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 GRADUATE SCHOOL OF ARTS AND SCIENCES

In Partial Fulfillment of the Requirements

for the Degree of

DOCTOR OF PHILOSOPHY

Molecular Pathology Program

May 2013

Winston-Salem, North Carolina

Approved By:

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.

 

ACKNOWLEDGMENTS

At 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

most in the future. Everyone on the floor is willing to help, and it is never a problem to ii

 

 

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.

iii

 

 

TABLE OF CONTENTS

LIST OF TABLES AND FIGURES...................................................................................v

LIST OF ABBREVIATIONS............................................................................................vii

ABSTRACT.....................................................................................................................ix

CHAPTER

I. INTRODUCTION.................................................................................................1

II. MOUSE ORGANIC SOLUTE TRANSPORTER α DEFICIENCY ALTERS FGF15 EXPRESSION AND BILE ACID METABOLISM (PUBLISHED IN THE JOURNAL OF HEPATOLOGY, 2012)........................................46 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) ..............................................92 IV. DISCUSSION...................................................................................................133

CURRICULUM VITAE.................................................................................................149

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LIST OF ILLUSTRATIONS AND TABLES

Page

Chapter II

Table I Hepatic gene expression (Relative to WT) 62 Table II Ileal gene expression (Relative to WT) 63 Table III Phenotypic changes in Fxr-/-, Ostα-/- and Ostα-/-Fxr-/- mice (Relative to WT) 64 Figure 1 BA metabolism, intestinal cholesterol absorption, and hepatic lipids 65 Figure 2 Intestinal length, weight, and villus morphology 67 Figure 3 Ileal FGF15 Expression 69 Figure 4 Ileal gene expression and BA accumulation 71 Supplemental Figure 1 Bile acid metabolism and intestinal cholesterol absorption in female wild type, Fxr-/-, Ostα-/-, and Ostα-/-Fxr-/- (DKO) mice 82 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 Figure 4 Intestinal expression of FGF15 and Apo A-I protein in male wild type and Ostα-/- mice 88

Chapter III

Figure 1 Effect of Asbt deficiency on lipid metabolism and atherosclerosis development in ApoE-/- mice 112 Figure 2 Effect of Asbt deficiency on hepatic and ileal gene expression in ApoE-/- mice 114 Figure 3 Effect of Ostα deficiency on lipid metabolism and atherosclerosis development in ApoE-/- mice 116

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Figure 4 Effect of Ostα deficiency on hepatic and ileal gene expression in ApoE-/- mice 118 Figure 5 Effect of Ostα deficiency on bile acid metabolism and de novo cholesterol synthesis in ApoE-/- mice 120 Figure 6 Effect of Ostα deficiency on reverse cholesterol transport 122 Supplemental Table I RT-PCR Primers 124 Table II Body weight and liver weight related to Asbt deficiency 125 Table III Hepatic and ileal gene expression 126 Table IV Body weight, liver weight and intestinal length and weight to Ostα deficiency 127 Supplemental Figure 1 Effect of Asbt deficiency on lipid metabolism and atherosclerosis development in LDLr-/- mice 128

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LIST OF ABBREVIATIONS

ASBT Apical Sodium Dependent Bile Acid Transporter

BARE Bile Acid Response Element

BAS Bile Acid Sequestrant

BSEP Bile Salt Export Pump

CA Cholic Acid

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

CM Chylomicron

COX Cyclooxygenase

FXR Farnesoid X 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

LCA Lithocholic acid vii

 

 

LDLr Low density lipoprotein receptor

LXR Liver X receptor

MDR Multiple drug resistance

MTP Microsomal triglyceride transport protein

NTCP Na+/taurocholate cotransporting polypeptide

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

SR Scavenger receptor

TNFα Tumor necrosis factor α

UDCA Ursodeoxychoic acid

WT Wild type

viii

 

 

ABSTRACT

Tian Lan

THE ROLE OF OSTα/β IN BILE ACID AND LIPID METABOLISM

Dissertation under the direction of Paul A. Dawson, Ph. D., Professor of Gastroenterology/Internal Medicine

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.

ix

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

bile acid signaling in cholesterol homeostasis and atherosclerosis development. The

 

 

x

 

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.

 

CHAPTER I

Introduction

Tian Lan prepared this chapter. Dr. Paul A. Dawson acted in an editorial and advisory capacity

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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 5-

or 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 β-

muricholic 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

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through the intestinal wall. The majority of bile acids are reabsorbed by a transporter-

mediated 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

3

 

 

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).

There are two pathways for bile acid synthesis in the liver (3, 5). In the

classic/neutral pathway, cholesterol is first hydroxylated at the C7 position by the

microsomal enzyme cholesterol 7α-hydroxylase (CYP7A1). The Δ5,6 double bond of 7α-

hydroxylcholesterol is then reduced by the microsomal 3β-hydroxy- Δ5-C27-steroid

dehydrogense/isomerase (HSD3B7). The resulting product can then take several

different routes. If hydroxylated at the C12 position by the sterol 12α-hydroxylase

(CYP8B1), the product will ultimately be converted to cholic acid (CA). If not 12α-

hydroxylated, the product is ultimately converted to chenodeoxycholic acid (CDCA) in

humans or muricholic acid in mice. Regardless, the cytosolic Δ4-3-oxosteroid-5β-

reductase (AKR1D1) and 3α-hydroxysteroid dehydrogenase (AKR1C4) enzymes

catalyze the next steps for either product. The intermediates 5β-cholestan-3α, 7α, 12α-

triol or 5β-cholestan-3α, 7α-diol are eventually subjected to C27 hydroxylation

(mitochondrial CYP27A1), Co-A ligation (bile acid CoA synthetase) and side chain β-

oxidation (peroxisomal enzymes).

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The alternative/acidic pathway is initiated by the sterol 27-hydroxylase

(CYP27A1), followed by 7α hydroxylation carried out by oxysterol 7α-hydroxylase

(CYP7B1) rather than CYP7A1. CDCA is the bile acid species derived from the

alternative pathway in humans. In mice, however, CDCA is further hydroxylated at the

C6 position and epimerized to form β-muricholic acid.

The classic pathway contributes approximately 75% of the total hepatic bile acid

synthesized in human and more than 90% in mice. In mice, the activity of the classic

pathway is low at birth and increases significantly after weaning (6). The alternative

pathway is active several weeks after birth in mice, and gradually reaches its peak level

at approximately 2-3 months of age. The classic pathway in mice is up-regulated by

cholesterol feeding, which provides more substrate for de novo synthesis, and down-

regulated by bile acid feeding (7). This regulation is mediated by controlling the

expression of Cyp7a1, the first committed and rate-limiting step in bile acid synthesis.

Details of Cyp7a1 regulation will be discussed in the next section. Unlike the classic

pathway, the alternative pathway is not responsive to dietary cholesterol or bile acid

depletion and is not thought to be tightly regulated. However, the alternative pathway

can serve as an important compensatory mechanism when the classic pathway is

compromised (6). In Cyp7a1 deficient mouse models and patients, Cyp27a1 expression

is up-regulated nearly 2-fold (8). The very high stool fat excretion observed in newborn

Cyp7a1-/- mouse significantly drops after day 21, correlating with the appearance of

oxysterol 7α-hydroxylation activity in liver. As Cyp27a1 is ubiquitously expressed, its

major function may be to stimulate cholesterol turnover in peripheral tissues by

synthesizing oxysterols for subsequent transport to the liver. In fact, CYP27A1 deficiency

in human is associated with cerebrotendinous xanthomatosis (CTX) characterized by

alterations in cholesterol homeostasis, including cholesterol deposits in tendon and other

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tissues, premature atherosclerosis, and gallstones (9). Although a minor player in bile

acid synthesis, the alternative pathway is physiologically indispensable.

2.2. Regulation of Cyp7a1 Expression

2.2.1. CYP7A1 protein and gene structure

CYP7A1 resides in the endoplasmic reticulum and is expressed exclusively in the

liver (7). The Cyp7a1 gene encodes proteins of 504 and 503 amino acids in human and

mouse, respectively. Like other members in the cytochrome P450 superfamily, CYP7A1

utilizes a heme cofactor for its oxidase activity.

As the first committed step of the bile acid synthesis pathway that links

cholesterol and bile acid metabolism, there is extensive regulation of Cyp7a1 (4).

Cyp7a1 is regulated by circadian rhythm, insulin and glucagon, enrichment or depletion

of the bile acid pool, and dietary cholesterol. The complicated hormonal and

physiological regulation is thought to occur primarily at the transcriptional level, which

has been the major focus of investigation. With regard to the regulation by cholesterol

and bile acids, two regions of the Cyp7a1 promoter have been implicated (namely bile

acid response elements, BARE-I and -II) (10). BARE-I contains a direct repeat separated

by 4 nucleotides (DR4) while BARE-II has overlapping DR1 and DR5. Briefly, COUP-

TFII and LXRα can interact with BARE-I and stimulate Cyp7a1 transcription. BARE-II

has specific affinity to HNF4α and LRH-1. Co-activators and co-repressors interact with

the transcription factors mentioned above to control Cyp7a1 expression.

2.2.2. Regulation of Cyp7a1 Promoter by Nuclear Receptors

LXRα: LXR is a nuclear receptor that specifically binds to oxysterols. Two

isoforms of LXR, LXRα and LXRβ, has been identified. LXRα and LXRβ are encoded by

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separate genes on different chromosomes and show distinct patterns of tisue expression.

The regulation of Cyp7a1 expression by LXRα was first demonstrated in Lxrα-/- mice (11).

Addition of 2% cholesterol to the diet significantly increases fecal bile acid excretion in

WT mice, a surrogate measurement of hepatic bile acid synthesis. Northern blot analysis

also revealed a 6-fold increase in hepatic Cyp7a1 mRNA levels. Genetic ablation of Lxrα

almost completely blocked the induction in Cyp7a1 expression, resulting in enormous

hepatic lipid accumulation and dysfunction. The presence of Lxrβ was unable to

compensate for the loss of Lxrα. The in vivo induction of mouse Cyp7a1 by Lxrα was

confirmed using in vitro reporter assays and Cyp7a1 promoter constructs.

Like other nuclear receptors, heterodimer formation with RXR is required for

LXRα activation of Cyp7a1. Rxr null mice display phenotypes similar Lxrα-/- mice with

regard to Cyp7a1. Notably, this feed-forward up-regulation of Cyp7a1 by cholesterol and

cholesterol metabolites acting through LXRα is present in rodents, but absent in humans.

The human CYP7A1 promoter lacks the LXR binding site and is not activated by LXRα

(4). Instead, LXRα in human hepatocytes activates expression of SHP (discussed in

detail in following sessions), a co-repressor of CYP7A1. The evolutionary significance of

this important metabolic difference (stimulation versus inhibition) between rodents and

humans is unknown.

HNF4α: HNF4α is abundantly expressed in hepatocytes. Direct binding of HNF4α

to the BARE-II element of rat Cyp7a1 promoter was demonstrated (12), and the diurnal

rhythm of Hnf4α expression positively correlates with that of Cyp7a1. In the liver specific

Hnf4α knockout mouse model, Cyp7a1 expression is significantly decreased, with a

corresponding decrease of fecal bile acid excretion, a measurement of hepatic bile acid

synthesis. HNF4α plays an important role in the regulation of Cyp7a1 in response to

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fasting-refeeding. During fasting, hepatic PPARγ activates expression of a series of

genes such as Pepck and Pgc1α. PGC1α association with HNF4α significantly activates

Cyp7a1 expression. HNF4α is also involved in controlling the diurnal rhythm of CYP7A1

expression by interaction with hepatic albumin promoter D-site binding protein (DBP)

(13). In hepatic Hnf4α knockout mice, the up-regulation of Cyp7a1 expression normally

observed in dark cycle is absent.

LRH-1 and SHP: Liver receptor homologue-1 (LRH-1) is an orphan receptor

expressed in pancreas, liver and intestine in the male mouse and is homologous to the

Drosophila Fushi Tarazu F-1 receptor (14). The specific binding of hepatic LRH-1 to the

Cyp7a1 promoter was first demonstrated in HepG2 cells in 1999 (15). LRH-1 appears to

be dispensable for baseline expression of Cyp7a1, but plays a role in the regulation by

bile acids. In the current model, activation of hepatic FXR, a bile-acid ligated nuclear

receptor, induces SHP. SHP is an atypical nuclear receptor that lacks a DNA binding

domain and associates with other transcription factors to repress gene expression. In

this model, SHP associates with LRH-1 to repress Cyp7a1 promoter activity (16).

Although later studies indicated this hepatic FXR pathway is subordinate to bile acid

feedback regulation mediated by the gut hormone FGF15/19 (which will be discussed

later), the hepatic FXR-SHP-LRH-1 pathway may be important under certain

pathophysiological conditions such as cholestasis.

Other Regulation Mechanisms: RXRα dimerizes with LXR and FXR before

binding to Cyp7a1 promoter. Mice with RXRα deficiency failed to respond to dietary

cholesterol by inducing hepatic bile acid synthesis (17). These mice also have higher

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basal levels of Cyp7a1 expression, indicating a lack of FXR-mediated feedback

regulation.

Disruption of normal hepatic bile secretion or bile flow can result in the

accumulation of toxic secondary bile acids such as lithocholic acid in hepatocytes. Under

these conditions, the nuclear receptors Pregnane X Receptor (PXR) and Constitutive

Androstane Receptor (CAR) are activated and function to repress Cyp7a1 expression.

PXR represses Cyp7a1 through interaction with HNF4α to prevent its interaction with

PGC1α (18). The activation of PXR and CAR also induce expression of other

cytochrome P450 and sulfotransferase genes to detoxify bile acids and protect

hepatocytes. PPARα also regulates Cyp7a1 expression by interfering with HNF4α. In

PPARα-/- mice, fibrates are unable to repress Cyp7a1 expression (19).

2.2.3 Other Regulatory Pathways

Cyp7a1 expression is also negatively regulated by cytokines such as TNF1α and

IL-1β. In vitro studies reveal that co-culturing of hepatoma cells with macrophages

decreases Cyp7a1 mRNA expression. Treatment of rosiglitazone, which activates

PPARγ and blocks cytokine secretion, blunts the inhibitory effect (20). The mediators of

this repressive pathway include hepatic PKC and JNK, which interfere with HNF4α

activation.

In 2005, an extra-hepatic signaling pathway mediated by intestinal bile acids was

identified (21). This pathway will be discussed at greater length in the following sections.

Briefly, intestinal bile acids activate FXR, which subsequently induces expression of the

protein hormone FGF15/19. Intestinal FGF15/19 is carried to the liver in the portal

circulation where it signals through its receptor FGFR4 to repress Cyp7a1 expression.

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Through this pathway, hepatic bile acid synthesis is regulated by the flux of bile acids

through the small intestine.

3. Enterohepatic Circulation of Bile Acids

Bile acids are the driving force for secretion of bile, a complex mixture of bile

acids, phospholipids, cholesterol, small ions (Na+, HCO3-, Cl-, etc), physiological waste

products such as bilirubin, and xenobiotics such as drugs and drug metabolites. Thus

bile is more than a digestive secretion to solubilize dietary lipids, and is continuously

secreted by the liver throughout the day. As intestinal reabsorption and re-circulation

back to the liver is very efficient (~95%), only about 5% of the bile acids secreted by the

liver each day are newly synthesized. This efficient recycling system conserves the

energy required for bile acid synthesis and ensures that sufficient bile acid is available

throughout the day to maintain hepatic bile secretion and absorption of dietary lipids.

The enterohepatic circulation also largely restricts bile acids to the intestinal and

hepatobiliary compartments, thereby reducing the potential for toxicity or altered

signaling. The enterohepatic bile acid circulation is maintained by a variety of

transporters residing on the enterocytes and hepatocytes.

3.1. Enterohepatic Circulation in Steps

3.1.1. Biliary Secretion and Excretion

Hepatic bile acids are secreted across the canalicular membrane by the Bile Salt

Export Pump (BSEP: ABCB11) (22). The concentration of biliary bile acids is much

higher than that inside the hepatocytes and ATP hydrolysis provides the energy for

BSEP to transport bile acids against this gradient. BSEP deficiency causes cholestasis

(23). Bile acids are the most abundant “primary solute” and provide much of the osmotic

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driving force for the movement of other solutes into bile. Apart from bile acids, bile

contains cholesterol, phosphatidylcholine, Cl-, HCO3-, bilirubin and xenobiotics. Biliary

cholesterol is excreted by the hepatic transporter ABCG5/ABCG8 and is the major route

for cholesterol disposal from the body. Hepatic bile is remodeled by the biliary epithelium

as it transits through the biliary tract and empties into the gallbladder during the inter-

digestive period. Bile is further concentrated approximately 5-10 fold in the gallbladder

by the absorption of water and biliary solutes, leaving a concentrated detergent solution

that can be delivered in a controlled fashion during a meal. Cholesterol in bile forms

micelles with bile acids and phospholipids. However, in the gallbladder, the

concentration of cholesterol may exceed its saturation index and lead to the formation of

cholesterol gallstones.

Phosphatidylcholine (PC) is an important solute in the bile. As mentioned above,

PC is transported from the canalicular cell membrane into bile by a flippase, ABCB4

(MDR3 in human, MDR2 in mouse) (24). PC has two major functions in bile: 1) PC

promotes micelle formation with bile acids and significantly decreases their CMC. 2) PC

prevents monomeric bile acids or pure bile acid micelle formation in the bile, which are

damaging to cell membranes. In fact, MDR2/MDR3 mutations lead to the development

of cholestasis (25).

Bile also contains sufficient amount of bicarbonate, which forms a protective

alkaline barrier (“umbrella”) on the surface of cholangiocytes (26). Bicarbonate increases

the pH of the cholangiocyte surface and prevents protonation of bile acids, which may

passively diffuse into and damage the cell. Bicarbonate is secreted by the biliary

epithelium in a two-step process involving Cl- secretion by CFTR followed by HCO3-/Cl-

exchange by AE2 (27). Bicarbonate secretion is regulated by biliary ATP levels and

TGR5, a cholangiocyte-surface bile acid receptor. Bicarbonate secretion is also up-

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regulated by UDCA, the active component of bear bile that had been used as part of

traditional Chinese medicine to “clear liver toxicity”. As a critical route for detoxification,

biliary excretion accounts for almost 100% of bilirubin, iron and copper disposal and the

excretion of many xenobiotics and drugs. The excretion of these solutes is mediated

primarily by ABC transporters (28).

3.1.2. Intestinal Reabsorption

Fatty acids and amino acids in meals trigger gallbladder contraction and bile flow

into the duodenum through release of cholecystokinin from enteroendocrine I-cells from

the duodenum (29). Bile acids join with pancreatic lipase and proteases for nutrient

digestion and absorption. Most absorption of nutrients takes place in the proximal to

middle part (jejunum) of the small intestine. By the time bile acids reach the distal

intestine (ileum), an active reabsorption system recycles bile acids back to the liver (30).

