«THE ROLE OF OSTα/β IN BILE ACID AND LIPID METABOLISM BY TIAN LAN A Dissertation Submitted to the Graduate Faculty of WAKE FOREST UNIVERSITY ...»
THE ROLE OF OSTα/β IN BILE ACID AND LIPID METABOLISM
A Dissertation Submitted to the Graduate Faculty of
WAKE FOREST UNIVERSITY GRADUATE SCHOOL OF ARTS AND SCIENCES
In Partial Fulfillment of the Requirements
for the Degree of
DOCTOR OF PHILOSOPHY
Molecular Pathology Program
Winston-Salem, North Carolina
Paul A. Dawson, Ph.D., Advisor Douglas S. Lyles, Ph.D., Chair Jonathan Mark Brown, Ph.D.
John S. Parks, Ph.D.
Gregory S. Shelness, Ph.D.
ACKNOWLEDGMENTSAt the very beginning, I would like to express my utmost gratitude to my advisor, Dr. Paul A. Dawson. I am greatly indebted to his mentorship for every single day throughout my graduate study. Without his help, I can never be the person I am. His care to me is not only in the academic field, but also extends to everyday life. From the very early days to my qualify exam, my preparation for conference talks, and the my postdoctoral interviews..., he is always there willing to give any suggestions and assistance I need. I regard him a role model of devotion to science, critical thinking, heart to students and lab members, and kindness to everybody.
I also would like to take this opportunity to thank Anu and Jamie in the lab. I still remember the days when I do not have a car, Anu will give me a ride anywhere anytime, even on weekends. She also taught me basic things of life and science. Jamie taught me hand by hand from every aspect of good lab techniques. Her perfection to keep data and run assays, her sharp eyes to discover problems, and her well-prepared plans of logistics, and so many other things, are all of great value for me to learn. No words can describe my gratitude to them.
My great thanks also goes to my committee members: Dr. Doug Lyles, Dr. Greg Shelness, Dr. John Parks and Dr. Mark Brown. Their critical comments, suggestions and questions enlightened me and broadened my view of how to look at a scientific question.
Dr. Shelness and Dr. Parks gave me a lot of suggetions for my experimental designs, and I would like to thank Dr. Brown for his time and effort during my preparation for the postdoctoral interview. I will never forget Dr. Lyles' comment on science: "Always think of alternative hypothesis of your study. If not, don't do it."
The "family-like" environment of our department is something that I will miss the
find things you want from another lab. The limited space on this page won't be enough for me to list everyone who gives me invaluable help. I will keep this experience as a treasure of my life.
I also would like to express my great thanks to my family. My parents are a great support to me. Nothing can explain how much I owe them for all of my life. Without their support I can never go this far. Their voice on the phone gives me strength, and their understanding gives me confidence. My grandparents and my uncle in Philadelphia makes me feel that home is close. I am very lucky to have them around. I wish my grandparents to be happy and in good health forever. In addition, I also want to wish happiness and health to Nana Barbara, who knows me since elementary school and always have me in her mind.
Allison, Missy, Shuai, Sinan, Mingxia, Xin, Swapnil, Helen, Riquita, Daniel and Bryan. You make me a better person and I am grateful to have you guys in my life. John (Owen and Hopson), Nikki, Peter, Austin, Paul, Carol and Kurt, I will miss the Contra Dance and our Blue Grass community band. We are the best!
Last but not least, I want to express my greatest thanks to the Barrys', especially Ryan, Mr. and Mrs. Barry. Your kindness and generosity is the sweetest memory I have.
With you all living nearby, I know the phone will ring when the holidays are on the way.
