epithelial cell proliferation and promoting apoptosis, which leads to epithelial injury, crypt loss and extensive ulceration and inflammation, predominantly localized to the distal colon (231, 232). The extent of colon damage increases with the amount of DSS administered (233, 234). Although, the precise mechanism of pathogenesis of DSS-induced colitis is unknown, DSS is known to be directly toxic to colonic epithelium (231, 235), and to subsequently activate inflammatory cells (236, 237) and alter intestinal microflora (238). DSS treatment also produces rectal bleeding, diarrhea, a decrease in colon length, and a reduction in total body weight (239), all of which are similar to the symptomatic findings in human ulcerative colitis (240).  1.5.4.2 The effect of SCD1 deficiency in experimental colitis Absence of SCD1 has been reported to exacerbate acute colitis in the DSSinduced colitis model (241). However, as noted above, SCD1-deficient mice with DBA/1LacJ and B6129S1F2 genetic backgrounds have been observed to consume increased amounts (~ 62% to 145%) of water, possibly due to increased transepidermal water loss (76, 80). This observation raised the question of whether the consequent increase in total DSS dose presented to the intestines of SCD1deficient mice might be responsible for the accelerated colitis reported previously (241). One report, with a limited sample size (n = 5), has concluded that minor  22  variations in fluid consumption do not affect the severity of DSS-induced colitis, based on Pearson's correlation analyses between clinical or histological results and total DSS intake (234). However, a more recent study reported significant correlations between histology score or neutrophil recruitment and total DSS intake in a sample size of 25 animals, supporting a conclusion that severity of DSS-induced colitis is dependent on total DSS intake in mice (233). Furthermore, these authors also found that a minimum DSS intake of 30mg/g body weight over seven days was required to reliably induce colitis in mice (233). In the only previous study of the effect of SCD1 deficiency on inflammatory bowel disease, Chen et al treated both SCD1-deficient mice and wild-type controls with 2% DSS for 7 days (241), a concentration that might be expected to deliver a total DSS intake of less than 30 mg/g to many of the mice in their study, particularly the wild-type controls that are expected to drink lower volumes of water (76, 80).  1.6 Thesis hypotheses and objectives In Chapter 1, I have discussed the evidence supporting a role for SCD activity in the metabolic syndrome. Earlier studies on the beneficial metabolic effects of SCD1 deficiency were confined to normolipidemic mice, and the role of SCD in the  associated  examined. investigate  development  of  atherosclerosis  has  not  been  previously  Therefore, the overall objective of this study was to the  role  of  SCD  in  modulating  susceptibility  to  atherosclerosis, including modulation of the atherogenic risk factors, metabolic syndrome and inflammation. The overarching hypothesis guiding this work was that SCD activity is atherogenic. The specific objectives of the work documented in this thesis were as follows: 1. To characterize the mouse Scd1ab-J allele In order to select an animal model in which to explore the function of SCD activity, I first performed a comparative analysis of vertebrate SCD gene families. A detailed characterization of the sequences and expression patterns of human and mouse SCD genes was undertaken to test my hypothesis that Scd1 is the most relevant mouse gene for metabolic studies modelling inhibition of human SCD activity.  Following this comparative analysis, the first objective of this  23  research was to characterize the mutation in Scd1 carried by the asebia-J mouse strain. 2. To determine the effect of absence of SCD1 on features of the metabolic syndrome in hyperlipidemic LDLR-deficient mice The second objective was to assess the effect of SCD1 deficiency on the metabolic syndrome in hyperlipidemic mice. I hypothesized that an absence of SCD1 would have a beneficial effect on characteristics of the metabolic phenotype of hyperlipidemic mice, including reduced hepatic and plasma TG, and reduced diet-induced weight gain and insulin resistance.  In order to test this  hypothesis, mice carrying the Scd1ab-J allele were crossed with the LDLRdeficient mouse strain and mice were challenged with a Western diet for 12 weeks. 3. To determine the effect of absence of SCD1 on inflammation and susceptibility to atherosclerosis in hyperlipidemic LDLR-deficient mice The same mouse cohort was used to assess the effect of SCD1 deficiency on atherosclerotic lesion area, the primary objective of this research.  I initially  hypothesized that an absence of SCD1 would reduce susceptibility to atherosclerosis in hyperlipidemic LDLR-deficient mice; however, my unexpected findings prompted me to explore the hypotheses that chronic inflammation has a role in mediating the proatherogenic effect of SCD1 deficiency. 4. To determine the effect of absence of SCD1 in a second inflammatory disease model: DSS-induced acute colitis To further explore the role of SCD in inflammation, a final objective was to determine the effect of SCD1 deficiency on DSS-induced acute colitis with dosing normalized to fluid consumption.  I hypothesized that SCD1 deficiency would  not impact on susceptibility to bowel inflammation, and that observations from a previous study were confounded by increased DSS intake in SCD1-deficient mice. The experimental design is illustrated in Figure 1.5 on page 25.  24  Figure 1.1: SCD in the de novo lipogenesis pathway. Schematic representation of the pathway controlling mammalian fatty acid synthesis. The initial starting molecule, acetyl-CoA, is produced by the catabolism of carbohydrates. Acetyl-CoA carboxylase (ACC) catalyzes the carboxylation of acetyl-CoA to generate malonyl-CoA. 2-carbon units, provided by malonyl-CoA, are sequentially added to acetyl-CoA by fatty acid synthase (FAS), resulting in the production of palmitic acid. SCD has a Δ9 desaturase activity that catalyzes the desaturation of 16- and 18-carbon saturated fatty acids. 16-carbon fatty acids are elongated by 2 carbons by the long-chain fatty acyl elongase enzyme (FACE, LCE, or ELOVL6). Polyunsaturated fatty acids (PUFA) are produced by additional elongation and Δ6/Δ5 desaturation reactions.  25  Figure 1.2: SCD in CE and TG synthesis. Schematic representation of the pathways controlling mammalian cholesteryl ester (CE) and triglyceride (TG) synthesis. Oleoyl-CoA, the product of the SCD reaction, is the major fatty acid found in CE and TG. Acyl-CoA:cholesterol acyltransferase (ACAT) produces CE from long-chain fatty acyl-CoA and free cholesterol. Glycerol-3-phosphate acyltransferase (GPAT) catalyzes the initial step in glycerolipid biosynthesis, forming lysophosphatidic acid, which is then acylated with another molecule of acyl-CoA to yield phosphatidic acid, a reaction catalyzed by 1-acylglycerol-3-phosphate-O-acyltransferase (AGPAT). Phosphatidic acid is then de-phosphorylated by phosphatidic acid phosphatase (PAP) to form diacylglycerol. Diacylglycerol acyltransferase (DGAT) catalyzes the terminal step in TG synthesis, adding the third fatty acid to diaglycerol.  26  Figure 1.3: SCD in lipoprotein metabolism. Dietary fat and cholesterol are absorbed by the intestine and converted to cholesteryl esters (CE) and triglycerides (TG) for incorporation into chylomicrons (CM). TG in CM and other TG-rich lipoproteins, including very-low-density lipoproteins VLDL, are hydrolyzed by lipoprotein lipase (LPL). CE on the CM remnants (CMr) is taken up by the liver and hydrolyzed by cholesteryl ester hydrolase (CEH) to free cholesterol (FC) in lysosomes. FC can be re-converted to CE by acyl-CoA cholesterol acyltransferase (ACAT) for storage. Oleic acid, the product of the SCD reaction, is the preferred FA substrate for ACAT. Such endogenously synthesized MUFA arising from SCD activity have also been proposed to be more available to liver and adipose glycerol phosphate acyltransferase (GPAT) for synthesis of TG than MUFA derived from the diet. TG and CE containing oleate are major constituents of liver-derived VLDL, which is initially converted to IDL and then LDL by LPL and hepatic lipase (HL). Modified LDL (e.g, by oxidation) is taken up by scavenger receptors in the macrophages and hydrolyzed by CEH. Excess FC may be effluxed by the ABCA1 transporter to nascent pre-β high density lipoprotein (HDL), where it is converted to CE by lecithin cholesterol acyltransferase (LCAT). Macrophage SCD may be necessary for optimal CE formation in cholesterol-loaded lesion macrophages. NEFA, nonesterified fatty acids.  27  Figure 1.4: Effect of SCD1 deficiency on tissue lipids. The most profound metabolic phenotype in SCD1-deficient mice is reduced liver TG, highlighting the important role of SCD1 activity in hepatic TG synthesis. Hepatic CE is also reduced. Similarly, SCD1-deficient mice are protected from weight gain in studies of high-fat feeding and genetic models of obesity, resulting in reduced storage of fatty acids as TG in adipose tissue. SCD1-deficient mice also display abnormal sebaceous cytodifferentiation, resulting in fewer lipid droplets in sebacous cells and a reduction in sebaceous gland lipids, including wax diesters and monoesters (WE) and sterol esters (CE). 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Cell Metab 2008;7:135-47.  46  CHAPTER 2 Comparative analysis of the vertebrate SCD gene families and characterization of the mouse Scd1ab-J allele1 2.1 Introduction Stearoyl-CoA desaturase (SCD) is a lipogenic enzyme that catalyzes the ratelimiting step in the synthesis of monounsaturated fatty acids. It introduces a cisdouble bond in the ∆-9 position of its substrates, thereby converting saturated fatty acids, palmitic (16:0) and stearic acid (18:0) to palmitoleic (16:1n7) and oleic acid (18:1n9), respectively (1).  The ratio of monounsaturated fatty acids to  saturated fatty acids, the “SCD index”, is frequently used as a surrogate measure of SCD activity in humans (2-4). Alterations in levels of SCD expression or SCD indices in liver (5), adipose (4, 6-8), and skeletal muscle (9, 9, 10) have been implicated in the metabolic syndrome and cardiovascular disease, suggesting that SCD may be a promising therapeutic target for these disorders. Several mammalian SCD genes have been reported, including two human genes.  The first human SCD gene to be identified is located on 10q24 and is  formally named SCD (11), although some publications continue to erroneously refer to it as SCD1 (7, 9, 12). The other human SCD gene is SCD5, also known as SCD2 and ACOD4, on 4q21 (13-15). The mouse genome contains a cluster of four genes on chromosome 19, Scd1 (16), Scd2 (17), Scd3 (18), and Scd4 (19), while two SCD genes have been described in rat on chromosome 1, Scd1 (20) and Scd2 (21). In non-mammalian vertebrates, two SCD genes have been identified in both chicken (SCD (22) and SCD5 (23)) and fugu (LOC777946 and fat-5 (24)), suggesting that there may an evolutionary advantage to expression of two or more SCD genes, such as divergent substrate specificity (25, 26, 26-28) or distinct tissue-specific (11, 15), developmental (13, 14, 29) or dietary regulation (30). 1A version of this chapter will be submitted for publication. MacDonald, M.L.E., Pape, T.D,  and Hayden, M.R. (2009). Characterization of the asebia-J allele: A 26kb deletion of Scd1 that does not encompass adjacent genes. The Genbank (http://www.ncbi.nlm.nih.gov/) accession number for the asebia-J sequence discussed in this chapter is GU017415.  47  Similar to other lipogenic genes, mammalian SCD genes are highly regulated and the diverse functions of SCD activity in normal lipid homeostasis and pathogenesis of the metabolic syndrome may be facilitated by distinct tissuespecific roles. Human SCD is expressed at highest levels in adipose tissue (15), with lower expression in liver (14, 15) and brain (11, 14, 15). Its transcription is repressed by polyunsaturated fatty acids and cholesterol via the sterol regulatory element-binding protein-1 (30). The expression pattern and dietary regulation of SCD suggests that this is the most relevant gene for studying the metabolic function of SCD activity in adult humans. In contrast, the highest levels of human SCD5 are in fetal brain (13), while lower expression in adult tissues has been mainly limited to brain and pancreas (13-15). Of the mouse genes, Scd1 has retained an expression profile that most closely mirrors that of human SCD, with expression primarily in adipose (16, 31), and some lower expression in the liver (18, 19), skin (18), lung (18, 19) and adrenal gland (31). In the adult mouse, Scd2 is expressed primarily in brain (1719) and the peripheral nervous system (32), with lower expression in lung (17, 18).  Scd3 and Scd4 are much more limited in their tissue distribution, with  unique expression in skin (18) and heart (19), respectively.  Genomic  organization and expression analyses of these highly similar SCD genes may provide understanding of their evolution and explain how they can catalyze the same type of reaction yet affect different physiological characteristics. New insights into the physiological role of SCD activity have come from recent studies of mouse strains with spontaneous or targeted mutations in SCD genes, with a particular emphasis on the asebia-J strain, which arose at The Jackson Laboratory in the BALB/cJ inbred strain and was subsequently out-crossed to C3H/ Di, back-crossed to F8, and maintained by brother-sister matings on the inbred strain ABJ/Le (33-35). The asebia-J strain contains a disruption in Scd1 (33) and possesses a phenotype similar to the six other mouse strains in which Scd1 mutant alleles have been identified. Of these, the asebia series of alleles (ABJ/LeScd1ab-J (33), BALB/c-Scd1ab (36), and DBA/1LacJ-Scd1ab-2J/J (35)) has been studied the most extensively.  Other Scd1 alleles include one additional spontaneous  mutation, Kunming-Scd1Xyk (37), one chemically-induced mutation, C57BL6/JScd1flk (38), and two targeted mutations, 129S-Scd1tm1Ntam (39) and B6129S1F2Scd1tm1Wst (40). While a targeted mutation of Scd2 has been produced (29), no  48  spontaneous mutations have been detected in any mouse SCD gene other than Scd1, highlighting the importance of this gene to lipogenesis in the mouse. Here we characterize the genomic structure and expression of human and mouse SCD genes, and perform an evolutionary analysis of the SCD loci in human, mouse, rat, chimp, dog, chicken, and fugu. We examine the role of gene duplication in the emergence of diverging SCD gene expression patterns and regulatory sequences, and propose a strategy to exploit mouse-specific paralogs to evaluate tissue-specific regulatory sequences. To facilitate mouse genotyping in future metabolic studies, we have sequenced the deletion of the ab-J allele in SCD1-deficient mice and confirmed the preservation of the neighbouring genes Scd4 and Wnt8b.  2.2 Methods 2.2.1 Genomic sequence analysis We analyzed genomic sequences for the presence of unidentified SCD genes using the TBLASTN and BLASTN algorithms against genomic sequence databases at  the  National  Center  for  Biotechnology  Information  (NCBI;  http://www.ncbi.nlm.nih.gov/). We compared human SCD genomic sequence to the paralogous SCD5 sequence as well as orthologous regions in mouse (Mus musculus), rat (Rattus norvegicus), chimp (Pan troglodytes), dog (Canis familiaris), chicken (Gallus gallus), and fugu (Takifugu rubripes). Relevant regions were identified, and coordinates of these regions were extracted from the genomic sequence. The coordinates of sequence used for making sequence comparisons are provided in Appendix Table A.1 on page 160.  Synteny was  initially assessed by manually comparing RefSeq genes and locations from http:// genome.ucsc.edu and then confirmed by reference to a published analysis of syntenic anchors (41).  Pseudogenes were confirmed by the presence of  premature stop codons or frameshifts in sequences derived from the genome assemblies and high-throughput genomic sequences at NCBI.  2.2.2 Phylogenetic analysis We computed sequence identities in inter- and intra-species pairwise global alignments of nucleotide and amino acid sequence using the Needleman-Wunsch  49  algorithm (42), as implemented by the needle program in the EMBOSS software package (43). Needle was used with default parameters of a gap open penalty of 10 and a gap extension penalty of 0.5. We aligned multiple protein sequences using ClustalX (44), performing manual editing when necessary. We constructed unrooted phylogenetic trees using the Neighbour-Joining method (45), and displayed trees using NJplot (46). Bootstrap values were calculated from 1000 resamplings of the alignment data. Details of the sequences used in the tree are summarized in Appendix Table A.2 on page 161.  We obtained full SCD coding sequences from NCBI.  Additional coding  sequences were obtained by sequence similarity searching of the expressed sequence tag (EST) database available at NCBI using the BLASTN algorithm. In the absence of EST data, the putative rat Scd4 ortholog was inferred by comparison with mouse Scd4 sequence.  Partial sequences were used for  phylogenetic reconstruction where the full coding sequence was not available.  2.2.3 Gene expression data We used human and mouse gene expression data from the version 2 Gene Expression Atlas of the Genomics Institute of the Novartis Research Foundation (GNF), obtained from human U133A+GNF1H, mouse GNF1M, and mouse MOE430 microarrays and processed using the GC content adjusted robust multi-array algorithm (47). EST counts from different tissue source libraries were extracted from the NCBI UniGene database.  2.2.4 Regulatory element analysis We carried out initial cross-species nucleotide sequence comparisons using the local alignment algorithm BLASTZ, implemented by MultiPipMaker (48). Genomic regions were then used for multiple alignments and further analysis using MLAGAN (49) and visualization using Vista (50).  Repeat elements in  genomic sequence were characterized using RepeatMasker (51).  2.2.5 Animals Mice carrying the Scd1ab-J null allele (33) were obtained from Rockefeller University (New York, NY) and were subsequently bred and maintained at the Wesbrook Animal Facility at the University of British Columbia.  All animal  50  breeding was in accordance with protocols approved by the University of British Columbia Animal Care Committee (Appendix B on page 162).  2.2.6 Genotyping and mutational analysis of Scd1ab-J DNA was extracted from tail biopsies by standard phenol-chloroform methods. Prior to mapping of the asebia-J mutation, genotyping of Scd1ab-J mice was performed by quantitative PCR using an ABI Prism 7700.  PCR primers for  quantitative mapping by sequence-tagged site were designed from genomic sequence masked with RepeatMasker. PCR amplifications were performed in a 15mL volume containing 20ng of genomic DNA, each dNTP at 0.2mM, each primer at 0.5uM, 2.5mM MgCl2, 1X PCR buffer (Invitrogen), and 0.75U Taq polymerase (Invitrogen).  The sample was  subjected to 35 cycles of denaturation at 95C for 30s, annealing at 60C for 30s, the extension at 72C for 1 min. Primers used to amplify the deletion break point and  for  subsequent  PCR  (5'AGGACAGAGCTGGTGAGATCC-3') GGCAGAAACATTATGCCTTGG-3').  genotyping and  were primer  primer B  A (5'-  PCR products spanning the deletion were  purified by PEG precipitation and sequenced on an ABI 3100 DNA sequencer using dye-labeled terminators.  Nucleotide numbering in descriptions of the  deletion uses the A of the ATG translation initiation start site described in reference sequence NM_009127.3 as nucleotide +1. In subsequent genotyping of Scd1ab-J mice, we used multiplex PCR reactions to amplify both Scd1+ and Scd1ab-J alleles from genomic DNA. Primer B and primer C (5'CCACAAGTTCTCAGAAACACACG-3') were used to amplify the wild-type allele in the same reaction in which primers A and B amplify the Scd1ab-J allele. The wildtype allele produces a 340 bp fragment, whereas the Scd1ab-J allele produces a 400 bp fragment.  2.3 Results 2.3.1 Multiple SCD gene duplications have occurred in the rodent lineage We compared sequences of human SCD genes with orthologous genomic sequences of other vertebrates in order to identify the most appropriate mouse SCD genes to target in metabolic studies modelling inhibition of human SCD 51  activity. A sequence similarity searching approach identified orthologs for human SCD in genomic sequences syntenic with the region spanning CHUK through WNT8B in each of the chimpanzee, dog and chicken genomes. The mouse genome shares conserved synteny with other land vertebrate genomes in the CHUK/SCD/WNT8B syntenic region, but there are 4 genes (Scd1, Scd2, Scd3 and Scd4) orthologous to SCD in the mouse genomic sequence, indicating that several tandem gene duplications likely occurred after the human/ mouse split.  Genes that are duplicated after a speciation event and are thus  othologous to a single gene in another species are referred to as co-orthologs. Global nucleotide alignments of the coding sequences of these genes (Table 2.1 on page 61) show high percent identity between human SCD and mouse Scd1, Scd2, Scd3, and Scd4 (78.9 to 82.9%), but low percent identity between human SCD5 and these mouse genes (60.7 to 61.5%).  Exons 1 to 6 are homologous  between mouse and rat Scd1, Scd2, Scd3 and Scd4 genes and the SCD genes of other land vertebrates.  However, the rat genomic sequence containing the  predicted Scd3 gene, supported by alignments with spliced ESTs from rat Harderian gland, contains a large gap. This gap would be expected to contain sequence orthologous to exons 3 to 6 of mouse Scd3.  The rat genome also  appears to have a putative Scd4 ortholog, deduced from alignments with the mouse Scd4 transcript.  However, we found no evidence for expression of the  putative rat Scd4 ortholog in the available rat EST sequence data, suggesting that this sequence may not contain a functional gene in the rat. In addition to the 4 mouse SCD orthologs, one unprocessed pseudogene, Scd-ps, was identified on mouse chromosome 19 in the CHUK/SCD/WNT8B syntenic region.  This  pseudogene contains homology to exons 2, 3 4 and 6 of human and mouse SCD genes, but is missing exons 1 and 5.  