The ileal reabsorption system is a very efficient and specific system: 1) the pH in

the ileal lumen is higher than the pKa of taurine or glycine conjugated bile acids. Thus

the bile acids are ionized and impermeable to the cell membrane. 2) Transporter-

facilitated bile acid reabsorption is a high capacity system, which relies on two major

transporters residing on the apical and basolateral membrane of enterocytes. Apical

sodium bile acid cotransporter (ASBT/SLC10A2) takes advantage of the concentration

gradient of sodium in the intestinal lumen to provide energy for bile acid cotransport and

exhibits a solute specificity that includes only bile acids (31, 32). The basolateral organic

solute transporter α/β (OSTα/β) is a heterodimeric transporter that requires expression of

both subunits for full function and may transport other substrates such as steroid

sulfactes in addition to bile acids (33). Both transporters are expressed at the highest

levels in the ileum, with ASBT expression almost undetectable in duodenum and

12

 

 

jejunum (33). Genetic ablation of either gene dramatically reduces bile acid transport

across the ileum, emphasizing the primary role of these transport systems (34). 3) Asbt

and Ostα/β are regulated in opposite directions by intracellular bile acid levels through

activation of the bile acid nuclear receptor, FXR (35, 36). Ostα/β expression is positively

regulated by bile acids to ensure sufficient export capacity. Conversely, Asbt is

negatively regulated by bile acids to prevent bile acid accumulation and toxicity. The

details of ASBT and OSTα/β distribution, regulation of physiological importance are

discussed in the following sections.

Although most intestinal bile acids are transported by Asbt in the ileum, there is

also passive absorption of bile acids down the length of the intestine. The passive

component is greater in humans than mice. The bile acid pool in humans is more

hydrophobic and includes mostly glycine-conjugated bile acids, which have a higher pKa

and are more membrane-permeable than the taurine-conjugates. Conversely, the mouse

bile acid pool is more hydrophilic and includes almost exclusively taurine-conjugated bile

acids species. In proximal intestine, a fraction of the glycine-conjugated bile acid may be

protonated (pKa ~ 5.0) and undergo passive non-ionic absorption. There may also be a

low level of transporter-mediated facilitative bile acid absorption in proximal small

intestine, most likely involving members of the OATP superfamily (37, 38).

Deconjugation of bile acids by the gut flora has complex effects on bile acid absorption.

Firstly, deconjugation of bile acids reduces their aqueous solubility, causing them to

precipitate and reducing their absorption. Secondly, the deconjugated and

dehydroxylated bile acids are also more hydrophobic and more likely to undergo passive

absorption, particularly during their long residence time in the colon.

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3.1.3. Systemic Circulation and Hepatic Bile Acid Uptake

Upon exiting the enterocytes, bile acids associate with albumin in the portal

circulation and are transported back to the liver. Hepatic uptake of bile acids is efficient

such that 50-90% (depending on the bile acid species) of the albumin-bound bile acids

are extracted during their first past through the liver. As a result, plasma bile acid levels

remain low (<4 µmol/L) in the fasting state in humans and only rise approximately 2 to 3-

fold in the immediate postprandial period (39).

Hepatic uptake of bile acids is mediated by both sodium-dependent and sodium-

independent mechanisms. The Na+ taurocholate cotransporting polypeptide

(NTCP/SLC10A1) accounts for more than 75% of hepatic uptake of conjugated bile

acids (30). The unconjugated bile acids, on the other hand, are taken up by members of

the organic anion transporting polypeptide (OATP1B1, OATP1B3; OATP2B1 in mouse).

NTCP expression is induced by RXR:RAR, HNF-1α, HNF-4α and FOX2α. Intracellular

bile acids repress NTCP through FXR activation of SHP expression.

3.1.4. The Role of Hepatic Bile Acid Synthesis in Enterohepatic Circulation

The total bile acid pool size is approximately 3g and 25mg in adult humans and

mice, respectively. In the fasting state, the majority of the bile acid pool is stored in the

gallbladder and proximal small intestine. Approximately 5% of the bile acids entering the

small intestine escape reabsorption and are excreted into the feces. This accounts for

almost half of the daily cholesterol disposal (3), secondary only to direct cholesterol

excretion by the liver and small intestine. This loss is balanced by new hepatic bile acid

synthesis. As such under steady state conditions, hepatic bile acid synthesis rate is

equal to fecal bile acid output. It is estimated that hepatic bile acid synthesis can be up-

regulated as much as 10-fold (40). As will be discussed later, inhibition of bile acid re-

14

 

 

absorption is a well-documented approach used to increase hepatic cholesterol turnover.

Understanding the regulation of intestinal bile acid transporters provides fundamental

information for pharmacological intervention of bile acid and cholesterol metabolism.

3.2. Intestinal Bile Acid Transporters

3.2.1. ASBT

The human and mouse ASBT genes encodes 348 amino acid proteins that share

80% identity, 89% similarity and a similar membrane topology (31). ASBT expressing

tissues include ileal enterocytes, renal tubule cells, and gallbladder epithelial cells (31,

41), all of which take part in bile acid recycling in the body. The observation that

mutations in the ASBT gene in humans cause bile acid malabsorption clearly

established its importance ileal bile acid reabsorption (42). Genetic inactivation of Asbt in

mice also increases fecal bile acid excretion and hepatic bile acid synthesis that is not

further elevated by administration of bile acid binding resins (40). ASBT in kidney

functions to prevent systemic loss of bile acid from renal filtration (30). ASBT in

cholangiocytes plays a role in the “cholehepatic shunt” where bile acids in bile are cycled

back to hepatocytes for resecretion.

Transcriptional regulation of Asbt expression has been extensively studied. The

ileum-specific expression of Asbt involves GATA4, a member of the zinc-finger family

with highest expression in the duodenum and jejunum. Intestine-specific knockout of

Gata4 results in an ileum-like phenotype in the jejunum with increased goblet cell

population, increased peptide YY levels and appearance of ASBT (43, 44). Clinically

targeting GATA4 may reverse the bile acid malabsorption observed following ileal

resection in patients.

15

 

 

HNF1α and LRH-1 maintain basal expression of Asbt (45, 46) and may be

involved in the regulation of Asbt expression by bile acids. Gene expression analysis

reveals bile acids feedback to down-regulate Asbt expression by activating FXR and its

target gene SHP, which antagonizes LRH-1 on the Asbt promoter (47). Apart from FXR-

SHP pathway, ileal FXR also activates the target gene FGF15, which acts as a paracrine

fashion on enterocyte-surface receptor FGFR4 to repress ASBT expression (48). This

feedback regulation of ASBT may serve to protect the enterocytes from bile acid

overload.

Asbt may also be regulated by SREBP2. Supplementation of the diet with

cholesterol and statins resulted in repressed and increased ASBT levels, respectively in

mice (49). The human and mouse ASBT promoters appear to lack SREBP2 binding

sites. In this mechanism, SREBP2 is thought to interact directly with HNF1α to induce

Asbt expression. Physiologically, this may be a mechanism to preserve the bile acid pool

and enhance cholesterol absorption under conditions of reduced cholesterol availability

or to enhance bile acid excretion under conditions of cholesterol excess.

Inflammatory cytokines also repress Asbt expression through two AP-1 binding

sites within the Asbt promoter (50). The upstream AP-1 site binds to c-Jun homodimers

and is responsible for the basal transcription of Asbt, whereas c-Jun/c-Fos heterodimers

negatively regulates promoter activity through binding to the downstream AP-1 site.

Inflammatory cytokines such as IL-1β promotes expression of c-Jun and c-Fos, which

functions to repress Asbt expression (51). The AP-1 sites are well conserved in mouse,

rat and human. c-Fos-/- mice displayed a paradoxical increase of ASBT expression in

response to indomethacin induced ileitis, possibly due to the activation of c-Jun

expression without corresponding antagonizing effect of c-Fos on the downstream AP-1

site.

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Post-transcriptional regulation of ASBT has been explored in several studies.

Apart from the AP-1/c-Fos mediated repression, inflammatory cytokines promote

ubiquitination and degradation of ASBT protein in cholangiocytes (52). Inflammation

associated PKC activation prevents localization of ASBT to cell surface (53). Depletion

of membrane cholesterol by β-cyclodextrin significantly inhibited bile acid transport

activity by ASBT, indicating functional ASBT requires association with lipid raft structure

(54).

Asbt expression is also regulated by hormones. Increased intestinal bile acid

absorption and expansion of bile acid pool is common in diabetic patients (55). Following

streptozotocin treatment of mice to induce diabetes, intestinal Asbt mRNA and protein

expression were both elevated (56). Ontogenic analysis of Asbt expression in rats

revealed a dramatic increase after weaning. Weaning at later times delays the increase

in Asbt expression whereas early weaning or administration of dexamethasone hastens

the increase for Asbt expression in rats. Activation of glucocorticoid receptor is

hypothesized to mediate Asbt expression changes at weaning (57, 58).

3.2.2. OSTα/β

OST is a heterodimeric transporter composed of an α and β subunit. In 2001, the

first ortholog was identified in the liver of skate (Raja erinacea) by expression cloning

approaches (59). Shortly after, the human and rodent orthologs of Ostα/β were identified

(60). Although relatively distant from the skate ortholog, the sequences of α and β are

well conserved in human, mouse and rat. The α subunit is 340 amino acids in length and

encompasses 7 predicted transmembrane domains, whereas the β subunit is 128 amino

acids in length and includes only 1 transmembrane domain. Both subunits have a

predicted extracellular N-terminus and intracellular C-terminus. In humans, moderate to

17

 

 

high OSTα/β expression is detected in testis, adrenal gland, kidney, liver and small

intestine. Low levels of expression are also found in brain, pituitary gland, heart, lung

and prostate. In mouse, OSTα/β was found in kidney, along the GI tract, but not

detected in liver under basal conditions.

Both subunits are required for trafficking of the protein complex to the plasma

membrane. In vitro studies showed that taurocholate is only transported when both

subunits were co-expressed (33). Protein expression of the β subunit is undetectable

when the α subunit is inactivated genetically (34). An extensive study of the OSTβ

protein (61) revealed the first amino acid of the transmembrane domain (Trp34) and the

two Arg immediately following the TM domain (Arg53, Arg54) directly regulates

maturation, cell-surface localization and taurocholate transport activity of OSTα/β.

Unlike the unidirectional, sodium-facilitated transport activity of ASBT, OSTα/β is

a bidirectional transporter. OSTα/β transports a wide range of substrates including bile

acids, estrone-3-sulfate, digoxin, prostaglandin-E2 and estradiol-glucuronide.

The expression of OSTα/β down the GI tract is similar to that of the ASBT, with

the highest levels in the ileum. Immunohistological studies localized OSTα/β to the

basolateral membrane of ileal enterocytes, clearly distinct from the apically expressed

ASBT (33). The abundance of OSTα/β in the ileum, its localization and its ability to

transport bile acids suggested a prominent role in intestinal basolateral bile acid re-

absorption. The exclusive involvement of OSTα/β in bile acid reabsorption is further

supported by everted gut-sac assays in wild type, Asbt-/-, and Ostα-/- mice (34) where

trans-ileal transport of radioactive taurocholate was blocked in gut sacs prepared from

Asbt-/- and Ostα-/- mice. Further knockout of another postulated basolateral bile acid

transporter, MRP3, had little effect on the residual taurocholate transport across the gut

sac.

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The regulation of OSTα and OSTβ by bile acids has been extensively

characterized. Transcription of both subunits is activated by the bile acid nuclear

receptor FXR (35) and repressed by the FXR target gene SHP acting at the LRH-1

binding sites. The dual role of FXR to both activate and repress Ostα/β expression is an

example of a push-pull fashion of regulation (30), which would function to carefully titrate

the expression of Ostα/β to match the bile acid load. In addition to FXR and LRH-1,

other transcription factors involved in controlling Ostα/β expression include LXR and

HNF4α. In intestinal specific LRH-1 knockout mice (46), ileal Ostα/β expression is

reduced. Luciferase activity assay revealed that LXR activated Ostα/β expression by

acting through the same IR-1 element used by FXR/RXR. HNF4α co-expression with

FXR/RXR further activates Ostα but not Ostβ promoter activity (62).

No diseases associated with mutations in the Ostα or Ostβ genes have yet been

reported. The physiological role of OSTα/β has been studied in animal models.

Compared to wild type mice, liver injury is much less severe in Ostα-/- mice after bile duct

ligation. The protective effect is attributed to an increase of urinary excretion of bile acids

following loss of renal Ostα/β (63). The effect of Ostα deficiency on bile acid metabolism

will be discussed in detail in the following sections. Briefly, Ostα-/- mice do not show a

compensatory increase in hepatic bile acid synthesis like the Asbt-/- mice (34). Fecal bile

acid excretion remains unchanged, bile acid pool is decreased, and intestinal cholesterol

absorption is decreased more severely than Asbt-/- mice. Phenotypically, the small

intestine is heavier and longer in Ostα-/- mice, and the change is more significantly in the

ileum, where Ostα/β is expressed at the highest level (64). In summary, OSTα/β is the

primary bile acid transporter on the basolateral membrane of ileal enterocytes, yet

Ostα/β deficiency does not phenocopy the loss of Asbt.

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3.3. FXR and Enterohepatic Circulation

3.3.1. Introduction of FXR

3.3.1.1. FXR Structure and Tissue Distribution

FXR was discovered in 1995 as one of the RXR Interacting Proteins (RIP14) by

Seol and colleagues (65). Using restriction enzyme digestion and partial sequencing, the

authors predicted that there are two forms of RIP14, which are distinguished by a

different N-termini and a 4-AA insert (MYTG) before the hinge domain. The name

“farnesoid X receptor” was given after the finding that the cholesterol biosynthetic

intermediate farnesol activated the RXR-RIP14 heterodimer. Further studies of FXR in

hamster, mouse and human genome revealed four isoforms of FXR: namely FXRα1, 2,

3 and 4, which are generated by differential promoter utilization and splicing (66, 67).

The amino acid sequences of the DNA and ligand binding domains are identical for the

four isoforms. However, FXRα1,2 and FXRα3,4 have unique N-terminal sequences

respectively, whereas FXRα1,3 includes the amino acids MYTG inserted before the

hinge domain. The transcriptional activity is different for the 4 isoforms, with the 2

isoforms that include MYTG sequences being weaker transcriptional activators (66).

FXR is expressed at the highest levels in liver, small intestine, kidney, and

adrenal glands. The function of FXR in bile acid metabolism will be discussed shortly.

FXR is also expressed in macrophages and at very low levels in heart and adipose

tissue. The physiological role of FXR in those tissues has not yet been explored in detail.

However, activation of FXR in macrophages was demonstrated to increase cholesterol

efflux and prevent atherosclerosis development (68). FXR regulates insulin sensitivity,

glucose uptake and differentiation of adipocytes (69), and a recent study showed FXR

may mediate apoptosis in heart myocytes (70).

20

 

 

3.3.1.2. FXR Ligands and DNA Binding

Although FXR was deorphanized by the identification of farnesol as a ligand.

However, the requirement of very high farnesol concentrations for activation prompted

pursuit of more potent natural ligands. In 1999, two groups used a yeast GAL4 fusion

protein assay (71) and luciferase reporter assay (72) to identify bile acids as natural FXR

ligand, with CDCA having the highest potency to activate FXR, (EC50 = 4.5µM). Using

synthetic promoter constructs, these groups showed that bile acids and FXR increased

and repressed the promoter activities of intestinal bile acid binding protein and Cyp7a1,

respectively. The feed-forward regulation of Cyp7a1 by cholesterol (through LXR) and

the feedback negative regulation by bile acids through FXR fit the anticipated direction

and indicated the central role of FXR in bile acid metabolism.

Several naturally occurring FXR agonists and antagonists were identified. These

include cafestol, a cholesterol-elevating neutral compound found in unfiltered coffee. It

was demonstrated that cafestol activates intestinal and hepatic FXR and PXR to induce

expression of genes related to cholesterol metabolism and detoxification and repress

Cyp7a1 expression (73). The FXR-mediated repression of Cyp7a1 by cafestol accounts

for its plasma cholesterol-raising effects. Conversely, gum resin from the guggle tree

contains appreciable amounts of guggulsterone, an FXR antagonist that induces Cyp7a1,

and is an ancient Indian medicine used to treat lipid disorders (74). Certain phytosterols

such as stigmasterol have also been identified as FXR antagonists. Interestingly,

stigmasterol is an abundant lipid in the soybean based formula used for parenteral

nutrition and antagonism of the protective effects of FXR by stigmasterol is thought to be

one of the mechanisms responsible for parenteral nutrition-associated cholestasis (75).

FXR dimerizes with RXR and binds to inverted repeat elements of AGGTCA with

one nucleotide spacing (IR-1) (76). FXR/RXR heterodimer also binds to IRs with multiple

21

 

 

spacing nucleotides (77), everted repeats (ER-8), and direct repeats with 1-5 spacing

nucleotides (DR-1 to DR-5). Whole genome chromatin-immunoprecipitation approaches

have recently been used to identify FXR binding sites and potential FXR target genes.

FXR-mediated transcriptional activation is regulated by coactivators. Steroid Receptor

Coactivator-1 (SRC-1) is recruited to FXR upon CDCA (78). Another member of the

SRC family, SRC-2, also binds to FXR during energy depletion, which increases BSEP

expression to maintain hepatic bile acid output for better lipid digestion and absorption

(79).

3.3.2. FXR is a Master Regulator of Enterohepatic Circulation of Bile Acids

The central role of FXR in bile acid synthesis and transport has been studied for

many years. [2H4] cholate infusion in Fxr-/- mice (80) revealed an increase in plasma bile

acid concentration, bile acid synthesis, biliary bile salt output, intestinal bile acid

reabsorption, bile acid pool size, and enrichment in bile acid species from the classic

synthesis pathway. No sign of hepatic toxicity was observed. However, FXR-/- mice on

cholic acid-containing diet showed significant increase of hepatic lipid accumulation,

urinary and plasma bile acid elevation, and a 40% decrease of body weight in as short

as 5 days on diet, whereas the WT littermates remained healthy (81). Feeding

cholesterol to FXR-/- mice significantly increases hepatic cholesterol and triglyceride

levels, and caused a general increase in all plasma lipoprotein cholesterol fractions (81).

In these mice, hepatic Cyp7a1 was unresponsive to dietary cholic acid levels. Hepatic

genes responsible for cholesterol metabolism including SR-BI, Abcg5/g8, HMG-CoA

reductase are generally decreased in FXR-/- mice (82). These studies demonstrated FXR

to be a critical regulator of the metabolism of other lipids besides just bile acids.

22

 

 

3.3.3. FXR Regulation of Bile Acid Transporters

The IR-1 FXRE within the promoter of Bsep was first identified by the lab of Dr.

Frederick Suchy (83). Following co-expression of FXR and RXR and addition of CDCA,

Bsep promoter activity was significantly induced. The mechanism for this induction has

been explored and involves recruitment of CARM1 and PRMT1, two histone methylases,

to enhance Bsep transcription (84, 85). BSEP activity is also regulated by ATP8B1

(FIC1), which is defective in familial intrahepatic cholestasis type 1. ATP8B1 may

regulate BSEP by an indirect mechanism involving FXR, whereby ATP8B1 stimulates

PKCζ phosphorylation of FXR. The phosphorylated FXR translocates to the nucleus and

activates its target genes including Bsep. In addition, ATP8B1 may directly modulate

BSEP activity in the canalicular membrane. ATP8B1 translocates phosphatidylserine

from the outer to inner leaflet of the canalicular membrane and functions to maintain

membrane cholesterol content, which is critical for BSEP function (86).

In hepatocytes, FXR represses Ntcp through Shp activation (87). This inhibition

is mediated through the RXR:RAR element. Under normal physiological conditions, the

expression of Ntcp is matched to bile acid load in the enterohepatic circulation. However,

under pathophysiological conditions such as cholestasis, this FXR-mediated repression

of Ntcp may be particularly important for protecting against bile acid accumulation and its

associated toxicity.