LIST OF TABLES AND FIGURES
LIST OF ABBREVIATIONS
CHAPTER I. INTRODUCTION
II. MOUSE ORGANIC SOLUTE TRANSPORTER α DEFICIENCY ALTERS FGF15
EXPRESSION AND BILE ACID METABOLISM(PUBLISHED IN THE JOURNAL OF HEPATOLOGY, 2012)
III. INTERRUPTION OF ILEAL APICAL VERSUS BASOLATERAL BILE ACID
TRANSPORT HAVE DIFFERENT EFFECTS ON CHOLESTEROL METABOLISM
AND ATHEROSCLEROSIS DEVELOPMENT IN APOE-/- MICE(TO BE SUBMITTED TO ATHEROSCLEROSIS, 2013)
Figure 2 Intestinal weight, DNA content, and RNA content of wild type, Fxr-/-, Ostα-/-, and Ostα-/-Fxr-/- (DKO) mice 84 Figure 3 Quantitative morphometric analysis of small intestine of wild type, Fxr-/-, Ostα-/-, and Ostα-/-Fxr-/- (DKO) mice 86
ASBT Apical Sodium Dependent Bile Acid Transporter BARE Bile Acid Response Element BAS Bile Acid Sequestrant BSEP Bile Salt Export Pump
CDCA Chenodeoxycholic Acid CMC Critical Micelle Concentration CYP7A1 Cholesterol 7 alpha-hydroxylase CYP7B1 25-hydroxysterol 7 alpha-hydroxylase CYP8B1 Sterol 12 alpha-hydroxylase CYP27A1 Sterol 27-hydroxylase CTX Cerebrotendineous xanthomatosis CAR Constitutive androstane receptor
FGF Fibroblast growth factor GLP Glucagon like peptide HDL High density lipoprotein HNF Hepatic nuclear factor HMGCR/S HMG-CoA Reductase/Synthase IL-1β Interleukin-1β IBABP Ileal bile acid binding protein
MDR Multiple drug resistance MTP Microsomal triglyceride transport protein Na+/taurocholate cotransporting polypeptide NTCP Ostα/β Organic Solute Transporter α/β PXR Pregnane X receptor PGE2 Prostaglandin E2 PPAR Peroxisome proliferator-activated receptors PEPCK Phosphoenolpyruvate carboxykinase PGC1α PPAR-gamma coactivator 1α RXR Retinoic X receptors SHP Short heterodimer partner SREBP Sterol regulatory element binding protein
Bile acids are synthesized from cholesterol in the liver. Fecal bile acid excretion accounts for nearly half of daily cholesterol disposal. It has been known for many years that ileal resection or blocking intestinal apical absorption of bile acids induces hepatic bile acid synthesis. However, inactivation of the intestinal basolateral bile acid transporter OSTα-OSTβ was associated with decreased hepatic bile acid synthesis. To further understand the underlying mechanisms responsible for this altered bile acid homeostasis, Ostα-Fxr double null mice were generated and studied. Inactivation of Ostα was associated with significant increases in small bowel length, small bowel mass predominantly in ileum, villus width and crypt depth, and villus epithelial cell number. Fxr deficiency in Ostα-/- mice reversed the increase in intestinal length, but not the increases in small bowel mass or villus hyperplasia. Fxr deficiency in Ostα-/- mice was associated with decreased ileal Fgf15 mRNA expression, increased hepatic Cyp7a1 expression, increased fecal bile acid excretion, increased bile acid pool size, and restoration of intestinal cholesterol absorption. Ileal enterocyte Fgf15 mRNA expression was not significantly increased, but ileal levels of FGF15 protein was increased almost 10-fold in Ostα-/- mice, and total ileal FGF15 protein levels are further elevated to 20-fold due to the increase in ileal enterocyte number.
The second part of this project took advantage of the differential regulation of ileal FGF15 protein expression in Asbt-/- versus Ostα-/- mice to study the effect of ileal
Asbt and Ostα null alleles were introduced into a hypercholesterolemic mouse background (LDLr-/- and ApoE-/- for Asbt; ApoE-/- for Ostα). The mice were then fed a diet containing 0.1% (w/w) cholesterol and bile acid metabolism, cholesterol metabolism and atherosclerosis development were examined. Mice deficient in Asbt exhibited the classic response to loss of bile acid reabsorption with significant reductions in hepatic and plasma cholesterol levels and aortic cholesteryl ester content. In contrast, despite increased levels of fecal neutral sterol excretion, plasma and hepatic cholesterol levels and atherosclerosis development were not reduced in mice deficient in Ostα.
Our results indicated that FXR-mediated expression of FGF15 is required for the paradoxical repression of hepatic bile acid synthesis in Ostα-/- mice. The atherosclerosis study suggested that decreases in ileal FGF15 expression and subsequent increases in hepatic Cyp7a1 expression and bile acid synthesis are essential for the plasma cholesterol-lowering effects associated with blocking intestinal bile acid absorption.
