Studies on the Hormonal Regulation of Bile Acid Synthesis

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

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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.

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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

______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 the liver to peripheral cells in the VLDL-IDL-LDL chain; the major fraction of LDL cholesterol returns to the liver through the uptake by LDL receptors. A particularly interesting concept is that of reverse cholesterol transport, where cholesterol is taken up from peripheral cells by HDL particles and carried to the liver both in HDL and after transfer to the other lipoproteins. This mechanism is supposed to explain the role of HDL as a protective agent in the process of atherosclerosis 4.

The BAs are transported from the liver via the bile ducts to the gallbladder 3. After the ingestion of a meal, the hormone cholecystokinin (CCK) is released from the intestine leading to contraction of the gallbladder and propelling of the stored BAs into the intestine. After contributing to fat and sterol absorption in the upper small intestine, the BAs are actively absorbed from the ileum and then returned to the liver via the portal

______vein, thereby completing their enterohepatic circulation. The intestinal absorption of BAs is efficient and >95% of the BAs return to the liver for reutilization. The continuous fecal loss of BAs is compensated for by new hepatic synthesis. Owing to their detergent like properties BAs are cytotoxic and their relative abundance and localization is therefore carefully controlled. The total pool of BAs in humans amounts to about 3-4 grams and traverses the enterohepatic circulation six to twelve times daily. The BA biosynthetic pathways in the liver involve at least 17 different enzymes and account for 90% of all breakdown of cholesterol in the body. Thus, the hepatic production of BAs is central for overall cholesterol homeostasis.

Figure 1.

Simplified graph on some major pathways in cholesterol and BA metabolism. The liver synthesises and exports VLDL particles rich in triglycerides that are delivered to peripheral tissues (muscle and fat). The particles are transformed into smaller cholesterol rich LDL particles that are taken up by the liver by the LDL receptors. Cholesterol from peripheral tissues is taken up by HDL for transport to the liver where one mechanism is uptake of cholesterol via SR-BI. Hepatic cholesterol is utilized for VLDL and BA synthesis where CYP7A1 is the rate limiting enzyme. BAs and cholesterol is secreted into the bile that is stored in the gallbladder. After a meal the gallbladder is contracted and bile enters the gut. >95% of the BAs are actively transported back in the portal vein to the liver for reuse.

8 T. Lundåsen ______1.2 BILE ACID SYNTHESIS

In humans, the primary BAs produced are cholic acid and chenodeoxycholic acid (CDCA), whereas in mice these are cholic acid and β-muricholic acid 2. Before leaving the hepatocyte the primary BAs are conjugated to the amino acids glycine and taurine, which enhances their solubility 3. The conjugated BAs are present as anionic salts under physiological pH 3. The primary BAs may undergo further modifications by anaerobic bacteria in the intestine. A multitude secondary BAs are produced by these bacteria. The predominant secondary BAs are deoxycholic acid and lithocholic acid, produced from cholic acid and CDCA, respectively 5.

The synthesis of BAs can occur either by the neutral, or “classic”, pathway or by the acidic, or “alternative” pathway. Schematically, the synthesis of a primary BA can be divided into different steps: 1) Initiation, generally involving the 7α-hydroxylation of a sterol precursor. 2) Additional modifications of the ring structure. 3) Oxidation and shortening of the side chain. 4) Conjugation of the BA to an amino acid, normally taurine or glycine 3. The classic pathway starts with the 7α-hydroxylation of cholesterol. This reaction is catalyzed by the liver specific microsomal enzyme cholesterol 7α-hydroxylase (CYP7A1), a member of the cytochrome p450 superfamily 6. Importantly, the reaction catalyzed by CYP7A1 is the rate limiting step of the classic BA biosynthesis pathway 6 7, 8.

Figure 2. Schematic display of the two major pathways to synthesise BAs. The classic neutral pathway is initiated by the hydroxylation of cholesterol at the 7α position by CYP7A1 whereas the alternative pathway CYP27A1 initiates hydroxylation at the 27 position of the cholesterol molecule. Both pathways ultimately utilizes CYP8B1 (12α-hydroxylation) for the formation of cholic acid. Dashed arrows indicate multiple enzymatic steps.

______After the 7α-hydroxylation step in the classic pathway the hydroxylated intermediate is converted to 7α−hydroxy-4-cholesten-3-one (C4) by action of the microsomal 2 C27βHSD enzyme . Optionally, the liver specific p450 enzyme CYP8B1, or 12α−hydroxylase will then hydroxylate the 12α position of 7α−hydroxy-4-cholesten-3- one 9, 10. A series of enzymatic steps will ultimately produce cholic acid. The CYP8B1 enzyme thus controls the ratio between cholic acid and CDCA 11. Since cholic acid and CDCA vary in hydrophobicity, CYP8B1 therefore determines the hydrophobic balance of BAs in the bile. If the 12α− hydroxylation step does not occur, CDCA will be the end product. After the modification of the steroid ring structure, the mitochondrial p450 enzyme member sterol 27-hydroxylase catalyzes the steroid side chain oxidation 12.

The alternative pathway for biosynthesis of BAs utilizes oxysterols as substrates for 7α−hydroxylation instead of cholesterol. The net contribution of oxysterols to BA production in humans has been estimated to as little as 5 to 10% 13-16. The main end product of this pathway is CDCA but cholic acid may also be generated. Hydroxylation of cholesterol can produce 24-hydroxycholesterol, 25-hydroxy-cholesterol and 27- hydroxycholesterol 17. The mitochondrial enzyme CYP27A1 catalyzes the conversion of cholesterol to 27-oxysterol. CYP27A1 is expressed in liver, lungs, vascular endothelium, atherosclerotic plaques and macrophages 14, 18-21. This enzyme can also produce 24- and 25-hydroxysterol 22. Of note is that all intermediates destined to become BAs must undergo modification by CYP27A1 since it participates in both the initiation and side chain oxidation steps. The 7α−hydroxylation step in the alternative pathway is carried out by either of the enzymes CYP39A1 or CYP7B1. The final steps of the alternative pathway are similar to those of the classic pathway.

Due to the wide substrate specificity and the participation of enzymes in multiple steps in the complex processes of BA biosynthesis a strict pathway definition may not always hold true. Mice with deletion of the CYP7A1 gene display liver failure, vitamin deficiency and lipid malabsorption. About 90% of these mice die within the first three weeks 23-25. The survivors have a BA pool that is only 25% of the normal size 24. These mice produce relatively more BAs via the alternative pathway where oxysterols serve as the initial substrate for 7α-hydroxylation. Only very few humans with CYP7A1 mutations have been identified. These individuals have hypercholesterolemia and presumably an increased risk to develop gallstone disease 16.

1.3 REGULATION OF BILE ACID SYNTHESIS

Since the classic (neutral) pathway of BA synthesis is the largest contributor to overall BA production in humans and operates with a much wider regulatory capacity, in both rodents and humans, studies on the regulation of BA synthesis have focused on this pathway 26-28. BA synthesis is regulated by the BAs themselves in a finely tuned manner exhibiting end-product feedback inhibition 29, 30. The use of BA binding polymers that inhibit BA uptake from the intestine and thus their recirculation indicated a regulatory role of BAs on their synthesis 31, 32. Further results have shown that in order for BAs to exert such

10 T. Lundåsen ______an inhibitory effect on BA synthesis they must be given orally and not parenterally 33. This fact, and the fact that ligation of the common bile duct results in an unexpected several fold stimulation of BA synthesis 34-36, has suggested that the intestine in some way could inhibit hepatic BA synthesis.

FXR. The transcriptional repression of CYP7A1 and CYP8B1 by BAs is dependent on the nuclear hormone receptor Farnesoid X Receptor (FXR) 37. FXR activates transcription upon binding to BAs and exists as an obligate heterodimer together with Retinoic acid X Receptor (RXR) when bound to DNA 38. When intracellular BAs accumulate, FXR initiates transcription of the atypical orphan nuclear hormone receptor Small Heterodimer Partner (SHP) 39, 40. SHP lacks a DNA binding domain 41.