The splice donor of the sequence  homologous to exon 2 of human SCD is not conserved, resulting in an early frameshift mutation that disrupts the open reading frame. The organization and duplicated regions of the human and mouse SCD loci are shown in Figure 2. In the fugu genome, we identified two SCD genes, LOC777946 and fat-5. These fugu SCD genes share a similar degree of sequence with human SCD and SCD5 genes and are both located near an ortholog of the PAX2 gene, also found in the human SCD genomic region, supporting previous evidence for ancient large-scale fish-specific duplications (52, 53).  52  2.3.2 SCD5 gene loss in the rodent lineage A sequence similarity searching approach also identified orthologs for SCD5 in in the region between HNRPDL and COPS4 in the chimpanzee, dog and chicken genomes.  In mouse, orthologs of  HNRPDL and  COPS4 are located on a  chromosome 5qE4 contig that is free of gaps and consists entirely of finished sequenced (Figure 2.1 on page 62). However, the candidate ortholog for human SCD5 that we identified in this syntenic region, Scd5-ps, contains premature stopcodons or frameshifts in 3 of the 5 exons that share homology with human SCD5, indicating rodent-specific gene loss. To identify additional human SCD paralogs, we searched the human genome with the BLASTN algorithm using the human SCD transcript. We detected a nonfunctional, processed SCD pseudogene at 17p11.2, as reported previously (11), and four additional pseudogenic sequences at 1p33, 5q33.2, 5p14.3, and 16q24.1 that share a high percent identity (>87%) with regions of exon 6 of human SCD between 185 and 350nt long. No additional paralogous transcribed human SCD genes were identified.  2.3.3 Phylogenetic analysis and expression patterns of vertebrate SCD genes Phylogenetic analysis of the known and predicted SCD proteins was performed (Figure 2.2 on page 63).  The phylogenetic tree derived from multiple protein  alignments shows the expected evolutionary relationship with mouse and rat orthologs clustering together with SCD proteins from other land vertebrates. In order to explore the functional implications of gene duplication and gene loss in the rodent lineage, we evaluated expression patterns of vertebrate SCD genes and overlaid this data on the phylogenetic tree. We used data from microarray experiments found in the GNF Gene Atlas (47), combined with previous experimental data in the literature (11, 13-16, 18, 19) and EST information from public databases to study divergence of transcription profiles of genes between human and mouse, compared across homologous tissues in the two species.  Four records for SCD were generated from the  GNF1H+U133A microarray experiment.  Consistent with the literature, the  expression data from the human SCD probes (200831_s_at, 200832_s_at, 211162_x_at, and 211708_s_at) reveal very high expression (greater than 30-fold  53  higher than the global median over all tissues tested) in adipocytes (15), with much lower expression in the liver (14, 15) and brain (11, 14, 15). Compared with human SCD reporters, the human SCD5 reporter (220232_at) showed relatively ubiquitous expression, with highest expression (3-fold to 11fold higher than global median) in skin, the peripheral nervous system (PNS; superior cervical ganglion, dorsal root ganglion, trigeminal ganglion, ciliary ganglion), peripheral blood mononuclear cells (CD19+ B cells, CD4+ T cells, CD8+ T cells,  CD14+ monocytes), and bone marrow-derived cells (CD33+  myeloid progenitors, CD71+ early erythroid progenitors and CD105+ endothelial cells).  None of these tissues were evaluated in previous experiments in the  literature, in which the highest SCD5 expression was identified in whole brain and pancreas (13-15). When compared with other tissues included in the Gene Atlas, only moderate expression (1- to 2-fold of global median) was observed in specific brain regions (prefrontal cortex, parietal lobe, temporal lobe, cingulate cortex, globus pallidus, subthalamic nucleus, medulla oblongata, pons, cerebellar peduncles) and pancreatic islets, with lower expression in whole brain and pancreas. The approximate expression pattern inferred from human SCD5 EST data  supports  the  micrarray  expression  profile,  demonstrating  relatively  ubiquitous expression, with much higher expression in peripheral nerve and brain tissue sources than in liver and adipose tissue. The rodent-specific tandem duplications of the ancestral SCD ortholog have led to divergence of expression profiles between these co-orthologous genes (Figure 2.2 on page 63).  Scd1 is the only mouse co-ortholog that retains an  expression pattern similar to human SCD, with expression primarily in adipose (16, 31), and some lower expression in the liver (18, 19), skin (18), lung (18, 19) and adrenal gland (31).  This expression pattern is consistent with expression  data for the mouse Scd1 probes (gnf1m00078_a_at, 1415964_at, 1415965_at), which reveal very high expression (greater than 30-hold higher than global median) in adipose tissue, with lower expression in liver, skin, lung and adrenal gland, as well as additional notable sites of expression in lymph nodes, mammary gland, and ovary (all more than 10-hold higher than global median). The expression of the other mouse co-orthologs has diverged.  Among adult  tissues, Scd2 is expressed primarily in brain (17-19) and the peripheral nervous system (32), with lower expression in lung (17, 18), observations that are consistent with microarray expression data for mouse Scd2 probes (1415822_at, 54  1415823_at, 1415824_at, gnf1m01722_a_at, and gnf1m09689_s_at). Compared to Scd1 and Scd2, Scd3 and Scd4 are very limited in their tissue distribution. Scd3 is expressed uniquely in skin (18), with probes 1423366_at and 1450956_at demonstrating more than 100-fold higher expression in skin than the global median for all tissues.  Similar to Scd3, expression data from the literature  indicates that Scd4 is expressed uniquely in one tissue; Scd4 is though to have evolved to be expressed uniquely in the heart (19), a  site of expression not  previously observed for other human or mouse SCD genes. No Scd4 probes are included in the Gene Atlas version datasets, but the mouse Scd4 EST data supports a similar expression pattern to the experimental data in the literature, with Scd4 expression limited to the heart and one additional site of expression, the uterus. When the primary sites of expression of vertebrate SCD genes sites are considered in concert with a phylogenetic analysis, it becomes apparent that subfunctionalization  and  neofunctionalization  following  duplication  of  the  ancestral SCD ortholog in the rodent lineage removed the selective constraints on the  SCD5  ortholog,  setting  the  stage  for  mutational  inactivation  and  pseudogenization (Figure 2.2 on page 63). That is, SCD5 function in skin, PNS, and brain may have become redundant in the presence of Scd2 expression in brain and PNS and Scd3 expression in skin.  2.3.4 Regulatory sequence analysis Diverging SCD expression patterns correlate with diversification of regulatory elements.  We used a comparative genomic approach to identify putative  regulatory sequences that may be responsible for the subfunctionalization and neofunctionalization of rodent genes.  The subfunctionalization model (54)  asserts that each duplicated gene accumulates degenerate mutations that reduce their expression patterns to that of the single ancestral gene. A global multiple alignment of genomic sequences from human, mouse, and rat suggests that rodent Scd1 and Scd2 have maintained different sequences from their common SCD ancestor in the 5' upstream genomic region, consistent with subfunctionalization (Figure 2.3 on page 64).  Scd1 has likely accumulated  mutations in sequences responsible for expression in brain, but has retained a conserved putative regulatory region that might be expected to be control expression in adipose and liver, sites of expression it shares with human SCD but 55  not its mouse co-orthologs (top panel in Figure 2.3).  This conserved region is  located at -309 to -128 bp relative to the translation start site of mouse Scd1 (+1) , and -307 to -147 bp from the translation start site of human SCD. Similarly, rodent Scd2 has accumulated mutations in sequences that may be responsible for expression in adipose and liver, but has an upstream sequence (8,537 to -8,445) with high similarity to sequence upstream of human SCD (-2,149 to -2,051) that may contain regulatory elements responsible for expression in brain (second panel in Figure 2.3).  However, this putative conserved regulatory  region consists of primate-specific Alu sequence (55) in human SCD and rodentspecific Alu-like B1 sequence (56) in mouse Scd2. Since these short interspersed elements (SINE) are believed to have arisen independently in the primate and rodent lineages, any analogous regulatory function of these similar sequences may have arisen independently by convergent evolution. The neofunctionalization model (57) argues that after duplication one coortholog retains the ancestral function, while another acquires new functions. Consistent with neofunctionalization, Scd3 and Scd4 are known to have acquired new sites of expression in skin (18) and heart (19), respectively, and do not show conservation in their 5' upstream genomic regions (Figure 2.3).  2.3.5 Characterization of the asebia-J mutation Of the four mouse SCD co-orthologs, Scd1 has the expression pattern that mostly closely mirrors the expression of human SCD in metabolically relevant tissues, including adipose and liver.  Therefore we selected Scd1 as the most  appropriate mouse gene to target in metabolic studies modelling inhibition of human SCD activity. We obtained four affected males from the asebia-J mouse strain, which has a spontaneous deletion in Scd1 (B6.Cg-Scd1ab-J) (33, 34).  This large deletion is  known to include exons 1-4 of Scd1, with a 3' boundary upstream of exon 5 (33), but the 5' boundary has not been mapped relative to Wnt8b, the gene immediately upstream of Scd1.  In order to determine whether Wnt8b is  disrupted by the asebia-J mutation, we mapped the extent of the deletion using novel sequence-tagged-site markers generated from mouse genomic sequence. After mapping the mutation to within 3kb, we amplified and sequenced genomic DNA across the deletion, identifying a deletion of 25,779 bp encompassing bp  56  44,295,709 to 44,321,487 (NCBI mouse assembly version 35; Figure 2.4 on page 65). The asebia-J mutation (NM_009127.3: c.-20205_492del), results in the deletion of the 5' end of the Scd1 gene, ending within exon 4 at nt 492 of the coding reference sequence (GenBank accession number NM_009127.3). The distal end of the deletion is 20,205 bp upstream of the start codon of Scd1 and 65,936 bp upstream  of  NM_011720.3).  the  start  codon  of  Wnt8b  (GenBank  accession  number  Though we cannot exclude the possibility that this deletion  includes some sequences that regulate the expression of  Wnt8b, this seems  unlikely given the distance from the start codon and the important role of Wnt8b in development (58).  2.4 Discussion An evaluation of genomic sequence and expression data reveals evidence for rodent-specific gene loss of the ancestral SCD5 ortholog, which may have become redundant subsequent to gene duplication in the rodent lineage and evolution of expression of Scd2 and brain/PNS and Scd3 in skin. Scd1 is the only mouse co-ortholog that retains an expression pattern similar to human SCD, and is therefore considered the most relevant mouse gene for metabolic studies modelling inhibition of human SCD activity. Toward this end, the mutation in the SCD1-deficient asebia-J mouse strain was defined as a 25,779 bp deletion that affects exons 1 to 4 of Scd1 but not adjacent genes. The recent emergence of genomic sequence from multiple species has led to many reports of new SCD loci.  Orthologs of the two human SCD genes,  designated SCD and SCD5 (11, 13-15), have now been identified in pig (23, 59), cow (60, 61), sheep (23, 62), and chicken (22, 23). However, despite reports of four SCD co-orthologs in mice (Scd1 (16), Scd2 (17), Scd3 (18), and Scd4 (19)), three SCD co-orthologs on hamster (Scd1, Scd2, and Scd3 (63)), and two coorthologs in rat (Scd1 (20) and Scd2 (21)), no SCD5 ortholog has been reported in any rodent species.  Our analysis of rat and mouse genomic sequence data  provides evidence for retention of putative Scd3 and Scd4 co-orthologs in rat and pseudogenization of the SCD5 ortholog in the rodent lineage. A single non-functional, processed SCD pseudogene at 17p11.2 has been described previously in humans (11). In addition to this known pseudogene, our  57  sequence similarity searching uncovered four additional pseudogenic sequences at 1p33, 5q33.2, 5p14.3, and 16q24.1 that share a high percent identity (>87%) with regions of exon 6 of human SCD between 185 and 350 nt long.  The  functional and evolutionary significance of these sequences is not known, but it is tempting to speculate that these sequences may hold a clue to the evolution of the unusually long 3'UTR sequences of SCD genes; 3'UTRs range from ~3.4 to 3.9 kb in SCD genes (64), relative to a mean length of ~500 nt in upper vertebrates (65). The long 3'UTRs of SCD genes are derived from a single exon and have been hypothesized to regulate the stability and/or translatability of the transcript (66). Mammalian SCD genes are highly regulated, with high levels of expression confined to specific tissues (11, 15), developmental periods (13, 14, 29), and dietary stimuli (30). However, reports of tissue-specific expression patterns have previously been limited to a small number of whole tissues. Our evaluation of data from microarray experiments found in the GNF Gene Atlas and EST information from public databases suggested that some important tissues have been missed in previous studies. For instance, in contrast to previous reports of highest SCD5 expression in whole brain and pancreas (13-15) microarray data indicates that human SCD5 is expressed at higher levels in tissues not previously evaluated, including skin, PNS, peripheral blood mononuclear cells, and bone marrow-derived cells. Recent studies have identified correlation of human SCD5 expression in peripheral blood leukocytes with plasma cholesterol (67) and upregulation of human SCD5 during macrophage differentiation (68), providing additional support for a role for SCD5 in human immune cells. Similarly, mouse Scd4 was previously thought to be expressed uniquely in heart (19).  However, EST data provides evidence for an additional site of  expression in the uterus, suggesting that future studies of lipogenesis in mouse uterine tissue would do well to consider a role for Scd4. Divergent SCD gene expression patterns and regulatory sequences in the rodent  lineage  suggest  that  subfunctionalization  and  neofunctionalization  following duplication of the ancestral SCD ortholog in the rodent lineage relaxed the selective constraints on the SCD5 ortholog, allowing for mutational inactivation and subsequence gene loss.  A comparative genomic approach  indicates that rodent Scd1 and Scd2 share different sequences in the 5' upstream genomic  region  subfunctionalization.  with  the  human  SCD  ortholog,  consistent  with  We identified a conserved region 309 to 128 bp upstream 58  of the translation start site of mouse Scd1 and 307 to 147 bp upstream of the translation start site of human SCD that may be responsible for the conserved expression of human SCD and Scd1 in adipose and liver. This region overlaps with the mouse Scd1 promoter described previously (16, 69-71), including the putative TATA box (16), fat-specific element 2 (69, 70), and unique SCD1-specific nuclear factor binding site (70), but not the putative Sp1-binding site (71), glucocorticoid regulatory element (69), cAMP response element (69), sterol regulatory elements (71), or nuclear factor Y binding sites (CCAAT box) (71). Our analysis is consistent with a previous report that highlighted the striking similarity of human SCD to mouse Scd1 as compared with mouse Scd2 in this proximal promoter region (72). We also identified a sequence upstream of mouse Scd2 (-8537 to -8445) with high similarity to sequence upstream of human SCD (-2149 to -2051) that may contain regulatory elements responsible for expression in brain. However, this putative conserved regulatory region of human SCD has not been associated with any known regulatory function (72) and consists entirely of SINE Alu sequence that is specific to the primate lineage (55).  Furthermore, the aligned region of  mouse Scd2 contains Alu-like SINE B1 sequence (56) that is specific to the rodent lineage.  Therefore, if these regions of high sequence similarity do contain  important regulatory elements in both the human SCD and mouse Scd2 genes, they may present an example of convergent evolution, rather than conserved sequence.  There is a known correlation between mouse B1 and human Alu  densities within the corresponding 5kb upstream regions of orthologous genes (73), which suggest a positive selection for retroposon insertion or maintenance in these regions. Moreover, Alu elements are known to contain binding sites for transcription factors (73), and we cannot exclude the possibility that functionally analogous cis-regulatory elements exist within this region of human SCD and mouse Scd2. The same comparative genomic approach revealed no strong evidence for sequence similarity between the 5' upstream genomic regions of human SCD and mouse Scd3 or Scd4.  Future studies that employ phylogenetic footprinting  techniques (74-76) may be useful to identify novel SCD regulatory regions in these rodent genes. Transcription factor binding sites shared between Scd3 or Scd4 and other co-expressed genes (i.e. expressed specifically in either skin or  59  heart) (77), but not shared with SCD/Scd1/Scd2 would be considered novel tissuespecific regulatory regions acquired after the gene duplication event. Consistent with previous studies (16, 18, 19, 31), our analysis of expression data confirms that Scd1 is the only mouse co-ortholog that retains an expression pattern similar to human SCD.  In order to facilitate future mouse studies  modelling inhibition of human SCD activity, we defined the mutation in the asebia-J mouse strain (33, 34). This strain has a spontaneous deletion of 25,779 bp that results in deletion of the 5' end of the Scd1 gene, including sequence 20,205 bp upstream of the start codon of Scd1 and terminating 65,936 bp upstream of the start codon of Wnt8b, the gene immediately adjacent to and upstream of Scd1. The delineation of this mutation confirms that Scd1 is the only gene likely to be affected by the asebia-J mutation and facilitates the design of DNA primers that can be used in a genotyping assay that distinguishes wild-type mice from heterozygous mice carrying the asebia-J mutation. This study provides evidence for pseudogenization of the ancestral SCD5 in the rodent lineage, suggesting that positive selective pressure was relaxed following gene duplication in the rodent lineage and evolution of expression of Scd2 and Scd3 in brain/PNS and skin, respectively. Our definition of the precise limits of the spontaneous deletion of Scd1 present in asebia-J mice will assist future studies of the role of SCD activity in metabolic syndrome and susceptibility to atherosclerosis.  60  Table 2.1: Comparison of coding sequences of human and mouse SCD genes. % Nucleotide Identity  % Amino Acid Identity  Human  Mouse  SCD  SCD5  Scd1  Scd2  Scd3  Scd4  SCD  100.0  58.9  82.3  82.9  82.6  78.9  59.0  55.1  SCD5  52.7  100.0  61.5  60.8  60.1  60.7  47.2  74.3  Scd1  84.1  53.2  100.0  89.8  90.6  84.5  63.8  56.2  Scd2  83.0  52.5  86.3  100.0  87.6  82.6  64.2  53.7  Scd3  82.7  51.5  88.0  78.3  100.0  83.4  63.7  55.1  Scd4  77.2  49.6  79.2  79.1  78.3  100.0  64.1  52.5  100.0  44.1  Scd-ps Scd5-ps  Scd-ps Scd5-ps  100.0  61  Figure 2.1: Schematic diagram of synteny and genomic organization of the SCD genes in human and mouse. Gene identities and orientations are indicated. Discontinuous lines indicate pseudogenes. Corresponding gene locations can be found in Table A.1 on page 160.  62  Figure 2.2: Phylogenetic relationships and sites of expression of SCD genes. Coding sequences for chimpanzee (PANTR) genes, dog (CANFA) genes and rat Scd3 and Scd4 were predicted from genomic sequence corresponding to alignments with ESTs or human and mouse SCD transcripts and predicted amino acid sequences were aligned. An unrooted tree was created using the neighbour-joining method. Bootstrap values calculated from 1000 resamplings of the alignment data are indicated. Sites of expression supported by EST data (E), previously reported expression experiments in the literature (L; (13)(14)(15)(11) (16)(18, 19)) and microarray experiments found in the GNF Gene Atlas (G) are indicated. SCDA, Takifugu rubripes (FUGRU) LOC777946 locus; SCDB, Takifugu rubripes fat-5 locus.  63  Figure 2.3: VISTA plots of the MLAGAN alignment of human SCD genomic sequence with coorthologous sequences from mouse and rat. Each of the horizontal panels shows alignment of human SCD with mouse (M) and rat (R). The y axis represents percentage sequence identify. Exons are shown in blue, UTRs are in light blue, and conserved non-coding sequences in pink. Repetitive sequences are marked as follows: LINE (red), SINE (green), LTR (pink). The grey arrow shows the direction of transcription of human SCD. We used a criterion of minimum 100-bp windows with 75% identify to define conserved non-coding sequences. Two putative regulatory elements that may be responsible for subfunctionalization are indicated by asterisks. 64  Figure 2.4: Analysis of the Scd1ab-J mutation. a, Sequence analysis of the PCR product spanning the Scd1ab-J deletion on chromosome 19 amplified from genomic DNA. Coordinates refer to positions on chromosome 19. b, Position of 25,779 bp deletion relative to Scd1 and Wnt8, the gene immediately adjacent to and upstream of Scd1. Gene identities and orientations are indicated in blue. 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The PAZAR database of gene regulatory information coupled to the ORCA toolkit for the study of regulatory sequences. Nucleic Acids Res 2009;37:D54-60. 