FXR regulation of the bile acid transporters in ileal enterocytes parallels that in

hepatocytes. The apical bile acid transporter ASBT is repressed by FXR-SHP-LRH-1

pathway (16). In this model, FXR stimulates the expression of SHP, which antagonizes

the transcriptional activity of LRH-1. As mentioned earlier, in vitro studies demonstrated

that LRH-1 activates Asbt promoter activity while SHP antagonizes it (47). With regard to

bile acid export from the ileal enterocyte, both subunits of the ileal basolateral bile acid

23

 

 

transporter Ostα/β are positively regulated by bile acids via one or more functional

FXREs. The discovery of LRH-1 elements in Ostα/β promoters suggests that bile acids

can negatively regulate their expression. This form of push-pull regulation will titrate the

expression of Ostα/β to closely match the bile acid load.

3.3.4. Regulation of CYP7A1: FXR-FGF15-FGFR4 Pathway

The negative feedback regulation of Cyp7a1 by bile acids has been appreciated

for many years. Bile acid feeding is accompanied by a reduction of hepatic bile acid

synthesis, whereas depletion of endogenous bile acids by feeding bile acid sequestrants

(BAS) up-regulates hepatic Cyp7a1 expression (7). Indeed, the bile acid pool is

expanded in FXR-/- mice and repression of Cyp7a1 by cholic acid feeding is significantly

impaired. The mechanism of Cyp7a1 regulation by bile acids was originally attributed to

the hepatic FXR-SHP-LRH-1 pathway (16), however more recent studies uncovered an

important role for the intestine-derived enterokine Fibroblast Growth Factor 15/19 in this

process.

Several observations have suggested bile acid accumulation in the liver and

activation of the FXR-SHP-LRH-1 is not sufficient to explain negative feedback up-

regulation of Cyp7a1 by bile acids. These includes: 1) hepatic CYP7A1 mRNA

expression is increased instead of decreased in cholestatic patients (88); 2) CYP7A1

activity in rat liver is increased after bile duct ligation, while infusion of lymph from sham

operated mice into bile duct ligated mice represses hepatic CYP7A1 activity (89), and 3)

duodenal but not intravenous infusion of bile acids would repress hepatic bile acid

synthesis. This later observation, in particular, suggested an intestine-derived factor was

involved. Such a mechanism would not be surprising since the intestine releases a

variety of other hormones such as cholecystokinin, peptide YY, gastro inhibitory peptide

24

 

 

and glucagon-like peptide-1 and 2 to regulate physiological functions including appetite,

bile and pancreatic juice release, insulin secretion and gastrointestinal mobility.

FGF15 (human ortholog FGF19) is an atypical members in the fibroblast growth

factor family (90). Together with FGF21 and 23, these atypical FGFs lack a heparin-

sulfate binding domain. Rather than act solely in an autocrine or paracrine fashion, the

lack of heparin sulfate binding allows FGF15/19 to function as an endocrine hormone,

entering the systemic circulation upon synthesis and affecting distinct organs through

binding to FGF receptors (FGFR).

Individual members of the FGF family have distinct binding specificities for the

different FGF receptors. For the endocrine FGFs, binding requires members of the

Klotho family (αKlotho or βKlotho), which function as co-receptor for specific FGFRs (91,

92). FGF15/19 activates downstream effectors (FRS2, ERK1/2) through FGFR1 and

FGFR4, when these receptors are coexpressed with βKlotho. FGFR4, which is

abundantly expressed in liver, only responds to FGF15/19 activation.

Several lines of evidence suggested the involvement of FGF15-FGFR4 signaling

in bile acid metabolism. In Fgfr4-/- mice, bile acid pool, fecal bile acid excretion, and

hepatic Cyp7a1 expression is significantly increased, a phenotype similar to Fxr-/- mice

(93). Transgenic expression of an active form of FGFR4 in mice significantly decreased

Cyp7a1 activity (94). Finally, human hepatocytes express FGF19 upon FXR activation

and FGF19 directly represses CYP7A1 expression through phosphorylated JNK (95).

These findings all pointed to some role of FGF15/19 in regulating hepatic bile acid

synthesis.

In 2005, the Kliewer/Mangelsdorf lab in Dallas directly demonstrated the

enterohepatic regulation of Cyp7a1 expression by ileal FGF15 (21). GW4064, a potent

FXR agonist, significantly increased Fgf15 expression in the ileum. Both GW4064 and

25

 

 

FGF15-expressing adenovirus infection significantly decreased hepatic Cyp7a1

expression. The inhibitory effect of GW4064 was absent in Fgf15-/- mice, and the

FGF15-expressing adenovirus mediated repression of Cyp7a1 was blocked in Fgfr4-/-

and Shp-/- mice. This study showed that ileal Fgf15 activation by FXR plays a major role

in the regulation of hepatic Cyp7a1 expression. It also emphasized the involvement of

hepatic SHP in this pathway. The mechanism of SHP activation by Fgfr4 is not entirely

clear, but some studies indicated kinases downstream can phosphorylate and stabilize

SHP protein by protecting it from ubiquitination and degradation (96).

Ileal FGF15 also regulates other physiological process and metabolic pathways

besides bile acid synthesis. Through interaction with FGFR3, FGF15 stimulates

gallbladder filling (97). Other hepatic effects of FGF15 include repression of

gluconeogenesis (98), induction of glycogen synthesis (insulin-like) (99), and repression

of fatty acid synthesis, a property distinct from insulin (100). The repression of fatty acid

synthesis is mediated by down-regulation of SREBP1c, a key transcriptional regulator of

fatty acid synthase, stearoyl-CoA desaturase-1, and acetyl-CoA carboxylase. Transgenic

FGF19 expression showed beneficial effect on insulin sensitivity, increase energy

expenditure, and slowed down body weight gain in response to a high fat diet (101, 102).

In summary, the postprandial uptake of bile acids by the ileum stimulates FXR

and FGF15 expression. FGF15 signals to the liver and exerts insulin-like effects

including suppression of bile acid synthesis. Time-controlled expression profile revealed

that intestinal FGF15 expression has a delayed peak relative to that of plasma insulin.

Thus FGF15 acts as a postprandial hormone in addition to insulin and may extend

intestinal control of anabolic processes after food intake.

26

 

 

3. Bile Acid Metabolism and Atherosclerosis

3.1. Atherosclerosis Pathology

Atherosclerosis is a chronic disease characterized by lipid deposition in the

cardiovascular system. The driving force behind the development of atherosclerosis is

high levels of cholesterol-rich, ApoB-containing lipoproteins, such as LDL, VLDL and

chylomicron remnants in the circulation (103). Progression of atherosclerosis is initiated

by retention of the ApoB-containing lipoprotein particles (mostly LDL) in the aortic intima.

This retention is mediated by both free diffusion and interaction between lipoprotein and

proteoglycans. The accumulation of lipoproteins results in release of chemokines from

endothelial cells, which subsequently stimulate monocyte entry to the intima, where the

lipoproteins are located (104). Monocytes then become macrophages and internalize

lipoproteins through cell surface receptors such as LDLr, SRA and CD36. LDLr-

mediated uptake is reduced after cellular cholesterol enrichment causes downregulation

of SREBP2, the major transcriptional regulator of LDLr expression. However, intimal

lipoproteins will become oxidized and then internalized through receptors such as

scavenger receptors A (SR-A) that are not feedback regulated by intracellular

cholesterol levels. Eventually monocytes become cholesterol loaded and are converted

to foam cells. In advanced atherosclerosis, apoptotic macrophages, cell debris, and

cholesterol crystals form the necrotic core of the atherosclerotic lesion, which is covered

with a thin fibrous layer. The advanced plaque is vulnerable to rupture and the exposed

surface results in thrombosis, which is the ultimate reason for heart attack, ischemia,

and stroke.

27

 

 

3.2. Lipoprotein Metabolism and Hypercholesterolemic Mouse Models

3.2.1. Metabolism of ApoB-containing lipoproteins and Hyperlipidemia

Chylomicrons (CM) serve to deliver dietary lipids from intestine to the systemic

circulation (105). After absorption of dietary lipids from the intestinal lumen, triglycerides,

phospholipids and cholesterol are packed into chylomicrons with ApoB48 and delivered

into lymph. While in lymph, chylomicrons acquired ApoE and finally enter the plasma

through the thoracic duct. Triglyceride carried by the CM is hydrolyzed by lipoprotein

lipases on various organs to release free fatty acids, converting the CM to a cholesteryl-

ester rich remnant (CR). ApoE on the CR binds to hepatic receptors such as LDLr and

LRP and is internalized (106).

The liver also delivers lipid to the other tissues through secretion of VLDL. VLDL

particles contain triglyceride, cholesterol, and the apolipoproteins B and E. Once

triglyceride is hydrolyzed, VLDL is converted to an IDL or LDL. ApoB100 on LDL binds to

LDLr and is internalized by hepatocytes.

As indicated earlier, the fundamental cause for development of atherosclerosis is

high levels of ApoB-containing lipoproteins. A variety of hyperlipidemias are associated

with defective lipoprotein-receptor interactions and the patients with the defect are more

susceptible for atherosclerosis development. For example, Type I hyperlipidemia is

characterized by defective lipoprotein lipase activity and accumulation of chylomicron;

Type II (familial hypercholesterolemia) is caused by defective LDLr; Type III

(dysbetalipoproteinemia) results from defective ApoE.

28

 

 

3.2.2. Hypercholesterolemic Mouse Models

Mice are ideal for biological studies due to their small size, short reproductive

cycle, and availability of genetically inbred strains (107). However important species

differences originally made mice a relatively poor model for the study of human

atherosclerosis. It was not until the advent of genetically modified models that mice

became widely used to study atherosclerosis. The limitations of mice with regard to

atherosclerosis research include: 1) Mice are HDL-predominant animals. A normal

mouse on chow carries almost all its plasma cholesterol in HDL particles. 2) Even when

fed an atherogenic diet such as the Paigen diet, the development of atherosclerosis is

limited. 3) Feeding of cholic acid, a component of the Paigen diet, causes hepatic

inflammation, which may confound interpretation of experimental results.

LDLr-/- and ApoE-/- mice are the most commonly used models for atherosclerosis

research. Mice lacking LDLr are unable to clear LDL particles. Examination of the

lipoprotein profile reveals dramatic accumulation of cholesterol in the LDL peak and

feeding a high cholesterol diet results in atherosclerotic lesions in the aorta and heart

valve. ApoE-/- mice exhibit defective clearance of lipoprotein remnants. As a result,

cholesterol accumulates in the chylomicron remnant/VLDL region. LDL can still be

cleared in ApoE-/- mice through binding of ApoB100 to LDLr. Compared to LDLr-/- mice,

ApoE-/- mice have higher plasma cholesterol levels and are prone to develop

atherosclerosis even on chow. High cholesterol-containing diets causes accumulation of

both ApoB100 and ApoB48 in plasma, while ApoE-/- mice only accumulates ApoB48.

Mice defective in both LDLr and ApoE (ApoE-/-LDLr-/-) do not show further increases of

TPC compared to ApoE-alone deficient mice (108).

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3.3. Interruption of Bile Acid EHC as a Therapeutic Approach to Hypercholesterolemia

3.3.1. Bile Acid Binding Resins Stimulate Hepatic CYP7A1 Expression

Bile acid sequestrants (BAS) bind negatively charged bile acids and facilitate

their fecal excretion (109). As discussed previously, increased fecal bile acid loss is

accompanied by a subsequent stimulation of hepatic Cyp7a1 expression and bile acid

synthesis. Conversion of cholesterol to bile acids depletes the hepatocyte cholesterol

pool, increasing levels of mature SREBP2, and inducing expression of its target genes

such as LDLr. LDLr clearance is thus increased and plasma ApoB-containing lipoprotein

levels are decreased. This is the basis for the action of BAS and early clinical trials using

these agents provided some of the first evidence that elevated levels of plasma

cholesterol levels are responsible for atherosclerosis.

Consistent with the BAS studies, human mutations in CYP7A1 also alter

cholesterol homeostasis. To date, there is only one report of a dysfunctional frame-shift

mutation in human CYP7A1 (8). In that family, homozygous carriers of the mutant

CYP7A1 allele displayed a nearly two-fold increase of hepatic cholesterol, increased

fecal lipid content and increased plasma cholesterol and triglyceride levels.

3.3.2. Effect of Bile Acid Binding Resins on Triglyceride Metabolism

An increase in plasma triglyceride levels after BAS has been appreciated for

many years (110). Patients with CYP7A1 mutations also displayed hypertriglyceridemia

(8). The mechanism responsible for the inverse relationship between plasma triglyceride

levels and the bile acid pool still remains to be elucidated. However, several hypotheses

have been proposed. 1) BAS prevent bile acid return to the liver and thus decreases

hepatic FXR activation. Hepatic FXR activation represses expression of microsomal

triglyceride transfer protein (MTP) and ApoB100, both of which are critical for VLDL

30

 

 

packaging and secretion. This mechanism involves an inhibitory effect of SHP on HNF4α

binding to the MTP promoter (111). FXR activation also inhibits expression of ApoCIII,

an inhibitor of lipoprotein lipase, probably through a similar HNF4α-mediated mechanism

(112). BAS decreases BA entry into the ileal enterocytes and FGF15 expression. In vitro

cell-based studies suggested that FGF15 reduces hepatocyte fatty acid synthesis (100),

while in vivo experiments using transgenic model or recombinant protein demonstrated

FGF15/19 protein administration can increase fatty acid oxidation (101, 102). Because of

this effect, BAS use is not recommended for patients with hypertriglyceridemia.

3.3.3. BAS and Hyperglycemia

The beneficial effects of BAS on hyperglycemia have been appreciated for

almost 20 years (109). A number of clinical trials showed that addition of BAS to anti-

diabetic therapy further reduces levels of fasting plasma glucose and glycated

hemoglobin (113, 114).

It is now thought that this effect of BAS is mediated by TGR5, a membrane BA

receptor belonging to the family of G protein coupled receptors (GPCR). TGR5 is

ubiquitously expressed, but with highest level in the gallbladder, intestine, liver, spleen,

and brown adipose tissue (115). Although found throughout the small intestine and colon,

TGR5 is not expressed by the enterocyte, but rather the enteroendocrine L cells and

other cell types in the submucosal compartment. Upon bile acid activation of TGR5,

enteroendocrine cells release intracellular GLP-1 into the circulation, which increases

insulin secretion, decreases glucagon secretion, and improves insulin sensitivity. In a

recent publication, Taoufiq Harach et al. demonstrated that BAS stimulate colonic

release of GLP-1 (116). Both artificial TGR5 agonists and BAS treatment in TGR5+/+

mice potentiated colonic GLP-1 release and improved insulin sensitivity, whereas these

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beneficial effects were lost in TGR5-/- mice. These studies supported the hypothesis that

BAS improves insulin sensitivity by increasing the colonic concentration of bile acids, a

TGR5 agonist, and stimulating GLP-1 release.

3.3.4. Alternative Approaches to Interrupt the EHC of Bile Acids

The enterohepatic circulation of bile acids can be interrupted by a variety of

approaches, with beneficial effects on plasma cholesterol levels. The POSCH (Program

on the Surgical Control of Hypercholesterolemia) study compared the mortality of

patients that underwent ileal bypass surgery with controls (117). Within 5 years of ileal

bypass, the patients had a significant decrease of plasma LDL levels and increase of

HDL levels. Coronary heart disease mortality was reduced moderately, and there were

significant decreases in overall mortality, atherosclerosis progression, as determined by

arteriogram, and coronary-artery bypass grafting.

Small molecule inhibitors of the ileal apical bile acid transport (ASBT) such as

SC-435 were shown to increase hepatic HMG-CoA reductase expression and reduce

plasma cholesterol levels in ApoE-/- mice (118). SC-435 administration to miniature pigs

increased hepatic CYP7A1 and HMG-CoA reductase activity and significantly increased

VLDL and LDL clearance (119).

4. Different Phenotypes in Asbt-/- and Ostα-/- Mice

Mouse models lacking either the ASBT and OSTα/β have been generated (34,

40). Everted gut sac assays revealed ileal mucosal-to-serosal taurocholate transport

was decreased more than 80% in both models, indicating a successful disruption of

intestinal bile acid re-absorption. The pups for both genotypes were smaller than wild

32

 

 

type littermates, but this was transitory and body weights were similar in adult wild type

and knockout mice.

Despite the similarities mentioned above, a closer examination revealed

significant phenotypic differences between the two models. Intestinal weight, length and

gross phenotype in Asbt-/- mice were no different from wild type littermates. However,

Ostα-/- mice exhibited longer and heavier small intestines. The increase in tissue weight

was particularly evident in the ileum, the major site of expression for OSTα/β.

Histological analysis also revealed blunted villi and deeper crypts in Ostα-/- mice ileum.

Bile acid metabolism was also different in the two mouse models. Consistent with

a block in ileal bile acid transport, Asbt-/- mice showed the hallmarks of intestinal bile acid

malabsorption including increased hepatic CYP7A1 activity (2.5-3 fold versus wild type),

a decreased bile acid pool (20% of wild type) enriched in cholic acid (from the classic

pathway), a significant increase in fecal fatty acid loss (4 fold versus wild type), and a

moderate decrease in intestinal ileal cholesterol absorption (70% of wild type). Ostα-/-

mice also exhibited a significant decrease in bile acid pool size (30% of wild type).

However, their bile acid pool was more enriched in β-muricholic acid compared to wild

type littermates, indicating that there was no induction of the CYP7A1 pathway.

Consistent with these findings, fecal bile acid excretion, another indicator of hepatic bile

acid synthesis, was not increased in Ostα-/- mice. In fact, Ostα-/- mice had reduced

expression of hepatic Cyp7a1 mRNA expression (20% of wild type) and increased ileal

Fgf15 mRNA expression (180% of wild type).

In summary, although ileal bile acid reabsorption was disrupted in both models,

the Asbt-/- and Ostα-/- mice showed very different changes in bile acid homeostasis. We

hypothesize that these phenotypic differences result from alterations in intestinal

33

 

 

signaling, which also alter lipid metabolism and development of lipid metabolism

associated diseases.

5. Statement of Research Intent

In the first set of research studies, we will test the hypothesis that the differential

regulation of hepatic Cyp7a1 expression and the corresponding changes in bile acid

metabolism in Ostα-/- mice result from constitutive activation of intestinal FXR and over-

expression of ileal FGF15. In this model, we hypothesize that blocking bile acid transport

at the basolateral membrane while leaving the apical transport intact in ileal enterocytes

of Ostα-/- mice results in the accumulation of bile acids in enterocytes and persistent

activation of the FXR-FGF15 signaling pathway.

In the second set of studies, we will test the hypothesis that blocking bile acid

transport at the ileal apical versus the basolateral side will differentially affect the

susceptibility to atherosclerosis. The finding that Ostα-/- mice have reduced bile acid

synthesis and intestinal cholesterol absorption raised important questions regarding

cholesterol homeostasis and susceptibility to the development of hypercholesterolemia.

Comparison of Asbt-/-ApoE-/- and Ostα-/-ApoE-/- mice will allow us to study the differential

effects of intestinal bile acid signaling on cholesterol metabolism and the development of

atherosclerosis.

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CHAPTER II

MOUSE ORGANIC SOLUTE TRANSPORTER ALPHA DEFICIENCY ALTERS FGF15

EXPRESSION AND BILE ACID METABOLISM

Tian Lan, Anuradha Rao, Jamie Haywood, Nancy D. Kock, and Paul A. Dawson

The following manuscript was published in Journal of Hepatology 2012, volume 57,

pages 359-365. Reprint permission of ths manuscript is from Elsevier. Stylistic variations

are due to the requirement of the journal. T. Lan performed all the experiments. A. Rao

performed some of the experiments. J. Haywood assisted with the breeding of mice and

some of the experiments. N. D. Kock performed histological analysis. T. Lan and P. A.