Figure 3. Bile acid mediated repression of CYP7A1 transcription. Bile acids induce the transcription of SHP (small heterodimer partner) through activation of FXR. SHP represses transcription of CYP7A1 by direct interaction with LRH-1. LRH-1 is necessary for CYP7A1 transcription. A binding site for LRH-1 is in addition located in the SHP promoter. LXRα can induce the CYP7A1 gene upon cholesterol accumulation in mice and rats. HNF4α also induces CYP7A1 transcription.

Instead SHP can act as a transcriptional repressor by direct protein-protein interaction with several members of the basic helix-loop-helix type transcription factors and a number of nuclear hormone receptors, including Liver Receptor Homologue 1 (LRH-1) and Hepatocyte Nuclear Factor 4α (HNF4α) 42. LRH-1 and HNF4α are normally positive regulators of both CYP7A1 and CYP8B1. Consequently, the BA-induced SHP expression initiated by FXR leads to decreased BA production due to SHP protein binding to LRH-1 and HNF4α. The importance of FXR and SHP in the regulation of BA synthesis is underscored by the observation that the BA-induced suppression of CYP7A1 is dampened in mice deficient in either FXR or SHP 37, 43, 44. The existence of several other BA-induced repressive pathways is evident from the fact that BA feeding to SHP deficient mice still represses CYP7A1 43, 44. The activation of the xenobiotic nuclear hormone receptor Pregnane X Receptor (PXR) is responsible for CYP7A1 repression in SHP deficient mice although the molecular mechanisms for this are unclear 44. The presence of a third pathway in the suppression of CYP7A1 was suggested from the use of a chemical c-jun N-terminal Kinase (JNK) inhibitor in cholic-acid-fed SHP null mice, where it was demonstrated that JNK inhibition ablated

______the BA suppressive effects on CYP7A1 independently of SHP and PXR 44. In addition, JNK inhibition in primary wt mouse hepatocytes increases CYP7A1 expression also in the absence of exogenous BAs 44, 45. Moreover, feeding SHP null mice with the BA binding resin cholestyramine induces CYP7A1 expression, which indicates the presence of other, not yet identified suppressive mechanisms 43.

Liver X Receptor α (LXRα). In mice and rats, dietary cholesterol induces the transcription of CYP7A1 and thereby enhances the conversion of cholesterol to BAs 6, 46. This effect is mediated via LXRα 47-49. An LXR-response element is located in the proximal promoter of mouse and rat CYP7A1, but not in CYP8B1 48. LXR is activated by oxysterols, dietary plant sterols such as stigmasterol, and late intermediates of the cholesterol synthesis pathway such as desmosterol 50, 51. LXRα null mice accumulate cholesterol in their livers upon cholesterol feeding and cannot induce CYP7A1 52, indicating that the presence of LXR feed-forward regulation of CYP7A1 is one of the reasons why rodents are resistant to dietary cholesterol. Importantly, this LXR-response element is not functional in the human CYP7A1 promoter and LXR induction in human hepatocytes decreases the expression of CYP7A1 53-57. In line with this, humans are clearly more sensitive to dietary cholesterol than are rodents.

Peroxisome Proliferator Activated Receptor α (PPARα). Activation of the nuclear hormone receptor PPARα has been demonstrated to decrease CYP7A1 expression in mice, rats and in humans 58, 59. Mechanistic studies indicated that PPARα suppresses CYP7A1 expression through antagonizing effects on HNF4α 60. PPARα deficient mice express normal levels of CYP7A1 which may be indicative of a minor role for this nuclear receptor on CYP7A1 basal expression 61. On the contrary, PPARα agonists increase the expression of CYP8B1, which would lead to an increased cholic acid content of the BA pool 62. In humans, treatment with fibrate drugs suppresses CYP7A1 activity and reduces BA synthesis which may contribute to the relative increase in biliary lipid cholesterol secretion predisposing for cholesterol gallstone development during such therapy 59.

1.4 HORMONAL EFFECTS ON BILE ACID SYNTHESIS

Several hormones have been shown to influence the synthesis of BAs, both in vitro and in vivo. Some of these are briefly discussed below. Insulin, with well characterized effects on glucose and lipid metabolism, has been shown to decrease the expression of CYP7A1 gene in rat hepatocytes 63. Other studies have suggested a role for the insulin regulated protein Foxo1 on the mouse and rat CYP7A1 promoter where it would repress transactivation 64. Recent studies using human primary hepatocytes suggested that insulin has dual effects on the human CYP7A1 expression, with an acute induction followed by a repression of the expression 64. Studies employing adenoviral mediated RNAi inhibition of hepatic insulin receptor substrates 1 and 2 (IRS1 and IRS2) showed increased CYP7A1 expression in fasting mice 65. The situation in diabetic humans with regards to BA synthesis has not been fully clarified, but some studies have indicated that increased BA pool size together with increased BAs excretion occurs in diabetic patients 66, 67.

12 T. Lundåsen ______

Thyroid hormone, 2,5,3’-triiodothyronine (T3) is a potent regulator of cholesterol 68-70 metabolism, and patients with hypothyroidism display hypercholesterolemia . T3 binds and activates the nuclear hormone receptors TRα and TRβ. TRβ is the dominant 71 variant in liver . Like insulin, T3 is increased in the fed state and decreased during 72 starvation . Administration of T3 to rats increases the expression of CYP7A1 but decreases the expression of CYP8B1 73-75. Studies on TR deficient mice have shown 76 that TRβ is responsible for the effects of T3 on CYP7A1 in mice . Hypothyroid mice express very low or undetectable levels of CYP7A1 mRNA, which can be reconstituted by T3 supplementation. Studies measuring BA synthesis in humans with hypothyroidism have resulted in variable results, but do not indicate a major 77, 78 importance of T3 for the basal level of BA production in man . An element in the human CYP7A1 promoter that confers to T3-induced repression has been described, however, and the human CYP7A1 gene is dose-dependently repressed by T3 in studies 79, 80 on primary human hepatocytes . Species differences in the T3-induced changes in BA synthesis may therefore need further attention.

Growth hormone (GH) has been shown to exert a stimulatory effect on CYP7A1 expression and activity in rats and mice 81, 82. However, this effect is lacking in man under conditions tested so far 83, 84. The physiologic role of GH mediated stimulation of BA synthesis in rodents is unclear but GH does not appear to be involved in the diurnal regulation of BA synthesis in rats 82.

Glucagon, like insulin and T3 is intimately linked to feeding-fasting. Glucagon levels are higher in the fasted state than in the fed state 72. Glucagon stimulates the accumulation of 3’,5’-cyclic monophosphate (cAMP) via binding to its cell surface receptors. In mice, CYP7A1 is induced under fasting conditions 85. Infusion of glucagon to rats was seen to increase the activity of CYP7A1 86. In contrast, it was shown that cAMP strongly inhibits the human CYP7A1 gene in primary hepatocyte cultures via a PKA dependent mechanism 87. The specific role of glucagon in the regulation of BA synthesis in vivo is thus not yet fully understood.

Leptin is a peptide hormone secreted from adipocytes. Its level in plasma is closely linked to adipose mass 88. Leptin is well known for its effect on food intake and has been described as a satiety factor 88. Ob/ob mice which have a sporadic mutation in the leptin gene, causing a non-functional protein, suffer from massive overweight due to overeating 89-91. The adipose tissue as well as the liver is dramatically enlarged in these mice due to fat accumulation. Ob/ob mice display hyperlipidemia, hyperglycemia and hyperinsulinemia, at least partially as a result of peripheral insulin resistance. In addition, leptin has effects on the immune system, the reproductive system and on the control of body temperature 88. Since leptin affects metabolic rate, food restriction alone will not fully correct the metabolic profile of ob/ob mice 92. Leptin acts through its cognate leptin receptor, which is an IL-6 family type receptor 88, 93. The db/db mouse and the Zucker fatty rat, which have a similar phenotype as the ob/ob mouse, lack a functional leptin receptor 94-96. The increased level of plasma cholesterol in ob/ob mice - as well as in db/db mice and Zucker fatty rats – is mainly due to higher plasma HDL cholesterol 97. It has been indicated that ob/ob mice have a

______defective HDL plasma clearence and an abrogated intracellular processing of cholesterol Tall 98, 99. In addition, fat Zucker rats show markedly reduced LDL receptor levels in their livers 100. The literature on the potential influence of leptin treatment on BA synthesis is limited and partly contradictory 101-103. Increased BA synthesis has been reported in human obesity 104-106. Leptin resistance is a common feature of obesity, which probably explains why the use of leptin as a weight reducing agent in obese humans has generally been of little success.