77. Ho Sui SJ, Mortimer JR, Arenillas DJ, Brumm J, Walsh CJ, Kennedy BP, et al. oPOSSUM: identification of over-represented transcription factor binding sites in co-expressed genes. Nucleic Acids Res 2005;33:3154-64.  71  CHAPTER 3 Absence of stearoyl-CoA desaturase-1 ameliorates features of the metabolic syndrome in LDLR-deficient mice1 3.1 Introduction Susceptibility to cardiovascular disease and diabetes is associated with the metabolic  abnormalities  of  obesity,  insulin  resistance,  dyslipidemia  and  hypertension (1-3). A key enzyme that has been implicated in these metabolic abnormalities is stearoyl-CoA desaturase (SCD), which introduces a cis-double bond in the ∆-9 position in its substrates, thereby converting palmitic (16:0) and stearic acid (18:0), to palmitoleic (16:1n7) and oleic acid (18:1n9), respectively (4, 5). These nutritionally and physiologically important monounsaturated fatty acids (MUFA) are the major fatty acids found in triglycerides (TG) and cholesteryl esters (CE) (6). A number of mammalian SCD genes have been reported, including a human SCD on 10q24 (7) and a cluster of four genes on mouse chromosome 19 (Scd1 (8), Scd2 (9), Scd3 (10), and Scd4 (11)). These appear to have arisen from local duplications after divergence of the primate and rodent genomes. An additional SCD gene, SCD5, which pre-dates separation of the primate and rodent lineages, has been identified recently and is expressed in the brain and pancreas (12-14). The most prominent site of expression for human SCD is in adipose tissue (14), with lower expression in liver and brain (7).  Like other lipogenic genes,  mammalian SCD genes are highly regulated. Human SCD is repressed in cultured cells by polyunsaturated fatty acids and cholesterol via sterol regulatory elementbinding protein-1 (SREBP-1) (15). Furthermore, SCD mRNA is elevated in skeletal muscle of obese humans (16), and several but not all (17) observational studies in humans have shown an association between increased indices of SCD activity and components of the metabolic syndrome, including insulin resistance (18-21), 1A version of this chapter has been published. MacDonald, M.L.E., Singaraja, R.R., Bissada,  N., Ruddle, P., Watts, R., Karasinska, J.M., Gibson, W.T., Fievet, C., Vance, J.E., Staels, B. et al. (2008). Absence of stearoyl-CoA desaturase-1 ameliorates features of the metabolic syndrome in LDLR-deficient mice. J Lipid Res. 49, 217-229.  72  obesity (16, 22-24), and hypertension (25). The expression of mouse Scd1 in adipose tissue and liver (8, 10) and its regulation by the lipogenic transcription factors SREBP-1 and LXR (26) define it as the most relevant murine ortholog for studying the metabolic function of SCD activity in humans. Seven Scd1 mutant alleles have now been described in mice, including four spontaneous mutations, BALB/c-Scd1ab (27), Scd1ab-2J/J (29),  and Kunming-Scd1Xyk (30),  C57BL6/J-Scd1flk (31),  ABJ/Le-Scd1ab-J (28),  DBA/1LacJ-  one chemically-induced mutation,  and two targeted mutations, 129S-Scd1tm1Ntam (32)  and  B6129S1F2-Scd1tm1Wst (33). The asebia series of alleles (ab, ab-J, and ab-2J) has been studied the most extensively.  Homozygosity for each is associated with  atrophic sebaceous glands, alopecia, and scaly skin, phenotypes which are also observed in mice carrying the targeted disruption of the gene (32). SCD1-deficient mice have been observed to exhibit reduced plasma TG, increased insulin sensitivity, an increased metabolic rate, and resistance to dietinduced obesity (34, 35).  However, the impact of SCD1 deficiency on  hyperlipidemic mice fed a Western diet is unknown. A recent study using homozygous 129S-Scd1tm1Ntam mice fed a chow diet has shown that absence of SCD1 protects against hypertriglyceridemia and increases plasma HDL-cholesterol induced by a synthetic LXR agonist (26), T0901317, which increases cholesterol efflux in hyperlipidemic mice (36, 37). However, LXR activation exacerbates hypertriglyceridemia (HTG) in hyperlipidemic mice (36, 37), and the role of SCD1 in regulating the severe LXR-induced HTG observed in hyperlipidemic mice has not yet been determined. Familial hypercholesterolemia (FH; OMIM 143890), characterized by markedly elevated LDL-cholesterol levels and premature atherosclerosis, was the first genetic disease of lipid metabolism to be clinically and molecularly defined (38). The risk to FH patients of developing coronary disease is further increased by the presence of metabolic syndrome (39) or its individual components, including low HDL-cholesterol(40-44), high triglycerides(40), and insulin resistance (45-47). The LDLR-deficient hyperlipidemic mouse mimics human FH and has now been used in numerous studies (48) as a model for the disrupted lipoprotein regulation and metabolic function that leads to diabetes and atherosclerosis. Unlike the most commonly used hyperlipidemic model, apoE-deficient mice (49), LDLR-deficient mice develop diet-induced diabetes and obesity when fed a  73  Western diet and also develop a lipoprotein phenotype similar to that seen in FH (48, 50-53). Earlier studies on the beneficial metabolic effects of SCD1 deficiency have been confined to normolipidemic mice (6, 26, 34, 54-58), and the influence on metabolic parameters in hyperlipidemic mice is unknown. Our results reveal that SCD1 deficiency reduces hepatic and plasma TG, inhibits diet-induced weight gain and insulin resistance, and reduces the hypertriglyceridemic effect of an LXR agonist in hyperlipidemic LDLR-deficient mice.  3.2 Methods 3.2.1 Animals and diet Mice carrying the Scd1ab-J (28) or Scd1ab-2J (29) null alleles were back-crossed to C57BL/6 for five generations then crossed to the B6.129S7-Ldlrtm1Her mutant strain (59). Mice carrying the Scd1ab-J allele were used in all experiments except those involving hepatocyte isolation in which mice carrying the Scd1ab-2J allele were used. Animals received a standard laboratory rodent chow diet (LabDiet 5010 Autoclavable Rodent Diet, PMI Nutrition International, Richmond, IN), or Western diet (TD.88137, Harlan Teklad, Madison, WI). For LXR agonist treatment, animals received T0901317 (60) (10 mg/kg body weight) daily by oral gavage for 3 days. The weight of the food contained in the food bin and any that had been spilled or buried in each cage (to the nearest 0.1g) was recorded every 2 to 3 days for 8 consecutive days and food intake for each mouse was averaged over the 8 days.  All studies were approved by the University of British Columbia  Animal Care Committee.  3.2.2 Adiposity measurements using magnetic resonance imaging and relaxometry For imaging, mice were anesthetized with isofluorane and imaged in a Bruker Biospec 70/30 7 Tesla Magnetic Resonance Imaging Scanner (Bruker Biospin, Ettlingen, Germany) with and without fat suppression. Images were acquired in the abdominal region in 1.5 mm transverse slices with a MSME T1-weighted pulse sequence, acquiring a field of view of 4 cm and a matrix size of 128 x128. The echo time was 12 ms and the repetition time was 300 ms. Magnetic resonance signal from the body of nonanesthetized mice was acquired with a quadrature 74  volume RF coil tuned to 300 MHz. Absolute fat and lean mass was calculated from the magnetic resonance data as described by Kunnecke et al. (61).  3.2.3 Fat pad measurements and histology Peri-uterine and peri-epididymal white adipose depots were dissected and weighed. For routine histology, similar areas from liver tissue from mice after 16h starvation were formalin-fixed, paraffin-embedded, sectioned at 4 μm, and stained with Oil Red O (counterstained with hematoxylin).  3.2.4 Lipid and lipoprotein analysis Fast protein liquid chromatography (FPLC) was preformed to separate the 3 major lipoprotein classes, VLDL, LDL, and HDL, in pooled plasma.  For hepatic  lipid analysis, liver tissue was homogenized in PBS and total lipids extracted using Folch solution (chloroform/methanol 2:1), dried under N2, and resuspended in 2% Triton X-100.  Unfractionated plasma, FPLC fractions, and tissue lipid extracts  were assayed for cholesterol and TG concentrations by enzymatic assays with the use of commercially available reagents. Plasma HDL cholesterol levels were determined  after  precipitation  of  apoB-containing  lipoproteins  with  phosphotungstic acid/Mg (Wako Diagnostics, Richmond, VA). Lipoproteins in the density < 1.21 g/mL fraction obtained by preparative ultracentrifugation were analyzed by SDS-PAGE on gradient gels (4-16%) for determination of apoB and apoE as described (62). Briefly, 10 μg of protein was added to each lane of the gel. Gels were stained with Coomassie blue and bands corresponding to apoB and  apoE were quantified by scanning using a densitometer. Unfractionated  plasma levels apoC-III were determined by immunonephelometry with the use of mouse-specific antibodies developed in rabbits. The distribution of lipids in plasma lipoproteins was assessed as described (63).  3.2.5 Hepatocyte isolation and radiolabeling with [3H]acetate Primary hepatocytes were isolated as described (64).  Briefly, mice fed a  Western diet for four weeks were anesthetized by intraperitoneal injection of Somnotol (22 µl/50 g body weight) and the livers were perfused with Hanks'-EGTA solution containing 1 mg/ml insulin followed by Hanks'-collagenase solution (100 units/ml) containing 1 mg/ml insulin. The hepatocytes were dispersed in Hanks'-  75  collagenase solution and washed three times in Dulbecco's modified Eagle's medium (DMEM), then suspended in medium containing 10% fetal bovine serum and plated on 60 mm collagen-coated dishes (1 x 10 6 cells/ml). Hepatocytes were incubated for up to 3 h with DMEM containing 25 µCi/ml [3H]acetate and then washed twice with DMEM. Lipids were extracted with chloroform: methanol (2:1, v/v) and then saponified by heating to 80°C in methanolic KOH. Non-saponifiable lipids were removed by extraction with diethyl ether.  The aqueous phase  containing released fatty acids was acidified and the fatty acids were extracted with hexane.  Incorporation of [3H]acetate into fatty acids was determined as  [3H]fatty acids per mg of total cell protein.  3.2.6 Physiological analysis Intraperitoneal glucose tolerance tests were performed on 12-hour fasted mice injected with 1.5 g/kg glucose. Blood samples were taken at 0, 15, 30, 60, and 90 minutes and blood glucose was measured with a glucometer (Lifescan, Milpitas, CA). Insulin tolerance tests were performed on 12-hour fasted mice injected with 0.75 U/kg human recombinant insulin (Novo Nordisk, Princeton, NJ). We measured plasma insulin by ELISA (Crystal Chem, Downers Grove, IL).  3.2.7 Real-time PCR and immunoblotting We extracted total RNA from liver using the TRIzol reagent according to manufacturer’s  instructions  (Invitrogen  Canada,  Burlington,  ON,  CA).  1  microgram of DNase-treated RNA was reverse-transcribed using Superscript II (Invitrogen Canada, Burlington, ON, Canada) to generate RNAse H-treated cDNA for real-time PCR using SYBR green PCR Master Mix (Applied Biosystems, Foster City, CA) in an ABI Prism 7700 Sequence Detection System. We used Gapdh as the invariant control. mRNA levels in control mice were arbitrarily set at 1. Western blotting was performed as previously described (65). Briefly, tissues were homogenized in low salt lysis buffer containing complete protease inhibitor (Roche Diagnostics, Laval, QC), and protein concentration was determined by the Lowry assay (66). Equivalent amounts of total protein were separated by SDSPAGE, transferred to polyvinylidene difluoride membranes, and probed with antiABCA1 (65) or anti-GAPDH antibodies.  76  3.2.8 Statistical analysis Data are presented as means plus or minus standard error of the mean. Initial analyses were performed by the unpaired two-tailed Student's t-test. If the data did not fit the constraints of this parametric test, data were analyzed with the Mann-Whitney test.  Data from body weight, [3H]acetate incorporation and  tolerance tests were analyzed by two-way analysis of variance (ANOVA; time, within subjects; genotype, between subjects), using repeated measures for body weight and tolerance tests, all followed by Bonferroni post-tests. Areas under the glucose curves (AUCglucose) were calculated by the trapezoid rule.  Statistical  analysis was performed GraphPad Prism software and with the open-source Rpackage (GraphPad, San Diego, CA; R Development Core Team, 2006 (67)). P < 0.05 was considered significant.  3.3 Results 3.3.1 SCD1 deficiency reduces weight gain and adiposity in Ldlr-/- mice An existing mouse strain with a spontaneous deletion in Scd1 (B6.Cg-Scd1ab-J) and an existing dyslipidemic mouse model (B6.129S7-Ldlrtm1Her)(59) were crossed to generate mice with deficiencies of both LDLR (Ldlr-/-) and SCD1 (Scd1-/-). Similar to previous descriptions of Scd1 null alleles in other genetic backgrounds, (30, 32, 33, 68, 69), SCD1-deficient mice exhibited progressive alopecia, a hunched posture, and a pronounced 'squinting' appearance of the eyes, but exhibited normal mobility and no other obvious adverse health effects. Mice at the age of 11-13 weeks were fed an atherogenic "Western" diet (70) for 12 weeks. Male Scd1-/-Ldlr-/- mice had a similar weight to the control Scd1+/ +  Ldlr-/- mice at the beginning of the diet study (28.4 g vs. 29.1 g, p = 0.51), but  they gained less weight after feeding a Western diet, (Figure 3.1a on page 86), despite tending to consume more food than controls (1.5g/day vs. 1.1g/day; p = 0.11, n = 4).  Female Scd1-/-Ldlr-/- mice also gained less weight than controls  after feeding a Western diet (Figure 3.1b).  After 12 weeks of a Western diet,  weights for male and female Scd1+/+Ldlr-/- mice were 44% and 54% higher than initial values, respectively, while neither male nor female Scd1-/-Ldlr-/- mice showed a significant increase in body weight. Both male and female Scd1-/-Ldlr-/77  mice had smaller peri-epididymal or peri-uterine fat pads than control Ldlr-/- mice (Figure 3.1c). To evaluate weight gain in terms of adiposity, fat mass and lean mass were determined  using  magnetic  resonance  relaxometry,  a  recently  validated  noninvasive method for the precise assessment of body composition (61) (Figure 3.2a-c on page 87).  Lean body mass was not different between SCD1-deficient  mice and controls (Figure 3.2b). However, SCD1-deficient mice had a significant 50% reduction in total fat mass compared to controls (males, p = 0.0006; females, p = 0.0043). Representative images in Figure 3.2c show a decrease in both visceral and subcutaneous lipid in SCD1-deficient mice.  3.3.2 SCD1 deficiency reduces hepatic steatosis in Ldlr-/- mice Nonadipose tissue also exhibited a marked decrease in lipid accumulation. Histological examination of the livers revealed protection from hepatic steatosis (Figure 3.3a, b on page 88), and hepatic TG levels were 5-fold higher in control Scd1+/+Ldlr-/- mice than in Scd1-/-Ldlr-/- mice (Figure 3.3c).  3.3.3 SCD1 deficiency reduces plasma lipids and improves lipoprotein profiles in Ldlr-/- mice To determine whether the reduced levels of tissue lipids in hyperlipidemic SCD1-deficient mice are reflected in plasma lipoprotein levels, fasting plasma lipid concentrations and lipoprotein profiles for Scd1-/-Ldlr-/Scd1  +/+  mice and control  -/-  Ldlr mice were evaluated (Figure 3.4 on page 89 and Table 3.1 on page  85). Total plasma TG is significantly reduced by approximately 51% in female SCD1-deficient mice (p = 0.021; Figure 3.4a).  Plasma TG also tended to be  reduced in male SCD1-deficient mice but this difference was not statistically significant (p = 0.23). Plasma total cholesterol (TC) was significantly reduced by approximately 26% in SCD1-deficient male mice (p = 0.023; Figure 3.4b), but this trend was not observed in females. HDL cholesterol was not significantly altered in SCD1-deficient mice, while non-HDL cholesterol paralleled the reduction observed in plasma TC (Figure 3.4c,d). FPLC analysis confirmed the decrease in VLDL-TG in SCD1-deficient mice with a small increase noted in VLDL cholesterol (Figure 3.4e-f).  78  3.3.4 SCD1 deficiency reduces plasma apolipoproteins in Ldlr-/- mice Reductions of plasma apoB (p = 0.0022), apoE (p = 0.0022), and apoC-III (p = 0.0005) in females (Figure 3.5a-c on page 90), consistent with a reduction in VLDL-TG, were evident.  Similar trends were observed in males, but these  reductions in plasma apolipoproteins were not significant (Figure 3.5).  3.3.5 SCD1 deficiency reduces fatty acid synthesis in Ldlr-/mice Decreases in hepatic and plasma lipid levels could result from decreased lipogenesis.  Therefore, to explore the mechanism by which SCD1 deficiency  decreases hepatic and plasma lipids in hyperlipidemic SCD1-deficient mice, we evaluated the incorporation of [3H]acetate into saponifiable lipids in primary hepatocytes isolated from Scd1-/-Ldlr-/- mice and control Scd1+/+Ldlr-/- mice (Figure 3.6a on page 91).  The incorporation of [3H]acetate into fatty acids in the  saponifiable lipid fraction over time was reduced in Scd1-/-Ldlr-/- hepatocytes relative to control Scd1+/+Ldlr-/- hepatocytes (p < 0.0001), and incorporation was significantly reduced by approximately 48 to 60% at all time points (p < 0.001). Acetyl-CoA carboxylase (ACC) and fatty acid synthase (FAS) are the two enzymes required for fatty acid synthesis (71, 72).  Thus, we hypothesized that  the reduced fatty acid synthesis in hepatocytes may result from reduced levels of transcripts encoding these enzymes, as well as reduced levels of the transcription factor SREBP-1c (73), an important regulator of these transcripts. We examined the hepatic expression of various lipid-sensitive mRNAs and observed significant reductions in the genes that encode ACC-1 (p = 0.0059) and FAS (p = 0.0020) in Scd1-/-Ldlr-/- mice relative to Scd1+/+Ldlr-/- mice (Figure 3.6b on page 91). However, SCD1 deficiency does not significantly alter the hepatic mRNA levels of genes that encode the transcription factors PPARα, LXRα, and SREBP-1c in LDLRdeficient mice challenged with a Western diet, although trends toward reduction are seen, particularly for PPARα (p = 0.060) and SREBP-1c (p = 0.082).  79  3.3.6 SCD1 deficiency reduces insulin resistance in Ldlr-/- mice The association between obesity, HTG, and diabetes is well documented (74, 75). To further investigate the effects of SCD1 on these parameters, the response to glucose challenge was assessed. Male gender is a predisposing diabetes susceptibility factor in most mouse strains (76), so we were not surprised to observe a clear sexual dimorphism in diabetes susceptibility of Scd1+/+Ldlr-/- mice. -/-  diet, male Scd1 Ldlr  -/-  evident in male Scd1  At 7 and 11 weeks on a Western  mice were protected from the impaired glucose tolerance +/+  Ldlr-/- mice (Figure 3.7a,b on page 92).  Female LDLR-  deficient mice were more successful than males at controlling their blood glucose when fed a Western diet, but by 11 weeks female Scd1-/-Ldlr-/- mice showed improved glucose tolerance relative to female Scd1+/+Ldlr-/- mice (p = 0.03; Figure 3.7c). Insulin sensitivity assays were performed on male mice mice fed a Western diet for 9 weeks and fasted overnight before intraperitoneal insulin injection (0.75 U/kg) and glucose was monitored over 90 min. Consistent with their lean phenotype, male Scd1-/-Ldlr-/- mice showed both an improved response 15min after insulin injection and reduced blood glucose (Figure 3.7d). Measurement of fasting glucose levels indicated that male Scd1-/-Ldlr-/- mice were protected from the hyperglycemia that had developed in Scd1+/+Ldlr-/- controls by 9 weeks on a Western diet (Figure 3.7e). Male Scd1-/-Ldlr-/- mice were also protected from the markedly elevated fasting plasma insulin that had developed in Scd1+/+Ldlr-/controls by 11 weeks on a Western diet (Figure 3.7f). These data indicate that the improved glucose tolerance that was observed in mice lacking SCD1 is attributable in part to increased insulin sensitivity.  3.3.7 SCD1 mediates the plasma lipid response to LXR agonist treatment in Ldlr-/- mice Treatment of mice with a synthetic LXR agonist, T0901317, has been shown to be antiatherogenic in hyperlipidemic LDLR-deficient mice (36, 37), but its therapeutic utility has been limited by the accompanying severe HTG (36, 37) and hepatic steatosis (60, 77). Recent studies have shown that the lipogenic effect of LXR agonists is mediated through SCD1 (26), but the role of SCD1 in regulating the severe LXR-induced HTG observed in hyperlipidemic LDLR-deficient mice has 80  not yet been determined. To determine whether SCD1 deficiency moderates the undesirable effects of LXR activation in hyperlipidemia, we fed female Scd1-/-Ldlr-/mice and Scd1+/+Ldlr-/- controls a Western diet for 12 days and treated with 10mg/ kg of T0901317 by oral gavage daily for the final 3 days. T0901317 treatment resulted in a 4.3-fold increase in plasma TG in LDLRdeficient mice (Figure 3.8a on page 93). However, plasma TG was reduced by 48% in T0901317-treated Scd1-/-Ldlr-/- mice relative to Scd1+/+Ldlr-/- controls, a similar relative reduction to that induced by SCD1 deficiency in hyperlipidemic mice prior to treatment with an LXR agonist. Plasma TC was ~ 10-30% lower at all time-points in SCD1-deficient mice compared with controls, primarily due to a reduction in non-HDL cholesterol (Figure 3.8b). Interestingly, plasma HDL-cholesterol was increased by 73% in T0901317treated Scd1-/-Ldlr-/- mice relative to Scd1+/+Ldlr-/- controls (Figure 3.8c).  To  explore the molecular mechanism by which SCD1 influences plasma lipids in T0901317-treated hyperlipidemic mice, we assessed hepatic expression levels of various genes (Figure 3.8d). We observed a 77% reduction in the level of FAS mRNA in SCD1-deficient mice (p = 0.0020).  A reduction of FAS indicates that  endogenous fatty acids are likely produced at a reduced rate and are less available for generation of triglycerides for secretion into the plasma.  Trends  toward decreases in transcripts of other lipogenic genes (acetyl-CoA synthetase, 78% reduction, p = 0.22; glycerol-3-phosphate acyltransferase, 51% reduction, p = 0.14) with SCD1 deficiency were also detected in mice fed the LXR agonist. Hepatic ABCA1 protein expression was increased by 45% in T0901317-treated Scd1-/-Ldlr-/- mice relative to Scd1+/+Ldlr-/- controls (Figure 3.8e, p = 0.002), which at least partly might explain the increased HDL levels (78, 79). However, we did not observe a significant alteration in the level of Abca1 mRNA (Figure 3.8d), suggesting that the increased ABCA1 protein level is not due to increased synthesis of ABCA1, but rather due to alterations in post-transcriptional regulation.  