Dawson prepared the manuscript.

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Abstract

Background & Aims: Blocking intestinal bile acid (BA) absorption by inhibiting or

inactivating the apical sodium-dependent BA transporter (Asbt) classically induces

hepatic BA synthesis. In contrast, blocking intestinal BA absorption by inactivation of the

basolateral BA transporter, Organic solute transporter alpha-beta (Ostα-Ostβ is

associated with an altered homeostatic response and decreased hepatic BA synthesis.

The aim of this study was to determine the mechanisms underlying this phenotype,

including the role of the farnesoid X receptor (FXR) and fibroblast growth factor 15

(FGF15).

Methods: BA and cholesterol metabolism, the intestinal phenotype, expression of genes

important for BA metabolism, and intestinal FGF15 expression were examined in wild

type, Ostα-/-, Fxr-/-, and Ostα-/-Fxr-/- mice.

Results: Inactivation of Ostα was associated with decreases in hepatic cholesterol 7α-

hydroxylase (Cyp7a1) expression, BA pool size, and intestinal cholesterol absorption.

Ostα-/- mice exhibited significant small intestinal changes including altered ileal villus

morphology, and increases in intestinal length and mass. Total ileal FGF15 expression

was elevated almost 20-fold in Ostα-/- mice as a result of increased villus epithelial cell

number and ileocyte FGF15 protein expression. Ostα-/-Fxr-/- mice exhibited decreased

ileal FGF15 expression, restoration of intestinal cholesterol absorption, and increases in

hepatic Cyp7a1 expression, fecal BA excretion, and BA pool size. FXR deficiency did

not reverse the intestinal morphological changes or compensatory decrease for ileal

Asbt expression in Ostα-/- mice.

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Conclusions: These results indicate that signaling via FXR is required for the

paradoxical repression of hepatic BA synthesis but not the complex intestinal adaptive

changes in Ostα-/- mice.

Key words: Bile acids; enterohepatic circulation; transporters; cholesterol; Cyp7a1; FXR

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Introduction

Regulation of hepatic expression of Cyp7a1, the rate-limiting enzyme for the

classical BA biosynthetic pathway, is complex and integrates responses to hormones,

cytokines, growth factors, oxysterols, BA, xenobiotics, and diurnal rhythm (1). It is well

established that the nuclear receptor FXR is essential for negative feedback regulation

of Cyp7a1 by BA (2,3). However, a critical role for gut-liver signaling via the endocrine

polypeptide hormone FGF15 (human ortholog: FGF19) has only recently been

appreciated (4,5). In that pathway, BA act as ligands for FXR in ileal enterocytes to

induce synthesis of FGF15. After its release into the circulation, FGF15 acts on

hepatocytes through its cell surface receptor, a complex of βKlotho protein and fibroblast

growth factor receptor-4 (FGFR4), to repress Cyp7a1 expression and BA synthesis (4-6).

After their hepatic synthesis and secretion into the small intestine, BA are

efficiently reabsorbed by active transport in the ileum and transported back to the liver

(7). In ileum, BA are transported across the apical brush border membrane via the Asbt

(gene symbol Slc10a2) (7), whereas the heteromeric transporter Ostα-Ostβ (gene

symbols: Osta, Slc51a1; Ostb, Slc51a1bp) is responsible for basolateral membrane

transport (8-10). Resection of terminal ileum, ileal disease, or inactivation of Asbt in

humans or mice interrupts the enterohepatic circulation of BA and results in increased

fecal levels of BA, induction of hepatic Cyp7a1 expression and BA synthesis, and partial

depletion of the BA pool (11-14). Although ileal BA transport is also impaired in Ostα-/-

mice, these animals do not exhibit the classical response to intestinal BA malabsorption

(9,10). The BA phenotype of Ostα-/- mice is more similar to that observed for Asbt-/- mice

treated with an FXR agonist or an FGF15-expressing adenovirus (13). Those

observations provide a possible explanation for the phenotype in Ostα-/- mice whereby

blocking basolateral export causes BA to accumulate in ileal enterocytes, leading to

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persistent activation of FXR, increased FGF15 expression, and the unexpected

repression of hepatic BA synthesis (9).

Portions of this work were presented at the 2010 Annual Meeting of the American

Association for the Study of Liver Disease and the 21st International Bile Acid Meeting

entitled “Falk Symposium 175: Bile Acids as Metabolic Integrators and Therapeutics”

and published in abstract and meeting proceedings form (15,16).

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Materials and Methods

Animals, Diets and Treatment Studies

The Institutional Animal Care and Use Committee approved these experiments.

Fxr-/- mice (Jackson Laboratory Stock#: 004144, Strain: B6;129X(FVB)-Nr1h4tm1Gonz/J)

were bred with Ostα-/- mice (C57BL/6J-129/SvEv) (9) to generate the required genotypes.

Unless indicated, the mice were fed ad libitum standard rodent chow. In selected studies,

mice were fed a basal diet (14) or basal diet supplemented with 0.2% (w/w) cholic acid

(CA) (Sigma-Aldrich). For analysis of ileal FGF15 protein expression, groups of non-

fasted WT mice were treated with a single oral gavage of 100 mg/Kg body weight of the

FXR agonist, GW4064 (a gift from Dr. Timothy Willson, GlaxoSmithKline) or vehicle

(Polyethylene glycol 400/Tween 80; 4:1, v/v). FGF15 protein expression was also

examined in ileal extracts from WT and Asbt-/- mice maintained on the basal diet (14)

and WT and Fgf15-/- mice (a gift from Dr. Steve Kliewer, University of Texas

Southwestern Medical Center) (17).

Histology

The small intestine was subdivided into 5 equal length segments and processed

for histology. Villus height and width, crypt depth, and muscle thickness were measured

for all intestinal segments in at least 20 well-oriented, full-length villus units per mouse.

Quantitative analyses were performed by AxioVision analysis of digitally acquired

images.

BA and lipid measurements

Feces were collected to measure total BA content by enzymatic assay and

neutral sterol content by gas-liquid chromatography. Intestinal cholesterol absorption 51

 

 

was measured using the fecal dual-isotope ratio method; BA pool size was determined

as the BA content of small intestine, liver, and gallbladder (14). BA composition was

determined by HPLC (18) and used to calculate the pool hydrophobicity (19). Plasma BA

were determined by enzymatic assay (Bio-Quant Inc.) and found to be similar between

the different groups (WT, 52+4 µM; Fxr-/-, 67+10 µM; Ostα-/-, 52+7 µM; Ostα-/-Fxr-/-,

66+13 µM; n = 3-5 male mice per group). To measure ileal-associated BA, small

intestine was divided into 5 equal segments, flushed with PBS, and ground under liquid

nitrogen using a mortar and pestle. Aliquots of ileal tissue were then used to isolate RNA

or extracted to measure tissue-associated BA (20). Plasma and hepatic levels of total

cholesterol, free cholesterol, and triglyceride were determined by enzymatic assay

(Roche Applied Science) (14).

Measurements of nucleic acid content and mRNA expression

Small intestinal DNA and RNA content was measured using modifications (21) of

the diphenylamine and orcinol colorimetric methods, respectively. Total RNA was

extracted from frozen tissue using TRIzol Reagent (Invitrogen). Real time PCR analysis

was performed as described (9); values are means of triplicate determinations and

expression was normalized using cyclophilin. The primer sequences used are provided

(Supplemental Table I). Whole intestinal segment mRNA expression was calculated by

normalizing to total RNA content. Unless otherwise indicated, expression levels are

plotted relative to WT mice.

Measurements of protein expression

A rabbit polyclonal antibody was raised against a glutathione-S-transferase

fusion protein (Amersham Biosciences) encompassing amino acids 171 to 218 of mouse

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FGF15, and purified by affinity chromatography using the same antigen coupled to

agarose beads (Amino-Link Immobilization Kit; Pierce). Sources of other antibodies were

as follows: anti-mouse Asbt (14), anti-mouse apoA-I (22), anti-β-actin (Sigma-Aldrich,

A5441), HRP-conjugated anti-rabbit (Sigma-Aldrich, A9169; or Cell Signaling, 7074),

HRP-conjugated anti-mouse (GE Healthcare, NXA931). Small intestinal extracts were

prepared, subjected to SDS-PAGE using 8% or 4-12% gradient (Bis-Tris Midi Gel,

Invitrogen) polyacrylamide gels, and analyzed by immunoblotting (9). Blots were stripped

and reprobed with antibody to β-actin to normalize for protein load. Protein expression

was quantified by densitometry using a Microtek ScanMaker i900 and FujiFilm

Multiguage 3 software, and expression data was normalized to levels of the β-actin

loading control.

Statistical analyses

Mean values ± SE are shown unless otherwise indicated. The data were

evaluated for statistically significant differences using the two-tailed Student’s t test or by

ANOVA (Tukey-Kramer honestly significant difference) (Statview; Mountain View, CA).

Differences were considered statistically significant at P<0.05 and are indicated by

different lowercase letters in the figures.

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Results

BA and cholesterol metabolism

Deletion of FXR in Ostα-/- mice resulted in a phenotype typically observed

following a block in intestinal BA absorption. Fecal BA excretion was similar between

Ostα-/- and WT mice, and induced by 2 to 3-fold in Ostα-/-Fxr-/- mice (Fig. 1A;

Supplementary Fig. 1A). The BA pool size was increased approximately 1.8-fold in Ostα-

/-Fxr-/- versus Ostα-/- mice (Fig. 1B; Supplementary Fig. 1B), but did not reach the levels

in WT or Fxr-/- levels due to increased fecal BA loss. The BA pool also became enriched

in taurocholate and more hydrophobic in Ostα-/-Fxr-/- versus Ostα-/- mice, as reflected by

an increase in the hydrophobicity index. The TβMC:TC ratio was also increased (Ostα-/-

versus Ostα-/-Fxr-/-; males: 0.42+0.07 versus 1.48+0.20; females 0.35+0.03 versus

0.70+0.10) (Figs. 1B, 1C; Supplementary Figs. 1B, 1C), suggesting a derepression of

hepatic BA synthesis via the Cyp7a1 pathway.

Since intestinal cholesterol absorption is greatly affected by luminal BA

concentration and BA pool hydrophobicity (14,23), fecal neutral sterol excretion and

cholesterol absorption were also examined in the different genotypes. Fecal neutral

sterol excretion was increased in Ostα-/- mice, reflecting a dramatic reduction in intestinal

cholesterol absorption (Figs. 1D, 1E; Supplementary Fig. 1D). This phenotype was

largely reversed by introducing FXR deficiency (Fig. 1E; Supplementary Fig. 1D) or by

feeding a diet containing a more hydrophobic BA (0.2% CA) (Fig. 1F; Supplementary Fig.

1E). Plasma cholesterol and triglyceride levels were not significantly altered in male

Ostα-/- mice; FXR deficiency tended to increase plasma cholesterol and triglyceride

levels, as observed previously (24), and this effect was maintained in the Ostα-/-

background (Supplementary Material and Methods). Hepatic cholesteryl ester (CE)

content was increased approximately 2.5-fold in Ostα-/- mice (Fig. 1G), in agreement with

54

 

 

the predicted decreased in hepatic conversion of cholesterol to BA. In contrast to Asbt-/-

mice (14), hepatic cholesteryl ester content was not decreased in Ostα-/-Fxr-/- mice.

Hepatic triglyceride levels tended to be higher in Ostα-/- but not Ostα-/-Fxr-/- mice (Fig. 1H).

Morphology of the small intestine

Ostα-/- mice were indistinguishable from adult WT or Fxr-/- mice with regard to

body weight or liver weight (data not shown), however there were significant small

intestinal changes that were not reversed by inactivation of FXR (Fig 2). The small

intestine was significantly heavier in both Ostα-/- and Ostα-/-Fxr-/- mice versus WT mice.

Since Ostα-Ostβ exhibits a gradient of expression along the small intestine length with

highest levels in ileum, the segmental distribution for intestinal mass was also examined.

In agreement with this pattern of expression, significant mass changes were evident in

distal small intestine of Ostα-/- mice, where intestinal weight per unit length was

increased approximately 66% (Supplementary Fig. 2A). Similar changes were observed

in female mice (data not shown). The ileal content of DNA and RNA was increased

approximately 2-fold in Ostα-/- mice as compared with WT mice (Supplementary Figs. 2B,

2C), reflecting an increase in cell number. These small intestinal changes persisted in

Ostα-/-Fxr-/- mice (Supplementary Fig. 2).

In Ostα-/- and Ostα-/-Fxr-/- mice, ileal but not jejunal morphology was significantly

altered (Figs. 2C, 2D), where the villi were blunted and thickened (fused), with evidence

of enterocyte dysplasia and occasionally dilated lacteals. Morphometric measurements

were performed using histological sections of small intestine from the four genotypes.

Muscle thickness was unchanged, but villi in ileum of Ostα-/- and Ostα-/-Fxr-/- mice were

shorter and wider, with deeper crypts (Supplementary Fig. 3). There was a slight

increase in lymphocytes and plasma cells in the lamina propria of Ostα-/- mice (Fig. 2C),

55

 

 

but no associated increases in expression of selected pro-inflammatory genes or genes

involved in ER stress (data not shown).

Hepatic and intestinal gene expression

Hepatic Cyp7a1 mRNA expression was decreased by 74% in Ostα-/- mice and

increased 2-fold in Ostα-/-Fxr-/- mice versus WT mice (Table I). Effects of Ostα

inactivation were greater for hepatic Cyp7a1 than Cyp8b1 mRNA expression, consistent

with previous findings demonstrating that Cyp7a1 is more strongly regulated by FGF15

signaling (25). Analysis of the hepatic mRNA expression of other genes involved in

hepatic bile acid or cholesterol metabolism revealed an increase in FXRα3,4 isoforms in

Ostα-/- mice. However there were few other significant changes with the exception of

decreases in MRP2 and SR-BI, which were not reversed by inactivation of FXR (Table I).

The decrease in hepatic Cyp7a1 expression was predicted to be secondary to an

induction of ileal FGF15 expression (9). Measurements revealed no significant change in

the ileocyte FGF15 mRNA (normalized to the housekeeping gene cyclophilin) but total

ileal FGF15 mRNA expression (normalized for increased ileal RNA content secondary to

intestinal morphological changes) was increased approximately 2-fold in Ostα-/- mice (Fig.

3A). The relatively small change in total FGF15 mRNA levels prompted us to examine

FGF15 protein expression (Figs. 3B, 3C). As a control, FGF15 protein expression was

determined under conditions known to alter ileal FGF15 mRNA expression (4,13). As

predicted, ileal FGF15 protein levels were increased in GW4064-treated WT mice,

reduced in Asbt-/- mice, and undetectable in Fgf15-/- versus WT mice (Fig. 3B).

Remarkably, immunoblotting revealed a more than 10-fold increase for ileal FGF15

protein expression in Ostα-/- mice (Fig. 3C). There was no accompanying change in the

intestinal gradient for FGF15 expression in Ostα-/- mice, nor does this increase appear to

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be due to a non-specific protein retention or delayed protein secretion by the ileal

enterocytes since ileal apo A-I protein levels were not increased (Supplemental Fig. 4).

Accounting for the elevated ileal protein content of Ostα-/- versus WT mice, total ileal

FGF15 protein is increased almost 20-fold (Fig. 3C). The mRNA and protein expression

for FGF15 were both dramatically reduced in ileum of Fxr-/- and Ostα-/-Fxr-/- mice.

Ileal gene expression and BA accumulation

The surprising finding that ileocyte FGF15 mRNA expression (normalized to

expression of the housekeeping gene cyclophilin) was not increased in Ostα-/- mice (Fig.

3A) prompted an examination of other FXR target genes. Ileal expression for Ibabp,

Ostα, PXR, Shp, and sodium-sulfate cotransporter (Slc13a1) were found to be reduced

rather then increased in Ostα-/- mice (Table II). A variety of mechanisms could modulate

ileal expression of FXR target genes in Ostα-/- mice including: 1) altered FXR expression

or activity, 2) decreased BA uptake by the ileal enterocyte, and 3) altered BA pool size or

composition. Measurements of ileal FXR mRNA levels revealed significant decreases for

individual FXR isoform and total FXR expression (Table II). Another possible adaptation

in Ostα-/- mice is decreased BA uptake. As shown in Fig. 4, ileal Asbt protein expression

is reduced almost 80% in both Ostα-/- and Ostα-/-Fxr-/- versus WT mice. The BA pool’s

decreased size and enrichment in muricholic acid, a poor FXR ligand (26), may also

contribute to the reduced expression of FXR target genes in Ostα-/- mice. To examine

this possibility, mice were fed a diet containing a 0.2% CA, an active FXR ligand, and

ileal BA accumulation and gene expression were measured (Figs. 4C to 4F). Expression

of ileal FXR target genes Ibabp and FGF15 were reduced in Ostα-/- mice versus WT

mice (Fig. 4E, 4F). Their reduced expression correlated with a decreased ileal BA

content , which is likely to be secondary to reduced Asbt expression. Inactivation of Ostα

57

 

 

was also associated with reduced expression of other non-FXR target genes such as

ABCA1, ABCG5, HNF4α, and NPC1L1. Interestingly, their expression was not restored

to WT levels in Ostα-/-Fxr-/- mice.

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Discussion

The major finding of this study is that FXR is required for the unanticipated

changes in BA homeostasis but not the complex intestinal morphological changes

observed in Ostα-/- mice. Moreover the original model (9), which proposed that the

unexpected repression of hepatic BA synthesis in Ostα-/- mice resulted from ileal

enterocyte BA accumulation and increased expression of FXR-target genes, must be

revised. The phenotypic changes in Ostα-/- mice are summarized in Table III.

Analysis of Ostα-/- mice revealed significant histological and morphometric

changes in ileum, including villi that were consistently blunted and fused. These changes,

which are typically associated with damage and subsequent healing, have not been

reported for patients or mouse models with primary BA malabsorption, a defect in apical

brush border membrane transport (14,27). The adult Ostα-/- mice do not exhibit

symptoms of persistent on-going intestinal injury, as indicated the absence of elevated

inflammatory gene expression, bleeding, or diarrhea. However, newborn Ostα-/- mice

exhibit a postnatal growth deficiency (9,10), and this may coincide with the onset of

injury or initiation of the adaptive response. Although the underlying mechanisms and

role of BA in this process remain to be determined, the finding of a similar intestinal

phenotype in Ostα-/- and Ostα-/-Fxr-/- mice argues against a role of the FXR-FGF15

pathway.

The classical physiological response to interruption of the enterohepatic

circulation is increased hepatic BA synthesis (13,14,28,29). However, despite evidence

of a block in intestinal BA absorption, Ostα-/- mice have decreased hepatic Cyp7a1

expression and no increase in fecal BA excretion (9,10). In order to further understand

how inhibiting BA transport at the enterocyte apical versus basolateral membrane

produces such different hepatic responses, the contribution of the FXR-FGF15 pathway

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to this unexpected BA phenotype was investigated. Inactivation of FXR in Ostα-/- mice

decreased ileal FGF15 expression and Ostα-/-Fxr-/- mice largely recapitulated the

classical BA malabsorption phenotype. In Ostα-/-Fxr-/- mice, the BA composition and

intestinal cholesterol absorption were restored to near WT levels, consistent with the

superior ability of TC versus TβMC to solubilize and deliver cholesterol to the intestinal

epithelial cell (23). The decreased intestinal cholesterol absorption in Ostα-/- mice is

balanced by reduced cholesterol demand for hepatic BA synthesis. As a result, hepatic

cholesteryl ester levels are only mildly elevated and plasma cholesterol levels are

unchanged in male Ostα-/- mice. Interestingly, there were few other significant changes

in the hepatic expression of genes involved in BA homeostasis in Ostα-/- mice and these

changes were not reversed by inactivation of FXR.