Fibroblast growth factors (FGFs) may be involved in the regulation of BA synthesis. Both the human FGF19 and its mouse orthologue FGF15 have been demonstrated to inhibit BA synthesis in the mouse 107, 108. Since these FGFs are expressed in the ileum and are driven by BA activated FXR, it has been suggested that intestinal FGF 15 may act as an intestinal hormone regulated by BA levels in the enterocyte 108. No information on whether such a mechanism may exist in humans has been available 109.

1.5 THE ENTEROHEPATIC CIRCULATION OF BILE ACIDS

Hepatic excretion of BAs The enterohepatic cycling of BAs is dependent on the presence of transporter proteins at the basolateral and apical membrane of cells in the liver and the small intestine. BAs are excreted from the hepatocyte into a network of small tubules (canaliculi). Canalicular excretion of bile salts is the rate limiting step in bile formation 110. In addition to serving as reservoir for bile, the gallbladder concentrates bile. The contents of the gallbladder are released into the intestine via the sphincter of Oddi, a muscular structure connecting the bile duct and the pancreatic duct with the duodenum. Secretion of bile is induced by gallbladder contraction in response to CCK, which induces nervous stimuli through the CCK receptors on the musclular cells surrounding the gallbladder 112. Once expelled into the duodenum, the BAs will solubilize lipophilic nutrients, cholesterol and fat soluble vitamins, thereby promoting their intestinal absorption.

Intestinal uptake of BAs Although passive uptake of BAs occurs through diffusion throughout all the intestine, the main mechanism of BA conservation is active uptake in the distal (terminal) ileum. This uptake is Na+-dependent and mediated by the ASBT protein, expressed at high levels at the apical brush border membranes of enterocytes in the distal ileum. The ASBT substrate affinity appears limited to bile salts only 113. Importantly, the ontogeny of ileal ASBT expression and ileal sodium dependent taurocholate uptake is strikingly similar 114. Moreover, inherited mutations in the human ASBT gene result in BA malabsorbtion and lead to interrupted enterohepatic circulation of BAs 113. Studies employing mice with genetically deleted ASBT gene have confirmed that ASBT mediates the predominant fraction of BA uptake in mice 115. The intracellular transport of BAs from the apical side of the enterocyte to the basolateral side is thought to be mediated by the ileal BA binding protein (IBABP). Basolateral efflux of BAs from the enterocyte to the portal blood has recently been shown to mainly occur via the heterodimeric organic solute transporter ostα/ostβ 116.

14 T. Lundåsen ______Another potential candidate for basolateral efflux of BAs from the enterocyte is the MRP3 transporter, which displays high expression in terminal ileum and also colon 117.

Figure 4. Schematic overview of some important steps in the enterohepatic circulation of BAs. BA synthesis from cholesterol is regulated by CYP7A1. In the liver BAs are pumped into the bile that is transported to the gallbladder where it is stored. After a meal the gallbladder contracts and bile is expelled into the intestine. In the terminal ileum BAs are pumped into the enterocytes by the ASBT protein. The BAs are pumped out of the enterocytes into the portal vein by OSTα / β and by MDR3. The enterohepatic cycle is completed upon active hepatic uptake of BAs from the portal vein by NTCP and the OATPs. ABCG5/8 transports cholesterol into the bile whereas phospholipids are transported into the bile by MDR3 and MRP2. The hepatocyte is a polarized epithelial cell with basolateral and apical plasma domains, where the apical side faces the caniculi. The concentration gradient of BAs is large between the canaliculus and the hepatocyte 111. The excretion of BAs from the hepatocyte to the canalicular system is ATP driven. The excretory transport proteins in the hepatocyte for BAs and other bile components are members of the large ABC (ATP Binding Cassette) superfamily 110.

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Hepatic uptake of BAs

The uptake of BAs from the portal circulation in the liver constitutes the final step of their enterohepatic circulation. This uptake process is mediated by both Na+-dependent and independent transport mechanisms 118. The first pass hepatic extraction rates of BAs from the portal blood are in the range of 75- 95%, thus representing a highly efficient process 119. The sodium dependent uptake is controlled by the NTCP protein (Sodium Taurocholate Co-transporting Peptide) at the basolateral membranes of hepatocytes. NTCP is, like ASBT, a member of the SLC superfamily and is also known as SLC10A1. Like ASBT, NTCP expression in development has a similar ontogeny to sodium dependent bile salt uptake 120. Sodium independent basolateral uptake of bile salts are governed by several members of the OATP-family of Organic Anion Transporting Polypeptides 121.

16 T. Lundåsen ______2 AIMS

The studies reported in this Thesis were performed to evaluate the roles of known as well as newly recognized hormones in the regulation of BA synthesis. A particular aim was to broaden our understanding of how extrahepatic structures can regulate BA synthesis in the liver. The following specific questions were addressed:

1. Does leptin have a role in the regulation of BA and cholesterol metabolism?

2. What are the molecular mechanisms which cause the lipid lowering effects observed following activation of hepatic TRβ? Can such therapy stimulate cholesterol excretion from the body, i.e. promote reverse cholesterol transport?

3. Is the newly described intestinal peptide FGF 19 present in the circulation of normal humans? Does FGF 19 have a role in the hormonal regulation of BA synthesis in man?

4. Can stimulation of BA synthesis through targeted deletion or pharmacological blockade of the intestinal ASBT protein alter other metabolic pathways apart from cholesterol and BA metabolism?

______3 MATERIALS AND METHODS

Animals Animal experiments were conducted in accordance with accepted standards of humane animal care and approved by the Animal Care and Use Committees at Karolinska Institutet and Göteborg University. Paper I. C57BL/6J-ob/ob male mice and littermate controls were purchased from Taconic, DK. For cholesterol feeding experiments, ob/ob and their wild-type controls were given either standard mouse chow or standard mouse chow supplemented with 2% cholesterol plus 10% oil (w/w) for 7 days. Leptin treatment was as outlined in detail in paper I. Paper II. Wt C57BL/6 mice were, purchased from Taconic, DK and kept under standard conditions. All treatments are described in detail in paper II. Paper IV. SLC10A2 -/+ and SLC10A2 -/- mice were generated at AstraZeneca R&D, Mölndal, as described in detail in supplemental experimental procedures of paper IV. A diet enriched in sucrose (D12329, Research Diets, NJ), and drinking water supplemented with 10% fructose was used for the sucrose feeding experiment. Animals had free access to the diet for two weeks. Control animals received standard mouse chow and regular tap water. In paper III, male C57BL/6J-ob/ob animals were purchased from Taconic, DK. Ob/ob animals were gavaged with a specific slc10a2 inhibitor (AZD7806, AstraZeneca R&D, Mölndal, Sweden) or a control vehicle for 11 days. Animals had free access to food and regular water. Human samples In paper III. human blood samples were used. They are described in paper III. and references therein. All subjects gave informed consent for participation in the studies, which had been approved by the Ethics Committee of Karolinska Institutet. Serum lipids and lipoproteins Total cholesterol or triglycerides were analysed using the commercially available reagents IL TestTM cholesterol 181618-10 and triglyceride 181610-60 kits (IL Scandinavia, Gothenburg, Sweden) on the Monarch 2000 system (IL Scandinavia, Gothenburg, Sweden). Lipoprotein cholesterol profiles were obtained by separation of 10 µl serum using a micro fast performance liquid chromatography (FPLC) system for the generation of lipoprotein profiles as described 76, 122. Serum marker for Cyp7a1 activity 7α-hydroxy-4-cholesten-3-one (C4) Serum levels of 7α-hydroxy-4-cholesten-3-one (C4) were used to monitor Cyp7a1 enzymatic activity (BA synthesis) according to the method of Gälman et al. 123. C4 levels were analyzed from serum samples after sample work up followed by high- pressure liquid chromatography as described in detail elsewhere 124. C4 values were corrected for total serum cholesterol according to Honda et al. 125 in paper III. The use of uncorrected values did not give significantly different results. Serum Bile acids Levels of serum bile acids were assayed by gas chromatography/mass spectrometry as previously described 126.

