Department of Endocrinology, Metabolism and Diabetes, Department of Medicine Karolinska University Hospital Huddinge, and Center for Nutrition and Toxicology, NOVUM, Karolinska Institutet, Stockholm, Sweden STUDIES ON THE HORMONAL REGULATION OF BILE ACID SYNTHESIS Thomas Lundåsen Stockholm 2007 ______________________________________________________________________ All previously published papers were reproduced with permission from the publishers. Published and printed by Karolinska University Press Box 200, SE-171 77 Stockholm, Sweden © Thomas Lundåsen, 2006 ISBN 91-7357-053-2 2 T. Lundåsen ____________________________________________________________________________________ ABSTRACT The maintenance of a normal turnover of cholesterol is of vital importance, and disturbances of cholesterol metabolism may result in several important disease conditions. The major route for the elimination of cholesterol from the body is by hepatic secretion of cholesterol and bile acids (BAs) into the bile for subsequent fecal excretion. A better understanding of how hepatic cholesterol metabolism and BA synthesis are regulated is therefore of fundamental clinical importance, particularly for the prevention and treatment of cardiovascular disease. In the current thesis, the roles of known and newly recognized hormones in the regulation of BA synthesis were studied with the aim to broaden our understanding of how extrahepatic structures regulate BA synthesis in the liver. In normal humans, circulating levels of the intestinal fibroblast growth factor 19 (FGF19) were related to the amount of BAs absorbed from the intestine. The results support the concept that intestinal release of FGF19 signals to the liver suppressing BA synthesis. Thus, in addition to the liver - which harbors the full machinery for regulation of BA synthesis - the transintestinal flux of BAs is one important factor in this regulation. In mice, abrogation of the BA enterohepatic circulation by targeted deletion or pharmacological inhibition of the intestinal BA transporter ASBT reduces triglycerides in plasma and in the liver, concomitantly with a reduced hepatic triglyceride synthesis. Sucrose feeding results in an increased intestinal expression of the mouse orthologue of FGF19, FGF15, which can at least partly account for the reduced BA synthesis observed when this diet is fed. The powerful hepatic metabolic regulator FGF21 is induced in ASBT deficient mice, and is strongly upregulated by dietary sucrose. Specific inhibition of ASBT in leptin-deficient ob/ob mice reduces plasma triglyceride and glucose levels concomitantly with increased hepatic FGF21 expression. The ob/ob mice display reduced levels of hepatic LDL and HDL receptors, as well as of the rate-limiting enzyme of BA synthesis, Cyp7a1. These findings may in part explain the elevated plasma (particularly HDL) cholesterol levels in these animals. The Cyp7a1 response to dietary cholesterol is attenuated in ob/ob mice, and - in contrast to wt animals - the plasma cholesterol levels are increased. The HDL receptor SR-BI is positively regulated by leptin treatment of ob/ob mice, whereas Cyp7a1 is not. Selective stimulation of hepatic thyroid hormone receptor β with the drug GC-1 decreases plasma cholesterol and triglycerides dose-dependently, and stimulates hepatic SR-BI and Cyp7a1 in normal mice. GC-1 also reduces elevated levels of plasma cholesterol in animals challenged with cholesterol and BAs. This indicates an important role for TRβ in reverse cholesterol transport, which might be useful in the treatment and prevention of atherosclerosis. It is concluded that hepatic BA synthesis in man is in part controlled from the intestine via FGF19, a novel pleiotropic metabolic regulator. Plasma glucose and triglycerides can be reduced by specific stimulation of BA synthesis or by the administration of a TRβ-specific agonist. Thus, modulation of BA synthesis is a promising approach for the metabolic control of lipid and glucose metabolism which may be important in our attempts to treat and prevent cardiovascular disease. 3 ______________________________________________________________________ LIST OF PUBLICATIONS This thesis is based on the following papers which will be referred to by their Roman numerals. I. Thomas Lundåsen, Wei Liao, Bo Angelin and Mats Rudling. Leptin Induces the hepatic high density lipoprotein receptor Scavenger Receptor B Type I (SR-BI) but not cholesterol 7α-hydroxylase (Cyp7a1) in leptin-deficient (ob/ob) mice. Journal of Biological Chemistry, 2003, 278, 43224-43228 II. Lisen Johansson, Mats Rudling, Thomas S. Scanlan, Thomas Lundåsen, Paul Webb, John Baxter, Bo Angelin, and Paolo Parini. Selective thyroid receptor modulation by GC-1 reduces serum lipids and stimulates steps of reverse cholesterol transport in euthyroid mice. Proceedings of the National Academy of Sciences USA, 2005;102,10297-10302 III. Thomas Lundåsen, Cecilia Gälman, Bo Angelin and Mats Rudling. Circulating intestinal fibroblast growth factor 19 has a pronounced diurnal variation and modulates bile acid synthesis in man. Journal of Internal Medicine. 