3.4 Discussion We have shown that an absence of SCD1 improves the metabolic phenotype of a mouse model of FH on a Western diet. Absence of the Scd1 gene product reduces hepatic and plasma TG, and strongly inhibits diet-induced weight gain in  81  LDLR-deficient mice. Absence of SCD1 also provides striking protection from dietinduced insulin resistance as measured by intraperitoneal glucose and insulin tolerance testing.  Finally, we have demonstrated that absence of SCD1 partially  reduces the undesirable hypertriglyceridemic effect of antiatherogenic LXR agonists in hyperlipidemic mice. Normolipidemic SCD1-deficient mice are known to be protected from insulin resistance and diet-induced obesity (34).  The role of SCD1 in resistance to  obesity has also been expanded to include the leptin-deficient model of obesity (35). We have now extended the findings and demonstrate that the absence of SCD1  provides  significant  protection  from  diet-induced  obesity  in  the  hyperlipidemic LDLR-deficient model. Liver TG are reduced by 40-65% in SCD1-deficient mice (6, 54) and TG synthesis is also reduced (35, 54, 80). The most profound impact of absence of SCD1 on the metabolic features of LDLR-deficient mice in this study was a 5-fold reduction in hepatic steatosis, a greater relative reduction than the reductions of ~65% observed previously in chow-fed SCD1-deficient genetic models of obesity (35) and lipodystrophy (58). Fatty liver is frequently observed in individuals with obesity, type 2 diabetes, and hyperlipidemia. Moreover, the degree of steatosis in non-alcoholic fatty liver disease is proportional to the degree of obesity (81), and insulin resistance is almost universally observed in non-alcoholic fatty liver disease (82, 83). Short term high-fat feeding in rodents, which leads to hepatic fat accumulation in the absence of increases in peripheral fat accumulation, has previously demonstrated a causal role for intracellular hepatic fat accumulation in the pathogenesis of hepatic insulin resistance (84). Furthermore, hepatic fat accumulation is often accompanied by a chronic, subacute state of inflammation, which can increase insulin resistance (85, 86). Thus the dramatic reduction in hepatic triglycerides that we observe in the absence of SCD1 may contribute in part to increasing insulin sensitivity.  However, overexpression of SCD1 (87) decreases insulin  signaling in muscle cells, suggesting that absence of SCD1 may also contribute to increased insulin sensitivity in skeletal muscle in addition to the liver. The role of SCD1 in regulating plasma TG has been evaluated in several studies and a reduction in plasma TG has not been consistently observed. Some studies have shown plasma TG reduced by over 50% (6, 55, 88), but two studies have shown no significant differences (56, 57). It is not known whether the 82  phenotypic differences could be attributed to the variations in age, sex, diet, fasting protocol, or genetic background of mice in the different studies. SCD1deficient mice have a markedly reduced rate of VLDL-TG production (35), and the effect on plasma TG levels may be more apparent in hyperlipidemic mice. Our results show that absence of SCD1 reduces plasma lipids (TC, TG) and improves lipoprotein profiles in LDLR-deficient mice. A potential mechanism for the reduction in hepatic and plasma lipids in SCD1deficient mice is reduced lipogenesis.  We found that SCD1 deficiency markedly  reduces fatty acid synthesis in hepatocytes and reduces hepatic mRNA levels of the two SREBP-1c regulated genes that encode enzymes required for long-chain fatty acid synthesis, ACC-1 and FAS.  These data are consistent with previous  studies that demonstrated reduced ACC-1 and FAS hepatic expression levels (34, 58, 89, 90) and reduced ACC activity (90) in SCD1-deficient chow-fed mice, and indicate that a reduction in hepatic de novo fatty acid synthesis is likely to be a major contributor to the decreased hepatic and plasma lipids we observe in hyperlipidemic SCD1-deficient mice. T0901317 is a synthetic LXR agonist which has been shown to be atheroprotective in hyperlipidemic mice (36, 37), an effect that is postulated to be mediated by stimulating cholesterol efflux from macrophages (36, 37, 77). However, LXR activation has been observed to lead to undesirable side effects, specifically HTG and hepatic steatosis (36, 37).  Our data demonstrate that  absence  plasma  of  SCD1  significantly  influences  the  lipid  response  to  antiatherogenic LXR agonist treatment, reducing non-HDL cholesterol and increasing beneficial HDL cholesterol. In addition, SCD1 deficiency is also able to partially improve LXR-induced HTG in the hyperlipidemic LDLR-deficient model. These data are consistent with data in a recent study of chow-fed mice that have shown that absence of SCD1 protects against hypertriglyceridemia and increases plasma HDL-cholesterol induced by a synthetic LXR agonist (26). Increased levels of SCD1 and its MUFA products inhibit cholesterol efflux mediated by ABCA1 (91-93) by a mechanism that may involve increased turnover rather than altered transcript levels (94).  In addition, SCD1 deficiency can  increase hepatic ABCA1 in SCD1-deficient mice fed a very-low fat diet (95). Our data now provide evidence that the increased plasma HDL cholesterol observed in SCD1-deficient LXR-treated  mice  relative  to  LXR-treated  controls  is  accompanied by increased levels of the ABCA1 protein but no significant 83  alteration in the level of Abca1 mRNA, consistent with a mechanism involving alterations in post-transcriptional regulation. In summary, our results establish the robust impact of SCD1 deficiency on the metabolic phenotype of the hyperlipidemic LDLR-deficient mouse  model,  including reduced hepatic and plasma TG, reduced diet-induced weight gain and insulin resistance, and a partially reduced hypertriglyceridemic response to an LXR agonist.  84  Table 3.1: Plasma lipid and apolipoprotein levels in Ldlr-/- mice lacking SCD1. Males  TG, mg/dL  Females  Scd1+/+Ldlr-/-  Scd1-/-Ldlr-/-  885 ± 673 (11)  497 ± 294 (8) 0.23  p  Scd1+/+Ldlr-/-  Scd1-/-Ldlr-/-  p  689 ± 414 (12)  350 ± 114 (9)  0.021  Cholesterol, mg/dL 2192 ± 635 (11) 1614 ± 307 (8) 0.023 1927 ± 611 (12) 1769 ± 269 (9) 0.86 HDL-C, mg/dL  58.5 ± 14.5 (11) 64.4 ± 8.3 (7) 0.34  41.7 ± 12.2 (12) 44.0 ± 10.9 (9) 0.66  Non-HDL-C, mg/dL 2133 ± 627 (11) 1555 ± 306 (8) 0.028 1885 ± 612 (12) 1725 ± 268 (9) 0.86 ApoB  1.00 ± 0.60 (6) 0.66 ± 0.15 (6) 0.18  1.35 ± 0.415 (6) 0.41 ± 0.15 (6) 0.0022  ApoE  1.00 ± 0.50 (6) 0.75 ± 0.18 (6) 0.24  1.29 ± 0.41 (6) 0.45 ± 0.14 (6) 0.0022  ApoC-III  1.00 ± 0.37 (11) 0.77 ± 0.26 (7) 0.17  0.97 ± 0.31 (12) 0.53 ± 0.15 (10) 0.0005  Apolipoprotein levels are expressed in relative units compared to to the levels of male Scd1+/+Ldlr-/- mice. Data represent mean ± SD. The number of animals in each subgroup is indicated in parentheses.  85  Figure 3.1: Total body and fat pad weights of Ldlr-/- mice lacking stearoyl-CoA desaturase (SCD1). Body weight was measured in (a) male (b) and female Scd1+/+Ldlr-/- and Scd1-/Ldlr-/- mice fed Western diet starting at 12 weeks of age (males, p < 0.0001; females, p = 0.01; repeated-measures analysis of variance (ANOVA)). n = 6-12 mice per group. c, Fat pad weights are shown. *p < 0.05. ***p < 0.001.  86  Figure 3.2: Adiposity in Ldlr-/- mice lacking SCD1. a, Total body fat mass, and b, total body lean mass from mice fed a Western diet. n = 3-10 mice per group. c, Transverse abdominal cross-sections of an SCD1deficient mouse and a control mouse obtained by magnetic resonance imaging. Slices (1.5 mm thickness) at the kidneys were identified in sagittal images from each mouse. Though the Scd1-/-Ldlr-/- mouse appears larger relative to the Scd1+/ + Ldlr-/- mouse, the absolute lean mass calculated from magnetic resonance data was not greater for this Scd1-/-Ldlr-/- mouse, and thus the apparent increase in cross-sectional area is attributed to an artifact of differences in restraint between the two mice. Fatty tissues are show as bright areas. Spinal muscle, kidneys and subcutaneous (SC) and visceral (V) fat are indicated.  87  Figure 3.3: Hepatic lipids in Ldlr-/- mice lacking SCD1. a and b, Livers of female Scd1+/+Ldlr-/- and Scd1-/-Ldlr-/- mice fed a Western diet stained with Oil Red O. C, Liver triglyceride (TG), free cholesterol (FC), and cholesteryl ester (CE) content of Scd1+/+Ldlr-/- and Scd1-/-Ldlr-/- mice fed a Western diet. n = 8 mice per group.  88  Figure 3.4: Plasma lipids and lipoprotein profiles in Ldlr-/- mice lacking SCD1. a, Plasma TG, b, total cholesterol (TC) content, c, HDL-cholesterol, and d, nonHDL-cholesterol content of Scd1+/+Ldlr-/- and Scd1-/-Ldlr-/- mice fed Western diet. n = 8-12 mice per group. e to h, Fast protein liquid chromatography lipoprotein profiles of pooled plasma samples from Scd1+/+Ldlr-/- and Scd1-/-Ldlr-/- mice fed Western diet. TG (e and f) levels were determined for each fraction from male (e) and female (f) mice. The lipoprotein peaks for VLDL, LDL, and HDL are indicated. 89  Figure 3.5: Plasma apolipoproteins in Ldlr-/- mice lacking SCD1. ApoB (a), apoE (b), and apoC-III (c) were measured in male and female Scd1+/ + Ldlr-/- and Scd1-/-Ldlr-/- mice fed Western diet. Apolipoprotein levels are expressed in relative units (RU) compared to to the levels of male Scd1+/+Ldlr-/- mice. n = 612 mice per group.  90  Figure 3.6: Fatty acid synthesis and hepatic gene expression in Ldlr-/- mice lacking SCD1. a, Assessment of fatty acid synthesis in hepatocytes from Scd1+/+Ldlr-/- and Scd1-/Ldlr-/- mice fed a Western diet. Primary hepatocytes were prepared from one mouse of each genotype and incubated for up to 3 hours with [3H]acetate, after which lipids were saponified and [3H]fatty acid content was measured (p < 0.0001; two-way ANOVA). Each value is the average of four independent dishes of hepatocytes and error bars are included unless obscured by symbols. ***p < 0.001. b, Relative amount of various mRNAs in livers of Scd1+/+Ldlr-/- and Scd1-/Ldlr-/- mice fed a Western diet. Each value represents the amount of mRNA relative to that in Scd1+/+Ldlr-/- mice (arbitrarily set at 1 for each transcript in these mice). n = 6-17 mice per group.  91  Figure 3.7: Glucose tolerance and insulin resistance in Ldlr-/- mice lacking SCD1. a to c, Intraperitoneal (IP) glucose tolerance tests (1.5 g/kg) were performed on mice fasted overnight after being fed a Western diet for 7 weeks (a, males; p < 0.0001, repeated measures ANOVA) or 11 weeks (b, males, p = 0.014; c, females, p = 0.03, repeated measures ANOVA). Insets show areas under the glucose curves (AUCglucose; mg/dL x 90 min). d, IP insulin tolerance tests (0.75 U/mL) were performed on male mice fasted overnight after being fed a Western diet for 9 weeks (p = 0.0085, repeated measures ANOVA). e, Glucose was measured in blood and f, insulin was measured in plasma obtained from the saphenous vein of mice fasted overnight after being fed a Western diet for 9 weeks and 11 weeks, respectively. n = 3-10 mice per group. Scd1+/+Ldlr-/- mice are indicated with a solid line, and Scd1-/-Ldlr-/- mice are indicated with a broken line. *p < 0.05. **p < 0.01. ***p < 0.001.  92  Figure 3.8: Plasma lipid response to LXR agonist treatment in Ldlr-/- mice lacking SCD1. a, Plasma TG and b, TC content of Scd1+/+Ldlr-/- and Scd1-/-Ldlr-/- mice at the age of 10-12 months at baseline, and after being fed a Western diet (WTD) for 9 days, and a Western diet plus 10mg/kg T0901317 for 3 additional days. c, Plasma HDL cholesterol at baseline and after T0901317 feeding. n = 7-8 mice per group. d, Relative amount of mRNA encoding acetyl-CoA synthetase (ACS), fatty acid synthase (FAS), glycerol-3-phosphate acyltransferase (GPAT) and ABCA1 in liver after T0901317 feeding . 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J Lipid Res 2006;47:2668-80.  100  CHAPTER 4 Despite antiatherogenic metabolic characteristics, SCD1deficient mice have increased inflammation and atherosclerosis1 4.1 Introduction Stearoyl-CoA desaturase (SCD) is the rate-limiting enzyme in the synthesis of monounsaturated fatty acids. It creates a cis-double bond in the ∆-9 position of palmitic (16:0) and stearic acid (18:0), thereby converting them to palmitoleic (16:1n7) and oleic acid (18:1n9) (1). Oleic acid is the major fatty acid found in triglycerides (TG) and cholesteryl esters (CE) (2), likely due to its status as the preferred fatty acid substrate of acyl-CoA:cholesterol acyltransferase (ACAT) (3), and the close proximity of SCD to diacylglycerol acyltransferase-2 in the endoplasmic reticulum (ER) (4). SCD1-deficient mice are protected from insulin resistance and diet-induced obesity (5) and have a markedly reduced rate of VLDL-TG production (6). We have recently shown (7) that SCD1 deficiency improves the metabolic phenotype of a hyperlipidemic LDLR-deficient mouse model of familial hypercholesterolemia (FH)(8). On a Western diet, LDLR-deficient mice develop diet-induced diabetes and obesity and develop atherosclerosis over 2 to 3 months (8). Absence of SCD1 reduces hepatic steatosis and plasma TG (by ~50%) and provides striking protection from diet-induced weight gain and insulin resistance in LDLR-deficient mice (7). A major unanswered question is whether the amelioration of these features in SCD1-deficient mice will lead to reduced susceptibility to atherosclerosis. In this study, we show that despite these antiatherogenic metabolic characteristics,  SCD1  deficiency  surprisingly  increases  lesion  size  in  hyperlipidemic LDLR-deficient mice and that this acceleration in atherosclerosis is  1A version of this chapter has been published. MacDonald MLE, van Eck M, Hildebrand RB,  Wong BWC, Bissada N, Ruddle P, Kontush A, Hussein H, Pouladi MA, Chapman MJ, Fievet C, van Berkel TJC, Staels B, McManus BM, Hayden MR (2009) Despite antiatherogenic metabolic characteristics, SCD1-deficient mice have increased inflammation and atherosclerosis. Arterioscler Thromb Vasc Biol. 29, 341-347.  101  likely to result from chronic inflammation primarily of the skin, which then leads to changes in markers of inflammation in plasma and proinflammatory changes in HDL.  4.2 Methods An extended Methods section is available in Appendix C on page 166. Mice carrying the Scd1ab-J (9) or Scd1ab-2J (10) null alleles were back-crossed to C57BL/6 for five generations to produce N5 incipient congenic mice and then crossed to the B6.129S7-Ldlrtm1Her mutant strain (11). The Scd1+/+Ldlr-/- control groups consisted of both littermates of Scd1-/-Ldlr-/- mice and additional age- and sexmatched Scd1+/+Ldlr-/- mice that were not littermates (~63% of all animals studied). Mice deficient in SCD1 with the Scd1ab-J allele were used in all experiments except those involving analysis of atherosclerotic lesions and paraoxonase-1 (PON1) activity, in which mice carrying a separately derived SCD1 deletion (the Scd1ab-2J allele) were also studied. Sections of the aortic root were stained as described in Singaraga et al (12).  4.3 Results 4.3.1 SCD1 deficiency increases atherosclerosis in Ldlr-/- mice Mice with a spontaneous deletion in Scd1 (B6.ABJ/Le-Scd1ab-J) were crossed with an existing dyslipidemic mouse model (B6.129S7-Ldlrtm1Her)(11) to generate mice with combined deficiencies of both LDLR (Ldlr-/-) and SCD1 (Scd1-/-). After 12 weeks of an atherogenic "Western" diet (13), weights for male and female Scd1+/ +  Ldlr-/- mice were 44% and 54% higher than initial values, respectively, whereas  neither male nor female Scd1-/-Ldlr-/- mice showed a significant increase in body weight, as described elsewhere (7). Total plasma TG was reduced by 44% and 51%, and non-HDL cholesterol was reduced by 8% and 27% in male and female Scd1-/-Ldlr-/- mice, respectively, relative to Scd1+/+Ldlr-/- controls. HDL cholesterol levels were unchanged by SCD1 deficiency. Absence of SCD1 also increased insulin sensitivity as measured by intraperitoneal glucose and insulin tolerance testing (7). Atherosclerotic lesion size was evaluated in multiple sections of the aortic root in this same cohort of Scd1-/-Ldlr-/- mice (males, n=6; females, n=10) and Scd1+/ 102  +  Ldlr-/- control mice (males, n=11; females, n=11) (Figure 4.1a and b on page  110). Unexpectedly, both male and female SCD1-deficient mice have significantly increased lesion size relative to controls. Lesion area was increased by 74% in males (p = 0.0002) and by 41% in females (p = 0.0004). In view of these observations in mice with the Scd1ab-J allele, we wished to examine whether these findings could be replicated in another cohort of mice carrying a different spontaneous null allele of Scd1 (B6.D1-Scd1ab-2J) (10). These mice were crossed with the same LDLR-deficient model and housed at a different specific pathogen-free barrier animal facility. Again, lesion area at the aortic root was increased in SCD1-deficient mice (129% increase in males; p < 0.0001; 70% increase in females; p < 0.0001; Figure 4.1b on page 110 and Appendix Figure D.1 on page 174), thus supporting our initial findings. The effect remained significant when all mice that were not littermates were excluded from the analysis (data not shown). Aortic root sections from the first cohort of mice were stained with Movat's pentachrome and hematoxylin and eosin (H&E) for histological examination. Extracellular matrix thickening and acellular areas containing cholesterol crystals were apparent in the deeper portion of the lesions from Scd1+/+Ldlr-/- mice (Figure 4.2a,b on page 111 and Appendix Figure D.2a on page 175). These findings were increased in the more advanced lesions of the Scd1-/-Ldlr-/- mice, with many extracellular cholesterol clefts in the large necrotic core underlying foam cell–rich regions. Staining for smooth muscle actin was evident in the media and fibrous caps of advanced lesions of both Scd1+/+Ldlr-/- and Scd1-/-Ldlr-/- mice (Appendix Figure D.2b). The increased lesion size in Scd1-/-Ldlr-/- mice fed the Western diet for 12 weeks was characterized by greater absolute areas of macrophage infiltration in these animals versus Scd1+/+Ldlr-/- controls. This macrophage infiltration was evident in both the large complex atheromatous lesions in the left coronary sinuses, as well as the smaller lesions of the right coronary and noncoronary sinuses. The majority of cells in early plaques were positive for monocyte/macrophage staining in both Scd1+/+Ldlr-/- and Scd1-/-Ldlr-/- mice (Appendix Figure D.2c). Semi-quantitative morphologic examination of sections stained with Movat's pentachrome and H&E was used to assign lesion severity scores on a 0 to 5+ scale based on the following parameters: foam cell characteristics, cholesterol clefts, presence of necrotic core, degree and composition of fibrous cap, 103  infiltration into the media, extracellular matrix deposition, calcification and plaque cellular characteristics. When examined in a blinded fashion, the aortic roots of Scd1-/-Ldlr-/- mice earned significantly higher lesion severity scores than Scd1+/+Ldlr-/- controls (p = 0.001; Figure 4.2c on page 111).  4.3.2 SCD1 deficiency promotes inflammation in Ldlr-/- mice Prior dermatological and immunological studies have indicated that SCD1deficient mice have skin that is rich in macrophages and mast cells (14, 15), indicative of chronic dermal inflammation. Icam1 mRNA was increased more than 2-fold in the skin of Scd1-/-Ldlr-/- mice relative to control Scd1+/+Ldlr-/- mice (males, p = 0.017; females, p = 0.093 Figure 4.3a on page 112) and skin ICAM-1 protein was increased more than 2-fold in Scd1–/–Ldlr–/– mice (males, p = 0.0091; females, p = 0.0022; Figure 4.3b). Scd1–/–Ldlr–/– mice have obvious skin abnormalities, including a hyperplastic epidermis and stratum corneum (Appendix Figure D.3a on page 176). Severe spontaneous  ulcerative  dermatitis  (Appendix  Figure  D.3b,c)  necessitated  euthanasia of 14-16% of SCD1-deficient mice by the end of the 12 weeks of feeding (Figure 4.3c). Diffuse inflammatory infiltration that included mast cells (Appendix Figure D.3c), exudation of inflammatory cells and fibrin onto the surface of the skin, and proliferation of fibrous tissue and granulation in the dermis was evident (Appendix Figure D.3b,c). We also observed mild to moderate lymphadenopathy, particularly protruding cervical and brachial lymph nodes, in mice with severe dermatitis. No Scd1+/+Ldlr-/- mice developed any skin lesions by the end of the study. These findings prompted us to examine whether SCD1-deficient mice also have  markers  of  systemic  inflammation  that  may  be  contributing  to  atherosclerosis. Interleukin (IL)-6 is increased by 67% (p = 0.043; Figure 4.4a on page 113), and soluble ICAM-1, an adhesion molecule that is elevated in serum of patients with the inflammatory skin disorders psoriasis (16) and atopic eczema (17) was also increased in Scd1-/-Ldlr-/- mice (p = 0.0035; Figure 4.4b). Two additional pro-inflammatory cytokines known to be elevated in psoriatric skin lesions, IL-1β (18) and IL-12p70 (19), were detected in plasma from Scd1-/-Ldlr-/mice but not Scd1+/+Ldlr-/- controls (Figure 4.4c,d). Circulating levels of MCP-1 were decreased 2-fold in Scd1-/-Ldlr-/- mice (p = 0.017; Figure 4.4e). White adipose tissue is the major source of MCP-1 in obese 104  mice (20), and the decreased levels of MCP-1 may be attributed to the significant decrease in white adipose tissue seen in Scd1-/-Ldlr-/- mice (7). The levels of RANTES (CCL5) were not significantly different (Figure 4.4f). In the absence of a pro-inflammatory dietary stimulus, several inflammatory indicators in plasma were below the limit of detection in both Scd1-/-Ldlr-/- and Scd1+/+Ldlr-/- mice. Weak trends toward increased inflammatory indicators were seen in Scd1-/-Ldlr-/- mice relative to Scd1+/+Ldlr-/- controls, but these increases were not significant (Appendix Figure D.4 on page 177).  