18 T. Lundåsen ______Serum Insulin Plasma insulin was analyzed using a commercially available rodent insulin Radio Immune Assay (RIA) according to manufacturer’s instructions (Linco, St. Charles, MI). Serum Leptin Plasma leptin levels were determined by a mouse specic leptin enzyme-linked immunosorbent assay kit (R&D Systems, Oxon, UK) following the manufacturer’s instructions. Serum FGF19 A sandwich enzyme-linked immunosorbent assay (ELISA) kit was used for colorimetric detection of FGF19 in serum (FGF19 Quantikine ELISA kit, Cat. No. DF1900; R&D Systems, Minneapolis, MN, USA), following the manufacturer's instructions. All serum samples were assayed in duplicate, the coefficient of variation was 2%. Fecal Bile Acids and Neutral Steroids In paper II feces were collected groupwise for 24 h. One gram of pooled dried feces was analyzed for neutral sterols (coprostanol, coprostanone, and cholesterol) and bile acids by quantitative gas-liquid chromatography as been previously described 127- 129. Ligand blot assay for the LDL receptor Pooled livers (0.5 g) were homogenized in 50 mM Tris, pH 6.8, 2 mM CaCl2, 0.5% Triton X-100, 1 mM phenylmethylsulfonyl fluoride, and 0.5 mM leupeptin. Samples were sonicated for 15 s and then centrifuged at 14,000 rpm for 10 min. The supernatant was centrifuged in an Airfuge® (Beckman Coulter AB, Bromma, Sweden) for 7 min, aliquoted, and stored at 80 °C until further analysis. Protein concentrations were determined with Lowry DC kit (Bio-Rad). All work was performed on ice. LDL receptor expression was assayed using liver membrane proteins that were electrophoresed under non-reducing conditions on 6% SDS-PAGE as outlined 130. Proteins were transferred to nitrocellulose membranes and incubated with 125I-labeled rabbit β-VLDL. The LDL receptor band (≈120 kDa) were analyzed and quantified using a Fuji BAS 1800 analyzer (Fuji Photo Film Co.) and Image Gauge software (Science Lab, 98, version 3.12, Fuji Photo Film Co.). Immunoblot assays Hepatic SR-BI protein was assayed by immunoblot using liver membrane proteins, as described for the LDL receptor ligand blot assay, and a rodent polyclonal antibody (Novus Biologicals Inc., Littleton, CO), as described previously in detail 131 employing standard immunoblotting techniques. The cyp7a1 antibody was generated by immunizing rabbits with the peptide NH2- KLH-Cys-Tyr-Lys-Leu-Lys-His-COOH corresponding to the last five amino acids in the mouse and rat cyp7a1 proteins. The peptide was coupled to an affinity column to purify cyp7a1 antibodies from rabbit serum. A cyp7a1 immunoblot was performed on hepatic microsomal proteins prepared as described previously, using 1 mM phenylmethylsulfonyl fluoride, and 0.5 mM leupeptin 132. To detect SREBP1 protein cytoplasmic and nuclear protein preparations from liver were performed by the NE-PER reagent (Pierce). A monoclonal antibody that was raised against the N’-terminus of SREBP1, detecting both the precursor and mature

______form of SREBP1 (Labvision Corporation, CA) was used as described in detail in paper IV. Enzymatic Activities HMGCoA reductase and cyp7a1 enzymatic activities were assayed in hepatic microsomal membranes as described previously 132. Determination of liver triglycerides and cholesterol Liver cholesterol content was determined as previously described and liver triglycerides were extracted 133 and determined employing a commercially available kit (Roche Applied Science,Indianapolis). In paper IV cholesterol was determined by GC/MS and related to total liver protein. Quantitative Real Time PCR Quantitative Real Time PCR was performed as described in each paper (I-IV). Briefly, total RNA was extracted from snap frozen tissues with Trizol reagent (Invitrogen) according to the manufacturer’s instructions. Total RNA was DNase-treated. cDNA synthesis was carried out with Superscript II or Superscript III (Invitrogen) according to the manufacturer’s instructions. Quantitative real time PCR was performed with the TaqMan or SYBR Green assays on the ABI Prism 7700 sequence detection system or the ABI 7500 Fast Real-Time PCR System (Applied Biosystems, Foster City, CA). Relative mRNA expression was calculated according to the using the deltaCt method following the guidelines in the user bulletin#2 (Applied Biosystems) on the “comparative Ct method.”

Statistical analyses All statistical analyses are described in the respective papers I-IV.

20 T. Lundåsen ______4 RESULTS

4.1 EFFECTS OF LEPTIN ON HEPATIC CHOLESTEROL AND BILE ACID METABOLISM IN OB/OB MICE (PAPER I)

Background: Increased BA synthesis has been reported in human obesity 104-106. Leptin resistance is a common feature of obesity. The literature on how leptin treatment may influence BA synthesis is contradictory, with both increased and unchanged expression of CYP7A1 reported in response to injection of the peptide in ob/ob mice 101, 102, 134. Also how leptin may influence HDL metabolism is controversial 99. Aim: To evaluate the possible role of leptin in the regulation of cholesterol and BA metabolism. Results: The effects of acute leptin treatment in leptin-deficient ob/ob mice were studied. The serum LDL and HDL fractions were increased compared to wt animals in the basal state, and increased further upon dietary challenge with 2% cholesterol. The serum lipid response to dietary cholesterol of ob/ob mice was opposite to that seen in wt mice, strongly indicating a deficiency in reverse cholesterol transport. In line with this observation, it was found that the the hepatic HDL receptor SR-BI was suppressed both at mRNA and protein level in ob/ob animals. The hepatic LDL receptor expression was also attenuated in ob/ob when assayed by ligand blot, while mRNA levels were not affected. Cyp7a1 was markedly suppressed in ob/ob animals both at mRNA and activity levels. In addition, the normal rodent stimulatory response of cyp7a1 upon cholesterol feeding was abrogated in the ob/ob animals. The hepatic SR-BI expression was dose-dependently increased upon short-term leptin treatment. Such an increase was however not found under the same conditions for cyp7a1 when assayed for the activity marker C4, protein or mRNA levels. Instead, cyp7a1 was actually reduced by leptin treatment of ob/ob animals. Conclusions: The ob/ob mice display reduced levels of hepatic LDL receptors, SR-BI and Cyp7a1. These findings may in part explain the elevated serum cholesterol levels, particularly in the HDL fraction. The Cyp7a1 response to dietary cholesterol is attenuated in ob/ob mice, and - in contrast to wt animals - the serum cholesterol levels are increased further The SR-BI is positively regulated by leptin treatment of ob/ob mice, whereas Cyp7a1 is not.

4.2 EFFECTS OF A SELECTIVE TRβ AGONIST ON HEPATIC LIPID AND BILE ACID METABOLISM IN NORMAL MICE (PAPER II)

Background: Since T3 stimulates the heart, mediated by TRα, its use as a lipid lowering agent has been hampered. The TRβ is the major TR in the liver. A TRβ−agonist may therefore show positive hepatic metabolic responses on lipid metabolism while sparing the heart. Aim: To study the effects of a TRβ-selective, liver-specific thyreoidomimetic, GC-1, on hepatic lipid and BA metabolism in normal mice, and to compare them to those of T3. Results: GC-1 treatment in normal mice lowered plasma cholesterol and triglycerides more efficiently than T3 especially at the lower doses tested. The plasma cholesterol

______profiles upon GC-1 treatment were similar to those of T3, displaying dose-dependent reductions particularly in HDL. Further, GC-1 efficiently reduced triglycerides in plasma VLDL and LDL. The levels of the hepatic HDL receptor SR-BI increased dose- dependently upon GC-1-treatment. T3 did not show the same positive effects on SR-BI expression. In line with the reduction of plasma cholesterol, cyp7a1 was induced by GC-1 treatment. A dramatic reduction of the mRNA levels for SREBP1c was evident in both GC-1 and T3-treated mice. GC-1 and T3 reduced plasma cholesterol levels in mice fed either a cholesterol or cholic-acid-enriched diet. Plasma triglycerides were markedly reduced in mice fed cholesterol and treated with either T3 or GC-1. This effect was not seen in mice fed cholic acid. Hepatic triglycerides in cholesterol-fed mice were not altered by GC-1 as compared to controls. However, T3 increased liver triglycerides on the same diet as compared to controls. Similar results were seen with the BA-containing diet. GC-1 induced SR-BI in the liver regardless of cholesterol or BA challenge. Likewise both T3 and GC-1 induced cyp7a1 enzyme activity under the same conditions. GC-1 and T3 treatment reduced the mRNA expression of hepatic SREBP1c both under cholesterol and BA-challenge. The fecal BA excretion was higher in animals treated with GC-1. Conclusions: Hepatic TRβ stimulation employing GC-1 decreases plasma cholesterol and triglycerides in a dose-dependent fashion concomitantly with increased levels of hepatic SR-BI and Cyp7a1 in normal mice. GC-1 also reduced elevated levels of plasma cholesterol in cholesterol and BA-challenged animals. The results of this study indicate an important role for TRβ in reverse cholesterol transport, and further points to hepatic TRβ as an attractive target for treating dyslipidemic disorders.