2006;260;530-536. IV. Thomas Lundåsen, Eva-Marie Andersson, Michael Snaith, Helena Lindmark, Sandra Schreyer, Ann-Margret Östlund-Lindqvist, Bo Angelin, and Mats Rudling. Interruption of bile acid circulation improves triglyceride metabolism and normalizes elevated plasma glucose levels. Manuscript. Submitted. 4 T. Lundåsen ____________________________________________________________________________________ LIST OF ABBREVIATIONS ABC ATP Binding Cassette ACC Acetyl Coenzyme A Carboxylase ACL Acidic Citrate Lyase BA Bile Acid BSEP Bile Salt Export Pump C4 7alpha-hydroxy-4-cholesten-3-one CDCA Chenodeoxycholic acid CYP7A1 Cholesterol 7α Hydroxylase CYP8B1 Cholesterol 12α Hydroxylase FAS Fatty Acid Synthase FGF Fibroblast Growth Factor FGFR Fibroblast Growth Factor Receptor FXR Farnesoid X Receptor HDL High Density Lipoprotein HMGCoAR β-hydroxy-methyl-glutaryl CoenzymeA reductase HNF4α Hepatocyte Nuclear Factor 4α IBABP Ileal Bile Acid Binding Protein JNK c-jun N-terminal Kinase LDL Low Density Lipoproteins LRH-1 Liver Receptor Homologue 1 LXRα Liver X Receptor α PPARα Peroxisome Proliferator Activated Receptor α PXR Pregnane X Receptor RXR Retinoic acid X Receptor SCD1 Stearoyl CoenzymeA Desaturase 1 SHP Small Heterodimer Partner SLC10A2 Solute Carrier 10A2, IBAT, ASBT SR-BI Scavenger Receptor B type I SREBP1 Sterol Regulatory Element Binding Protein 1 SREBP2 Sterol Regulatory Element Binding Protein 2 T3 Thyroid hormone VLDL Very Low Density Lipoprotein wt wild type 5 ______________________________________________________________________ CONTENTS ABSTRACT 3 LIST OF PUBLICATIONS 4 LIST OF ABBREVIATIONS 5 1. INTRODUCTION 7 1.1 Bile acids and cholesterol 7 1.2 Bile acid synthesis 9 1.3 Regulation of bile acid synthesis 10 1.4 Hormonal effects on bile acid synthesis 12 1.5 The enterohepatic circulation of bile acids 14 2. AIMS 17 3. MATERIALS AND METHODS 18 4. RESULTS 21 5. GENERAL DISCUSSION 25 6. CONCLUSIONS 32 7. ACKNOWLEDGEMENTS 33 8. REFERENCES 35 6 T. Lundåsen ____________________________________________________________________________________ 1 INTRODUCTION The maintenance of a normal turnover of cholesterol is of vital importance for all organisms. In humans, disturbances of cholesterol metabolism may result in several important disease conditions, such as dyslipidemia and atherosclerosis, gallstones, malabsorption and neurological disorders. The liver is a key organ in cholesterol metabolism being responsible for both synthesis and elimination of cholesterol. The liver is also the major site of uptake of cholesterol from blood thereby being the major regulator of cholesterol levels in the circulation. The major route for the elimination of cholesterol from the body is by hepatic secretion of cholesterol and bile acids (BAs) into the bile for subsequent fecal excretion. A better understanding of how hepatic cholesterol metabolism and BA synthesis may be regulated is therefore of fundamental clinical importance, in particular in order to prevent and treat cardiovascular disease. Hormones are substances that are secreted into body fluids and transported to other organs, where they produce specific metabolic effects. In the current studies the roles of known as well as newly recognized hormones in the regulation of BA synthesis have been studied. A particular aim was to broaden our understanding of how extrahepatic structures can regulate BA synthesis in the liver. 1.1 BILE ACIDS AND CHOLESTEROL The secretion of bile is an important route for the excretion not only of excess lipids, but also of toxins and many drugs and their metabolites, as well as endogenous hormones. The major constituents of bile are BAs, cholesterol, phospholipids, bilirubin and water. In human hepatic bile the molar ratio between cholesterol and BAs is about 1:20 1. BAs are formed from cholesterol and are amphipathic molecules with a hydroxylated sterol core and a side chain terminated by a carboxyl group 2. They are essential for the absorption of dietary lipids and fat-soluble vitamins in the small intestine where they act as emulsifiers 3. The liver is the only site where BAs are produced. Hepatic cholesterol for BA synthesis can be derived both from de novo synthesis from acetate and from delivery by various lipoproteins (Figure 1). In the postprandial state, absorbed cholesterol reaches the liver in chylomicron remnants which are taken up with high efficiency. Cholesterol is also transported from