4.3.3 SCD1 deficiency alters HDL-associated proteins in Ldlr-/mice Inflammation has been shown to have a proatherogenic effect on the composition of HDL particles, such that they become depleted in specific proteins, such as apoA-I, apoA-II, and PON1, while enriched in serum amyloid A (SAA)(21). Indeed, in SCD1-deficient mice, plasma SAA is dramatically increased (females, 52-fold increase, p = 0.0087; males, 2.9-fold increase, p = 0.0043; Figure 4.5b on page 114), while plasma apoA-I (females, ~37% reduction, p = 0.0017; males, ~16% reduction, p = 0.44; Figure 4.5c) and apoA-II are decreased (~40% reduction; females, p = 0.0007; males, p = 0.0003; Figure 4.5d). The changes in plasma SAA and apoA-II were paralleled by significant changes in mRNA encoding these genes (Saa1, p = 0.017; Saa2, p = 0.017; Apoa2, p = 0.039 Figure 4.5a). Furthermore, hepatic mRNA levels of Pon1, the gene that encodes PON1, an enzyme that contributes to the antioxidant properties of HDL (22), are decreased by nearly 75% (p < 0.001; Figure 4.5a) while mRNA levels of Clu, the gene that encodes apoJ/clusterin, an acute phase HDL-associated protein (23), are increased by more than 2-fold (p = 0.0001; Figure 4.5a). No changes were observed in mRNA levels of Lcat (Figure 4.5a). SCD1-deficient mice also had a significantly lower serum PON1 activity (p = 0.0028; Figure 4.5e). These data indicate that SCD1 deficiency has a proatherogenic effect on HDL protein composition that may be attributed to chronic inflammation.  4.3.4 SCD1 deficiency does not alter macrophage function The atherogenic effect of SCD1 deficiency could also result from a direct effect of SCD1 deficiency on macrophage function. If the increased macrophage  105  infiltration is due to a direct effect of SCD1 deficiency in macrophages, we would expect an increased inflammatory response in SCD1-deficient macrophages. We therefore evaluated the effect of SCD1 deficiency on the inflammatory response of thioglycollate-elicited peritoneal exudate cells (Figure 4.6a on page 115). Inflammatory gene expression was induced by lipopolysaccharide (LPS), an agonist of toll-like receptor 4 signalling, and mRNA levels of several LPS-induced inflammatory proteins were assessed. No significant differences were observed in genes encoding IL-6, TNF-α, IL-1β, IL-12p35, iNOS, IP-10, GARG-16 or COX2, suggesting that the increased atherosclerosis observed with SCD1 deficiency is not due to an altered macrophage inflammatory response.  4.3.5 Macrophage SCD1 deficiency does not alter atherosclerosis in Ldlr-/- mice Another way to examine whether the atherogenic effect of SCD1 deficiency results from a direct effect on SCD1 deficiency in macrophages is to evaluate the effect of SCD1 deficiency in bone marrow-derived cells on atherosclerosis in vivo. Bone marrow from Scd1-/- and Scd1+/+ mice was transplanted into LDLR-deficient mice. At 6 weeks after bone marrow transplantation, the diet was switched from regular chow diet to Western diet. After 6 weeks on the Western diet, no skin lesions were observed in the transplanted mice, nor did SCD1 deficiency in bone marrow-derived cells have an effect on serum lipids (Appendix Table A.1 on page 160). Most importantly, no effect was observed between the two transplanted groups on atherosclerotic lesion size (0.207±0.018 mm 2 in Scd1+/+→Ldlr-/- mice versus 0.189±0.029 mm2 in Scd1-/-→Ldlr-/- mice; Figure 4.6b,c), indicating that loss of macrophage SCD1 does not directly play a significant role in atherogenesis in these mice. Overexpression of SCD1 has been reported to result in decreased cholesterol efflux in HEK293 and CHO cells (24). SCD1 deficiency had no effect on cholesterol efflux to apoA-I or HDL under our experimental conditions (Figure 4.6d), further supporting the fact that SCD1 deficiency is not associated with altered macrophage function.  106  4.4 Discussion Despite an antiatherogenic lipid and metabolic profile, absence of SCD1 promotes inflammation and atherosclerosis in a mouse model of FH on a Western diet. Absence of SCD1 also increases plasma IL-6, IL-1β, IL-12p70 and sICAM-1 levels and has a proinflammatory effect on the components of HDL particles, increasing SAA and apoJ/clusterin and reducing apoA-I, apoA-II, and PON1. Specific deficiency of SCD1 in bone-marrow derived cells does not influence atherosclerotic lesion size. We have recently shown that SCD1-deficient mice have relatively reduced plasma triglycerides and are protected from obesity and insulin resistance (7), phenotypic components of the metabolic syndrome that have been linked to increased susceptibility to atherosclerosis (25, 26). These surprising data suggested that SCD1-deficient mice must have some proatherogenic stimulus that overcomes the antiatherogenic metabolic characteristics expected to reduce lipid accumulation in the aorta. Chronic inflammation has been reported in the skin of chow-fed SCD1-deficient mice, indicated by increased mRNA encoding ICAM-1 (15) and increased infiltration of macrophages and mast cells but only rare lymphocytes or neutrophils in the dermis (14). In addition, subcutanous cyclosporine A can inhibit ICAM-1 expression and reduce mast cell numbers in the skin, restoring the wild-type skin phenotype (15). Histopathological studies in these mice have demonstrated that the chronic inflammatory reaction is a foreign body response, with extreme sebaceous gland hypoplasia in SCD1deficient animals resulting in hair fiber perforation of the follicle base and a foreign body response to fragments of hair fiber in the dermis (10). Inflammation is recognized to play a major role in all stages of atherogenesis (27),  and  plasma  markers  of  systemic  inflammation  are  predictive  for  cardiovascular events in humans (28, 29). Indeed, standard preventive drug therapies such as aspirin and statins are known to have antiinflammatory properties and have been shown to be most beneficial in individuals with elevated inflammatory markers at baseline, even in those with relatively low serum cholesterol levels (30). In the LDLR-deficient mouse model, used by several groups to study the link between chronic inflammation and atherosclerosis (31, 32), plasma markers of systemic inflammation increase in response to dietary cholesterol, and these 107  markers are associated with increased lesion area independent of plasma lipoprotein levels (33). Our observations of increased plasma IL-6, a marker of systemic inflammation that is associated with atherosclerosis (28), sICAM-1, an adhesion molecule that is elevated in serum of patients with inflammatory skin disorders (16, 17), and IL-1β and IL-12p70, pro-inflammatory cytokines known to be elevated in psoriatric lesional skin (18, 19), suggest that chronic inflammation of the skin may be contributing to the proatherogenic profile of SCD1 deficient mice. During an inflammatory response, HDL particles are known to become depleted in apoA-I, apoA-II, and PON1 and enriched in SAA, a liver-derived protein increased by Western diets and correlated with lesion area in LDLR-deficient mice (21, 33). Our results show that absence of SCD1 has a proatherogenic effect on HDL composition. Atherosclerotic lesion size and macrophage cholesterol efflux are not altered in LDLR-deficient mice transplanted with bone marrow from SCD1-deficient mice. These observations, in addition to the lack of altered LPS-induced inflammatory response in SCD1-deficient peritoneal macrophages, suggest that macrophage SCD1 does not play a significant role in atherogenesis in this model. However, it should be noted that these results are from early lesions only and a longer term study is needed of SCD1 in lesion macrophages. The SCD1-deficient mouse model affords a unique opportunity to compare and contrast directly the effects of an antiatherogenic metabolic profile with proinflammatory pathways. In this instance, proinflammatory pathways overcome the favourable metabolic profile. However, significant unanswered questions remain regarding possible atherogenic effects of circulating lipoproteins or tissue lipids with increased saturated fatty acids (SFA) or decreased monounsaturated fatty acids (MUFA), as shown in SCD1-deficient mice (2, 34). The relevance of these findings to the development of SCD inhibitors for treatment of the metabolic syndrome in humans is unclear. Observational studies in humans have shown an association between increased indices of SCD activity and components of the metabolic syndrome (35-37), inflammatory markers (38), and potentially coronary heart disease (39), suggesting that the atherogenic inflammation observed in this mouse model of SCD1 deficiency may not extend to humans with reduced SCD1 activity. The findings in this study represent the effects of long-term complete SCD1 deficiency in all tissues in mice. Antisense oligonucleotides (ASO) may also be expected to result in near complete 108  deficiency of SCD1 expression in some extra-hepatic tissues (40), which could lead to atherogenic inflammation in rodent models similar to that observed here. By contrast, pharmaceutical compounds are generally not used at levels that would cause complete inhibition of the target enzyme through a 24-hour cycle, and would not be distributed throughout all tissues in the body. While a manuscript related to this work was in review, two other research groups reported on the relationship between atherosclerosis and SCD1 deficiency mediated by ASO. Both groups treated mice with identical SCD1-targeted ASO, but the experiments yielded different results: increased atherosclerosis in the Ldlr-/-Apob100/100  model  (41)  and  reduced  atherosclerosis  in  the  chronic  intermittent hypoxia (CIH) model (42). One clue to the discrepancy could be model-specific  effects  on  HDL-cholesterol  levels,  as  SCD1  deficiency  is  accompanied by a ~50% reduction of HDL-cholesterol levels in the dyslipidemic Ldlr-/-Apob100/100 model (41), a change in HDL expected to be associated with increased atherosclerosis (43). However, ASO-mediated SCD1 deficiency in the CIH model is accompanied by an atheroprotective ~20% increase in HDLcholesterol levels (42). Our results indicate that in the absence of changes in HDL-cholesterol levels, complete and chronic SCD1 deficiency is likely to exacerbate atherosclerosis. These studies provide strong support for the role of chronic inflammation in promoting atherosclerosis, even in the presence of antiatherogenic biochemical and metabolic characteristics.  109  Figure 4.1: Lesion area in Ldlr-/- mice lacking SCD1. a, Lesions in aortic roots of Scd1+/+Ldlr-/- (left) and Scd1-/-Ldlr-/- (right) mice carrying the Scd1ab-J allele were stained with Oil Red O to detect accumulation of lipids and photographed. b, Quantitation of atherosclerotic lesion area in the aortic root. n = 6-11 mice per group.  110  Figure 4.2: Lesion morphology Ldlr-/- mice lacking SCD1. a and b, Necrotic cores (absence of purple/black nuclei, NC) and extracellular cholesterol clefts (needle-shaped lucencies; C) were observed with Movat pentachrome staining. The absence of yellow staining indicates the lack of significant collagen deposition. c, Semi-quantitative assessment of lesion severity. n = 16-22 mice per group.  111  Figure 4.3: Skin of Ldlr-/- mice lacking SCD1. Levels of ICAM-1 mRNA (a) and protein (b). n = 5-6 mice per group. c, Dermatitis in Scd1-/-Ldlr-/- mice but not Scd1+/+Ldlr-/- mice (Scd1ab-J , p = 0.016; Scd1ab-2J, p = 0.023). n = 28-35 mice per group.  112  Figure 4.4: Inflammation in Ldlr-/- mice lacking SCD1. Plasma IL-6 (a), sICAM-1 (b), IL-1β (c), IL-12p70 (d), MCP-1 (e), and RANTES (f) levels were determined in mice fed a Western diet. n = 8-12 mice per group.  113  Figure 4.5: HDL phenotype in Ldlr-/- mice lacking SCD1. a, Relative amount of liver mRNAs. n = 12 mice per group. Plasma SAA (b), apoA-I (c), and apoA-II (d). Relative units (RU). n = 6-12 mice per group. e, Serum PON1 activity. n = 3 mice per group.  114  Figure 4.6: Macrophages from mice lacking SCD1. a, LPS-induced mRNAs in thioglycollate-elicted peritoneal macrophages. Lesion area (b) and representative aortic root sections (c) of Ldlr-/- mice reconstituted with Scd1+/+ (left) and Scd1-/- (right) bone marrow and fed a Western diet for 6 weeks. n = 12 mice per group. d, Cholesterol efflux assays from bone-marrow macrophages from Scd1+/+ and Scd1-/- transplanted mice. n = 4 mice per group.  115  4.5 References 1. Miyazaki M, Ntambi JM. Role of stearoyl-coenzyme A desaturase in lipid metabolism. Prostaglandins Leukot Essent Fatty Acids 2003;68:113-21. 2. Miyazaki M, Kim YC, Gray-Keller MP, Attie AD, Ntambi JM. The biosynthesis of hepatic cholesterol esters and triglycerides is impaired in mice with a disruption of the gene for stearoyl-CoA desaturase 1. J Biol Chem 2000;275:30132-8. 3. Landau JM, Sekowski A, Hamm MW. Dietary cholesterol and the activity of stearoyl CoA desaturase in rats: evidence for an indirect regulatory effect. Biochim Biophys Acta 1997;1345:349-57. 4. Man WC, Miyazaki M, Chu K, Ntambi J. 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Circulation 1999;100:1816-22.  118  CHAPTER 5 Absence of stearoyl-CoA desaturase-1 does not promote DSSinduced acute colitis1 5.1 Introduction In chronic inflammatory diseases, including atherosclerosis, inflammatory skin disorders (psoriasis and eczema) and inflammatory bowel disease (Crohn disease and ulcerative colitis), cytokines recruit leukocytes to the site of the lesions, thereby amplifying the inflammatory state and perpetuating the tissue damage. Genetic  and  environmental  factors,  specific  cytokine  involvement,  and  inflammatory infiltrate differ with the tissue involved, but the inflammatory processes are common to all chronic inflammatory diseases (1). Stearoyl-CoA desaturase (SCD) is a lipogenic enzyme that has been recently implicated in inflammatory disease. SCD activity creates a cis-double bond in the ∆-9 position of palmitic (16:0) and stearic acid (18:0), converting them to palmitoleic (16:1n7) and oleic acid (18:1n9), respectively (2).  However, the  mechanism whereby the specific lipids altered by SCD activity contribute to inflammatory processes in vivo remains unknown. Similar to other lipogenic genes, the expression of mammalian SCD genes is highly regulated (3, 4).  Human SCD is expressed most abundantly in adipose  tissue (5), with lower expression in liver and brain (6). Of the SCD co-orthologs in mice, Scd1 is the most relevant murine ortholog for studying the metabolic and inflammatory functions of SCD activity in humans, due to its expression in adipose tissue and liver (7, 8). SCD1-deficient mice exhibit extreme sebaceous gland hypoplasia, which leads to hair fibre perforation of the follicle base and a foreign body response to fragments of hair fibre in the dermis (9). In addition to this chronic inflammation of the skin (10, 11), absence of SCD1 increases markers of systemic inflammation and susceptibility to atherosclerosis (12).  Absence of SCD1 has also been  1A version of this chapter has been published. MacDonald, M.L.E., Bisssada, N., Vallance,  Bruce A., and Hayden, M.R. (2009). Absence of stearoyl-CoA desaturase-1 does not promote DSS-induced acute colitis. Biochim Biophys Acta. Epub 2009 Aug 17. doi:10.1016/j.bbalip.2009.08.001  119  reported to exacerbate acute colitis in mouse model for human inflammatory bowel disease, i.e. dextran sulfate sodium (DSS)-induced colitis (13).  These  findings may have implications for the ongoing development of SCD inhibitors for treatment of the metabolic syndrome in humans, and invite further exploration of pro-inflammatory mechanisms in SCD1-deficient mice. DSS is a sulfated heparin-like polysaccharide; depending on the time course of oral administration in drinking water, it can induce both acute and chronic colitis, inhibiting epithelial cell proliferation and promoting apoptosis, which leads to epithelial  injury,  crypt  loss  and  extensive  ulceration  and  inflammation,  predominantly localized to the distal colon (14-17). The extent of colon damage increases with the amount of DSS administered (18, 19). SCD1-deficient mice with DBA/1LacJ and B6129S1F2 genetic backgrounds have been observed to consume increased amounts (~ 62% to 145%) of water, possibly due to increased trans-epidermal water loss (9, 20). This observation raised the question of whether the consequent increase in total DSS dose imbibed by the SCD1-deficient mice might be responsible for the accelerated colitis in these mice reported by Chen et al (13). One group has concluded that minor variations in fluid consumption do not affect the severity of DSS-induced colitis, based on Pearson's correlation analyses between clinical or histological results and total DSS intake (19).  However, a more recent study reported  significant correlations between histology scores or neutrophil recruitment and total DSS intake, supporting a conclusion that severity of DSS-induced colitis is dependent on total DSS intake in mice (18).  Furthermore, these authors also  found that a minimum DSS intake of 30mg/g body weight over seven days was required to reliably induce colitis in mice (18). In the only previous study of the effect of SCD1 deficiency on inflammatory bowel disease, Chen et al treated both SCD1-deficient mice and wild-type controls with 2% DSS for seven days (13), a concentration that might be expected to deliver a total DSS intake of less than 30 mg/g to many of the mice in their study, particularly the wild-type controls that are expected to drink lower volumes of water (9, 20).  Under these experimental conditions, Chen et al  observed increased weight loss, shorter colon length, and more severe diarrhea and rectal bleeding with SCD1 deficiency (13). In contrast, our results reveal that SCD1 deficiency does not accelerate inflammation in the DSS-induced model of acute colitis when total DSS intake is increased and DSS dosing is adjusted to 120  account for genotypic differences in fluid consumption. Therefore, these findings suggest that clinical use of SCD inhibitors should not impact on susceptibility to bowel inflammation.  5.2 Methods 5.2.1 Animals and diet Mice carrying the Scd1ab-2J (9) null allele were back-crossed to C57BL/6 for 10 generations to produce congenic mice (21). Animals were allowed free access to a standard laboratory rodent chow diet (Prolab Isopro RMH 3000, PMI Nutrition International, Richmond, IN) with a maximum of five mice per cage. Animals were maintained in a specific pathogen-free barrier facility that is free of common murine viruses and Mycoplasma pulmonis on serology testing, free of significant bacterial pathogens on culture of trachea or intestine, and free of ectoparasites and endoparasites.  Sentinel mice were also confirmed by polymerase chain  reaction amplification to be free of common Helicobacter species in fecal samples. All studies were approved by the University of British Columbia Animal Care Committee (Appendix B on page 162). To determine the average rate of water consumption over 24 hours, a bottle was filled with water and the change in water weight was measured over several days.  Mice were 20-22 weeks of age.  Females were housed 1-3 to a cage  (Scd1+/+, n = 6; Scd1-/-, n = 5), and males were housed individually because siblings of the same genotype were not available (Scd1+/+, n = 5; Scd1-/-, n = 3).  5.2.2 Induction of colitis Clinically healthy age-matched female mice were used for induction of experimental colitis. Experimental colitis was induced by giving sterile filtered DSS (mol wt 36,000-50,000; MP Biomedicals, Solon, OH) in drinking water for five days ad libitum.  Treatment with 3.5% (wt/vol) DSS for five days has been  previously established to induce moderate to severe colitis while minimizing mortality (15), and can be expected to exceed the minimum DSS intake of 30mg/ g body weight required to reliably induce colitis in mice (18). In the initial evaluation of colitis severity in C57Bl/6 mice, wild-type mice were treated with 3.5% DSS (n = 4) or water (n = 3) for five days. To control for genotype  121  differences in fluid consumption (9, 20), mice were grouped by genotype (Scd1+/+, n = 10 Scd1-/-, n = 7) and water consumption was monitored over three consecutive days immediately prior to the 5-day treatment period.  The drug  concentration was adjusted to obtain equivalent daily dosages in each genotype. We employed DSS from the same lot (9135J) throughout this study. None of the mice in this study died before termination of the experiment after five days of DSS treatment.  5.2.3 General assessment of colitis Animals were assessed daily and mean DSS/water consumption and body weights were recorded.  Stool consistency was assessed daily using a 0 to 3  scale: 0 = normal well-formed fecal pellets, 1 = loosely shaped moist pellets that do not adhere to the anus, 2 = amorphous, moist, sticky pellets, 3 = liquid stool. The presence of blood in the stools was assessed by a guaiac paper test (ColoScreen Occult Blood Test, Helena Laboratories, Beaumont, TX) using a 0 to 4 scale: 0 = negative, 1 = faintly blue, 2 = moderately blue, 3 = dark blue, 4 = fecal blood visible to the eye.  After 5 days of DSS treatment, the mice were  aenaesthetized by intraperitoneal injection of 250 mg/kg 2,2,2-tribromoethanol (Sigma-Aldrich, Oakville, ON, Canada) and euthanized by exsanguination via cardiac puncture and cervical dislocation.  5.2.4 Histological assessment of colitis The entire colon was excised, extending from the ileocecal junction to the anus, and the length was recorded.  Colons were subsequently opened  longitudinally along the mesenteric border and fecal matter was removed. The colons were divided into three sections: distal, mid-colon, and proximal colon. The mid-colon section was rolled, formalin-fixed, embedded in paraffin, sectioned at 5 μm thickness, and stained with hematoxylin and eosin (H&E) in a standard manner. Semi-quantitative  assessment  of  colon  damage  was  performed  in  a  randomized and blinded fashion using a scoring system modified from Dieleman et al (22), by estimating: 1) the severity of inflammation; 2) the extent of inflammation; and 3) the amount of crypt damage. For each of these features, the percentage of area involved by the disease process was scored on a 1 to 4 scale  122  as follows: 1 = 1-25%; , 2 = 26-50%; 3 = 51-75%; 4 = 76-100%.  The severity of  inflammation was scored on a scale from 0 to 3 as follows: 0 = none; 1 = slight; 2 = moderate; 3 = severe. The depth of inflammation was scored on a scale from 0 to 3 as follows: 0 = rare inflammatory cells in the lamina propria; 1 = increased numbers of granulocytes in the lamina propria; 2 = inflammatory cells extending into the submucosa; 3 = transmural extension of the infiltrate. For crypt damage: 0 = intact crypts; 1 = loss of basal one third of crypts; 2 = loss of basal two thirds of crypts; 3 = entire crypt loss with the surface epithelium remaining intact; and 4 = entire crypt loss with change of epithelial surface (erosion). Sections from each animal were scored for each feature separately by establishing the product of the grade for that feature and the percentage involvement (in a range from 0 to 12 for each of inflammation severity and infiltrate extent, and in a range from 0 to 16 for crypt damage). All scores on the individual parameters together could result in a total score ranging from 0 to 40.  