Is it really that simple? Alyssa Hasty, PhD Associate Professor Molecular Physiology and Biophysics

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go wrong in humans Animal models dlto study lipoprotein metabolism and

1 Alyssa Hasty, PhD Associate Professor Molecular Physiology and Biophysics Why we care about hepatic lipogenesis Control of lipid synthesis What can go wrong in humans Animal models dlto study lipoprotein metabolism and atherosclerosis The Simplified Model The Simplified Model Is it really that simple? 1

2 Synthesized Cholesterol (900 mg/day) Bile Fecal Sterols (1,200 mg/day) SREBP 1 and SREBP 2 LXR PPAR α, β, γ1, and γ2 HNF 4 RXR Adapted with permission from the American Journal of Clinical Nutrition. Am J Clin Nutr. American Society for Clinical Nutrition. Dietschy JM. Am J Clin Nutr. 1997;65(suppl):1581S 1589S. 2

Transcriptionally activate a cascade of enzymes involved in synthesis of Cholesterol Fatty acids Triglycerides Phospholipids Acetyl CoA SREBP HMG- CoA Sterol Response Element Binding Protein

Endocytosis Members of the basic helix loop helix leucine zipper family of transcription factors Have 3 domains: NH 2 DNA binding 2 hydrophobic transmembrane spanning segments COOH regulatory domain

3 Transcriptionally activate a cascade of enzymes involved in synthesis of Cholesterol Fatty acids Triglycerides Phospholipids Acetyl CoA SREBP HMG- CoA Sterol Response Element Binding Protein Mevalonate Squalene synthase Farnesyl pyrophosphate Farnesyltransferase Farnesylated proteins Squalene Geranylgeranylated proteins Dolichol Geranylgeranyl pyrophosphate Cholesterol Ubiquinones -R Endocytosis Members of the basic helix loop helix leucine zipper family of transcription factors Have 3 domains: NH 2 DNA binding 2 hydrophobic transmembrane spanning segments COOH regulatory domain Precursor bound to ER in inactive form Translocated to Golgi upon sterol deprivation, cleaved and sent to nucleus for lipid synthesis Bind to SRE and E boxes SREBP 1a and 1c activate fatty acid synthesis and SREBP 2 activates cholesterol synthesis J. Clin. Invest :

natural and synthetic ligands Natural ligands are sterol metabolites such as 22(R),24(S) hydroxycholesterol LXRs are constitutively

4 Chawla et al. Science 2001 LXRβ is expressed ubiquitously LXRα is expressed in liver, adipose tissue and macrophages Both respond to the same natural and synthetic ligands Natural ligands are sterol metabolites such as 22(R),24(S) hydroxycholesterol LXRs are constitutively nuclear Cholesterol 7α hydroxylase (CYP7A1) rate limiting step in conversion of cholesterol into bile acids LXRα KO mice lose ability to upregulate CYP7A1 and develop fatty livers Mice lacking LXRα/β have a more severe phenotype Mice lacking LXRβ are normal 4

Dietary and secreted biliary cholesterol enters intestinal lumen and is absorbed by enterocytes LXR agonist reduce cholesterol l absorption by upregulation of LXREcontaining genes ABCA1, ABCG5 and

5 Dietary and secreted biliary cholesterol enters intestinal lumen and is absorbed by enterocytes LXR agonist reduce cholesterol l absorption by upregulation of LXREcontaining genes ABCA1, ABCG5 and ABCG8 pump cholesterol back into the intestinal lumen Peroxisome proliferator activated receptors Zhang and Mangelsdorf Mol Interv Apr;2(2):78-87 Members of nuclear hormone receptor superfamily Ligand dependent Heterodimerize with RXR and bind to PPARresponse elements (PPREs) in target gene promoters There are 3 isoforms: PPARγ, PPARα, and PPARδ (with 60 80% homology in ligand and DNA binding domains) PPARγ and PPARα synthetic ligands have been produced for treatment of hyperlipidemia and diabetes Lipid uptake Lipid transport Lipid storage Lipid disposal 5

Ligands are native and oxidized unsaturated long

Thiazolidinediones ld d (TZDs) are synthetic ligands

or other disorders that affect lipoprotein metabolism

monogenic disorders that may involve triglyceride or

6 Ligands are native and oxidized unsaturated long chain fatty acids 15 deoxy Δ 12,14 prostaglandin J2 Thiazolidinediones ld d (TZDs) are synthetic ligands Secondary to diet, lifestyle, obesity, medications, or other disorders that affect lipoprotein metabolism (e.g., diabetes) and are polygenic in origin Primary monogenic disorders that may involve triglyceride or cholesterol metabolism Martin MJ et al. Lancet. 1986;II:

Low-density lipoprotein particles -Familial hypercholesterolemia -Familial defective apob-100 -Autosomal dominant hypercholesterolemia -Familial sitosterolemia -Hypobetalipoproteinemia

7 Low-density lipoprotein particles -Familial hypercholesterolemia -Familial defective apob-100 -Autosomal dominant hypercholesterolemia -Familial sitosterolemia -Hypobetalipoproteinemia -Abetalipoproteinemia Lipoprotein (a) Familial Lp(a) hyperlipoproteinemia Remnant lipoproteins Dysbetalipoproteinemia type III Hepatic lipase deficiency Triglyceride-rich lipoproteins Lipoprotein lipase deficiency ApoC-II deficiency Familial hypertriglyceridemia Familial combined hyperlipidemia High-density lipoprotein particles ApoA-I deficiency Familial HDL deficiency/tangier Disease Familial LCAT Deficiency syndromes CETP deficiency R ApoB PCSK9 ARH ABCG5/ABCG8 ApoB MTP APOA APOE HL LPL APOCII Polygenic Polygenic APOAI ACAT LCAT CETP Familial hypercholesterolemia disorders involving and R Tangier s Disease and CETP Deficiency disorders involving HDL Receptor mutations Familial defective apolipoprotein B (FDB) PCSK9 (proprotein convertase subtilisin like like kexin type 9) Autosomal Recessive Hypercholesterolemia (ARH) 7