Another goal of this study was to identify additional protective mechanisms

engaged in Ostα-/- mice. Surprisingly, inactivation of Ostα did not lead to ileal BA

accumulation. The mechanisms responsible for the decreased BA accumulation and

reduced expression of FXR target genes appear to be multi-fold and may include

decreased ileal expression of FXR (30). Changes in BA pool size and composition are

unlikely to be major factors since expression of FXR target genes remained lower in

Ostα-/- mice fed a CA-containing diet. However, a major protective mechanism in Ostα-/-

mice appears to be reduced Asbt expression (30). This response does not require FXR,

nor does it appear to be due to FGF15 acting in a paracrine fashion (31), since Asbt

expression remains suppressed in the Ostα-/-Fxr-/- mice where FGF15 expression is

markedly reduced. Another surprising finding was that the increase in ileal FGF15

production in Ostα-/- mice results from a combination of elevated FGF15 protein

expression and increased number of FGF15-expressing enterocytes.

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These results affirm the critical role for Ostα-Ostβ in BA absorption and

metabolism and the importance of FGF15 for regulating hepatic BA synthesis. The

significance of these findings with regard to human hepatic or gastrointestinal disease is

unknown. However, dysregulation of FGF19 production and hepatic BA overproduction

have been implicated in the etiology of idiopathic BA malabsorption (32) and a similar

phenotype of decreased BA absorption coupled with an inability of the liver to synthesize

additional BA was previously described for gallstone patients (33).

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Table I. Hepatic gene expression (Relative to WT) Genotype Gene Fxr-/- Ostα-/- Ostα-/-Fxr-/- Ostα-/- / WT ABCA1 0.98±0.03 0.79±0.03* 0.94±0.06 ↓ ABCG5 0.77±0.02 1.04±0.08 0.94±0.17 ↔ ASBT 0.91±0.22 1.14±0.13 0.82±0.20 ↔ BSEP 0.61±0.06* 0.92±0.03 0.74±0.04* ↔ CYP7A1 1.43±0.28 0.26±0.07* 2.01±0.33* ↓ CYP8B1 1.93±0.18* 0.72±0.12 2.96±0.36* ↔ CYP27A1 0.92±0.60 0.96±0.13 0.93±0.03 ↔ FGFR4 1.04±0.07 0.80±0.07 1.35±0.12* ↔ FXRα ND 1.11±0.05 ND ↔ FXRα1,2 ND 0.84±0.03 ND ↔ FXRα3,4 ND 1.39±0.08* ND ↑ HMGCR 0.83±0.13 0.87±0.10 1.86±0.29* ↔ HMGCS1 1.44±0.23 1.69±0.37 4.25±0.77* ↔ HNF4α 0.84±0.06 0.79±0.06 0.74±0.05* ↔ MRP2 0.96±0.13 0.49±0.05* 0.50±0.11* ↓ MRP3 0.97±0.19 0.91±0.08 0.98±0.08 ↔ MRP4 1.49±0.12* 0.95±0.13 2.75±0.12* ↔ NTCP 0.77±0.03* 1.07±0.06 1.04±0.05 ↔ PXR 0.69±0.06* 1.07±0.17 0.64±0.07* ↔ SR-BI 0.64±0.03* 0.63±0.04* 0.40±0.04* ↓ SHP 0.69±0.16 0.78±0.07 0.47±0.12 ↔

Data are shown + SEM (n=5 mice per group). Significant changes (P < 0.05) versus WT are indicated by an asterisk. Arrows indicate the direction of changes in Ostα-/- mice versus WT. ND, not determined.

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Table II. Ileal gene expression (Relative to WT) Genotype Gene Fxr-/- Ostα-/- Ostα-/-Fxr-/- Ostα-/- / WT ABCA1 1.66±0.27 0.40±0.06* 0.45±0.02* ↓ ABCG5 1.26±0.24 0.44±0.07* 0.65±0.16 ↓ ASBT 0.90±0.15 0.74±0.10 0.64±0.13* ↔ FGF15 0.19±0.07* 0.93±0.14 0.12±0.03* ↔ FGFR4 1.11±0.08 2.11±0.23* 2.53±0.35* ↑ FXRα ND 0.66±0.06* ND ↓ FXRα1,2 ND 0.64±0.07* ND ↓ FXRα3,4 ND 0.72±0.05* ND ↓ HMGCR 1.08±0.11 1.34±0.06* 1.33±0.20 ↑ HMGCS1 1.10±0.13 1.13±0.09 1.31±0.32 ↔ HNF1α 1.14±0.10 1.03±0.11 0.96±0.09 ↔ HNF4α 0.93±0.09 0.68±0.02* 0.62±0.08* ↓ IBABP 0.03±0.01* 0.91±0.12 0.07±0.01* ↔ MRP3 1.12±0.14 0.96±0.08 0.83±0.03 ↔ MRP4 1.16±0.14 1.31±0.06* 1.04±0.07 ↑ NPC1L1 1.01±0.18 0.34±0.08* 0.43±0.09* ↓ Ostβ 0.40±0.05* 0.50±0.03* 0.21±0.02* ↓ PXR 0.86±0.09 0.72±0.06* 0.80±0.07* ↓ SHP 0.13±0.08 0.20±0.07 0.17±0.11 ↔ SLC13A1 0.67±0.03* 0.29±0.06* 0.29±0.05* ↓ Data are shown + SEM (n=5 mice per group). Significant changes (P < 0.05) versus WT are indicated by an asterisk. Arrows indicate the direction of changes in Ostα-/- mice versus WT.

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Table III. Phenotypic changes in Fxr-/-, Ostα-/- and Ostα-/-Fxr-/- mice (Relative to WT) Genotype Parameter Fxr-/- Ostα-

/-Ostα-/-Fxr-/-

Morphology Small Intestinal Length ↔ ↑ ↔ Small Intestinal Weight ↔ ↑ ↑ Ileal Weight ↔ ↑↑ ↑↑ Ileal DNA Content ↔ ↑↑ ↑↑ Ileal RNA Content ↔ ↑↑ ↑↑ Ileal Villus Height ↔ ↓↓ ↓↓ Ileal Villus Width ↔ ↑↑ ↑↑ Ileal Crypt Depth ↑ ↑↑ ↑↑ BA & Chol Metabolism BA Excretion ↑ ↔ ↑↑ BA Pool Size ↑↑ ↓↓ ↔ Plasma BA ↑ ↔ ↑ Hydrophobicity Index ↔ ↓↓ ↔ Total Plasma Chol ↑ ↔ ↔ Hepatic CE ↔ ↑ ↔ Hepatic Cyp7a1 ↑ ↓ ↑ Hepatic Cyp8b1 ↑ ↓ ↑↑ Ileal FGF15 Protein ↓ ↑↑ ↓ Arrows indicate the direction of changes versus WT mice.

64

 

 

Figure 1.

BA metabolism, intestinal cholesterol absorption, and hepatic lipids. Male mice were

analyzed. (A) Fecal BA excretion (n = 10). (B) BA pool size and composition (n = 5). (C)

Calculated hydrophobicity index of the BA pool (n = 5). (D) Fecal neutral sterol excretion

(n = 10). (E) Intestinal cholesterol absorption (n = 5). (F) WT and Ostα-/- mice (3 to 5

months of age) were fed control diet or a diet containing 0.2% CA for 7 days prior to

measuring intestinal cholesterol absorption (n = 3-4). (G) Hepatic cholesteryl ester

content (n = 5). (H) Hepatic triglyceride content (n = 5). Different lowercase letters

indicate significant differences (P<0.05) between groups. DKO, Ostα-/-Fxr-/- double

knockout; TC, taurocholate; TβMC, tauro-β-muricholate; Con, control diet; CA, 0.2% CA-

containing diet. CE, cholesteryl ester.

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Figure 1.

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Figure 2.

Intestinal length, weight, and villus morphology. (A) Length and (B) weight of small

intestine was measured in male mice (n = 10). (C) Representative light micrographs of

hematoxylin-eosin-stained transverse sections of jejunum and ileum from WT and Ostα-/-

mice. Images are shown at 20x and 40x magnification. (D) Representative light

micrographs of hematoxylin-eosin-stained transverse sections of jejunum and ileum from

the indicated genotypes. Images are shown at 20x magnification. Black bar, 100 µm.

67

 

 

Figure 2.

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Figure 3.

Ileal FGF15 Expression. Male mice were analyzed (n = 5). (A) Ileal FGF15 mRNA

expression. Total ileal FGF15 mRNA expression was calculated by normalizing the

mRNA expression determined by real time PCR to the total RNA yield from ileum for

each animal. (B) ileal FGF15 protein expression in individual mice from the indicated

genotypes. As a control, ileal extracts from WT mice treated with vehicle or GW4064 (n

= 2-4; pooled) and from WT, Asbt-/- (n = 5; pooled), and Fgf15-/- mice (n = 6; pooled)

were also analyzed. (C) Ileal FGF15 protein expression was quantified by densitometry.

Values for individual mice were normalized to β-actin expression and are expressed

relative to WT mice. Total ileal FGF15 protein expression was calculated by normalizing

the protein expression determined by immunoblotting to total protein yield from ileum for

each animal.

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Figure 3.

70

 

 

Figure 4.

Ileal gene expression and BA accumulation. RNA and protein were isolated from ileum

(segment 5) of individual male mice (n = 5) and used for real-time PCR analysis and

immunoblotting. (A) Protein extracts from individual mice were pooled (50 µg) and

subjected to immunoblotting. (B) Ileal Asbt protein expression was analyzed in individual

mice. Values are expressed relative to WT mice. Different lowercase letters indicate

significant differences (P<0.05) between groups. (C) Measurement of ileal tissue-

associated BA in WT and Ostα-/- mice fed a control diet or diet containing 0.2% CA for

4.5 days. (D) Ileal mRNA expression of Asbt, (E) Ibabp, and (F) FGF15. An asterisk

indicates significant differences (P<0.05) as compare to WT mice for that diet group.

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Figure 4.

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Supplemental Methods and Figure Legends

Expanded Materials and Methods

Animals

The Institutional Animal Care and Use Committee approved these experiments.

Fxr-/- mice were purchased from the Jackson Laboratory (Stock#: 004144, Strain:

B6;129X(FVB)-Nr1h4tm1Gonz/J) and bred with the Ostα-/- mice (C57BL/6J-129/SvEv) to

generate Ostα-/-Fxr-/- mice. All mice were group-housed in a temperature-controlled room

(22° C) with 12-h (6 AM to 6 PM) light/dark cycling. Unless indicated, the mice were fed

ad libitum standard rodent chow. Both male and female mice were initially analyzed

since previous studies suggested that gender influences the phenotype of the Ostα-/-

mice (1). Unless indicated, mice between two and three months of age were used to

measure intestinal cholesterol absorption and fecal excretion of bile acids and neutral

sterols. At 3 months of age, the mice were fasted for approximately 4 h at the end of the

dark phase prior to being euthanized to isolate plasma and tissues for lipid, mRNA, and

protein measurements. Mice used for histological analysis were approximately 2 months

of age and not fasted before being euthanized.

Diet and Treatment Studies

For the cholic acid feeding experiments, the Ostα-/- mice had been backcrossed

onto a C57BL/6J background for 8 generations and were compared with wild type mice

on the same background. Mice were fed a basal diet containing 16% fat, 20% protein,

64% carbohydrate (% of Calories) and 0.017 mg/Calorie of cholesterol (0.006% w/w) (2)

or the basal diet supplemented with 0.2% (w/w) cholic acid (Sigma-Aldrich) for the

indicated time period. For the analysis of ileal FGF15 protein expression, groups of non-

fasted wild type mice (females; 3-4 months of age; C57BL/6J background) were treated 76

 

 

with a single oral gavage of 100 mg/Kg body weight of a selective, synthetic FXR

agonist, GW4064 (3) (a gift from Dr. Timothy Willson, GlaxoSmithKline) or vehicle

(Polyethylene glycol 400/Tween 80; 4:1, v/v) at 9:00 PM and euthanized 16 h later (4).

FGF15 protein expression was also examined in ileal extracts from wild type and Asbt-/-

mice (males; 3-4 months of age; 129SvEv background) maintained on the basal diet (2)

and WT and Fgf15-/- mice (tissue kindly provided by Dr. Steve Kliewer, University of

Texas Southwestern Medical Center) (5).

Histology

Since the epithelium exhibits great functional diversity along the cephalocaudal

axis of the mammalian small intestine (6) and the bile acid absorption is primarily

restricted to the distal small intestine (7), the small intestine was subdivided into 5 equal

length segments for analysis. The most proximal segment is designated “Segment 1”

and includes duodenum and proximal jejunum; “Segment 2” and “Segment 3”

corresponds to mid-jejunum; “Segment 4” corresponds to distal jejunum; “Segment 5”

corresponds to ileum (8,9). Unless indicated otherwise, jejunum refers to segment 2 and

ileum refers to segment 5. The intestinal segments were flushed with PBS, fixed

overnight in 10% neutral formalin (Sigma-Aldrich), and stored in 60% ethanol until

processed for histology. Each segment was cut into longitudinal strips, stacked, and

encased in 2% agarose prior to being embedded in paraffin and processed. Histological

sections (5 µm) were cut and stained with hematoxylin and eosin. Villus height, villus

width, crypt depth, and muscle thickness were measured in all intestinal segments for at

least 20 well-oriented, full-length villus units per mouse. Quantitative analyses were

performed by AxioVision analysis of digitally acquired images.

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Bile acid and lipid measurements

Mice were individually housed in wire bottom cages and feces were collected for

three days to measure total bile acid content by enzymatic assay (2) and neutral sterol

content by gas-liquid chromatography (10). Intestinal cholesterol absorption was

measured using a modification of the fecal dual-isotope ratio method (2). Individually

housed mice were gavaged with 0.1 µCi of [4-14C]cholesterol (GE Healthcare Life

Sciences) and 0.2 µCi of [22,23-3H]sitosterol (PerkinElmer) dissolved in 200 µl of

vegetable oil. Feces were then collected for three days and used to measure fractional

cholesterol absorption as described (11). Bile acid pool size was determined as the bile

acid content of small intestine, liver, and gallbladder. Total bile acids were extracted in

ethanol (12) and bile acid composition was determined by HPLC (13). Individual bile acid

species were measured using an evaporative light scatter detector (Alltech ELSD 800)

and quantified by comparison to authentic standards purchased from Steraloids

(Newport, RI). The hydrophobicity indices of the bile acid pool were calculated as

described by Heuman (14). Plasma bile acids were determined by enzymatic assay (Bio-

Quant Inc.). To measure ileal-associated bile acids, mice were fed the indicated diets ad

libitum and killed at 9 PM during the dark phase of the diurnal rhythm. The small

intestine was divided into 5 equal segments, flushed with PBS, and ground under liquid

nitrogen using a mortar and pestle. Aliquots of ileal tissue were then used to isolate RNA

or extracted to measure tissue-associated bile acids (7). Plasma and hepatic levels of

total cholesterol, free cholesterol, and triglyceride were determined by enzymatic assay

(Roche Applied Science) (15,16). Plasma cholesterol levels were increased in Fxr-/- but

not Ostα-/- or Ostα-/-Fxr-/- mice versus WT mice; (WT, 83+6 mg/dl; Fxr-/-, 124+9 mg/dl;

Ostα-/-, 81+4 mg/dl; Ostα-/-Fxr-/-, 99+4 mg/dl; n = 5 male mice per group). Plasma

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triglyceride levels were similar among genotypes; (WT, 39+6 mg/dl; Fxr-/-, 55+7 mg/dl;

Ostα-/-, 36+8 mg/dl; Ostα-/-Fxr-/-, 52+5 mg/dl; n = 4-5 male mice per group).

Measurements of nucleic acid content and mRNA expression

The DNA and RNA content of the small intestine was measured using modifications (17)

of the diphenylamine (18) and orcinol (19) colorimetric methods, respectively. For

quantitation of mRNA expression, total RNA was extracted from frozen tissue using

TRIzol Reagent (Invitrogen). Real time PCR analysis was performed as described

(20,21); values are the mean of triplicate determinations and expression was normalized

using cyclophilin. The primer sequences used are provided (Supplemental Table I). The

pro-inflammatory genes examined in ileum included: TNFα, IL1β, IL-6, and IL-12; the ER

stress genes examined in ileum included Xbp1, Xbp1-p, Atf6, Ddit3, and Hspa5. Whole-

segment mRNA expression was calculated by normalizing the mRNA expression

determined by real time PCR to the total RNA yield. Unless otherwise indicated, the

expression levels are plotted relative to wild type mice.

Measurements of protein expression

In order to raise an antibody against mouse FGF15, cDNA encoding amino acids

171 to 218 of mouse FGF15 was PCR-amplified, subcloned into pGEX-3X (Amersham

Biosciences), and sequenced. The glutathione S-transferase (GST)-FGF15 fusion protein

was expressed in E. coli (BL21) and purified from cytosol by glutathione affinity

chromatography. Two New Zealand White rabbits were immunized with 500 µg of GST-

FGF15 fusion protein in Freund’s complete adjuvant (Lampire Biological Laboratories,

Pipersville, PA). The anti-FGF15 antibody was partially purified by ammonium sulfate

precipitation, and then isolated by affinity chromatography using the GST-FGF15 fusion

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protein (amino acids 171-218) coupled to agarose beads (Amino-Link Immobilization Kit;

Pierce). The affinity-purified antibodies were stored at –80° C in PBS and subjected to

only one freeze-thaw cycle. Sources of the other antibodies used for these studies were

as follows: anti-mouse Asbt (2), anti-mouse apoA-I (22) (kindly provided by Dr. John

Parks, Wake Forest University School of Medicine), anti-β-actin (Sigma-Aldrich, A5441),

HRP-conjugated anti-rabbit (Sigma-Aldrich, A9169; or Cell Signaling, 7074), HRP-

conjugated anti-mouse (GE Healthcare, NXA931).

For immunoblotting analysis of small intestine, intestinal segments were

homogenized on ice in 25 mM Tris-HCl (pH 7.4), 300 mM NaCl, 1% (v/v) Triton X-100

0.5% (w/v) sodium deoxycholic acid, and 0.1% (w/v) SDS in the presence of a protease

inhibitor cocktail (1 mM PMSF, 10 µg/ml pepstatin, 10 µg/ml aprotonin, 10 µg/ml

leupeptin, 10 mM EDTA) using a Polytron (Model PT 1200C, Kinematia AG,

Switzerland). After homogenization, samples were centrifuged twice at 10,000 x g for 10

minutes each at 4°C, and the supernatant was aliquoted and stored at -70° C. Samples

were diluted into laemmli sample buffer containing 50 mM Tris-HCl pH 6.8, 4% (w/v)

SDS, 10 mM EDTA, 10% (v/v) glycerol, 100 mM DTT, and 0.004% bromophenol blue,

heated at 37° C for 20 minutes and subjected to SDS-PAGE using 8% or 4-12% gradient

(Bis-Tris Midi Gel, Invitrogen) polyacrylamide gels. After transfer to nitrocellulose

membranes, blots were blocked and incubated with antibody as described previously

(20). Blots were stripped and reprobed with antibody to β-actin to normalize for protein

load. Antibody binding was detected using an enhanced chemiluminescence technique

(SuperSignal West Pico; Pierce). Protein expression was quantified by densitometry

using a Microtek ScanMaker i900 and FujiFilm Multiguage 3 software, and expression

data was normalized to the levels of the β-actin loading control.