5.2.5 Statistical analysis Data are presented as means plus or minus standard error. Initial analyses were performed by the unpaired two-tailed Student's t-test.  Data that did not  follow a normal distribution as judged by Kolmogorov-Smirnov tests were analyzed with the Mann-Whitney test for unpaired data. Body weight data were analyzed by two-way analysis of variance (ANOVA; time, within subjects; genotype, between subjects) using repeated measures, followed by Bonferroni post-tests. Statistical analysis was performed with GraphPad Prism software and with the open-source R-package (GraphPad, San Diego, CA; R Development Core Team, 2006 (23)). P < 0.05 was considered significant.  5.3 Results 5.3.1 SCD1 deficiency increases water consumption in C57BL/ 6 mice Water consumption was evaluated in male and female congenic C57BL/6 mice homozygous for a spontaneous deletion in Scd1 (Scd1-/-). Both male and female SCD1-deficient mice consumed significantly more water compared to controls (Figure 5.1 on page 130). Water consumption was increased by 67% in males (p  123  = 0.036; Scd1+/+, n = 5; Scd1-/-, n = 3) and by 100% in females (p = 0.0043; Scd1+/+, n = 6; Scd1-/-, n = 5).  5.3.2 SCD1 deficiency does not accelerate DSS-induced acute colitis Wild-type mice (n = 4) were treated with 3.5% DSS for five days to confirm that this dose can reliably induce acute colitis in the C57BL/6 strain, which has previously been demonstrated to be relatively resistant to DSS-induced colitis (15). Control wild-type mice (n = 3), receiving water only, did not exhibit any of the clinical signs associated with acute colitis (diarrhea, fecal occult blood, and weight loss).  After a 5-day course of 3.5% DSS, wild-type mice lost 12.7% of  their initial body weight, while wild-type mice receiving water only showed no significant change in body weight (Figure 5.2a on page 131). DSS-treated mice all developed diarrhea (Figure 5.2b) and hemoccult-positive stools (Figure 5.2c), which were also often bloody.  Colon length was decreased by 21% in DSS-  treated mice compared to controls (p = 0.0044; Figure 5.2d). survived until euthanized on day 5.  All animals  Histological analysis revealed a patchy  pattern of inflammatory cell infiltration in the lamina propria and submucosa with affected areas showing mucosal damage that ranged from erosion of surface epithelium to total loss of crypts (Figure 5.3a versus b on page 132). 9-week-old female SCD1-deficient mice (n = 7) and wild-type control mice (n = 10) were treated with different DSS concentrations adjusted to control for total DSS intake. In the three days prior to treatment, SCD1-deficient mice consumed ~38% more water than control mice, so DSS concentration was accordingly decreased to 2.5% for SCD1-deficient mice. After 5 days of ad libitum exposure to DSS, all mice treated with DSS displayed clinical and macroscopic signs of acute colitis. Prior to DSS consumption, SCD1-deficient mice tended to weigh more than wild-type controls (19.3±0.5 g vs. 18.5±0.2 g; p = 0.075), but this difference was not significant. Over the course of DSS treatment, SCD1 deficient mice lost less weight than controls (p = 0.0079; Figure 5.4a on page 133). After reaching a peak body weight at day 3 of treatment, weights of SCD1-deficient mice decreased by 9.0% (19.4±0.2 g, p < 0.0001; Figure 5.4a), a similar proportion of weight as that lost by wild-type mice over the 5-day course of DSS treatment (9.5% decrease; 16.7±0.3 g, p = 0.0002).  DSS treatment reduced stool 124  consistency to a lesser extent in SCD1-deficient mice than wild-type controls (p = 0.042; Figure 5.4b), but fecal occult blood was similar between the two groups (p = 0.16; Figure 5.4c). Colon length was not significantly different between the two genotypes at the end of DSS treatment (46±1 mm vs. 47±2 mm; p = 0.71; Figure 5.4d). Microscopic examination of colon tissues indicated that DSS-induced colon damage was similar in SCD1-deficient mice (Figure 5.3c on page 132) and controls (Figure 5.3b). A patchy pattern of severe mucosal damage characterized by a loss of crypts and surface epithelium and infiltration of Inflammatory cells into both the mucosa and submucosa was observed in H&E-stained colon tissues from both SCD1-deficient and wild-type control mice after 5 day DSS treatment. The inflammatory cell infiltrate within the mucosa consisted of a mix of mononuclear cells and granulocytes, while significant edema was observed in the submucosa. Semi-quantitative histological assessment of sections stained with H&E was used to assign histological colon damage scores on a 0 to 40+ scale based on the following  parameters:  the  percentage  of  involved  area,  the  amount  of  inflammatory infiltrate, the depth of inflammation, and the degree of crypt damage. When examined in a blinded fashion, there was no significant difference between the histological colon damage scores of SCD1-deficient mice and wildtype control mice (Figure 5.3d on page 132 and Table 5.1 on page 129). Over the 5-day treatment period, the average volume of DSS-treated water consumed in two cages of SCD1-deficient mice was 32.3 mL/mouse, while the average volume consumed in the two cages of wild-type control mice was 17.7 mL/mouse. This resulted in a total intake 808 mg DSS (41.9 mg/g initial body weight) by SCD1-deficient mice, and 621 mg DSS (33.7 mg/g initial body weight) by wild-type controls. These observations demonstrate that the beneficial effect of SCD1-deficiency on DSS-induced weight loss and diarrhea cannot be explained on the basis of decreased intestinal exposure to DSS.  Indeed, SCD1-deficient  mice may even be somewhat protected from DSS-induced colitis.  5.4 Discussion Despite chronic inflammation of the skin and increased susceptibility to atherosclerosis, SCD1 deficiency does not accelerate inflammation in the DSS-  125  induced model of acute colitis. The total amount of DSS-supplemented water per gram body weight is known to affect the induction and severity of experimental colitis (18).  Therefore we used a total DSS intake greater than 30 mg/g body  weight and adjusted dosing to account for increased fluid consumption in SCD1deficient mice.  In contrast to a prior report (13), absence of SCD1 does not  worsen fecal occult blood, colon shortening, or colonic damage, and in fact may ameliorate other colitis-induced sequelae, including weight loss and diarrhea. We have recently shown that SCD1-deficient mice have chronic inflammation of the skin, and when exposed to a pro-inflammatory dietary stimulus these mice also demonstrate increased susceptibility to atherosclerosis and increased plasma markers of systemic inflammation (12). Several pro-inflammatory markers, including IL-6, IL-1β, IL-12p70, soluble intercellular adhesion molecule-1, and serum amyloid A are elevated in these mice (12), and intriguingly, most are also upregulated in both human inflammatory bowel disease (24, 25) and the DSS model of colitis in mice (26-29). Our previous observations (12) suggested that the systemic pro-inflammatory profile in SCD1-deficient mice might contribute to inflammation at other sites, including the gastrointestinal tract, and one recent study has reported that absence of SCD1 exacerbates DSS-induced colitis (13), an observation attributed to reduced SCD1-mediated hepatic oleic acid biogenesis and subsequent reduction in oleoyl-lysophosphatidylcholine (LPC) (13). Chen et al. (13) used a low dose of DSS that may not reliably induce colitis (18) and did not control for the increased fluid consumption associated with SCD1-deficiency (9, 20).  This  study raised the question of whether colonic inflammation is also exacerbated in SCD1-deficient mice when total DSS intake is controlled. Our results show that absence of SCD1 does not exacerbate DSS-induced colitis when total DSS intake is increased and DSS dosing is adjusted to account for genotypic differences in fluid consumption. Colonic inflammation, colon length and fecal blood are not altered by SCD1-deficiency in DSS-induced colitis, while diarrhea and total weight loss are ameliorated by SCD1 deficiency. Therefore, although SCD activity and oleoyl-LPC levels are decreased by DSS treatment (13), reduced SCD activity does not play a significant causal role in DSS-induced colonic inflammation. In addition to the differences in DSS dosing between this study and that reported by Chen et al (13), it should be noted that there were also differences in 126  age, sex, diet, and genetic background between mouse cohorts used in the two studies, which could have effects on colonic inflammation or the immune response. Both studies used inbred C57BL/6 mice carrying Scd1 null alleles, but the DSS-treated mice used in this study were 9-week old females carrying the Scd1ab-2J allele (9), while Chen et al (13) used 5- to 8-week old males carrying the Scd1tm1Ntam allele (30).  No comparisons of these two alleles on the  C57BL/6  genetic background have been reported, however, a male bias for severity of DSS-induced colitis has been previously described (15, 31) and might have played a role in exacerbating the accelerated colitis in SCD1-deficient mice reported previously (13). While SCD1-deficient mice are protected from weight gain in studies of highfat feeding or genetic models of obesity (32, 33), studies of chow-fed lean female but not male mice have demonstrated increased body weight with SCD1 deficiency (33, 34). In this study, female SCD1-deficient mice tended to weigh more than wild-type controls at the start of DSS treatment, suggesting that differences in body weight may be influenced by phenotypic effects of SCD1 deficiency other than susceptibility to DSS-induced colitis. The modest beneficial effect of SCD1-deficiency is not due to the lower percentage of DSS in their water, since SCD1-deficient mice consumed a total intake of DSS that was actually greater than that of wild-type controls. These observations suggest that chronic inflammation of the skin and a modest systemic pro-inflammatory profile do not play a significant role in colonic inflammation in this model. The SCD1-deficient mouse continues to provide a useful model in which to explore inflammatory pathways as they relate to lipid synthesis and metabolic characteristics. However, the specific characteristics of each disease model must be taken into consideration when drawing extensions to human pathology. While this manuscript was in preparation, two different studies reported on the relationship between SCD1 deficiency and inflammatory liver disease (35, 36). SCD1 deficiency provided protection from concanavalin-A-induced nonalcoholic steatohepatitis  (35),  but,  SCD1  deficiency  increased  liver damage  in  a  methionine-choline-deficient dietary model of nonalcoholic steatohepatitis(36). In the former model, steatohepatitis is induced by a T lymphocyte mitogen, while phosphatidylcholine synthesis and VLDL secretion are blocked in the latter model (37).  Additional  experiments  are  needed  to  test  the  effect  of  SCD1  overexpression and inhibition on inflammation in various cell types, but these 127  conflicting results in the different mechanisms of liver damage in these two models suggest that SCD1 deficiency may protect against fatty liver and liver inflammation under normal or high-fat dietary conditions, and that SCD1 activity plays an important role in preventing lipotoxicity in the context of hepatic accumulation of free fatty acids. Various mechanisms have been suggested to explain a pro-inflammatory response in cells and tissues exposed to increased saturated fatty acids and/or reduced unsaturated fatty acids, the conditions induced by endogenous SCD activity (38, 39). These proposed mechanisms include altered toll-like receptor signalling (40, 41), activation of nuclear factor-κB (42-44), and altered production and fatty acid saturation of lysophosphatidylcholine (13, 45, 46). SCD1-deficient mice have also been demonstrated to have increased tissue levels of arachidonic acid (38, 43, 47, 48), a key intermediate in the production of pro-inflammatory eicosanoids (49).  Further studies are needed to fully explore the role of SCD  activity in inflammation and the in vivo contribution of these mechanisms to the chronic inflammation observed in SCD1 deficient mice. The relevance of the SCD1-deficient mouse model to the role of human SCD activity in inflammation is unclear. Observational studies in humans have shown an association between increased SCD expression and indexes of SCD activity and inflammatory markers (50-52), suggesting that the chronic inflammation observed in this mouse model of SCD1 deficiency may not extend to humans with reduced SCD1 activity. This study indicates that despite chronic inflammation of the skin (10-12), SCD1 deficiency does not accelerate inflammation in the DSS-induced model of acute colitis when total DSS intake is increased and DSS dosing is adjusted to account for genotypic differences in fluid consumption.  A previous report of  accelerated DSS-induced colitis with SCD1-deficiency (13) may now be attributed to a genotype-related increase in the intake and delivery of DSS to the gut, rather than to a role for SCD1 in inflammatory bowel disease.  128  Table 5.1: Histological assessment of DSS-induced colon damage in SCD1deficient mice. Scd1+/+  Scd1-/-  p  Inflammation severity  4.3 ± 0.4 (10)  3.6 ± 0.7 (7) 0.36  Inflammation extent  5.7± 0.6 (10)  6.0 ± 1.1 (7) 0.89  Crypt damage  7.2 ± 1.2 (10)  6.0 ± 1.1 (7) 0.60  Total histological score  17.1 ± 2.0 (10) 15.6 ± 2.7 (7) 0.47  Data represent mean ± SEM. The number of animals in each subgroup is indicated in parentheses.  129  Figure 5.1: Water consumption in C57BL/6 mice lacking SCD1. SCD1-deficient (Scd1-/-) and wild-type control (Scd1+/+) mice were housed individually (males; Scd1+/+, n = 5; Scd1-/-, n = 3) or in cages of 1-3 mice (females; Scd1+/+, n = 6; Scd1-/-, n = 5) and given free access to water.  130  Figure 5.2: DSS-induced acute colitis in C57BL/6 mice. a-c, Body weight (a), stool consistency (b), and fecal blood (c) were measured in wild-type control (Scd1+/+) C57BL/6 mice treated with 0% (n = 3) or 3.5% DSS (n = 4) in the drinking water over the course of 5 days (a, p = 0.0008; b,c, p < 0.0001; repeated-measures analysis of variance (ANOVA)). 0% DSS is indicated with a solid line, and 3.5% DSS is indicated with a broken line. **p < 0.01. ***p < 0.001. d, Colon lengths at the end of the 5 days are shown.  131  Figure 5.3: DSS-induced colon damage in SCD1-deficient mice. a-c, No colon damage was observed in control wild-type mice that received water only (a), but a patchy pattern of severe mucosal damage characterized by a loss of crypts and infiltration of Inflammatory cells into both the mucosa and submucosa was observed in hematoxylin and eosin-stained colon tissues from wild-type control (b; Scd1+/+; DSS = 3.5%; n = 10) and SCD1-deficient (c; Scd1-/-; DSS = 2.5%; n = 7) mice after 5 day DSS treatment. Mucosa (M), submucosa (SM), and muscularis propria (MP). d, Semi-quantitative histological assessment of disease severity.  132  Figure 5.4: DSS-induced acute colitis in SCD1-deficient mice. a-c, Body weight (a), stool consistency (b), and fecal blood (c)were measured in SCD1-deficient (Scd1-/-; n = 7) and wild-type control mice (Scd1+/+; n = 10) treated with DSS in the drinking water over the course of 5 days (a, p = 0.0079; b, p = 0.042; c, p = 0.16; repeated-measures analysis of variance (ANOVA). *p < 0.05. **p < 0.01. ***p < 0.001. d, Colon lengths at the end of the 5 days are shown.  133  5.5 References 1. Barnes PJ, Karin M. Nuclear factor-kappaB: a pivotal transcription factor in chronic inflammatory diseases. N Engl J Med 1997;336:1066-71. 2. Miyazaki M, Ntambi JM. Role of stearoyl-coenzyme A desaturase in lipid metabolism. Prostaglandins Leukot Essent Fatty Acids 2003;68:113-21. 3. Bené H, Lasky D, Ntambi JM. 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Atherosclerosis 2009;203:298-303.  137  CHAPTER 6 Conclusions, perspectives, and future directions 6.1 Summary of findings The primary objective of this thesis was to investigate the effect of decreased SCD activity on susceptibility to atherosclerosis. despite  an  antiatherogenic  metabolic  profile,  We demonstrate here that SCD1  deficiency  increases  atherosclerosis in hyperlipidemic LDLR-deficient mice. These findings reinforce the crucial role of chronic inflammation in promoting atherosclerosis, even in the presence of antiatherogenic metabolic characteristics. In order to facilitate mouse studies that assessed the phenotypic effects of SCD1 deficiency, we first characterized the mutation in Scd1 that is carried by the asebia-J mouse strain, defining a 25,779 bp deletion that does not affect adjacent genes. This finding allowed us to distinguish wild-type mice from mice carrying one copy of the Scd1ab-J allele, which had not been possible in previous studies involving this strain (1-3). We then employed the Scd1ab-J strain to demonstrate that a constitutive reduction of SCD activity improves the metabolic phenotype of LDLR-deficient mice. We show that absence of the Scd1 gene product reduces plasma TG and reduces weight gain in severely hyperlipidemic LDLR-deficient mice challenged with a Western diet. Absence of SCD1 also increases insulin sensitivity, as measured by intraperitoneal glucose and insulin tolerance testing. SCD1 deficiency dramatically reduces hepatic lipid accumulation while causing more modest reductions in plasma apolipoproteins, suggesting that in conditions of sustained hyperlipidemia, SCD1 functions primarily to mediate lipid storage. In addition,  absence  of  SCD1  partially  ameliorates  the  undesirable  hypertriglyceridemic effect of antiatherogenic LXR agonists. Unexpectedly, examination of lesion area in the aortic root revealed that despite antiatherogenic metabolic characteristics, atherosclerosis is significantly increased in males and females in two models of SCD1 deficiency. This finding prompted a search for a possible proatherogenic mechanism.  Inflammatory  changes are evident in the skin of these mice, including increased intercellular  138  adhesion molecule (ICAM)-1 and ulcerative dermatitis. Increases in ICAM-1 and IL6 are also evident in plasma of SCD1-deficient mice. HDL particles demonstrate changes associated with inflammation, including decreased plasma apoA-II and apoA-I  and  paraoxonase-1  and  increased  plasma  serum  amyloid  A.  Lipopolysaccharide-induced inflammatory response and cholesterol efflux are not altered in SCD1-deficient macrophages. In addition, when SCD1 deficiency is limited to bone marrow-derived cells, lesion size is not altered in LDLR-deficient mice. We propose that the acceleration in atherosclerosis in SCD1-deficient mice is likely to result from chronic inflammation primarily of the skin, leading to increased systemic inflammatory markers and proinflammatory changes in HDL (Figure 6.1 on page 152). These results suggested that a systemic proinflammatory profile in SCD1deficient mice may also contribute to chronic inflammatory diseases other than atherosclerosis, such as inflammatory bowel disease induced by DSS. Wild-type controls were treated with 3.5% DSS for 5 days to induce moderately severe colitis, while the concentration of DSS given to SCD1-deficient mice was lowered to 2.5% to control for increased fluid consumption. Although SCD1-deficient mice consumed a total intake of DSS that was greater than that of wild-type controls, colonic inflammation, colon length and fecal blood were not altered by SCD1deficiency in DSS-induced colitis, while diarrhea and total weight loss were modestly improved. Our analysis indicates that a previous report of accelerated DSS-induced colitis with SCD1-deficiency (4) should be attributed to a genotyperelated increase in the intake and delivery of DSS to the gut, rather than to a role for SCD1 in inflammatory bowel disease.  6.2 Mammalian SCD gene families Chapter 2 provides the first evidence demonstrating pseudogenization of the ancestral SCD5 gene in the rodent lineage. Further study is now needed to better understand the function of the SCD5 gene that remains in other land vertebrates, including humans.  Our analysis of publicly available microarray data suggests  that SCD5 is likely to have a previously unexplored function in skin, PNS, peripheral blood mononuclear cells, and bone marrow-derived cells. A transgenic mouse that expresses human SCD5 could be crossed with the existing SCD2deficient mouse model (5) to determine whether human SCD5 plays a role in lipid  139  synthesis in during early skin and liver development.  Human peripheral blood  leukocytes hemapoietic stem cells could be employed in order to identify the stimuli that activate SCD5 expression during macrophage differentiation (6), as well as the consequences for fatty acid composition. Another intriguing future direction is the investigation of the putative brainspecific regulatory element we identified in sequence upstream of mouse Scd2 (8537 to -8445) that has high similarity to sequence upstream of human SCD (2149 to -2051).  The next step would be to employ phylogenetic footprinting  techniques (7, 8) in an attempt to identify transcription factor binding sites in these genes, followed by experimental validation in cell culture expression systems.  6.3 Role of SCD in ameliorating the metabolic syndrome in hyperlipidemic mice Despite a growing body of evidence supporting a key role for dysregulated lipogenesis in the metabolic syndrome, the underlying mechanisms by which alterations in lipid composition in various tissues influence systemic metabolic homeostasis are not well understood.  