8 Normal Coronary Artery Coronary Artery Disease 8

Reductase, the rate limiting enzyme in cholesterol biosynthesis Inhibition i of cellular l cholesterol l biosynthesis i results in up

9 Lifestyle changes diet and exercise Statins treatment of choice for C reduction Bile acid sequestrants t block bile acid reabsorption forcing upregulation of receptors in liver Cholesterol absorption inhibitors such as ezetimibe Dietary supplements Inhibit HMG CoA Reductase, the rate limiting enzyme in cholesterol biosynthesis Inhibition i of cellular l cholesterol l biosynthesis i results in up regulation of receptors Increased receptors lead to increased uptake of plasma and decreased plasma cholesterol apheresis Liver transplantation Gene transfer 9

10 Endothelial Cell ApoB lipolysis Space of Disse lipolysis ApoB ApoE R LRP HSPG Hepatocyte Internalization FPLC of R(-/-) Mouse Serum Endothelial Cell ApoB lipolysis Space of Disse lipolysis Hepatocyte ApoB ApoE R LRP HSPG Accumulation of 10

Endothelial Cell lipolysis Space of Disse lipolysis Hepatocyte lipo ApoB ApoE R LRP HSPG Cholesterol (mg/dl) 40 35 30 25 20 15 10 5 V ApoE(-/-) HDL C57BL/6 Accumulation of lipoproteins 0 Fraction

11 Endothelial Cell lipolysis Space of Disse lipolysis Hepatocyte lipo ApoB ApoE R LRP HSPG Cholesterol (mg/dl) V ApoE(-/-) HDL C57BL/6 Accumulation of lipoproteins 0 Fraction Number Agouti Mice Yellow coat color (AP blocks binding of α MSH to MCR1) Hyperphagia (AP binds to and inactivates MCR4) Moderate adult onset obesity R / Mice Increased levels Do not develop atherosclerosis unless on a WD Crossed obese Agouti mice with R / mice to study hyperlipidemia and obesity. 11

4 months old a/a;r-/- Ay/a;R-/- Western Diet (WD) or Chow diet (CD) 7 months old Body Composition Plasma Lipids ATM Accumulation holesterol (mg/dl) Ch 1500 1200 900 600 300 Cholesterol * ** Tri

12 4 months old a/a;r-/- Ay/a;R-/- Western Diet (WD) or Chow diet (CD) 7 months old Body Composition Plasma Lipids ATM Accumulation holesterol (mg/dl) Ch Cholesterol * ** Tri glyceride (mg/dl) Triglyceride * ** 4 Experimental Groups 1. a/a;r-/- on CD (Lean) 2. Ay/a;R-/- on CD (GIO) 3. a/a;r-/- on WD (DIO) 4. Ay/a;R-/- on WD (GIO/DIO) * ** 0 Lean GIO DIO GIO/DIO P<0.001 vs Lean and GIO P<0.05 vs DIO NEFA (meq/ml) NEFA * ** 0 Lean GIO DIO GIO/DIO * ** P<0.01 vs All P<0.001 vs All 0.0 Lean GIO DIO GIO/DIO * ** P<0.001 vs Lean and GIO P<0.05 vs DIO ol (mg/dl) Cholestero 150 V Lean GIO 125 DIO GIO/DIO HDL Fraction Number es (mg/dl) Triglyceride Lean 450 V GIO 400 DIO 350 GIO/DIO HDL Fraction Number In the 1950s two independent lines of obese mice (ob/ob and db/db) developed from spontaneous mutations Excessively obese Early onset Hyperphagia Inappropriately decreased energy expenditure In the 1970s it was determined that the ob/ob mice were missing a circulating satiety factor and that db/db mice were missing the receptor 12

13 Nishina et al. Metabolism 43:554 FPLC of db/db;r(-/-) Mouse Serum Genotype N Cholesterol (mg/dl) Triglyceride (mg/dl) R(+/+) R(-/-) ob/ob;r(+/+) 4 81 ± ± ± ± ± ± 12 ob/ob;r(+/-) ± ± 8 ob/ob;r(-/-) ± ± 172 Hasty et al. JBC 276:

14 Production Rates (mmol trigs/hour/kg) C57BL/6 140 R(-/-) ob/ob ob/ob;r(-/-) Lipoprotein Clearance in ob/ob;r(-/-) Mice Turnover of 125 I V (d<1.019) Turnover of 125 I (d= ) Time (hours) 14

15 apoe(-/-) Obese apoe(-/-) En face Lesion Area db/db db/db;r -/- db/db;apoe -/- 0% 3.2% 7.3% Acknowledgements: Some slides from Lipids Online Slide Library, 15

Cholesterol metabolism. Function Biosynthesis Transport in the organism Hypercholesterolemia

Cholesterol metabolism. Function Biosynthesis Transport in the organism Hypercholesterolemia Cholesterol metabolism Function Biosynthesis Transport in the organism Hypercholesterolemia - component of all cell membranes - precursor of bile acids steroid hormones vitamin D Cholesterol Sources: dietary