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Statistical analyses

Mean values ± SE are shown unless otherwise indicated. The data were

evaluated for statistically significant differences using the two-tailed Student’s t test or by

ANOVA (Tukey-Kramer honestly significant difference) (Statview; Mountain View, CA).

Differences were considered statistically significant at P<0.05.

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Supplemental Figure 1.

Bile acid metabolism and intestinal cholesterol absorption in female wild type, Fxr-/-,

Ostα-/-, and Ostα-/-Fxr-/- (DKO) mice. Female mice of the indicated genotypes were

analyzed. (A) Fecal bile acid excretion was measured by enzymatic assay (n = 10). (B)

Bile acid pool size and composition was determined by HPLC (n = 5). (C) Hydrophobicity

index of the bile acid pool (n = 5). (D) Intestinal cholesterol absorption was measured by

a dual-isotope fecal assay (n = 5). (E) Female wild type and Ost�-/- mice (3 to 5 months

of age) were fed a control diet or a control diet containing 0.2% cholic acid for 7 days

prior to measuring Intestinal cholesterol absorption using the dual-isotope fecal assay (n

= 3 to 4 mice per group). Mean values ± SE of the indicated number of mice per group

are shown. Different lowercase letters indicate significant differences (P<0.05) between

genotypes or diets groups; bw, body weight; TC, taurocholate; TβMC, tauro-β-

muricholate.

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Supplemental Figure 1.

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Supplemental Figure 2.

Intestinal weight, DNA content, and RNA content of wild type, Fxr-/-, Ostα-/-, and Ostα-/-

Fxr-/- (DKO) mice. (A) The small intestine was divided into five equal length segments

and the weight of each segment is indicated as mg per cm length. (B) Aliquots of ground

tissue for each segment were extracted for measurement of the DNA content using a

colorimetric assay. (C) Aliquots of ground tissue for each segment were extracted for

measurement of the RNA content using a colorimetric assay. The nucleic acid content is

expressed as µg per cm length (n = 5). Mean values ± SE are shown. Different

lowercase letters indicate significant differences (P<0.05) between genotypes for an

individual segment of small intestine.

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Supplemental Figure 2.

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Supplemental Figure 3.

Quantitative morphometric analysis of small intestine of wild type, Fxr-/-, Ostα-/-, and

Ostα-/-Fxr-/- (DKO) mice. The small intestine of male mice (2 months of age) of the

indicated genotypes was divided into five equal segments. Hematoxylin-eosin-stained

sections from each segment were used to measure (A) Villus length, (B) villus width, (C)

crypt depth, and (D) muscle layer thickness in all intestinal segments in at least 20 well-

oriented, full-length villus units per mouse. Data are expressed as the means (n = 2 mice

per group).

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Supplemental Figure 3.

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Supplemental Figure 4.

Intestinal expression of FGF15 and Apo A-I protein in male wild type and Ostα-/- mice. (A)

Gradient of intestinal FGF15 and apo A-I protein expression. Protein extracts were

prepared from the indicated segments of individual mice (n = 5), pooled, and subjected

to immunoblotting analysis. The blots were probed using antibodies to FGF15, apo A-I,

and β-actin. (B) Analysis of FGF15 and apo A-I protein expression in ileum from mice of

the indicated genotypes. Protein extracts from ileum (segment 5) of individual mice were

subjected to immunoblotting analysis. The blots were probed using antibodies to FGF15,

apo A-I, and β-actin. Ileal apo A-I protein expression was quantified by densitometry.

Values for individual mice (n = 5) were normalized to β-actin expression and are

expressed relative to wild type mice. Ileal apo A-I protein level were reduced in Ostα-/-

mice versus wild type mice (wild type, 1.00 + 0.06, versus Ostα-/-, 0.77 + 0.09; P>0.05).

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Supplemental Figure 4.

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References

1. Rao, A., J. Haywood, A. L. Craddock, M. G. Belinsky, G. D. Kruh, and P. A. Dawson. 2008. The organic solute transporter alpha-beta, Ostalpha-Ostbeta, is essential for intestinal bile acid transport and homeostasis. Proc Natl Acad Sci U S A 105: 3891-3896.

2. Dawson, P. A., J. Haywood, A. L. Craddock, M. Wilson, M. Tietjen, K. Kluckman,

N. Maeda, and J. S. Parks. 2003. Targeted deletion of the ileal bile acid transporter eliminates enterohepatic cycling of bile acids in mice. J Biol Chem 278: 33920-33927.

3. Maloney, P. R., D. J. Parks, C. D. Haffner, A. M. Fivush, G. Chandra, K. D.

Plunket, K. L. Creech, L. B. Moore, J. G. Wilson, M. C. Lewis, S. A. Jones, and T. M. Willson. 2000. Identification of a chemical tool for the orphan nuclear receptor FXR. J Med Chem 43: 2971-2974.

4. Inagaki, T., A. Moschetta, Y. K. Lee, L. Peng, G. Zhao, M. Downes, R. T. Yu, J.

M. Shelton, J. A. Richardson, J. J. Repa, D. J. Mangelsdorf, and S. A. Kliewer. 2006. Regulation of antibacterial defense in the small intestine by the nuclear bile acid receptor. Proc Natl Acad Sci U S A 103: 3920-3925.

5. Potthoff, M. J., J. Boney-Montoya, M. Choi, T. He, N. E. Sunny, S. Satapati, K.

Suino-Powell, H. E. Xu, R. D. Gerard, B. N. Finck, S. C. Burgess, D. J. Mangelsdorf, and S. A. Kliewer. 2011. FGF15/19 regulates hepatic glucose metabolism by inhibiting the CREB-PGC-1alpha pathway. Cell metabolism 13: 729-738.

6. Gordon, J. I., G. H. Schmidt, and K. A. Roth. 1992. Studies of intestinal stem

cells using normal, chimeric, and transgenic mice. Faseb J 6: 3039-3050. 7. Beuling, E., I. M. Kerkhof, G. A. Nicksa, M. J. Giuffrida, J. Haywood, D. J. aan de

Kerk, C. M. Piaseckyj, W. T. Pu, T. L. Buchmiller, P. A. Dawson, and S. D. Krasinski. 2010. Conditional Gata4 deletion in mice induces bile acid absorption in the proximal small intestine. Gut 59: 888-895.

8. Kararli, T. T. 1995. Comparison of the gastrointestinal anatomy, physiology, and

biochemistry of humans and commonly used laboratory animals. Biopharm Drug Dispos 16: 351-380.

9. Battle, M. A., B. J. Bondow, M. A. Iverson, S. J. Adams, R. J. Jandacek, P. Tso,

and S. A. Duncan. 2008. GATA4 is essential for jejunal function in mice. Gastroenterology 135: 1676-1686 e1671.

10. Temel, R. E., R. G. Lee, K. L. Kelley, M. A. Davis, R. Shah, J. K. Sawyer, M. D.

Wilson, and L. L. Rudel. 2005. Intestinal cholesterol absorption is substantially reduced in mice deficient in both ABCA1 and ACAT2. J Lipid Res 46: 2423-2431.

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11. Dietschy, J. M., S. D. Turley, and D. K. Spady. 1993. Role of liver in the maintenance of cholesterol and low density lipoprotein homeostasis in different animal species, including humans. J Lipid Res 34: 1637-1659.

12. Schwarz, M., D. W. Russell, J. M. Dietschy, and S. D. Turley. 1998. Marked

reduction in bile acid synthesis in cholesterol 7alpha-hydroxylase-deficient mice does not lead to diminished tissue cholesterol turnover or to hypercholesterolemia. J Lipid Res 39: 1833-1843.

13. Belinsky, M. G., P. A. Dawson, I. Shchaveleva, L. J. Bain, R. Wang, V. Ling, Z. S.

Chen, A. Grinberg, H. Westphal, A. Klein-Szanto, A. Lerro, and G. D. Kruh. 2005. Analysis of the in vivo functions of Mrp3. Mol Pharmacol 68: 160-168.

14. Heuman, D. M. 1989. Quantitative estimation of the hydrophilic-hydrophobic

balance of mixed bile salt solutions. J Lipid Res 30: 719-730. 15. Carr, T. P., J. S. Parks, and L. L. Rudel. 1992. Hepatic ACAT activity in African

green monkeys is highly correlated to plasma LDL cholesteryl ester enrichment and coronary artery atherosclerosis. Arterioscler Thromb 12: 1274-1283.

16. Carr, T. P., C. J. Andresen, and L. L. Rudel. 1993. Enzymatic determination of

triglyceride, free cholesterol, and total cholesterol in tissue lipid extracts. Clin Biochem 26: 39-42.

17. Williams, D. L., T. C. Newman, G. S. Shelness, and D. A. Gordon. 1986.

Measurement of apolipoprotein mRNA by DNA-excess solution hybridization with single-stranded probes. Methods Enzymol 128: 671-689.

18. Burton, K. 1956. A study of the conditions and mechanism of the diphenylamine

reaction for the colorimetric estimation of deoxyribonucleic acid. Biochem J 62: 315-323.

19. Almog, R., and T. L. Shirey. 1978. A modified orcinol test for the specific

determination of RNA. Anal Biochem 91: 130-137. 20. Dawson, P. A., M. Hubbert, J. Haywood, A. L. Craddock, N. Zerangue, W. V.

Christian, and N. Ballatori. 2005. The heteromeric organic solute transporter alpha-beta, Ostalpha-Ostbeta, is an ileal basolateral bile acid transporter. J Biol Chem 280: 6960-6968.

21. Frankenberg, T., A. Rao, F. Chen, J. Haywood, B. L. Shneider, and P. A.

Dawson. 2006. Regulation of the mouse organic solute transporter alpha-beta, Ostalpha-Ostbeta, by bile acids. Am J Physiol Gastrointest Liver Physiol 290: G912-922.

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22. Timmins, J. M., J. Y. Lee, E. Boudyguina, K. D. Kluckman, L. R. Brunham, A. Mulya, A. K. Gebre, J. M. Coutinho, P. L. Colvin, T. L. Smith, M. R. Hayden, N. Maeda, and J. S. Parks. 2005. Targeted inactivation of hepatic Abca1 causes profound hypoalphalipoproteinemia and kidney hypercatabolism of apoA-I. The Journal of clinical investigation 115: 1333-1342.

 

 

CHAPTER III

INTERRUPTION OF ILEAL APICAL VERSUS BASOLATERAL BILE ACID

TRANSPORT HAVE DIFFERENT EFFECTS ON CHOLESTEROL METABOLISM AND

ATHEROSCLEROSIS DEVELOPMENT IN APOE-/- MICE

Tian Lan, Jamie Haywood and Paul A. Dawson

The following manuscript will be submitted to Atherosclerosis. Stylistic variations are due

to the requirement of the journal. T. Lan and J Haywood performed all the experiments.

P. A. Dawson assisted with the sacrificing of mice. J. Haywood assisted with the

breeding of mice and some of the experiments. T. Lan and P. A. Dawson prepared the

manuscript.

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Abstract

Objective: Bile acid sequestrants have been used to treat hypercholesterolemia for

many years. Interruption of intestinal bile acid absorption classically decreases intestinal

cholesterol absorption, upregulates hepatic bile acid synthesis and increases clearance

of ApoB-containing lipoproteins in plasma. However, the role of intestinal bile acid

signaling in this atheroprotective effect has not been studied. Inactivation of apical

(ASBT) or basolateral (OSTα/β) bile acid transporters in hypercholesterolemic mouse

models (LDLr-/- and ApoE-/-) provides a unique opportunity to determine how altering

intestinal bile acid signaling affects cholesterol metabolism irrespective of deficient bile

acid re-absorption.

Approach and Results: At 6 weeks of age, LDLr-/- or ApoE-/- mice deficient in Asbt or

Ostα were fed an atherogenic diet for 16 weeks. Bile acid metabolism, cholesterol

metabolism, gene expression and development of atherosclerosis were measured. 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 cholesterol

ester content. In contrast, despite reduced levels of intestinal cholesterol absorption,

plasma and hepatic cholesterol levels and atherosclerosis development were not

reduced in mice deficient in Ostα.

Conclusions: Our data demonstrated 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.

Key Words: Bile acids; atherosclerosis development; ASBT; OSTα/β; FGF15; CYP7A1

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Introduction

Lipoprotein particles are packaged and secreted from intestine (chylomicrons)

and liver (VLDLs) to deliver fatty acids and cholesterol to peripheral tissues. Following

intravascular metabolism, the resultant remnant particles CR and LDL are cleared by the

liver. The clearance of LDL and VLDL involves the interaction of hepatic LDL receptor

(LDLr) or LDLr Related Protein (LRP) with ligands such as Apolipoprotein B (ApoB) and

ApoE residing on the particle surface (1). In contrast, CR carries a truncated form of

ApoB (ApoB 48) that lacks the LDLr binding site, and is cleared primarily through ApoE-

LRP interaction (2). Hyperlipidemia is characterized by over-enrichment of cholesterol

and triglyceride in circulation (3). In hypercholesterolemic patients, the elevated levels of

plasma cholesterol are associated with chylomicron remnants (CR), very low density

lipoprotein (VLDL), and low density lipoprotein (LDL) fractions. Both increased

production and decreased clearance can increase plasma levels of lipoprotein particles.

Several genetic forms of hyperlipidemia are associated with defects of either the ligands

or the receptors for lipoprotein clearance. Genetic inactivation of LDLr and ApoE in mice

generates the two most commonly used animal models for the study of atherosclerosis

(4).

Treatment approaches to lower plasma cholesterol levels include 1) blocking

intestinal bile acid absorption using bile acid sequestrants, 2) inhibiting cholesterol

synthesis using HMG-CoA reductase inhibitors (statins), 3) blocking intestinal cholesterol

using NPC1L1 inhibitors (ezetimibe), and 4) increasing dietary intake of ω-3 fatty acids,

which affects hepatic cholesteryl ester synthesis and VLDL secretion (5). Recently, a

novel trans-intestinal cholesterol excretion pathway was identified as a potential

alternative mechanism to facilitate cholesterol elimination (6).

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Inhibition of intestinal bile acid reabsorption is a well-established method to treat

hypercholesterolemia. Bile acids are synthesized from cholesterol in the liver by a

pathway where the first and rate-limiting enzyme is catalyzed by the cytochrome P450

enzyme, cholesterol 7α-hydroxylase (CYP7A1). CYP7A1 expression is regulated by a

negative feedback regulatory pathway involving bile acid induction of

expression/secretion of the FXR target gene FGF15/19 in ileum (7). Daily fecal loss of

bile acids accounts for ~45% of cholesterol disposal. Ileal bile acid reabsorption is a very

efficient system and accounts for approximately 95% of daily input (8). Blocking this

system results in a corresponding increase of hepatic CYP7A1 and bile acid synthesis,

which subsequently increases hepatic clearance of lipoproteins from plasma. The critical

role of CYP7A1 in the control of plasma cholesterol levels has been demonstrated.

Transgenic expression of Cyp7a1 in LDLr-/- mice prevented diet-induced

hypercholesterolemia (9). Inherited mutations of the CYP7A1 gene in humans are

associated with hyperlipidemia (10). Common methods to block intestinal bile acid re-

absorption includes bile acid sequestrants, ileal resection, and small molecule inhibitors

of the ASBT (11, 12). In previous animal studies, ASBT inhibitors such as SC-435 were

effective in reducing plasma cholesterol levels and impeding the development of

atherosclerosis (12). Although induction of hepatic LDL receptor expression is thought to

be an important part of the mechanism responsible for lowering plasma cholesterol

levels, administration of the ASBT inhibitor PR835 significantly decreased VLDL and

LDL levels in LDLr-/-ApoE-/- double knockout mice, suggesting that alternative

mechanisms involving lipoprotein clearance may also be involved (13).

Ileal bile acid transport is mediated at the enterocyte level by apical uptake via

the ASBT and export by the basolateral transporter OSTα/β. Although it was initially

hypothesized that genetic inactivation of either transport system would yield a similar bile

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acid malabsorption phenotype, important phenotypic differences were observed between

the Asbt-/- and Ostα-/- mice (14, 15). The bile acid pool is reduced in both animal models,

but fecal bile acid excretion was not increased in Ostα-/- mice, as is typically observed

with a block in intestinal bile acid absorption. Hepatic CYP7A1 expression and bile acid

synthesis is dramatically increased in Asbt-/-, but not Ostα-/- mice. The differential

regulation of CYP7A1 expression in Ostα-/- mice is consistent with an increase in FGF15

expression in ileum (16). Because of the important role of hepatic CYP7A1 in controlling

plasma cholesterol levels, we hypothesized that introducing Asbt versus Ostα deficiency

into an atherogenic background will result in differential effects on cholesterol

metabolism and the development of atherosclerosis, despite the inhibition of ileal bile

acid re-absorption and a decrease of intestinal cholesterol absorption in both models.

Our result demonstrated that Asbt deficiency phenocopies the protective effects of bile

acid sequestrants, whereas Ostα deficiency failed to protect against

hypercholesterolemia and atherosclerosis.

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Materials and Methods

Animals and Diets

The Institutional Animal Care and Use Committee at Wake Forest School of

Medicine approved these experiments. Female mice were used for all studies. For Asbt

related atherosclerosis studies, Asbt-/- mice (129SvEv) were bred with ApoE-/- (C57/Bl6,

Jackson Laboratory) or LDLr-/- mice (C57BL/6, Jackson Laboratory) to generate the

desired genotypes. Littermate controls were used. For the Ostα/β related atherosclerosis

studies, Ostα-/- mice (backcrossed to C57BL/6 background for more than 8 generations)

were bred with ApoE-/- mice (C57BL/6, Jackson Laboratory) to generate the desired

genotypes. At 6 weeks, mice were switched from a cereal-based chow diet to a semi-

purified atherogenic diet containing 10% of calories of fat and 0.1% (w/w) cholesterol

(Diet A). The diet was prepared at the Diet Laboratory at the Wake Forest University

Primate Center and included all essential nutrients. Mice were sacrificed after 16 weeks

on this diet. For studies of reverse cholesterol transport, 5-month old wild type (WT),

Ostα-/- and Asbt-/- mice (all backcrossed to C57BL/6 for more than 8 generations) were

switched to the atherogenic diet 2 weeks prior to analysis.

Hepatic Lipids, Plasma Lipids and Lipoprotein Profile Analysis

Hepatic lipids were extracted as previously described (17, 18). At 0, 4, 8, 12 and

16 weeks on diet, mice were fasted for 4 hours and blood was collected from the

submandibular vein and immediately supplemented with 1% protease inhibitor cocktail.

Total plasma cholesterol (TPC) and triglyceride (TG) concentrations were determined by

a colorimetric assay as previously described (17). The cholesterol concentration in the

VLDL, LDL and HDL fractions was determined by High Pressure Liquid Chromatography

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(HPLC) using a Superose-6 column (GE Healthcare) run at a flow rate of 0.5 ml/min as

previously described (17).