The studies described herein were  designed to explore the metabolic function of endogenous MUFA production, catazlyzed by SCD. Scd1 is the only mouse co-ortholog that retains an expression pattern similar to human SCD (9-14), and is therefore considered the most relevant mouse gene for metabolic studies modelling inhibition of human SCD activity. The results described in Chapter 3 establish the robust impact of SCD1 deficiency on the metabolic phenotype of the hyperlipidemic LDLR-deficient mouse model, including reduced hepatic and plasma TG, reduced diet-induced weight gain and insulin resistance, and a partially reduced hypertriglyceridemic response to an LXR agonist. An obvious limitation of this approach derives from the different SCD genes present in humans and mice. A genetic deficiency of mouse Scd1 reduces SCD activity in adipose, skin, lung, adrenal gland, and lymph nodes (9-12), but spares expression of the other mouse SCD genes, Scd2, Scd3, and Scd4, in brain/PNS, skin, and heart, respectively.  Unlike the specific reduction in activity of the  mouse SCD1 enzyme studied here, phamacological inhibition of SCD activity in humans would be expected to reduce activity of both human isoforms, SCD and  140  SCD5, depending on the inhibitor's tissue distribution.  Additionally, the SCD1-  deficient mouse model represents a complete deficiency of SCD activity in affected tissues throughout development, and may not accurately reflect the phenotypic effect of acute inhibition of SCD activity in adult animals.  6.3.1 Relationship between fatty liver and insulin resistance There remains much uncertainty about the mechanism by which SCD activity and its product, MUFA, modulate metabolism and insulin signalling in particular. Despite a well-known correlation between fatty liver and insulin resistance (15), it is not yet clear whether a causal relationship exists between these two metabolic characteristics. Although the accumulation of hepatic TG is widely believed to result in insulin resistance, recent studies have shown a clear dissociation between hepatic steatosis and insulin resistance (16-19), suggesting that hepatic TG themselves are not toxic and may instead protect the liver from lipotoxicity by buffering the accumulation of non-esterified fatty acids (20, 21). Two of the examples of dissociation of fatty liver and insulin resistance come from studies of mouse models with deficiencies in other lipogenic genes.  For  instance, on a low-fat diet, a liver-specific deficiency of FAS results in both fatty liver and increased hepatic insulin signaling (22).  A second example of  dissociation of hepatic lipid accumulation from insulin resistance comes from mice with a null mutation in the gene Elovl6, which encodes the long chain fatty acyl elongase enzyme (FACE or LCE) that extends 16-carbon SFA and MUFA (23). In this case, mice with a disruption in Elovl6 become obese and develop fatty liver when fed a high-fat diet, and these mice also show marked protection from hepatic insulin resistance (17). These data from deficiencies in the two enzymes that precede SCD in the DNL pathway suggest that the improved insulin resistance that we observe with SCD1 deficiency does not result from reduced hepatic or adipose TG, but rather from some other mechanism. Further evidence to support this hypothesis comes from a study of short-term ASO-mediated SCD1 deficiency in rats fed a high-fat diet, which demonstrated both improved hepatic insulin sensitivity and increased hepatic TG (24). It is not yet clear why a short-term reduction of SCD1 would result in increased hepatic TG, while most other reports of ASO-mediated and genetic deficiencies of SCD1, including the one described in Chapter 3, demonstrate reduced hepatic TG (1, 25, 26); however, an important clue may lie in another mouse model that possesses 141  disruptions in two genes that encode adipose fatty-acid binding proteins, FABP4 and FABP5 (27). FABP4/5-deficient mice exhibit a striking increase in adiposespecific DNL, mediated by increased levels of mRNAs encoding FAS, SCD1, and FACE; they are also resistant to obesity, insulin resistance, and fatty liver induced by a high-fat diet (27).  Quantitative lipidomic analyses and intralipid infusion  studies led to the discovery that dramatically increased plasma palmitoleate (16:1n7) secreted by adipose tissue is responsible for the metabolic phenotype of this model, increasing insulin signalling and reducing fat accumulation in liver (28).  Plasma palmitoleate is also able to down-regulate liver SCD1 via  suppression of Scd1 promoter activity and destabilization of the SCD1 protein (27). This new information about the endocrine, or “lipokine”, effects of a product of adipose SCD activity suggests that in the presence of intact SCD genes and a high fat diet, it may be beneficial to increase SCD activity in adipose, while also decreasing SCD activity in liver. That is, if SCD activity is completely absent in both liver and adipose, a high-fat diet cannot lead to significant fat accumulation in either tissue (26, 29). On the other hand, if SCD is inhibited in both tissues but not completely absent, reduced plasma palmitoleate may signal that adipose is not capable of sufficient TG synthesis to buffer levels of circulating fatty acids and that the liver must respond by increasing its lipid storage capacity, perhaps by increasing fatty acid esterification, or reducing VLDL secretion; with some liver SCD1 continuing to be expressed, endogenous MUFA is still available for incorporation into TG, resulting in a net accumulation of hepatic TG, at least in the short term (24).  This intriguing hypothesis could be tested in vivo by  evaluating hepatic VLDL-TG secretion and lipid flux in the presence of both shortand long-term treatment with SCD1-ASO inhibitors compared with complete SCD1 deficiency.  6.3.2 Tissue-specific function of SCD A mouse model that can be manipulated to disrupt Scd1 in specific cell types has recently become available and is helping to elucidate the role of SCD activity in various parts of the body (30). deficiency  of  accumulation  SCD1 in  liver  are and  Unexpectedly, mice with a liver-specific  protected adipose,  from but  high-carbohydrate-induced not  from  high-fat-induced  lipid lipid  accumulation and insulin resistance, highlighting the importance of SCD activity 142  in extrahepatic tissues in responding to dietary fat (30). Interestingly, the fatty acid composition of liver TG in mice with a liver-specific deficiency of SCD1 is similar to that of control mice after high-fat feeding or standard chow (17% of calories from fat), but not low-fat/high-carbohydrate-feeding (30). This suggests that hepatic SCD activity only contributes to DNL at significant levels under conditions of limiting dietary fat. The contribution of hepatic SCD to insulin resistance, however, remains uncertain.  Unlike mice with a complete deficiency of SCD1, mice with a  disruption in Scd1 specific to hepatocytes do not exhibit improved glucose clearance and insulin sensitivity in the presence of a high-fat diet or any other diet tested (30).  This appears to be inconsistent with the effect of a liver-  selective partial repression of SCD1 activity in rats achieved by a slow intraportal infusion of ASO, in which high-fat-induced insulin resistance is normalized by SCD1 ASO (24). It is not clear at this time whether the phenotypic differences should be attributed to a species difference between rats and mice, the different levels of remaining hepatic SCD1 activity, low levels of ASO leakage to extrahepatic tissues, or some difference in the way insulin resistance is evaluated in these two studies. Future studies employing multiple doses of ASO and the gold standard hyperinsulinemic-euglycemic clamp will be necessary to address this unsettled issue. A mouse model with a disruption of Scd1 in cells that express keratin-14, including sebocytes and other cells of the epidermis, has recently been reported (31).  Surprisingly, these mice have significantly increased energy expenditure  and are protected from high-fat diet-induced obesity, fatty liver and insulin resistance (31).  Adipose and plasma fatty acid composition and expression of  lipogenic genes in adipose were not reported, so this aspect of the phenotype cannot be compared to FABP4/5-deficient mice; however, on a standard chow diet, hepatic expression of Scd1 and other lipogenic genes is reduced, while on a high-fat diet, the lipogenic response to high-fat feeding is intact (31). Therefore, the reduction in diet-induced fatty liver and obesity with skin-specific SCD1 deficiency cannot be explained by a decrease in hepatic lipogenesis, but may instead be explained by increased expression of genes encoding proteins involved in fatty acid oxidation, lipolysis and thermogenesis, including carnitine palmitoyltransferase 1a (CPT-1a), acyl-CoA oxidase 1 (AOX), PPAR-γ coactivator1α (PGC-1α), lipoprotein lipase, hormone-sensitive lipase, and uncoupling 143  proteins (31), similar to animals with a global deficiency of SCD1 (25, 29, 32-35). Parallel studies of insulin resistance and diet-induced obesity on mice with a complete deficiency of SCD1 were not included, so it is not yet possible to quantify the contribution to global energy homeostasis of SCD1 in the skin relative to other peripheral tissues. This study indicates that a major part of the metabolic phenotype of whole-body SCD1 deficiency in mice is mediated by loss of SCD1 in skin, and not liver or some other extrahepatic tissue (31).  6.3.3 Relationship between insulin resistance and inflammation One seemingly paradoxical finding in this work is the observation of increased insulin sensitivity in SCD1-deficient mice (Chapter 3) coincident with mild chronic inflammation (Chapter 4).  Plasma markers of subclinical inflammation are  associated with insulin resistance in humans (36-38), and evidence from mice indicates that inflammatory cytokines can trigger insulin resistance (39). Futhermore, cytokines such as TNF-α and IL-6 are able to decrease insulin action at the cellular level (40, 41). One important difference between our mouse model and other models of inflammation-induced insulin resistance may be the status of adipose tissue: SCD1 deficiency dramatically reduces the mass of white adipose tissue in severely hyperlipidemic LDLR-deficient mice challenged with a Western diet (Chapter 3), and actually reduces levels of plasma MCP-1 (Chapter 4), an inflammatory marker produced by adipose tissue (42). Our results suggest that chronic mild inflammation does not play a significant role in promotion of insulin resistance in the presence of reduced adiposity and reduced secretion of inflammatory markers by adipose tissue, supporting the role of adipose tissue as the primary pathogenic site of inflammation-induced insulin resistance (43, 44).  6.3.4 Fate of excess fatty acids The observation of increased expression of genes involved in fatty acid oxidation in mice with a deficiency of SCD1 in skin (31) highlights an important unresolved issue requiring more attention, namely, the role of SCD in controlling the fate of excess cellular fatty acids in the liver.  Non-esterifed fatty acids,  whether derived by DNL or exogenously, are cytotoxic, and can be dealt with by the cell in one of three ways: esterification to TG for storage as lipid droplets, secretion as part of VLDL for transport to other tissues, or disposal via fatty acid 144  oxidation in mitochondria.  It is the relative rate of these three pathways that  determines whether fat accumulates in the liver and other tissues. Though not a quantitatively significant contributor to VLDL triglyceride production under most dietary conditions (45-47), DNL appears to be an important marker of the relative rate of fatty acid esterification and oxidation (46). Given the function of SCD in the synthesis of MUFA, SCD would be expected to mediate its effects on fat accumulation through TG synthesis. When in vivo hepatic TG synthesis has been evaluated in SCD1-deficient mice, significant reductions have been observed, particularly in the presence of high-carbohydrate/low-fat diets (26). In addition, TG synthesis in primary hepatocytes from SCD1-deficient mice is decreased by ~56% (48). Investigations into the expression of genes involved in DNL have also uncovered reductions in hepatic mRNAs encoding SREBP-1c (29, 33), FAS (24, 29, 35, 49), ACC (24, 25, 32, 33, 50), fatty acyl elongase (35), and glycerol phosphate acyltransferase (29). Furthermore, these reductions are exacerbated by high-fat diets (33, 35). Despite this evidence supporting a role for SCD1 in hepatic DNL and TG synthesis, several early reports on the phenotype of SCD1deficient mice emphasized an increase in the expression of genes involved in fatty acid oxidation in liver (29), muscle (51), and brown adipose tissue (32). Although hepatic fatty acid oxidation rates have only rarely been evaluated (32, 50), there have been several reports of increased mRNAs encoding acyl-CoA oxidase 1, carnitine palmitoyltransferase 1 in liver (25, 29, 32) and other tissues (34, 51). Further investigation into the relative contribution of changes in DNL, TG synthesis, and fatty acid oxidation to the phenotypic effect of SCD1 deficiency is needed. Our observations of reduced fatty acid synthesis in primary hepatocytes from SCD1-deficient mice and reduced levels of mRNAs encoding SREBP-1c, ACC1 and FAS suggest that a reduction in hepatic DNL is likely to be a major contributor to the decreased hepatic and plasma lipids we observe in hyperlipidemic SCD1-deficient mice. We also determined that hepatic fatty acid oxidation is not likely to be increased by SCD1 deficiency in hyperlipidemic LDLRdeficient mice, as suggested by reductions in mRNA levels of genes encoding PPAR-α (36% reduction; p = 0.060), CPT-1a (54% reduction; p = 0.013), and AOX1 (69% reduction; p = 0.0056; data not shown). An absence of up-regulation of fatty acid oxidation with SCD1 deficiency is not entirely unexpected, as hepatic mRNAs for fatty acid oxidation genes are not altered in mice with a liver-specific 145  deficiency of SCD1 (30), and reductions in genes encoding PPAR-α, PGC-1 α, AOX, long-chain acyl-CoA dehydrogenase, and CPT-1a have even been observed in SCD1-deficient mice fed a high-fat diet for a prolonged period (33). It is not clear why fatty acid oxidation is reduced rather than increased in our model of SCD1 deficiency,  but  we  hypothesize  that  increased  systemic  inflammation,  exacerbated by severe hyperlipidemia, may be responsible for reduced PPAR-α and its target genes involved in fatty acid oxidation (52, 53), similar to the effects of the acute phase response on PPAR-α  in other models (54, 55).  Future  experiments might involve parallel analyses of DNL, TG synthesis, and fatty acid oxidation in liver, adipose, and muscle from mice with a complete deficiency of SCD1, compared with mice with disruptions in Scd1 limited to liver (30), adipose, skeletal muscle, or skin (31). Additional information on the role of SCD activity in responding to dietary stimuli could be gained by subjecting these mice to diets high in fat or carbohydrates.  6.4 Role of SCD in susceptibility to atherosclerosis Our observation of increased inflammation and atherosclerosis in SCD1deficient mice (Chapter 4) provides strong support for the role of chronic inflammation  in  promoting  atherosclerosis,  even  in  antiatherogenic biochemical and metabolic characteristics.  the  presence  of  While we have not  ruled out the possibility that the increased systemic inflammation with SCD1 deficiency is a consequence of increased atherosclerosis rather than its cause, alterations in multiple  indicators of inflammation  are  consistent with a  proatherogenic role for inflammation in the absence of murine SCD1.  Further  studies are needed to clarify the mechanisms by which SCD1 deficiency increases inflammatory processes and the relevance of these findings to the development of SCD inhibitors for treatment of the metabolic syndrome in humans. The LDLR-deficient mouse model was used in this work due to its human-like lipoprotein phenotype and its propensity to develop diet-induced diabetes, obesity, and atherosclerosis (56-58).  While mouse models, and the LDLR-  deficient model in particular, have proved extremely useful for studying atherosclerotic lesion development, it should be noted that no mouse model is able to precisely mirror human atherosclerotic cardiovascular disease (59). For instance, mice differ from humans in the location of atherosclerotic plaques and  146  the absence of end-stage ischemic lesions, as well as the lack of progression to occlusive coronary artery disease, myocardial infarction, cardiac dysfunction, and premature death, which are all characteristics of human coronary heart disease (59).  Therefore, our observations of the effect of SCD1 deficiency on  susceptibility understanding  to  experimental  role  of  SCD  atherosclerosis  activity  and  are  chronic  primarily  relevant  inflammation  in  for  lesion  development, but are of limited use for drawing conclusions about the role of SCD activity in more advanced stages of human cardiovascular disease.  6.4.1 Function of SCD in immune cells Understanding the function of SCD activity in macrophages and other immune cells that are involved in lesion development is an area that requires more detailed analysis. Our work demonstrated that when SCD1 deficiency is limited to bone marrow-derived cells, lesion size is not altered in LDLR-deficient mice (Chapter 4).  We also demonstrated that the lipopolysaccharide-induced  inflammatory response is not altered in SCD1-deficient macrophages.  These  observations suggest that macrophage SCD1 does not play a significant role in atherogenesis in this model; however, our results are from early lesions only and a longer term study of SCD1 deficiency in lesion macrophages is still needed. It should be noted that our observations on the effect of macrophage SCD1 deficiency differ from those reported in a recent study by Brown et al, which indicated that SCD1 inhibition mediated by ASO exacerbates the proinflammatory response in macrophages (60). There are several differences between the two experiments, including degree of SCD1 inhibition, mouse strain, proinflammatory stimulus, diet, length of time in culture, length of pre-incubation in serum-free media, and length of time for cytokine expression. Additional experiments that systematically control for each of these variables will be needed to fully explore the role of SCD activity in the inflammatory response of thioglycollate-elicited peritoneal macrophages. Tissue profiling and immunohistochemical staining will also be valuable to elucidate the relative expression of human and mouse SCD genes in other cells involved in atherosclerotic lesion development, including vascular endothelial cells and smooth muscle cells.  147  6.4.2 Mechanism of inflammation in SCD1 deficiency The very recently reported mouse strain with a deficiency of SCD1 limited to the epidermis (31) would be a useful model in which to test our hypothesis that accelerated atherosclerosis in mice with a global SCD1 deficiency is due to chronic dermal inflammation. If this hypothesis is correct, then hyperlipidemic LDLR-deficient mice with an absence of SCD1 in the skin would be expected to exhibit the same degree of systemic inflammation and atherosclerosis as the SCD1-deficient model described in Chapter 4. It is also possible that the absence of hepatic SCD1 may be at least partly responsible for the proinflammatory effect of global SCD1 deficiency on atherosclerosis and steatohepatitis (61).  Indeed,  current evidence suggests that TG synthesis promoted by hepatic SCD activity may  in  fact  protect  against  lipotoxicity,  liver  injury,  and  progressive  steatohepatitis (61), perhaps by buffering the accumulation of fatty acids in liver (20). Future experiments involving mice with a liver-specific deficiency of SCD1, whether achieved by liver-specific genetic disruption (30) or acute ASO-mediated partial suppression (24), will be useful to clarify the role of hepatic SCD activity in inflammation in vivo. Our observations of a systemic pro-inflammatory profile in SCD1-deficient mice suggested that SCD1 deficiency may also contribute to other chronic inflammatory diseases in addition to atherosclerosis, such as inflammatory bowel disease induced by DSS. In a previous report, absence of SCD1 was observed to exacerbate DSS-induced experimental colitis in mice (4); however, this study did not control for increased fluid consumption in SCD1-deficient mice (35, 62). The experiments described in Chapter 5 indicate that despite chronic inflammation of the skin (63-65), SCD1 deficiency does not accelerate DSS-induced colitis when total DSS intake is increased and dosing is normalized to fluid consumption.  A  precise replication of the previous study that also increased total DSS intake to levels that reliably induce colitis (66) and normalized dosing to fluid consumption would have required multiple doses of DSS and was beyond the scope of this project.  Therefore, we cannot exclude the possibility that SCD1-deficient mice  are more sensitive than wild-type mice to doses of DSS lower than those used here, and that there is ceiling effect. It is also possible that at higher doses of DSS, SCD1-deficient mice will have high mortality.  148  Attention should now turn to the careful characterization of the role of SCD activity in the cellular inflammatory response.  The mild chronic dermal  inflammation observed with SCD1 deficiency is thought to be secondary to structural defects of the hair follicle (62-64, 67, 68), rather than a direct result of SCD1 deficiency in the epidermis. Nevertheless, there is accumulating evidence that supports a pro-inflammatory effect of the SCD substrate, palmitic acid, in several cell types, including muscle (69) adipose (70), macrophage (71) and endothelial (72) cells, and this pro-inflammatory effect is reversed by the presence of the SCD product, oleic acid (69). Additional studies are now needed to test the effect of SCD1 overexpression and inhibition on cytokine induction in these various cell types. Various mechanisms have been proposed to explain a pro-inflammatory response in cells exposed to reduced SCD activity or increased palmitic acid. One possibility is the altered levels or fatty acid saturation of lipid signalling molecules, such as  diacylglycerol, lysophosphatidylcholine (LPC), or ceramide  might alter the activities of regulatory proteins that are involved in the cellular inflammatory response.  For example, in muscle cells, oleic acid reduces  induction of IL-6 by increasing TG synthesis and channelling palmitic acid away from diacylglycerol and consequent activation of the protein kinase C/nuclear factor-κB pathway  (69).  