4.3 FGF19 AND BILE ACID SYNTHESIS IN HUMANS (PAPER III)

Background: FGF19 and its mouse orthologue FGF15 have been demonstrated to inhibit BA synthesis in the mouse. Both these genes are driven by the BA activated nuclear receptor FXR 135, 136. FGF19 and FGF15 are expressed in the ileum but not in the liver 108, 137. FGF15 and FGF19 have been shown to repress the expression of the CYP7A1 gene in mice. However, the presence of the mouse FGF15 protein in the blood or lymph has not been demonstrated, and there is no information on circulating FGF19 in man 109. Aim: To determine if evidence could be found for a role of FGF19 in the regulation of BA synthesis in man. Results: A tissue survey for FGF19 mRNA by RT PCR showed that FGF19 mRNA was detectable in small intestine, cervix, thymus and testes. The FGF19 expression in small intestine was 5-fold and that in cervix 8-fold higher, respectively, relative to the expression in testes. FGF19 was readily detected in the serum of 15 normal fasting volunteers and showed a 12-fold interindividual variation. Serum samples from subjects that had received treatment with the BA-binding resin cholestyramine were assayed for FGF19 protein by ELISA and the serum marker for BA synthesis, C4. It was found that the FGF19 levels were strongly reduced during treatment with cholestyramine. In contrast, in patients that had undergone treatment with the BA CDCA, the serum levels of FGF19 were strongly increased. The serum C4 was inversely related to the FGF19 levels during the respective treatments. These results

22 T. Lundåsen ______support the concept that intestinal FGF19 is synthesized and released into the circulation following the passage of BAs over the intestinal wall. BA synthesis in humans has a marked diurnal rhythm with two major peaks during the day 126. Assay of FGF19 in samples collected from normal subjects every 90 minutes during a 24 hour period revealed that also FGF19 showed a similar diurnal variation. However, the FGF19 peaks preceded the C4 peaks by approximately 90 minutes. Thus, the highest serum FGF19 levels occurred during the periods when hepatic BA synthesis was leveling off. FGR19 levels were preceded by increases in serum BAs, reflecting the transintestinal flux of BAs. When food was omitted the FGF19 diurnal changes disappeared indicating an obligate dependence on BA intestinal input. Conclusions: Circulating FGF19 levels are influenced by the amount of BAs being absorbed in the small intestine. The intestinal release of FGF19 in turn signals to the liver leading to a suppression of BA synthesis. Thus, in addition to the liver, harboring all machinery for the control of the BA pool, the transintestinal flux of BAs may also be one important process in this regulation.

4.4 INHIBITION OF ASBT (SLC10A2) MODULATES TRIGLYCERIDE AND GLUCOSE METABOLISM (PAPER IV)

Background: The SLC10A2 gene, coding for the ASBT protein, is responsible for the major part of the ileal absorption of BAs. Both genetic ablation and pharmacological inhibition of ASBT leads to increased fecal loss of BAs and a compensatory induction of BA synthesis 115, 124. Treatment with BA-binding resins is known to cause elevations of plasma triglycerides in humans. The mechanisms behind this response are not clear. Treatment of mice with FXR agonists, including BAs, has been shown to decrease plasma triglycerides and glucose levels. Aim: To investigate the effects of an interrupted BA circulation on triglyceride and glucose metabolism. Results: When BA metabolism was investigated in SLC10A2 heterozygous (+/-) and homozygous (-/-) mice, the rate limiting enzyme CYP7A1 was found to be increased as expected. A clearly smaller increase for CYP7A1 activity than for mRNA and protein was noted. In line with an induced transcription of this enzyme SHP mRNA was reduced, most pronounced in the SLC10A2 -/- animals. No major difference in serum cholesterol was found between the groups, but when the serum triglyceride levels were analyzed, a reduction was found in the SLC10A2 -/+ and SLC10A2 -/- animals. The reduction was confined to the LDL fraction in both heterozygous and homozygous animals and also to the VLDL fraction in the homozygotes. No change in the expression of the hepatic receptors for LDL or HDL were detected, but the rate-limiting enzyme for cholesterol synthesis was increased in the livers of both SLC10A2 -/+ and SLC10A2 -/- mice in a dose-dependent fashion. Notably, the mRNA for the transcription factor SREBP1c was suppressed in SLC10A2 -/+ and SLC10A2 -/- animals, with the lowest expression in the latter group.

When SLC10A2 -/- mice were challenged with a sucrose-rich diet they displayed lower liver cholesterol and triglyceride contents as compared to controls. This occurred in parallel with reduced hepatic mRNA expression of enzymes in the fatty acid synthesis

______pathway (ACL, ACC, FAS and SCD1) in SLC10A2 -/- mice when fed the sucrose-rich diet. Concomitantly, the protein expression of a major transcriptional determinant for these enzymes, SREBP1, was suppressed in SLC10A2 -/- mice. In addition, an altered response to dietary sucrose was seen in SLC10A2 -/- mice for the LPK and G6PDH genes, involved in glucose handling. No changes in plasma glucose were seen in this experiment. The elevated levels of CYP7A1 mRNA and activity were found to be normalized in SLC10A2 -/- mice fed sucrose diet. The FXR-induced genes FGF15, SHP and IBABP in the distal ileum of SLC10A2 -/- mice were all markedly suppressed. The FGF15 mRNA expression was strongly increased in SLC10A2 -/- animals receiving the sucrose diet. The hepatic mRNA expression of the metabolic regulator FGF21 was higher in SLC10A2 -/- mice compared to wt controls. A most striking dietary effect was found for FGF21. In both wt and SLC10A2 -/- mice receiving the sucrose diet, the hepatic FGF21 mRNA levels were increased drastically by 25–30-fold. When ob/ob mice were treated with a specific inhibitor of SLC10A2, serum insulin, glucose and triglycerides were all reduced. The hepatic mRNA levels for FGF21 displayed a 100% increase in ob/ob animals receiving the inhibitor. The SLC10A2 inhibitor had the expected effects on FXR target genes in the terminal ileum and on CYP7A1 in liver. Conclusions: Abrogation of the enterohepatic circulation by a targeted deletion of SLC10A2 in mice causes lowered levels of serum VLDL and LDL triglycerides. The decreased levels of hepatic triglycerides and levels of fatty acid synthesis enzymes is likely to depend, at least partly, on reduced levels of SREBP1c. Cyp7a1 is normalized by sucrose feeding in SLC10A2-/- animals. An increased expression of FGF15 could partly account for this repression of Cyp7a1. The metabolic regulator FGF21 in liver tissue is induced in SLC10A2 deficiency. FGF21 in liver is highly upregulated by dietary sucrose. Inhibition of SLC10A2 in ob/ob leads to reduced serum triglycerides and glucose levels concomitantly with increased mRNA FGF21 levels in the liver.

24 T. Lundåsen ______5 GENERAL DISCUSSION

The conversion of cholesterol to BAs in the liver represents a major pathway of cholesterol excretion from the body. Perturbations of the enterohepatic circulation of BAs induce compensatory response mechanisms of cholesterol metabolism which may lead to positive effects on plasma lipoprotein metabolism and atherosclerosis. In the present thesis, some aspects on how the regulation of this important pathway can be regulated have been studied. Particular emphasis is put on how the gut-liver interaction is exerted, and on how circulating hormones may affect hepatic cholesterol metabolism.