Tian Lan prepared this chapter. Dr. Paul A. Dawson acted in an editorial and advisory capacity
1. Chemistry and Physiologic Function of Bile Acids
1.1. Structure of Bile Acids and Bile Alcohols Bile alcohols and bile acids are amphipathic metabolites of cholesterol (1). The A/B cis configuration of the saturated cyclopentanophenanthrene nucleus (with A, B, C and D ring) gives these molecules a curved shape with hydrophilic (concave/α) and hydrophobic (convex/β) sides (2). The various bile acid and bile alcohol species are distinguished by differences in the: 1) hydroxylation position on the nucleus ring, 2) orientation (α or β) of the hydroxy groups, 3) number of carbons on the side chain, and 4) carboxy or alcoholic group at the end of the side chain. Bile acids may have either a 5or 8-carbon length side chain (C24 or C27, respectively), but all bile alcohols are C27 species (1). C27 bile alcohols predominate in fish and ancient endothermic organisms (such as the manatee). Bile acids can be C24 or C27 species, with C27 bile acids being found in amphibians, reptiles, and more advanced birds and mammals exclusively using C24 bile acids. In human and mouse, the most abundant bile acid species include cholic acid (3α, 7α, 12α hydroxylation on the nucleus), chenodeoxycholic acid (3α, 7α) and βmuricholic acid (3α, 6β, 7β), all of which are denoted “primary bile acids” since they are synthesized directly from liver. Other common bile acids include deoxycholic acid (3α, 12α) and lithocholic acid (3α), which are the 7α-deoxylated products of CA and CDCA, respectively and produced by the gut microflora.
Bile acids are conjugated to taurine or glycine at their C24 position prior to being secreted into bile. The conjugation process involves a bile acid-CoA intermediate.
Addition of taurine is the most common form of conjugation in most vertebrates including mice, whereas conjugation with glycine predominates in human liver. Taurine and glycine conjugation lowers the pKa of bile acids and ensures that they will remain charged under pH conditions of the intestinal lumen to prevent non-ionic diffusion through the intestinal wall. The majority of bile acids are reabsorbed by a transportermediated system in the distal small intestine (the ileum), which will be discussed later.
Sulfation is also observed in bile alcohols and bile acids. Bile alcohols are sulfated at their C27 alcoholic group, which serves a similar purpose as taurine/glycine conjugation.
Sulfation of cytotoxic bile acids, such as LCA, promotes hepatic and urinary elimination and prevents their intestinal reabsorption.
1.2. Bile Acids and Micelle Formation Bile acids are amphipathic molecules with hydrophilic (α) and hydrophobic (β) surface divided by the planar nucleus. Bile acids form micelles within a specific concentration range, defined as the critical micellization concentration (CMC).
Temperature and pH are also critical for micelle formation. The CMC of each bile acid species is inversely correlated to the hydrophobic area of the molecule. Thus dihydroxy bile acids have lower CMC compared to trihydroxy bile acids. CMC drops significantly in the presence of sodium (0.15 M). Pure bile acid micelles (simple micelles) are rare under normal physiological conditions. Bile acids form mixed micelles with phosphatidylcholine in bile, which is important for preventing bile acids from damaging the membranes of the hepatic canaliculus and biliary epithelium. The phosphatidylcholine is provided by the flippase MDR2 (ABCB4). The protective role of phospholipid in bile is illustrated by Type 3 Progressive Familial Intrahepatic Cholestasis, a disease caused by mutations in the ABCB4 gene. In that disorder, membranes of the hepatocyte and biliary epithelium are progressively damaged by the simple bile acid micelles in bile, ultimately resulting in cholestasis. In the intestine, bile acids play a critical role solubilizing fatty acids, monoglycerides, cholesterol and fat-soluble vitamins, and promote lipid digestion by pancreatic lipase. The concentration required to form micelles in the presence of other lipids is much lower than CMC.
2. Hepatic Synthesis of Bile Acids
2.1. Classic and Alternative Pathways for Bile Acid Synthesis In adult humans, bile acid synthesis (~500 mg/day) accounts for approximately 45% of daily cholesterol turnover (other routes include biliary secretion with fecal excretion and to a lesser extent, cell sloughing and synthesis of steroid hormones) (3).
Bile acid synthesis is tightly controlled by negative feedback inhibition to maintain the body’s bile acid pool size within a narrow range. Bile acid synthesis is also regulated by metabolic hormones such as insulin and glucagon, and by inflammatory cytokines (4).