Similarly, palmitic acid (73) and SCD1 deficiency (4)  promote production of saturated LPC, a component of oxidized LDL that induces an inflammatory response (74-76) and stimulates lipoapoptosis (73), while oleic acid decreases hepatocyte LPC content and inhibits the deleterious effects of palmitoyl-LPC (73). Yet another intracellular signaling molecule that may be affected by SCD activity is ceramide, a sphingolipid that induces apoptosis  (77) and cytokine  expression (78). Palmitic acid, but not palmitoleic or oleic acid, is a precursor to ceramide (79), and ceramide accumulates in muscle cells treated with palmitic acid (80) and in livers of mice with a hepatocyte-specific disruption in SCD1 fed a diet high in SFA (30).  Further investigation will be required to determine the  relative significance of these lipid signaling molecules in mediating the proinflammatory effects of SCD deficiency.  149  6.5 Pharmacological inhibition of SCD Evidence from early studies of SCD1 deficiency in mice (2, 29) and associations between increased indices of SCD activity and components of the metabolic syndrome in humans (81-87) has raised the possibility that SCD activity can be inhibited in humans for the treatment of the metabolic syndrome, particularly obesity and insulin resistance. In addition to efforts to inhibit SCD activity via ASO (24, 25, 60), several small molecule SCD inhibitors have now been reported (88-92). The findings in Chapter 4 support the utility of SCD inhibitors in treatment of the metabolic syndrome and fatty liver in humans with dyslipidemia; however, the specific tissues that need to be targeted by inhibitors in order to mediate these therapeutic effects while avoiding deleterious effects of SCD inhibition in the skin (93) and eyelids (64, 94) remain uncertain. The phenotype of mice with a liver-specific deficiency of SCD1 indicates that chronic pharmacological inhibition of SCD in liver should have beneficial effects on carbohydrate-induced obesity, but that the prevention of HF-induced obesity and even insulin resistance may require inhibition of SCD in some extrahepatic tissue (30). Until recently, it was generally assumed that the extrahepatic target for SCD inhibitors might be adipose tissue (30), but the phenotype of mice with a skin-specific deletion of SCD1 suggests that a major part of the metabolic phenotype of whole-body SCD1 deficiency in mice, including protection from high-fat-induced obesity and insulin resistance, is mediated by loss of SCD1 in skin, and not adipose or some other extrahepatic tissue (31). Extensive evaluation of SCD inhibitors will be necessary to fully characterize the acute and chronic effects of pharmacological inhibition of SCD on body weight, insulin sensitivity, and fatty liver in vivo. The relevance of the findings in Chapter 5 to the development of SCD inhibitors for treatment of the atherosclerotic risk factors in humans is unclear. Studies in humans have been limited, but the single prospective observational study of CVD that evaluates the SCD index as a surrogate for SCD activity reports an association between the 16:1n7:16:0 SCD index in serum CE and both serum C-reactive  protein  (95)  and  CVD  (96),  suggesting  that  the  atherogenic  inflammation observed in our mouse model of complete SCD1 deficiency may not extend to humans with reduced SCD activity.  150  The findings in this study represent the effects of long-term complete SCD1 deficiency in all tissues in mice. ASO result in near complete deficiency of SCD1 expression in extrahepatic tissues (24, 60), which leads to atherogenic inflammation in rodent models similar to that observed here (60). By contrast, pharmaceutical compounds are generally not used at levels that would cause complete inhibition of the target enzyme through a 24-hour cycle (88), and would not be distributed throughout all tissues in the body (88). An evaluation of the effect of small molecule SCD inhibitors on atherosclerosis in hyperlipidemic LDLRdeficient mice will allow us to determine whether inhibition of SCD1 in hepatic and adipose tissues is sufficient to increase systemic inflammation and susceptibility to atherosclerosis, even in the absence of SCD1 inhibition in the skin. In so doing, such mouse studies will provide a valuable prediction of “ontarget” proinflammatory effects of SCD inhibitors in humans. In the absence of studies that directly assess the effect of SCD inhibitors on atherosclerosis and other chronic inflammatory diseases, the findings reported here indicate that inflammation must be closely monitored in future studies of SCD inhibitors for treatment of the metabolic syndrome in humans.  151  Figure 6.1: Effect of SCD1 deficiency on susceptibility to atherosclerosis. We propose that acceleration in atherosclerosis in SCD1-deficient mice is likely to result from chronic inflammation primarily of the skin. Sebaceous gland hypoplasia produces a hair shaft that is unable to shed its sheath, and instead grows in reverse toward the subcutis. When the hair fibre perforates the follicle base, this results in a foreign body response to fragments of hair fibre in the dermis. This mild chronic dermal inflammation, indicated by increased vascularity, increased skin histamine, increased mast cells and macrophages, and increased ICAM-1, is thought to be secondary to structural defects of the hair follicle, rather than a direct result of SCD1 deficiency in the epidermis. Increases in ICAM-1 and IL-6 are also evident in plasma of SCD1-deficient mice, suggesting that chronic inflammation of the skin may be contributing to the proatherogenic profile of SCD1 deficient mice. 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Am J Clin Nutr 2008;88:203-9.  159  APPENDIX A Sequence information  Table A.1: Genomic sequences used for analysis of vertebrate SCD gene families. Species  Locus  Chromosome  Coordinates  Genome Assembly Source  Human  SCD  10q24.31  102081079-102122747  NCBI Build 35  Human  SCD5  4q21.22  83708942-84169579  NCBI Build 35  Human  SCD6PS†  17p11.2  20165069-20740045  NCBI Build 35  Human  SCD7PS  †  1p33  46518410-46591536  NCBI Build 35  SCD8PS  †  5q33.2  153834726-154174652  NCBI Build 35  SCD9PS  †  5p14.3  19508914-22889488  NCBI Build 35  16q24.1  85169831-85908523  NCBI Build 35  Human Human  †  Human  SCD10PS  Chimpanzee  SCD  10  100687566-100700426  NCBI Build 2  Chimpanzee  SCD5  4  47575721-47744164  NCBI Build 2  Mouse  Scd1  19qC3  44240615-44387346  NCBI Build 35  Mouse  Scd2  19qC3  44155319-44226965  NCBI Build 35  Mouse  Scd3  19qC3  44086281-44142045  NCBI Build 35  Mouse  Scd4  19qC3  44200733-44288328  NCBI Build 35  Mouse  Scd-ps*  19qC3  44142301-44187961  NCBI Build 35  Mouse  Scd5-ps*  5qE3  99435221-99569900  NCBI Build 35  Rat  Scd1  1q54  249663748-249803138  RGSC version 3.1  Rat  Scd2  1q54  249599025-249651648  RGSC version 3.1  Rat  Scd3‡  1q54  249272476-249306385  RGSC version 3.1  Rat  Scd4  1q54  249630001-249717544  RGSC version 3.1  Rat  Scd5-ps  chr14_random  283572-375836  RGSC version 3.1  Dog  SCD  28  16093871-16141250  NCBI v1.0 (July 2004)  Dog  SCD5  32  9442725-9722695  NCBI v1.0 (July 2004)  Chicken  SCD  6  16673481-16689544  WashU (February 2004)  Chicken  SCD5  4  46948575-46965654  WashU (February 2004)  Fugu  LOC777946  Unknown  126366080-126368963  JGI v3.0 (August 2002)  Fugu  fat-5  Unknown  158905039-158907656  JGI v3.0 (August 2002)  NCBI, National Center for Biotechnology Information; RGSC, Rat Genome Sequencing Consortium; WashU, Washington University School of Medicine in St. Louis; JGI, Department of Energy Joint Genome Institute.* predicted unprocessed pseudogene; †predicted processed pseudogene; ‡partial sequence;  160  Table A.2: Coding sequences used for phylogenetic analysis of vertebrate SCD gene families. Species  Locus  Transcript Accession No.  Human  SCD  NM_005063.4  Human  SCD5  NM_001037582.2  Chimpanzee SCD*  AU297975†  Chimpanzee SCD5*  DC520222†  Mouse  Scd1  NM_009127.3  Mouse  Scd2  NM_009128.2  Mouse  Scd3  NM_024450.2  Mouse  Scd4  NM_183216.3  Rat  Scd1  NM_139192.2  Rat  Scd2  NM_031841.1  Rat  Scd3*‡  CK221378†  Rat  Scd4*  (predicted)  Dog  SCD*  DN374598†  Dog  SCD5*  BM539753†  Chicken  SCD  NM_204890.1  Chicken  SCD5  EU275768  Fugu  LOC777946 AY741383  Fugu  fat-5  AY741384  *predicted transcript; † EST; ‡ partial sequence  161  APPENDIX B Animal care certificates  162  163  164  165  APPENDIX C Supplemental methods C.1 Animals and diet Animals received a standard laboratory rodent chow diet (LabDiet 5010 Autoclavable Rodent Diet, PMI Nutrition International, Richmond, IN), or Western diet (TD.88137, Harlan Teklad, Madison, WI). All studies except those involving bone marrow transplantation were approved by the University of British Columbia Animal Care Committee.  C.2 Histological analysis At 11-13 weeks of age, Scd1-/-Ldlr-/- and Scd1+/+Ldlr-/- mice were placed on the Western diet. After a period of 12 weeks, the mice were fasted overnight, then anaesthetized by intraperitoneal injection of 250 mg/kg 2,2,2-tribromoethanol (Sigma-Aldrich, Oakville, ON, Canada). After exsanguination, mice were perfused transcardially with phosphate-buffered saline (PBS). Hearts with attached aortas were then removed and fixed in a 4% solution of paraformaldehyde in phosphatebuffered saline prior to embedding and freezing in Tissue-Tek OCT (Sakura Finetek USA Inc., Torrance, CA).  Sixteen consecutive 10-μm sections were  obtained working from the apex of the heart towards the aortic origin (1), beginning from the point where all three aortic valve cusps became clearly visible. Four slides were made from each animal. From the 16 sections, every fourth section (40 μm apart) was stained for Oil Red O and hematoxylin to visualize neutral lipid.  Adjacent sections were stained with hematoxylin and  eosin or Movat pentachrome as described in Singaraja et al (2). pentachrome  staining  was  used  for  visualization  of  Movat  proteoglycan-rich,  extracellular matrix thickening of the intimae (sea-green colour) and necrotic cores (absence of purple/black nuclei). Image analysis was performed with Image-Pro Plus (Media Cybernetics, Silver Springs, MD) or ImageJ (version 1.41b; National Institutes of Health, Baltimore, MD). Measurements were made at a magnification of 4× after calibration of the image analysis software using a micrometer image scanned at a magnification identical to that used for the aortic root tissue.  166  It was critical to manually evaluate lesion area from the luminal edge to the intima-media border rather than using threshold-based quantification of Oil Red O staining of neutral lipids, as we observed inconsistent staining between two studies and within lesions that contain regions of extracellular matrix and unesterified cholesterol. Values reported represent the mean lesion area from 4 sections for each animal. Semi-quantitative assessment of lesion severity and inflammatory cell infiltration was performed in a randomized and blinded fashion by a human and experimental cardiovascular pathologist (BMM) using a 0 to 5+ scale. To assess reproducibility of this analysis, randomly selected slides were used to assess intra-observer variability on two separate occasions. Qualitative morphologic assessment of lesion complexity was performed with light microscopy for the following parameters: foam cell characteristics, cholesterol clefts, presence of necrotic core, degree and composition of fibrous cap, infiltration into the media, extracellular matrix deposition, calcification and plaque cellular characteristics. Sections were graded (0-5+) based on the following scale: 0, few or no apparent foam cells and no apparent intimal lesion; 1+, small, foam-cell predominant plaque; 2+, intermediate plaque with multilayered or diffuse foam cells, occasional cholesterol clefts, and few or no apparent acellular degenerative areas; 3+ mixed plaque with fibrous cap that may have cholesterol clefts and an atheromatous core superficially covered by smooth muscle cells; 4+, mixed plaque composed of smooth muscle cells, collagen, and elastic fragments, with a consistent acellular core, fibrous cap, possible calcification and common cholesterol clefts; 5+, advanced complex lesion with multilayered and diffuse foam cells, many deep cholesterol clefts, consistently, large acellular cores, a large amount of extracellular matrix, a consistent fibrous cap and consistent calcification. Dorsal skin tissue was embedded and frozen in Tissue-Tek OCT and 10-μm sections were prepared and stained with hematoxylin and eosin. Skin samples from severe dermatitis lesions were fixed, embedded in paraffin, and then sectioned for staining with hematoxylin and eosin or toluidine blue.  167  C.3 Immunohistochemical studies For the assessment of smooth muscle cells, cryosections were immunolabeled with a primary mouse monoclonal antibody against smooth muscle α-actin, clone 1A4 (Thermo Fisher Scientific, Fremont, CA). For the assessment of macrophage infiltration in early lesions, mice were euthanized after 5 weeks on the Western diet, hearts with attached aortas were removed and embedded in OCT, and cryosections were immunolabeled with a primary rat monoclonal antibody against mouse monocytes/macrophages, clone MOMA-2 (AbD Serotec, Raleigh, NC).  C.4 Quantitative RT-PCR We extracted total RNA from dorsal skin, liver tissue, and cells from mice fed a Western diet using the TRIzol reagent according to manufacturer’s instructions (Invitrogen Canada, Burlington, ON, CA). 1 μg of DNase-treated RNA was reversetranscribed using Superscript II (Invitrogen Canada, Burlington, ON, Canada) to generate RNAse H-treated cDNA for real-time PCR using Power SYBR green PCR Master Mix (Applied Biosystems, Foster City, CA) in an ABI 7500 Fast Real-Time PCR System. We used Gapdh as the invariant control. mRNA levels in control mice were arbitrarily set at 1.  C.5 Measurements of inflammatory molecules. For whole skin ICAM-1 protein analysis, dorsal skin tissue was homogenized in PBS containing complete protease inhibitor (Roche Diagnostics, Laval, Quebec, CA) and stored at -20°C overnight. Supernatants were collected by centrifugation (2000g, 5min) and protein concentration was determined by the assay of Lowry et al. (3). Levels of murine inflammatory protein molecules in plasma and whole skin homogenates from mice fed a Western diet were measured using commercial immunoassay kits (R&D Systems, Minneapolis, MN). The threshold of detection for these assays was 12, 29, 2, and 2pg/mL for interleukin-6 (IL-6), intercellular adhesion molecule (ICAM-1), monocyte chemoattractant protein 1 (MCP-1) and regulated upon activation, normal T expressed and presumably secreted protein (RANTES), respectively. Levels of interleukin (IL)-1β and IL12p70 in all mice, and levels of  MCP-1 and RANTES in mice before  commencement of Western diet were measured using Milliplex multi-analyte  168  profiling assays (Millipore, Billerica, MA) and quantified using a Luminex100 instrument.  C.6 Apolipoprotein analysis Unfractionated plasma levels apoA-I and apoA-II in male and female mice fed Western diet were determined by immunonephelometry with the use of mousespecific antibodies developed in rabbits. Levels of murine SAA in plasma were measured using a commercial immunoassay kit (BioSource, Camarillo, CA), which had a threshold of detection of 270 ng/mL.  C.7 Paraoxonase (PON1) activity After a period of 12 weeks on a Western diet, blood was collected from female Scd1+/+Ldlr-/- mice and Scd1-/-Ldlr-/- mice (homozygous for the Scd1ab-J  allele)  following a four-hour fast. Blood was allowed to clot for 1h on ice and then serum was separated by centrifugation at 4°C.  Serum was mixed with sucrose (final  concentration 0.6%) as a cryoprotectant and frozen at -80°C under nitrogen for less than 3 months. Serum PON1 activity toward phenyl acetate (arylesterase activity) was determined photometrically in the presence of CaCl2 (1 mM)(4), where one unit = 1 µmol phenylacetate hydrolyzed per min.  C.8 Bone marrow transplantation Bone marrow transplantation (BMT) experiments were performed at the Gorlaeus laboratories of the Leiden/Amsterdam Center for Drug Research in Leiden, The Netherlands in accordance with the national laws.  BMT protocols  were approved by the Ethics Committee for Animal Experiments of Leiden University. To induce bone marrow aplasia, female Ldlr–/– recipient mice were exposed to a single dose of 9 Gy (0.19 Gy/min, 200 kV, 4 mA) total body irradiation using an Andrex Smart 225 Röntgen source (YXLON International) with a 6-mm aluminum filter 1 day before the transplantation. Bone marrow was isolated by flushing the femurs and tibias from female Scd1-/- and Scd1+/+ mice. Irradiated recipients received 0.5x107 bone marrow cells by tail vein injection. Animals received a standard laboratory rodent chow diet or Western diet (Diet W, Special Diet Services, Witham, UK).  The hematologic chimerism of the Ldlr–/–  169  mice was determined using genomic DNA from bone marrow by polymerase chain reaction (PCR) at 12 weeks after transplant.  C.9 Macrophage functional studies After 5 days on a Western diet, thioglycollate-elicited peritoneal macrophages obtained from Scd1-/-Ldlr-/- and Scd1+/+Ldlr-/- mice were counted and plated in 24well plates at a density of 400,000 cells per well and the media was changed after 2h. After an additional 24 h, the media was changed to fresh DMEM/10% FBS or  DMEM/10% FBS containing 100 ng/mL lipopolysaccharide (E. coli  O113:H10; Associates of Cape Cod). After 6 h, RNA was isolated from at least two independent wells from each animal for each condition. For cholesterol efflux studies, bone marrow-derived cells were labeled with 0.5 µCi/mL [3H]cholesterol in DMEM/0.2% bovine serum albumin for 24 h. Cholesterol efflux was studied by incubation of the cells with DMEM/0.2% BSA alone or supplemented with 10 µg/mL apoA-I or 50 µg/mL human HDL. [3H]Cholesterol released to HDL after 24h incubation was measured by liquid scintillation counting.  Cholesterol efflux is expressed as the radiolabel released as a  percentage of [3H]cholesterol within cells before addition of acceptor.  C.10 Statistical analysis Data are presented as means plus or minus standard error. Initial analyses were performed by the unpaired two-tailed Student's t-test.  Data that did not  follow a normal distribution as judged by Kolmogorov-Smirnov tests were analyzed with the Mann-Whitney test for unpaired data. For cytokine data the minimum detectable limit was assigned to those values below the limit of detection and the Wilcoxon signed-rank test was used.  Analyses of the  cumulative frequency of dermatitis were performed by a two-sided log rank test. Statistical analysis was performed with GraphPad Prism software and with the open-source R-package (GraphPad, San Diego, CA; R Development Core Team, 2006 (5)). P < 0.05 was considered significant.  170  C.11 References 1. Paigen B, Morrow A, Holmes PA, Mitchell D, Williams RA. Quantitative assessment of atherosclerotic lesions in mice. Atherosclerosis 1987;68:231-40. 2. Singaraja RR, Fievet C, Castro G, James ER, Hennuyer N, Clee SM, et al. Increased ABCA1 activity protects against atherosclerosis. J Clin Invest 2002;110:35-42. 3. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein measurement with the Folin phenol reagent. J Biol Chem 1951;193:265-75. 4. Tsimihodimos V, Karabina SP, Tambaki AP, Bairaktari E, Goudevenos JA, Chapman MJ, et al. Atorvastatin preferentially reduces LDL-associated plateletactivating factor acetylhydrolase activity in dyslipidemias of type IIA and type IIB. Arterioscler Thromb Vasc Biol 2002;22:306-11. 5. R Development Core Team. R: A language and environment for statistical computing [computer program]. Vienna, Austria: R Foundation for Statistical Computing; 2008. Available from:URL:http://www.R-project.org.  171  APPENDIX D Supplemental tables & figures  Table D.1: Conversions between conventional units and SI units. Substance  Conventional Unit Conversion Factor SI unit  Cholesterol  mg/dL  0.0259  mmol/L  Triglycerides mg/dL  0.0113  mmol/L  Glucose  0.0555  mmol/L  mg/dL  172  Table D.2: Serum lipid levels in Ldlr-/- mice transplanted with bone-marrow derived cells lacking SCD1. Chow  Western Diet  Scd1+/+→Ldlr-/-  Scd1-/-→Ldlr-/-  p  Scd1+/+→Ldlr-/-  Scd1-/-→Ldlr-/-  p  TC, mg/dL  285 ± 6 (12)  283 ± 12 (12)  0.92  1025 ± 55 (12)  997 ± 68 (12)  0.75  CE, mg/dL  339 ± 8 (12)  335 ± 14 (12)  0.81  1333 ± 73 (12)  1301 ± 90 (12)  0.79  FC, mg/dL  84.3 ± 2.1 (12)  85.2 ± 3.9 (12)  0.83  237 ± 13 (12)  227 ± 14 (12)  0.61  PL, mg/dL  490 ± 33 (12)  531 ± 29 (12)  0.37  668 ± 31 (12)  696 ± 23 (12)  0.47  TC, total cholesterol; CE, cholesterol esters; FC, free cholesterol; PL, phospholipids. Data represent mean ± SEM. The number of animals in each subgroup is indicated in parentheses.  173  Figure D.1: Lesion area in Ldlr-/- mice lacking SCD1. Lesions in aortic roots of Scd1+/+Ldlr-/- (left) and Scd1-/-Ldlr-/- (right) mice carrying the Scd1ab-2J alleles were stained with Oil Red O to detect accumulation of lipids and photographed. Scale bar, 0.5 mm.  174  Figure D.2: Lesion morphology in Ldlr-/- mice lacking SCD1. For the assessment of lesion complexity, lesions in aortic roots of Scd1+/+Ldlr-/(left) and Scd1-/-Ldlr-/- (right) mice fed a Western diet for 12 weeks were stained with hematoxylin and eosin (H&E). Images of representative sections from the aortic root were captured at a magnification of 20× (a). Smooth muscle cell content (b) in mice fed a Western diet for 12 weeks and macrophage content (c) in mice fed a Western diet for 5 weeks was determined by immunohistochemical staining for α-actin and MOMA-2, respectively.  175  Figure D.3: Skin of Ldlr-/- mice lacking SCD1. Skin sections of Scd1-/-Ldlr-/- mice (a) or Scd1-/-Ldlr-/- mice with dermatitis (b,c) were stained with hematoxylin and eosin (H&E) (a,b) or toluidine blue (c) to visualize mast cells (arrows). Stratum corneum (SC), epidermis (E), dermis (D), fat tissue(F), and ulceration (U).  176  Figure D.4: Inflammation in Ldlr-/- mice lacking SCD1. Plasma cytokine concentrations were determined before commencement of Western diet. Data are represented as proportion of the mean plasma cytokine concentration relative to that in Scd1+/+Ldlr-/- mice. n = 8 mice per group.  177  

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