In paper III, we show for the first time that the intestinal peptide FGF19 is present in the circulation in normal humans. We demonstrate a strong correlation between the levels of serum BAs and FGF19 protein in serum in humans. Thus, when humans were treated with the BA binding resin cholestyramine the levels of FGF19 in serum were drastically reduced, while treatment with CDCA resulted in an opposite response. These results are thus fully compatible with FGF19 being transcriptionally activated and released upon BA entry into the ileal enterocytes. When serum FGF19 levels were monitored during 24 hours, it was clear that their variation was closely related to the serum levels of BAs, with a delay of about 90-180 min. Since the serum levels of BAs reflect the amount of BAs returning via the portal vein after intestinal uptake, this is in line with findings where the expression of FGF19 and its mouse orthologue FGF15 has been shown to be positively regulated by FXR at the mRNA level 135, 136, 138. In humans, the mRNA expression of FGF19 was evident in the ileum but not in the liver; these data are in agreement with previous observations 137. Our findings are thus compatible with the concept that the transintestinal flux of BAs regulates the synthesis of FGF19 and its secretion from the intestine into the circulation. How the peptide is transported in the blood has not yet been studied in detail. In preliminary studies, we have not seen any differences in FGF19 levels between arterial, portal venous or hepatic venous blood, which may indicate that the peptide is cleared by other routes. Ongoing studies will address the possible importance of renal excretion, as well as the possibility that lymph flow represents a transport pathway. As expected, BA synthesis was strongly induced by cholestyramine treatment and reduced by CDCA feeding. BA synthesis has previously been demonstrated to be subject to a pronounced diurnal variation 126. When the diurnal variation of serum FGF19 was analysed, a two-peak pattern similar to what has been observed for BA synthesis could be demonstrated. Moreover, the decline of the serum marker for BA synthesis, C4, was preceded by a rise in FGF19 serum levels. This strongly indicates a role for circulating FGF19 in the regulation of BA synthesis in humans, not only at pharmacological but also at physiological conditions. It is important to note that the increase in BA synthesis takes place during the day also under fasting conditions, whereas, consistent with a role for FGF19 in suppressing the synthesis of BAs in humans, no increases in FGF19 levels were observed during the fasting experiment.

Taken together, these results suggest that BAs, via FXR, activate FGF19 expression in the ileum resulting in release of the peptide into the circulation, where it may then

______repress Cyp7a1 by hepatic FGFR4 activation. Thus, FGF19 can be said to act as an intestinal hormone regulated by the transintestinal flux of BAs that in turn influences hepatic BA synthesis. This mechanism is in agreement with the proposed model described by Inagaki et al, based on experiments in the mouse 108, 109. However, no endogenous FGF15 protein has so far been detected in mouse serum.

In addition to its role in regulating BA synthesis, FGF19 has been ascribed a role in regulating metabolic rate 107, 139. It was demonstrated that mice overexpressing human FGF19 display resistance to the diabetogenic effects of high fat feeding, including markedly lowered levels of glucose and insulin as well as resistance to weight gain despite higher food consumption 139. The FGF19 transgenic animals showed decreased levels of hepatic ACC2 and SCD1, and increased levels UCP1 in adipose tissue. The hepatic triglyceride content was reduced in these animals regardless of diet. Furthermore, FGF19 increases the metabolic rate and reduces glucose, insulin and plasma triglyceride levels and reduces hepatic cholesterol and triglyceride content in ob/ob mice 107. Administration of BAs has also been demonstrated to decrease endogenous triglyceride production and plasma levels in humans as well as in mice and hamsters 140-142. This effect has been suggested to be mediated through a pathway involving FXR mediated SHP repression of hepatic SREBP1c transcription 142.

FXR deficient mice display elevated levels of plasma triglycerides 37. Additionally, activation of FXR has been demonstrated to be involved in pathways lowering triglycerides and controlling plasma glucose levels 37, 85, 143-147. Administration of BAs to mice have been demonstrated to modulate metabolic rate. This effect was suggested to be dependent on BA binding to the G-coupled receptor TGR5, or GPBAR1, in adipose tissue of mice 148. On the other hand, humans treated with cholestyramine respond with an acute increase in plasma triglycerides which levels off after prolonged treatment and return to (even below) base levels 140, 149, 150. Since cholestyramine treatment of humans was shown to strongly suppress the FGF19 serum levels (Paper III), and since elevated levels of FGF19 have been demonstrated to lower plasma triglycerides in mice, the near absence of FGF19 in cholestyramine-treated subjects could speculatively contribute to the observed increase in plasma triglycerides observed in humans treated with BA binding resins. The role of FGF15/19 in these BA-induced effects on lipid and carbohydrate metabolism should now be investigated, as should the possibility that interindividual (genetic) variations in circulating (fasting or postprandial) FGF19 levels, and/ or FGF19 sensitivity, may contribute to interindividual variations in energy metabolism and sensitivity to dietary lipid and carbohydrate

In light of these connections between triglyceride, glucose and BA metabolism, it was of interest to investigate how interruption of the enterohepatic circulation of BAs may influence lipid and glucose metabolism. For this purpose, we used mice ablated for the BA transporter SLC10A2 gene, or ASBT or IBAT. Such mice have been created previously and are characterized by dramatically increased fecal losses of BAs due to their diminished uptake in the ileum, and by a compensatory increase in BA synthesis, reflected by elevated levels of Cyp7a1 and Cyp8b1 mRNAs 115. In our newly

26 T. Lundåsen ______constructed mice, increased levels of Cyp7a1 were indeed found in the SLC10A2-/- mice at mRNA, protein and activity levels. The discrepancy found between Cyp7a1 gene expression and level of activity may imply a substrate deficiency for BA synthesis. The levels of the hepatic SHP mRNA were dose-dependently decreased in heterozygous and homozygous mice.

The lack of induction of the LDL receptor and SR-BI, together with increased levels of the hepatic HMGCoA reductase, suggests that substrate supply to BA synthesis in SLC10A2-/- animals is mediated only via new synthesis of cholesterol in the liver. When serum triglyceride levels were analyzed, proportionally decreased levels were found in heterozygous and homozygous mice. This reduction was due to diminished VLDL levels in both heterozygous and homozygous animals, with an additional reduction seen in the homozygotes in the LDL triglyceride fraction. In line with these results, a reduction in the hepatic SREBP1c mRNA was evident in the SLC10A2-/+ and SLC10A2-/- mice. Importantly, the ablation of the SLC10A2 gene did not cause hypoglycemia. These results are thus in the opposite direction to what has been observed for humans treated with BA-binding resins, although few long term studies have been performed with respect to plasma triglyceride dynamics. In most studies, the increase in plasma triglycerides upon treatment with BA binding resins declines with time 140, 149-151. The effects of SLC10A2 ablation on triglyceride metabolism was further substantiated when these animals were fed a sucrose-enriched diet in order to promote hepatic lipogenesis. Hepatic triglycerides as well as mRNA levels of enzymes critical to hepatic fatty acid synthesis were reduced in SLC10A2-/- mice fed the sucrose-rich diet compared to wt controls. Consistently the SREBP1 protein levels were reduced in SLC10A2-/- mice. No changes in blood glucose levels were detected in wt or SLC10A2 deficient mice. The mechanisms underlying the reduction in hepatic triglyceride metabolism in the SLC10A2-/- mice should be different and not likely explained by FXR agonism since the activity of FXR is reduced under conditions of BA deficiency. A more plausible explanation to the altered triglyceride metabolism involves mechanisms for substrate and energy preservation. Cholesterol and triglyceride synthesis share the common substrate acetyl CoA. To simplify, under conditions of substrate restriction the output from the cholesterol synthesis must be balanced by the output from the fatty acid synthesis pathway. SLC10A2 deficient mice have a reduced BA pool. For the liver to be able to fully compensate for this reduced pool size by providing de novo synthesized cholesterol to meet the demand for increased BA synthesis, the ultimate net effect may be an increased hepatic uptake of glucose with reduced plasma glucose as a consequence. A reduction in plasma glucose is potentially lethal and powerful mechanisms are therefore present to avoid this. Since no differences in plasma glucose were seen in the SLC10A2-/- mice, these animals “prioritized” the conservation of plasma glucose before further increasing BA synthesis. The reduced hepatic expression of liver pyruvate kinase and glukokinase is in line with such a “saving” of plasma glucose Surprisingly, when excess of sucrose was given, and hence the capacity to produce acetyl CoA should be increased, the initially high levels of Cyp7a1 mRNA in the SLC10A2-/- animals were normalized instead of being increased as we had expected. The molecular mechanism for this regulation is presently unknown, although similar