Biliary Lipids Analysis

Gall bladder bile lipids were extracted and analyzed as described for the analysis of liver

lipids. The aqueous phase from the extraction was used to measure bile acid levels by

the same enzymatic assay used for fecal bile acid analysis (16). For analysis of biliary

bile acid species, the aqueous phase of biliary lipid extraction was mixed with methanol,

spiked with Nor-DCA as an internal standard and dried down under N2. The samples

were then re-dissolved in a measured volume of methanol and analyzed by HPLC

alongside with standards containing 100 µM, 500µM and 1 mM of nor-DC (Steraloids),

TC (Steraloids), TDC (Sigma), TβMC (Steraloids), TCDC (Sigma), and TUDC

(Calbiochem). HPLC was performed using a Symmetry C18 column (GE Healthcare,

WAT054275) coupled to an evaporative light scattering detector (Alltech, ELSD 800)

Fecal Neutral Sterol and Fecal Bile Acid Measurement

Feces were collected over a 72 hour period at 6 weeks of age just prior to initiating the

atherogenic diet, and at 8 and 14 weeks on diet. Fecal bile acids were measured using

an enzymatic assay (19), and fecal neutral sterols were measured by gas-liquid

chromatography as previously described (18).

Atherosclerosis Measurement

As previously described, the extent of atherosclerosis was quantified by lesion area (20)

and cholesterol content of the entire aorta, as measured from the heart to the iliac

bifurcation (21). Briefly, the aorta was isolated when mice were sacrificed and fixed in

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formalin. The adventitia was cleaned and the aorta was opened to measure the

percentage of total area covered by atherosclerotic lesions. Total cholesterol was then

extracted with chloroform-methanol, using 5α-cholestane as internal standard. The

extracted cholesterol was saponified and re-dissolved in hexane. Cholesterol mass was

measured by gas-liquid chromatography as previously described (22). The delipidated

aorta was digested in 1N NaOH and protein mass was measured by Lowry assay (23).

Quantitative Real-time PCR

RNA extraction and quantitative PCR (with pooled or individual RNA samples) was

conducted as previously described (14). Expression was calculated as means of

triplicate measurements and normalized to cyclophilin. The primer sequences are

provided (Supplemental Table I). mRNA expression of the whole ileum was calculated

by normalizing mRNA expression to total RNA content of the tissue. Unless otherwise

indicated, expression levels were plotted relative to Asbt+/+ or Ostα+/+ mice.

Measurement of Protein Expression

A rabbit polyclonal antibody against FGF15 was raised and purified as previously

described (16). Ileal protein was extracted and immunoblotting was performed as

previously described (14). Protein expression was quantified by densitometry using a

Microtek ScanMaker i900. Sources of other antibodies are as follows: anti-β-actin

(Sigma-Aldrich, A5441), HRP-conjugated anti-rabbit (Cell Signaling, 7074), HRP-

conjugated anti-mouse (GE Healthcare, NXA931).

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Reverse Cholesterol Transport

Reverse cholesterol transport was performed according to methods previously

described (24, 25). J774 cells were maintained in RPMI-1640 containing 2g/L D-glucose

(Gibco, 11875-093), supplemented with 10% (v/v) fetal bovine serum (Invitrogen), 100

U/ml penicillin (Invitrogen), and 100 µg/ml streptomycin (Invitrogen) (Media A). Cells

were radiolabeled for 56 hours in Media A containing 1 µCi/ml 3H-Cholesterol (American

Radiolabeled Chemicals, ART0257) and acetylated LDL (generously provided by Dr.

Lawrence L. Rudel at Wake Forest University) at a protein concentration of 100 µg/ml.

The foam cells were then washed with PBS and equilibrated for 12 hours in RPMI-1640

containing 100 U/ml penicillin, 100 µg/ml streptomycin, and 0.2% fat free BSA (Sigma,

A6003). The cells were then digested by Versene (Gibco, 15040-066), harvested, and

re-suspended in plain RPMI-1640 at a density of 3-10 X 106 cells/ml and radioactivity of

1-4 X 106 dpm/ml.

WT, Ostα-/- and Asbt-/- mice at 5 months of age were fed an atherogenic diet for 2

weeks prior to the assay. 0.5 ml of the re-suspended cells were injected intraperitoneally

to individually housed recipient mice on wire bottom cages. Feces was collected for 48

hours. Blood was collected at 24 hours (submandibular bleeding) and at 48 hours (heart

puncture). Mice were sacrificed at 48 hours for tissue collection.

Radioactivity in the liver was extracted in methanol. The methanol extraction was

dried down under N2 and re-suspended in scintillation cocktail. The total liver weight was

used to calculate the liver-associated radioactivity.

Fecal radioactivity as neutral sterols and bile acids were measured separately by

methods described above.

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Statistical Analyses

Mean values ± SE are shown unless otherwise indicated. The data were evaluated for

statistically significant differences using the two-tailed Student’s t test (Statview;

Mountain View, CA). Differences were considered statistically significant at p<0.05 and

are indicated by an asterisk in the figures.

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Result

Asbt Inactivation Prevented Atherosclerosis Development in ApoE-/- mice

After 16 weeks on diet (22 weeks of age), Asbt-/-ApoE-/- mice had a small but

significant decrease in body weight, liver weight and liver to body weight ratio versus

ApoE-/- mice (Supplemental Table II). No other differences in the gross phenotype were

observed. Feeding the atherogenic diet increased total plasma cholesterol levels in both

genotypes, but the levels at the end of diet feeding were significantly lower in Asbt-/-

ApoE-/- mice versus ApoE-/- mice (Figure 1A, 1599 ± 126 vs. 1101 ± 116, respectively).

Plasma triglyceride levels remained similar between the two genotypes (Figure 1B).

Further analysis of plasma lipoprotein profile revealed a significant reduction of

cholesterol associated with the VLDL and LDL fractions in Asbt-/-ApoE-/- versus ApoE-/-

mice (Figure 1C and D). Similarly, hepatic cholesteryl-ester levels were significantly

decreased in Asbt-/-ApoE-/- mice (Figure 1E ), although hepatic free cholesterol and

triglyceride levels remained similar between the two genotypes. Consistent with changes

in plasma ApoB-containing lipoprotein fractions, atherosclerosis development, as

measured by aortic total cholesterol (Figure 1F) or cholesteryl ester content (16.6 ± 2.5

and 2.7 ± 0.8 µg/mg protein in Asbt-/-ApoE-/- and ApoE-/- mice, respectively) is reduced

more than 50% in Asbt-/-ApoE-/- versus ApoE-/- mice.

Hepatic and Intestinal Gene Expression Changes in Asbt-/-ApoE-/- mice

Similar to administration of bile acid binding resins, ASBT inhibitors, or ileal

resection, hepatic Cyp7a1 expression is increased significantly by 2.4-fold in Asbt-/-

ApoE-/- mice versus ApoE-/- mice (Figure 2A). Hepatic Cyp8b1, which is critical for CA

synthesis, is up-regulated by 4.8-fold in Asbt-/-ApoE-/- mice. Consistent with the increase

for hepatic cholesterol conversion to bile acids, expression of hepatic HMG-CoA

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synthase and HMG-CoA reductase were increased by 3.8 and 1.9-fold, respectively. in

Asbt-/-ApoE-/- mice.

Inactivation of the Asbt suppressed expression of FXR target genes in the ileum

(Figure 2B). Ibabp, Shp, and Fgf15 were reduced 66%, 98% and 78% in Asbt-/-ApoE-/-

mice. The protein levels of FGF15, the negative regulator of Cyp7a1, followed the same

trend as Fgf15 mRNA, and decreased ~80% in Asbt-/-ApoE-/- mice (Figure 2C). There

was a strong correlation (rs = 0.8452) between ileal Fgf15 mRNA levels and

atherosclerosis development (Figure 2D).

Effect of Asbt Deficiency in LDLr-/- Mice

Inactivation of Asbt in LDLr-/- mice mirrored the atherosclerosis protective effects

observed in Asbt-/-ApoE-/- mice (Supplemental Figure 1), except the hepatic triglyceride

content was also reduced by ~50% in Asbt-/-LDLr-/- mice (Supplemental Figure 1E). Liver

weight was significantly lower in ASBT-/-LDLr-/- mice after 16 weeks on diet while there is

no change in body weight (Supplemental Table II). The changes of hepatic and ileal

genes responsible for hepatic and ileal bile acid and cholesterol absorption in Asbt-/-

LDLr-/- mice paralleled the trends in Asbt-/-ApoE-/- mice in comparison to their respective

controls (Supplemental Table III).

Ostα Inactivation Failed to Prevent Atherosclerosis in ApoE-/- Mice

Consistent with previous reports in Ostα-/- mice, the small intestine was heavier

and longer in Ostα-/-ApoE-/- compared to ApoE-/- mice (Supplemental Table IV). No

differences were observed regarding body weight and liver weight, while colon length is

significantly decreased in Ostα-/-ApoE-/- mice. In contrast to Asbt deficiency, total plasma

cholesterol levels were not reduced in Ostα-/-ApoE-/- versus ApoE-/- mice (Figure 3A).

103

 

 

Analysis of the lipoprotein cholesterol profile revealed a small but significant increase of

LDL associated cholesterol in Ostα-/-ApoE-/- mice (Figure 3C & D). There were also no

differences in hepatic cholesterol or cholesteryl ester levels in Ostα-/-ApoE-/- versus

ApoE-/- mice (Figure 3E). Consistent with the data in plasma cholesterol levels, Ostα

deficiency did not protect these mice against the development of atherosclerosis, as

measured by either aortic total cholesterol levels (Figure 3F), aortic cholesteryl ester

levels (33.3 ± 3.61 vs 34.9 ± 5.1 µg/mg protein in Ostα-/-ApoE-/- and ApoE-/- mice,

respectively), or aortic lesion area (Figure 3G).

Hepatic and intestinal gene expression was also analyzed. Hepatic Cyp7a1 and

Cyp8b1 expression were not affected in Ostα-/-ApoE-/- mice versus ApoE-/- mice (Figure

4A). Hepatic HMG-CoA reductase was repressed significantly (38%) in Ostα-/-ApoE-/-

mice. Consistent with previous studies in Ostα-/- mice, Ostα inactivation in ApoE-/- mice

results in a 60% decrease of ileal Asbt mRNA expression (Figure 4B). There were no

differences of ileal FXR target genes such as Ostβ, Shp and Ibabp between the two

genotypes. Ileal Fgf15 mRNA did not change in Ostα-/-ApoE-/- mice, but when normalized

to total RNA yield of the ileum, total Fgf15 mRNA levels had a 3.6-fold increase (Figure

4C).

Bile Acid and Cholesterol Metabolism in Ostα-/-ApoE-/- Mice

As previously reported in Ostα-/- mice, fecal bile acid excretion was not different

between Ostα-/-ApoE-/- and ApoE-/- mice at 8 and 14 weeks on diet (Figure 5A). Fecal

neutral sterol excretion was elevated 4-fold in Ostα-/-ApoE-/- mice, indicating a deficiency

of cholesterol absorption.

The lack of atherosclerosis prevention despite a defective cholesterol absorption

in Ostα-/-ApoE-/- mice prompted us to analyze other aspects of cholesterol metabolism.

104

 

 

Cholesterol concentration in gall bladder bile, as an indicator of biliary cholesterol

secretion, was not changed in Ostα-/-ApoE-/- mice. Biliary phospholipid and bile acid

concentration were also similar between the two genotypes (Figure 5C).

A decrease in hepatic bile acid synthesis may account for the maintenance of

plasma cholesterol levels in Ostα-/-ApoE-/- mice is. However, there was no change of

Cyp7a1 mRNA expression in Ostα-/-ApoE-/- mice despite an increase in total ileal Fgf15

mRNA expression. As an alternative measurement of the Cyp7a1-initiated classic

pathway of bile acid synthesis, the ratio of TCA to TβMCA in gall bladder bile was

analyzed. As anticipated, the ratio was decreased by approximately 20% in Ostα-/-ApoE-

/- mice (Figure 5D).

The possibility of an increase in de-novo cholesterol synthesis to compensate for

defective cholesterol absorption prompted us to measure expression of HMG-CoA

synthase and HMG-CoA reductase in the intestine and liver of Ostα-/-ApoE-/- mice. The

expression of HMG-CoA synthase was significantly increased in the proximal intestine

(segment 1) and the ileum (segment 4 and 5), and there was a trend for increased HMG-

CoA reductase expression (Figure 5E). In agreement with the data for hepatic

cholesterol content, there was no change regarding hepatic expression of both genes in

Ostα-/-ApoE-/- mice (Figure 4A).

Reverse cholesterol transport (RCT) serves as an important pathway for

"centripetal" cholesterol turnover from the peripheral tissue to the liver and the intestine.

HDL plays a central role as the carrier of cholesterol effluxed from macrophages residing

in the atherosclerotic lesion, and a defective RCT is associated with increased

atherosclerosis susceptibility. To test whether such a mechanism is impaired following

Ostα/β inactivation, 3H-cholesterol loaded J774 macrophages were injected into WT and

Ostα-/- mice. In contrary to what was expected, the appearance of macrophage-derived

105

 

 

radiolabel was significantly increased in Ostα-/- mice in the plasma (Figure 6A) and the

feces as fecal neutral sterols and bile acids (Figure 6C). There was no difference of

radiolabel appearance in the liver (Figure 6B).

106

 

 

Discussion

We previously reported that hepatic bile acid synthesis was differentially

regulated in Asbt-/- versus Ostα-/- mice, secondary to changes in ileal FGF15 expression

(16). In light of the close relationship between bile acid and cholesterol homeostasis, we

further examined the differential regulation of cholesterol metabolism following

inactivation of Asbt versus Ostα in mouse models of hypercholesterolemia and

atherosclerosis development. Our results suggested that blocking ileal bile acid re-

absorption at the apical membrane effectively decreases plasma cholesterol levels and

retards the development of atherosclerosis, whereas blocking at the basolateral

membrane is not protective.

The phenotypes observed in Asbt-/-ApoE-/- and Asbt-/-LDLr-/- mice was consistent

with the effects of bile acid binding resins and ASBT inhibitors, including a decrease of

plasma ApoB containing lipoproteins, hepatic cholesteryl ester content, and aortic

cholesterol deposition. Hepatic and ileal gene expression analysis following ASBT

deficiency supported the mechanism involving increased clearance of plasma

lipoproteins in response to the increased hepatic cholesterol demand for bile acid

synthesis. This was reflected by the increase in the expression of the major genes

responsible for hepatic cholesterol (HMG-CoA reductase, HMG-CoA synthase) and bile

acid (Cyp7a1 and Cyp8b1) synthesis. The ileal-derived enterokine, Fgf15, is significantly

down-regulated in Asbt-/-ApoE-/- mice both at the mRNA and protein levels. Correlation

analysis revealed ileal Fgf15 mRNA is a good predictor of aortic total cholesterol levels,

which is in agreement with the hypothesized involvement of the FGF15-CYP7A1

pathway in the regulation of plasma cholesterol levels and atherosclerosis development.

Although the increase of hepatic LDLr is attributed to these beneficial effects, Ldlr

mRNA expression was not increased in Asbt-/-ApoE-/- mice. One possibility is that

107

 

 

despite being a SREBP-2 target gene, Ldlr transcription is not as responsive as other

genes regulated by SREBP-2, such as HMG-CoA synthase. Another possibility is up-

regulation of alternative mechanisms for lipoprotein clearance. A similar conclusion was

suggested in a study where administration of an ASBT inhibitor decreased ApoB

containing lipoproteins in plasma of LDLr-/-ApoE-/- mice, with the largest decreases being

observed in the VLDL fraction. In agreement with this observation, plasma VLDL levels

showed the greatest decreases in our study of Asbt-/-ApoE-/- and Asbt-/-LDLr-/- mice.

Potential receptors for VLDL include the VLDL receptor (VLDLr) and LRP (1, 26). VLDLr

is usually expressed at very low levels in the liver, but its expression is up-regulated in

certain conditions, such as Ldlr deficiency (27). LRP is unlikely to play a role since ApoE

is required for its binding to VLDL.

One important finding of this study is that Ostα/β inactivation did not prevent

atherosclerosis development in ApoE-/- mice despite a defect in intestinal cholesterol

absorption. Cholesterol absorption is decreased in Ostα-/-ApoE-/- mice (data not shown),

which is also indicated by a significant increase of fecal neutral sterol excretion (4-fold,

Figure 5B). A smaller effect on intestinal cholesterol absorption has previously been

reported in female Asbt-/- mice (~13%). In that same study, fecal neutral sterol excretion

was increased 5~6-fold in female Asbt-/- mice maintained on a synthetic basal diet (19).

These data indicated that cholesterol absorption changes did not contribute to the

differential effect of Asbt versus Ostα deficiency on plasma cholesterol and

atherosclerosis development.

Several aspects related to cholesterol homeostasis were studied. Cholesterol

concentration in gall bladder bile was not different between ApoE-/- and Ostα-/-ApoE-/-

mice, indicating comparable biliary cholesterol secretion. Alternatively, an increase of de

novo cholesterol synthesis in Ostα-/-ApoE-/- mice may compensate for the absorption

108

 

 

defect. Significant increase of HMG-CoA reductase mRNA was observed in the proximal

intestine (segment 1) and the ileum (segment 4 & 5), where ASBT and OSTα/β are

expressed at the highest levels. However, the extent of up-regulation is limited. Further

experiments are necessary to directly measure de novo synthesis. Hepatic cholesterol

synthesis genes were not increased Ostα-/-ApoE-/- mice, consistent with their sustained

hepatic cholesterol concentration. Atherosclerosis development is related to both the

entry of LDL into the lesions and the efflux of cholesterol from foam cells through HDL

(reverse cholesterol transport, RCT). To test whether RCT rate was decreased with Ostα

deficiency, the efflux of cholesterol from pre-loaded J774 cells was determined in WT

and Ostα-/- mice. In contrast to the hypothesis, RCT was significantly increased in Ostα-/-

mice.

The failure of Ostα deficiency to prevent development of atherosclerosis further

supported the central role of hepatic Cyp7a1 in cholesterol homeostasis. Whereas

activating Cyp7a1 improves hepatic clearance of plasma cholesterol, several previous

studies showed that a defect in Cyp7a1 is associated with a defect in the control of

plasma cholesterol levels and atherosclerosis. In the wild type background, Cyp7a1

inactivation results in an almost complete block of intestinal cholesterol absorption, while

plasma cholesterol levels remained stable in these mice due to increased cholesterol

synthesis in liver and intestine (28). Mutation of CYP7A1 in human patients is associated

with hyperlipidemia (10). Introducing CYP7A1 deficiency into ApoE3-leiden mice (a

hyperlipidemic model resulting from defective ApoE binding to receptors) resulted in

significantly decreased fecal bile acid excretion and a 2-fold increase of fecal neutral

sterols. However, Cyp7a1-/-ApoE3-leiden mice displayed 4-fold elevation of hepatic

cholesterol levels and similar plasma cholesterol levels compared to ApoE3-leiden

control littermates (29). Consistent with these studies, Cyp7a1 expression in Ostα-/-

109

 

 

ApoE-/- mice did not increase in parallel to a sustained development of atherosclerosis.

The disconnect between increased ileal FGF15 expression and Cyp7a1 expression in

Ostα-/-ApoE-/- mice may result from the diurnal rhythm of Cyp7a1 mRNA which has the

lowest levels in the middle of the light circle (30), when in our study hepatic tissue was

collected. The ratio of biliary TC/TβMC trended to decrease in Ostα-/-ApoE-/- mice, which

may indicate a decrease in CYP7A1 activity. In Ostα-/- versus WT mice, we also

observed that TC/TβMC ratio in the bile acid pool tended to decrease in female Ostα-/-

mice versus wild type littermates (data not shown).