______findings with reduced Cyp7a1 expression have been reported in fructose fed hamsters 141. As expected, FGF15 mRNA was reduced in the distal ileum of the SLC10A2-/- mice. Thus, FGF15 signalling from the gut does not explain the repressed triglyceride metabolism in these animals. Interestingly, however, the SLC10A2-/- animals displayed a several fold increase in mRNA of the FGF15 in distal ileum in response to the sucrose rich diet, which is fully in line with the concomitantly reduced hepatic BA synthesis. This represents a previously not recognized dietary regulation of the FGF15 gene and in addition provides a hypothetical explanation to the reduced Cyp7a1 levels under these conditions. In search for other putative mediators of the suppressed triglyceride levels in SLC10A2 deficiency, we assayed hepatic mRNA levels of FGF21. This peptide has been shown to reduce plasma levels of glucose and triglycerides under diabetic conditions in rodents and monkeys but not in normoglycemic animals 152, 153. FGF21 mRNA was found to be increased in the livers of SLC10A2 -/- mice compared to wt. Strikingly, the sucrose diet inferred strong positive regulatory action of the hepatic FGF21 gene. Thus, FGF21 participates in a response when the liver is loaded with carbohydrates in excess. The mechanisms underlying the regulation of the FGF21 gene is under intensive study. When a potent pharmacological inhibitor of SLC10A2 was employed in experiments on ob/ob mice as a model of dyslipidemia and diabetes, the expected induction of the Cyp7a1 mRNA was seen. More interestingly, interruption of the BA enterohepatic circulation by this compound, under non-weight reducing conditions, caused a large decrease in plasma triglycerides, glucose, as well as insulin. This was accompanied by doubling of hepatic FGF21 mRNA levels in the treated animals. If FGF21 is solely responsible for these effects is not known. However, substrate deficiency should not be present in the ob/ob animals. The positive effects on glucose and lipid metabolism in the ob/ob animals indicate that therapeutics aimed at interruption of BA enterohepatic circulation may be of clear interest in the treatment of subjects with overweight, insulin resistance and dyslipidemia, all representing common metabolic disturbances in the population. The ob/ob mice represent an animal model with complex metabolic disturbances including hyperlipidemia, hyperglycemia with peripheral insulin resistance and fatty liver. The primary cause for the dysregulated metabolic phenotype is overeating due to a lack of functional leptin. In paper I, it was shown that ob/ob mice display reduced hepatic LDL receptor activity as compared to lean controls when fed regular chow, although the LDL receptor mRNA levels were increased. This finding is identical to what has been described for the LDL receptor regulation in mice and obese Zucker rats, which have a non-functional leptin receptor 100. The hepatic levels of the HDL receptor, SR-BI, were decreased both at mRNA and protein levels in ob/ob animals compared to lean controls. The latter finding is in contrast to what has been described by Silver et al. 99 who could not detect any change in SR-BI expression in ob/ob mice. The reason for this discrepancy is not clear. However, in the same study Silver et al. found that livers from ob/ob mice had a four-fold reduced uptake of radiolabeled HDL, in concert with our finding of reduced SR-BI expression.

The finding that the rate limiting enzyme in BA synthesis, CYP7A1, was reduced in ob/ob under basal conditions together with reduced hepatic expression of lipoprotein

28 T. Lundåsen ______receptors highlights the presence of a generally reduced capacity for hepatic cholesterol catabolism in these animals, which may partly explain the elevated levels of plasma lipoproteins seen in the LDL and HDL fractions. This was confirmed by cholesterol feeding experiments where both LDL and HDL cholesterol were further elevated in the ob/ob mice, which was in strong contrast to what was observed for their lean littermates where HDL cholesterol was reduced upon cholesterol feeding. The difference in hepatic lipoprotein receptor expression between ob/ob mice and lean controls persisted during cholesterol feeding. One important finding was that the normal response of CYP7A1 to cholesterol was severely attenuated in the ob/ob animals. In the lean controls the CYP7A1 activity increased from 25 to 80 pmol/min/ mg protein when challenged with dietary cholesterol, whereas the CYP7A1 activity of ob/ob mice only increased from 12 to 22 pmol/min/mg prot. Thus, the CYP7A1 response to dietary cholesterol in ob/ob is only 20% to that of the lean littermates. This response to dietary cholesterol, present in rodents, is important to protect against accumulation of toxic levels of lipids in the liver as shown in LXRα deficient mice 52.

In order to evaluate whether the absence of leptin in the ob/ob mice is directly responsible for the reduced expression of SR-BI and CYP7A1, short term treatment with increasing concentrations of leptin was performed. SR-BI mRNA and protein were increased in a dose dependent manner when leptin was injected into ob/ob mice, except for the highest concentrations tested, which were not physiological. This may imply a direct role for leptin in regulating SR-BI. Importantly, the treatment period was only two days to avoid secondary effects due to reductions in food intake and subsequent weight loss. Neither the expression nor the enzymatic activity of CYP7A1 was found to increase upon short term leptin treatment of ob/ob mice. Instead, CYP7A1 was repressed during these conditions. A putative explanation to the latter results could be the increased sensitivity to insulin induced by the leptin treatment. Insulin has been demonstrated to repress the CYP7A1 gene transcription 64, 154. The finding that CYP7A1 is not increased as a response to leptin treatment is supported by other studies showing that long term administration of leptin leads to a smaller pool of BAs in ob/ob mice without affecting CYP7A1 activity 102. The ob/ob mice are relatively resistant to gallstone formation when exposed to a lithogenic diet, but treatment with leptin has been shown to increase the susceptibility to gallstone formation under such conditions 134. No increase in CYP7A1 expression was found in the latter study. In another study comparing different mouse models of obesity it was concluded that obesity per se does not confer to gallstone susceptibility in mice 155. The ob/ob or db/db mice in that study were more resistant to gallstone formation. Obesity in man is the second largest risk factor for the development of cholesterol gallstone disease. The link between leptin and gallstone formation is less well characterized in man. Some studies have indicated an expanded BA pool together with an increase in both hepatic cholesterol synthesis and CYP7A1 activity in obesity 104-106. It is known that during weight loss in man when the incidence of gallstone formation further increases, the BA pool size decreases, which should occur concomitantly with an increase in the sensitivity for leptin 106.

The concept of directly stimulating CYP7A1 activity in order to promote cholesterol breakdown to BAs and subsequent net cholesterol elimination from the body is an

______attractive principle for therapeutic development. T3 participates in regulating many of the processes involved in obesity and dyslipidemia. The beneficial effects of T3 include reduction of plasma LDL cholesterol and triglycerides and induction of weight loss 70, 71. However, therapeutic attempts with T3 have been hampered by the fact that they also display unwanted side effects like tachycardia, atrial arythmia, congestive heart failure, bone and muscle loss, fatigue and also negative psychological effects 70. Studies in mice deficient in either of the two T3 receptors, TRα or TRβ, indicated that the deleterious effects on the heart is mediated via TRα and the beneficial effects on plasma cholesterol and triglycerides are mediated via TRβ 71, 76, 156-159. The compound GC-1 is a TRβ selective agonist that in addition accumulates selectively in the liver 160, 161. GC-1 has been demonstrated to reduce plasma cholesterol levels in cholesterol-fed rats and in cynomolgus monkeys 161, 162. We were now able to show that such plasma cholesterol and triglyceride reducing effects occur in a dose-dependent fashion also in normal mice (paper II). The effect of GC-1 on plasma cholesterol was more pronounced than for T3, particularly at the lower doses tested. The effect on plasma triglycerides could in part be mediated via the observed inhibition on SREBP1c transcription in the livers of GC-1 treated animals. SREBP1c is known to be an essential activator of genes participating in hepatic triglyceride synthesis 163. This effect on the SREBP1c mRNA was not specific for GC-1, as the same effect was observed with T3. Part of the explanation for the difference between the two compounds in triglyceride-reducing effect may also be related to the fact that T3 but not GC-1 has extrahepatic effects on tissues such as muscle and adipose tissue. A marked dose-dependent reduction in plasma HDL cholesterol levels was observed in GC-1 treated animals. Notably, this reduction correlated to an increase of the hepatic HDL receptor SR-BI. The activity and expression of cyp7a1 increased in GC-1 and T3 treated animals in conjunction with a reduced hepatic expression of SHP. The positive regulation of cyp7a1 by GC-1 is in line with previous in vivo studies demonstrating an 73, 164 increased cyp7a1 activity in response to T3 . The exact mechanism for TRβ mediated induction is not clear but one binding site for TRβ on the mouse CYP7A1 promoter involves the LXR response element and in addition, two thyroid response elements that confer positive regulation have been identified in the 5’-part of the mouse Cyp7a1 promoter 165, 166. Treatment with the liver-selective, TRβ-specific T3 mimetic GC-1 resulted in enhanced CYP7A1 expression together with increased SR-BI expression, concomitant with an augmented fecal excretion of BAs. These results thus imply that TRβ stimulation in the liver stimulates the elimination of cholesterol from the body through stimulation of reverse cholesterol transport. In addition, GC-1 treatment could suppress plasma cholesterol levels from increasing in response to feeding either a cholesterol or cholic acid containing diet. It may be speculated that these effects would result in prevention and/or regression of atherosclerotic plaques through the removal of cholesterol. Further studies should now be performed in order to evaluate if selective TRβ stimulation by drugs of this kind exert similar effects in humans, and if the concept of hormone- induced stimulation of BA synthesis can be an effective principle of therapy for atherosclerosis and its complications.

30 T. Lundåsen ______

Figure 5 Some major findings in the present studies

______6 CONCLUSIONS

Extrahepatic tissues, such as the intestine, the thyroid gland, and the adipose tissue, exert important regulatory control on the hepatic synthesis of BAs from cholesterol.

1. From studies in ob/ob mice, it could be shown that leptin exerts important effects on the hepatic HDL receptor, SR-BI, resulting in concomitant changes in plasma HDL levels. Ob/ob mice also display reduced levels of hepatic LDL receptors and Cyp7a1, and have an attenuated response to dietary cholesterol. Leptin does not directly regulate Cyp7a1. 2. Hepatic TRβ stimulation employing the compound GC-1 decreases plasma cholesterol and triglycerides in a dose-dependent fashion concomitantly with increased levels of hepatic SR-BI and Cyp7a1 in normal mice. GC-1 also reduced elevated levels of plasma cholesterol in cholesterol and BA-challenged animals. This implies an important role of TRβ in the promotion of reverse cholesterol transport, and further points to hepatic TRβ as an attractive target for treating dyslipidemic disorders. 3. Circulating FGF 19 levels in man are influenced by the amount of BAs being absorbed in the small intestine. The intestinal release of FGF19 in turn signals to the liver leading to a suppression of BA synthesis. Thus, in addition to the transhepatic flux, the transintestinal flux of BAs may also be one important process in this regulation. 4. Stimulation of BA synthesis through targeted deletion or pharmacological blockade of the intestinal ASBT protein causes lowered levels of plasma and hepatic triglycerides and lowered expression of enzymes of fatty acid synthesis in the liver. Sucrose feeding reduces Cyp7a1 expression at least partly due to an increased expression of FGF15 in the intestine. FGF21 in liver is highly upregulated by dietary sucrose. Inhibition of ASBT in ob/ob mice leads to reduced plasma triglycerides and glucose levels concomitantly with increased mRNA FGF21 levels in the liver.

32 T. Lundåsen ______7 ACKNOWLEDGEMENTS

I would like to express my sincere gratitude to all the people who have contributed, directly or indirectly, to the completion of this work. In particular I wish to thank

Mats Rudling, my supervisor, for invaluable help, advice, and unrestricted support throughout this work. For teaching me about the many aspects of liver and enterohepatic biology, for being stubborn, and pushing me uphill when needed, and for being a free-thinker. Thank you!!

Bo Angelin, my co-supervisor, for being an enormous invaluable source of knowledge of all scientific matters, and for encouragement and unrestricted support at all situations, when needed. Thank You !!

Jan Bolinder, Head of the department for medicine at Huddinge, for providing an excellent inspiring scientific work environment.

Jan-Åke Gustafsson, for contributing greatly to make NOVUM an international stronghold in nuclear receptor biology with an inspiring scientific atmosphere.

Curt Einarsson and Ingemar Björkhem for contributing both practically and theoretically to these studies. Gösta Eggertsen for educating me on bile acid science, science and society in general and for many helpful tips and comments.

Ann-Margret Östlund-Lindqvist and Eva-Marie Andersson at the AstraZeneca R&D, Mölndal for making the IBAT, ASBT (or SLC10A2) studies possible and for all the shared knowledge, scientific discussions, patience, continued interest and many other things. Thank you ! Michael Snaith, Sandra Schreyer and Helena Lindmark and all other crew at AstraZeneca who were involved in one way or another in the IBAT studies.

Past and present members of the unit for molecular nutrition, at NOVUM. Camilla Pramfalk for not throwing me out of the writing room when I was just plain boring, and the many helpful advices and comments on science and life matters. Manuela Matasconi for always being positive, late night discussions, and the wonderful sightseeing trip in Copenhagen. Lisen Johansson for a happy attitude and hard co-work. Cecilia Gälman for the positive norrländsk attitude and the enormous C4 assay work, many scientific advices, and the continued FGF19 story. Paolo Parini for the many advices on everything, from the other sex to science and particulary the GC-1 work. All the smiling people in the the corridors of NOVUM for discussions and help, including Knut Steffensen for sharing the many PCR reagents and answering LXR questions, Sanyal Sabayashi for interesting discussions on the latest FXR issues and for technical help and friendly advices, Eckardt Treuter for providing SHP expertise and Anders Ström for friendly advices on many technical and scientific matters.

______The people at Huddinge hospital particularly Metabollab, past and present, Lilian Larsson for the lots of help in these studies and for friendly chats about science and non- science. Ingela Arvidsson for all invaluable technical help and the hard work on the C4 assay among things, Lisbeth Benthin for professional help with many things including gallbladders and to keep things in order. Zhao Yan Jing for teaching me about gallbladder physiology and discussions. Lisa-Mari Nilsson, Ewa Ellis, Catharina Sjöberg, Katarina Hertel, Sabine Süllow-Barin, Pia Gustafsson, Britt-Marie Lejonhufvud, Wiveka Åberg, Ewa Steninger, Janne Holm, Mats Eriksson, Stefan Sjöberg and Peter Arner for additinal help with many various things. Lena Emtestam and Åse Walters for excellent administrative help, kindly reminding me of things I tend to forget. Mikael Rydén (not only for the primary Schwann cultures)

The service crew : Inger Moge, Inger Palm, Inger Ericson and Bernt without whose help and work NOVUM would stop. All the administrative and technical crew at NOVUM. Computer dept. guys at NOVUM including Jonas, Anders and Erik for always providing good service and instant help with computer problems.

…and from the pre-metabolic lab eras : Kristinn P. Magnússon for teaching me the noble art of PCR and how to survive in Stockholm. Ulf Klangby, Ismail Okan, Yisong Wang, Lazlo Szekely. Dan A. Lidholm for crucial advice at the cross roads. Rizaldy Scott, Svend Kjaer, Jonas Anders, Andries Blokzijl, Susanna Eketjäll, Joe Wagner, Beth-Ann Sieber and Peter Åkerud.

Per Lingensjö for support and the concept of Blue Out, nice landings and discussions on serious matters.

My great Uncle Per Caruso Österberg, for the strong and unrestricted support, crucial for my Huddinge endeavors and for never surrender.

My sister Susanne for being a lead star to follow and her family Mats, Alice and Astrid.

And not least my parents Valter and Gunnel, without whose continuous unrestricted strong support in all kinds of situations and complications, and for taking so good care of Hannes, this work would never lead to completion. Thank you!!!

And finally my little son Hannes for enlightening the days and making me realize other things are also of importance, for keeping asking me the questions I can not answer and for standing the weekend sleeping competitions.

34 T. Lundåsen ______8 REFERENCES

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