Our study indicated that the moderate decrease of intestinal cholesterol

absorption in Ostα-/-ApoE-/- mice can be compensated by a lack of Cyp7a1 activation and

possibly an increase of cholesterol synthesis from organs such as the small intestine.

The beneficial effects of inhibiting intestinal cholesterol absorption and atherosclerosis

have been studied (31). The dietary cholesterol content (0.15%) in these studies was

comparable to our study (0.1%), and diet-fed ApoE-/- mice used in these studies have

comparable plasma cholesterol levels as ours (~800mg/dl). However, the inhibition of

cholesterol absorption with ezetimibe or Npc1l1 deficiency was much greater than that in

Ostα-/-ApoE-/- mice, which may exceed the compensatory induction of cholesterol

synthesis in liver and the intestine (31). It should be noted that Ostα-/-ApoE-/- mice had

significantly lower plasma cholesterol levels on chow, and the cholesterol-binding

features of chow results in much higher fecal neutral sterol excretion in both genotypes

with much greater difference than diet-fed condition (data not shown). Nonetheless,

under diet-feeding conditions, the moderate decrease in cholesterol absorption versus

contrasting changes in Cyp7a1 expression in Asbt-/-ApoE-/- and Ostα-/-ApoE-/- mice

emphasized the pivotal role of bile acid synthesis in the control of cholesterol

homeostasis.

110

 

 

To summarize, our studies demonstrated that blocking of apical versus

basolateral bile acid transport results in differential responses of the FGF15-CYP7A1

signaling pathway, and subsequently differential changes in plasma cholesterol levels

and atherosclerosis. In several other studies, the metabolic effect of FGF15/19 signaling

was extended to the control of hepatic glucose homeostasis (32, 33). A recent study

demonstrated that bile acid activation of a colonic receptor, TGR5, also had beneficial

effects on glycemic control through GLP-1 secretion (34). All of these studies suggested

that bile acid signaling from the GI tract may have broader metabolic effects apart from

controlling hepatic bile acid synthesis. The differences in bile acid pool composition, bile

acid input (as measured by fecal bile acid excretion), and intestinal intracellular bile acid

levels in Asbt-/- and Ostα-/- mice may promise further studies to find other metabolic

targets of intestinal bile acid signaling.

111

 

 

Figure 1.

Effect of Asbt deficiency on lipid metabolism and atherosclerosis development in ApoE-/-

mice. Female mice were analyzed. (A) Total plasma cholesterol and (B) plasma

triglyceride levels were measured by enzymatic assays (n = 13-16). (C) Lipoprotein

cholesterol profile (n = 5) was measured by HPLC, and (D) the area under curve (AUC)

of each lipoprotein peak was calculated. (E) Hepatic lipid levels (n = 5) were analyzed by

enzymatic assays. (F) Aortic total cholesterol was extracted and analyzed by gas

chromatography (n = 13-16). Mean value ± SE were shown. Significant differences

(p<0.05) relative to ApoE-/- mice were indicated by an asterisk. TC, total cholesterol; CE,

cholesteryl ester; FC, free cholesterol; TG, triglyceride; AU, arbitrary unit.

112

 

 

Figure 1.

113

 

 

Figure 2.

Effect of Asbt deficiency on hepatic and ileal gene expression in ApoE-/- mice. (A)

Hepatic and (B) ileal RNA of individual mice (n = 5) were isolated for real-time PCR

analysis. (C) Ileal protein was extracted from individual mice (n = 10) and subjected to

immunoblotting. FGF15 protein was analyzed in individual mice. Values are expressed

relative to ApoE-/- mice. (D) Ileal Fgf15 mRNA levels were plotted against aortic total

cholesterol levels. Significant differences (p<0.05) relative to ApoE-/- mice were indicated

by an asterisk.

114

 

 

Figure 2.

115

 

 

Figure 3.

Effect of Ostα deficiency on lipid metabolism and atherosclerosis development in ApoE-/-

mice. Female mice were analyzed. (A) Total plasma cholesterol and (B) plasma

triglyceride levels (n = 10), (C) Lipoprotein cholesterol profile (n = 5), (D) lipoprotein peak

AUC and (E) hepatic lipid levels (n = 9-17) were measured as mentioned above.

Atherosclerosis was measured by either (F) gas chromatography of aortic total

cholesterol or (G) en face percent lesion area of the whole aorta (n = 9-13). Significant

differences (p<0.05) relative to ApoE-/- mice were indicated by an asterisk.

116

 

 

Figure 3.

117

 

 

Figure 4.

Effect of Ostα deficiency on hepatic and ileal gene expression in ApoE-/- mice. (A)

Hepatic and (B) ileal RNA of individual mice (n = 5) were isolated for real-time PCR

analysis. (C) Cyclophilin normalized Ileal Fgf15 mRNA levels was presented as

expression per cell. Total ileal Fgf15 mRNA expression was calculated by normalizing

the mRNA expression determined by real time PCR to the total RNA yield from ileum.

Significant differences (p<0.05) relative to ApoE-/- mice were indicated by an asterisk.

118

 

 

Figure 4.

119

 

 

Figure 5.

Effect of Ostα deficiency on bile acid metabolism and intestinal de novo cholesterol

synthesis genes in ApoE-/- mice. (A) Fecal bile acid excretion was measured by an

enzymatic assay (n = 10). (B) Fecal neutral sterols was measured by gas

chromatography (n = 10). (C) Biliary lipid levels were measured by enzymatic assays

(phospholipid and bile acid) and gas chromatography (cholesterol), and (D) the biliary

bile acid extracted was subjected to HPLC for composition analysis (n = 6). (E)

Expression of cholesterol synthesis-related genes in the small intestine. Significant

differences (p<0.05) relative to ApoE-/- mice were indicated by an asterisk.

120

 

 

Figure 5.

121

 

 

Figure 6.

Effect of Ostα deficiency on reverse cholesterol transport (n = 10). (A) Time course of

3H-cholesterol recovery in plasma. (B) 3H-cholesterol recovery in the liver 48 hours after

dose administration. (C) 3H appearance in feces as neutral sterols and bile acids 48

hours after dose administration. Data was expressed as percentage of the dose injected.

Significant differences (p<0.05) were indicated by an asterisk. FNS, fecal neutral sterols;

FBA, fecal bile acids.

122

 

 

Figure 6.

123

 

 

Supplemental Table I: RT-PCR Primers

Gene Primers ABCG5 (Forward) 5’-TCCTGCATGTGTCCTACAGC-3’

(Reverse) 5’-ATTTGCCTGTCCCACTTCTG-3’ ABCG8 (Forward) 5’-AACCCTGCGGACTTCTAGG-3’ (Reverse) 5’-CTGCAAGAGACTGTGCCTTCT-3’ ASBT (Forward) 5’-TGGGTTTCTTCCTGGCTAGACT-3’

(Reverse) 5’-TGTTCTGCATTCCAGTTTCCAA-3’ ACC (Forward) 5’-TGACAGACTGATCGCAGAGAAAG-3’

(Reverse) 5’-TGGAGAGCCCCACACACA-3’ CYP7A1 (Forward) 5’-AGCAACTAAACAACCTGCCAGTACTA-3’

(Reverse) 5’-GTCCGGATATTCAAGGATGCA-3’ CYP8B1 (Forward) 5’-GCCTTCAAGTATGATCGGTTCCT-3’

(Reverse) 5’-GATCTTCTTGCCCGACTTGTAGA-3’ FAS (Forward) 5’-GCTGCGGAAACTTCAGGAAAT-3’

(Reverse) 5’-AGAGACGTGTCACTCCTGGACTT-3’ FGF15 (Forward) 5’-GAGGACCAAAACGAACGAAATT-3’

(Reverse) 5’-ACGTCCTTGATGGCAATCG-3’ HMG-CoA Reductase (Forward) 5’-CTTGTGGAATGCCTTGTGATTG-3’

(Reverse) 5’-AGCCGAAGCAGCACATGAT-3’ HMG-CoA Synthase (Forward) 5’-GCCGTGAACTGGGTCGAA-3’

(Reverse) 5’-GCATATATAGCAATGTCTCCTGCAA-3’ IBABP (Forward) 5’- CAAGGCTACCGTGAAGATGGA-3’

(Reverse) 5’-CCCACGACCTCCGAAGTCT-3’ LDLr (Forward) 5’-AGGCTGTGGGCTCCATAGG-3’

(Reverse) 5’-TGCGGTCCAGGGTCATCT-3’ OSTβ (Forward) 5’-GTATTTTCGTGCAGAAGATGCG-3’

(Reverse) 5’-TTTCTGTTTGCCAGGATGCTC-3’ PCSK9 (Forward) 5’-GAAGACCGCTCCCCTGAT-3’

(Reverse) 5’-GCACCCTGGATGCTGGTA-3’ SCD-1 (Forward) 5’-TTCCCTCCTGCAAGCTCTAC-3’

(Reverse) 5’-CAGAGCGCTGGTCATGTAGT-3’ SREBP-1c (Forward) 5’-GGCTCTGGAACAGACACTGG-3’

(Reverse) 5’-TGGTTGTTGATGAGCTGGAG-3’ SHP (Forward) 5’- CGATCCTCTTCAACCCAGAT-3’

(Reverse) 5’- AGCCTCCTGTTGCAGGTGT-3’

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Supplemental Table II.  Body Weight and Liver Weight Related to Asbt Deficiency

Genotype Body Weight (g) Liver Weight LW/BW Asbt+/+ApoE-/- (n=15) 33.22±1.30 2.29±0.17 6.79±0.35 Asbt-/-ApoE-/- (n=17) 27.92±1.05* 1.58±0.10* 5.59±0.22* Asbt+/+LDLr-/- (n=20) 27.83±0.75 1.61±0.07 5.79±0.17 Asbt-/-LDLr-/- (n=16) 28.23±1.20 1.26±0.06* 4.21±0.11* Mean value + SEM were shown (sample size is indicated after genotype). Significant changes (P < 0.05) versus Asbt+/+ are indicated by an asterisk.

125

 

 

Supplemental Table III. Hepatic and Ileal Gene Expression

Genotype Gene

Asbt-/-ApoE-/-

(Relative to ApoE-/-) Asbt-/-LDLr-/-

(Relative to LDLr-/-)

Hepatic CYP7A1 2.39 2.42 CYP8B1 4.76 1.69 HMGCS 3.70 2.82 HMGCR 2.04 1.63 LDLr 1.19 ND PCSK9 1.89 1.61 SR-BI 0.78 1.09 ABCG5 0.63 0.95 ABCG8 0.73 1.09 SREBP-1c 1.51 1.19 ACC 1.04 1.11 FAS 1.13 1.19 SCD-1 0.83 0.90 Ileal OSTα 0.76 1.15 OSTβ 0.57 0.87 IBABP 0.44 0.70 SHP 0.02 0.51 NPC1L1 1.00 0.73

Data of pooled sample from 5 mice are shown. ND, not detected.

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Supplemental Table IV. Body Weight, Liver Weight and Intestinal Length and

Weight Related to Ostα Deficiency

Genotype Body

Weight (g)

Liver Weight

(g)

SI Weight

(g)

SI Length (cm)

Colon Weight (mg)

Colon Length(cm)

Ostα+/+ApoE-/- (n=13)

25.3 ± 0.4

1.4 ± 0.1

0.83 ± 0.02

31.7 ± 0.4

152 ± 3

6.99 ± 0.10

Ostα-/-ApoE-/- (n=13)

24.6 ± 0.5

1.3 ± 0.1

1.07 ± 0.03*

34.6 ± 0.5*

159 ± 3

6.42 ± 0.07*

Mean value + SEM were shown (sample size is indicated after genotype). Significant changes (P < 0.05) versus Ostα+/+ are indicated by an asterisk.

127

 

 

Supplemental Figure 1.

Effect of Asbt deficiency on lipid metabolism and atherosclerosis development in LDLr-/-

mice. Female mice were analyzed. (A) Total plasma cholesterol and (B) plasma

triglyceride levels were measured by an enzymatic assay (n = 15-19). (C) Lipoprotein

cholesterol profile (n = 5) was measured by HPLC, and (D) the area under curve of each

lipoprotein peak was calculated. (E) Hepatic lipid levels (n = 5) were analyzed by

enzymatic assays. (F) Aortic total cholesterol was extracted and analyzed by gas

chromatography (n = 15-19). Significant differences (p<0.05) relative to ApoE-/- mice

were indicated by an asterisk.

128

 

 

Supplemental Figure 1.

129

 

 

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Acyl-CoA:cholesterol O-acyltransferase (ACAT) 1 and lecithin:cholesterol acyltransferase, ACAT2 displays the greatest capacity to differentiate cholesterol from sitosterol. J Biol Chem 278: 47594-47601.

23. Lowry, O. H., N. J. Rosebrough, A. L. Farr, and R. J. Randall. 1951. Protein

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2003. Overexpression of apolipoprotein A-I promotes reverse transport of cholesterol from macrophages to feces in vivo. Circulation 108: 661-663.

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28. Schwarz, M., D. W. Russell, J. M. Dietschy, and S. D. Turley. 1998. Marked

reduction in bile acid synthesis in cholesterol 7alpha-hydroxylase-deficient mice does not lead to diminished tissue cholesterol turnover or to hypercholesterolemia. J Lipid Res 39: 1833-1843.

29. Post, S. M., M. Groenendijk, K. Solaas, P. C. Rensen, and H. M. Princen. 2004.

Cholesterol 7alpha-hydroxylase deficiency in mice on an APOE*3-Leiden background impairs very-low-density lipoprotein production. Arterioscler Thromb Vasc Biol 24: 768-774.

30. Zhang, Y. K., G. L. Guo, and C. D. Klaassen. 2011. Diurnal variations of mouse

plasma and hepatic bile acid concentrations as well as expression of biosynthetic enzymes and transporters. PLoS One 6: e16683.

31. Davis, H. R., Jr., L. M. Hoos, G. Tetzloff, M. Maguire, L. J. Zhu, M. P. Graziano,

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32. Potthoff, M. J., J. Boney-Montoya, M. Choi, T. He, N. E. Sunny, S. Satapati, K.

Suino-Powell, H. E. Xu, R. D. Gerard, B. N. Finck, S. C. Burgess, D. J. Mangelsdorf, and S. A. Kliewer. 2011. FGF15/19 regulates hepatic glucose metabolism by inhibiting the CREB-PGC-1alpha pathway. Cell Metab 13: 729-738.

33. Kir, S., S. A. Beddow, V. T. Samuel, P. Miller, S. F. Previs, K. Suino-Powell, H. E.

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34. Harach, T., T. W. Pols, M. Nomura, A. Maida, M. Watanabe, J. Auwerx, and K.

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Chapter IV

Discussion

Tian Lan prepared this chapter. Dr. Paul A. Dawson acted in an editorial and advisory

capacity.

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Bile acids play a critical role in lipid metabolism: 1) Bile acids in the intestinal

lumen facilitate micelle formation and cholesterol and fatty acid absorption. 2) Hepatic

bile acid synthesis, which equals to fecal bile acid excretion, account for almost half of

the daily cholesterol elimination (1). 3) Bile acid signaling through Fxr affects lipid

homeostasis. For example, hepatic Fxr activation increases expression of Abcg5, Abcg8,

and SR-BI, and significantly increases hepatic cholesterol excretion and reverse

cholesterol transport, another pathway for cholesterol elimination (2). Fxr activation in

macrophages increases expression of Abcg1, increases cholesterol efflux from

macrophages, and impedes the development of atherosclerosis (3). Moreover, as

mentioned earlier, Fxr activation reduces hepatic VLDL secretion and its target gene

Fgf15/19 increases fatty acid oxidation and subsequently improves hypertriglyceridemia.

The synthesis and enterohepatic cycling of bile acids are tightly regulated to

compartmentalize these important detergents and signaling molecules. Under normal

physiological conditions, the liver is sensitive to changes in the bile acid pool and

correspondingly shifts the rate of bile acid synthesis to maintain the pool size. Discovery

of Fxr and the subsequent characterization of its involvement in the regulation of every

step of bile acid metabolism, such as intestinal bile acid transporters, hepatic bile acid

transporters, the regulation of hepatic Cyp7a1 through intestinal Fgf15/19 signaling,

marked Fxr as the master regulator of bile acid homeostasis. Consistent with these

findings, impaired Fxr activity leads to alteration in bile acid metabolism accompanied by

dyslipidemia.

Blocking intestinal bile acid absorption at the basolateral membrane yields a

phenotype very different from the responses observed following ileal resection,

administration of BAS or Asbt inhibitors, or inactivation of the Asbt gene. The Ostα null

mice fail to upregulate hepatic Cyp7a1 expression, and exhibit a decreased bile acid

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pool that is enriched in β-muricholic acid. We hypothesized that this dysregulation of bile

acid metabolism in Ostα-/- mice results from an abnormal activation of intestinal Fxr-

Fgf15 signaling. Meanwhile, we further hypothesized that this altered intestinal signaling

would differentially affect cholesterol homeostasis and related diseases such as

atherosclerosis.

In Chapter II, we tested the first hypothesis by introducing Fxr deficiency into

Ostα-/- mice. Indeed, hepatic bile acid synthesis is de-repressed in Ostα-/-FXR-/- mice.

These mice showed a phenotype similar to the classic response to interruption of

intestinal bile acid reabsorption. Compared to Ostα-/- mice, Ostα-/-Fxr-/- mice has 5-fold

increase of hepatic Cyp7a1, 1.5-fold increase of fecal bile acid excretion, 80% increase

of bile acid pool size enriched in cholic acid, a corresponding increase of pool

hydrophobicity, and restoration of intestinal cholesterol absorption (25%) to wild type

levels.

The decrease of intestinal cholesterol absorption in Ostα-/- mice can be explained

by a decrease in bile acid pool size (50%) as compared to wild type littermates and

enrichment in β-muricholic acid, which decreases the pool hydrophobicity. Similar effects

of β-muricholic acid on cholesterol absorption have been observed in previous studies.

Wang et al. demonstrated that dietary supplementation with β-MCA significantly reduces

intestinal cholesterol absorption (from 37% in control to 11% in β-MCA fed mice) (4). In

our study β-MCA was enriched due to a repression of the classical pathway, which also

results in a reduction in the bile acid pool size. In Cyp7a1-/- mice, the bile acid pool was

reduced to the similar levels as the Ostα-/- (13 µmole/100g BW), and intestinal

cholesterol absorption was also greatly reduced. However, fat absorption is still

preserved in Cyp7a1-/- mice (5). These studies indicated that changes of the bile acid

pool, both size and composition, has a more profound effect on cholesterol absorption.

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In addition, the finding that feeding a CA-enriched diet largely reversed the decrease in

intestinal cholesterol absorption further supported the effect of bile acid pool changes on

cholesterol absorption.

Repression of hepatic Cyp7a1 results from increased intestinal Fgf15 signaling

(6). However, contrary to the hypothesized model, intestinal Fgf15 mRNA in Ostα-/- mice

is not elevated in Ostα-/- mice. Other Fxr target genes such as Ibabp and Slc13a1 were

actually reduced in Ostα-/- mice. There are several potential explanations for his

phenomenon: 1) repression of Fxr expression; 2) β-MCA enrichment in the bile acid pool,

which is a poor Fxr agonist; and 3) adaptive reduction of Asbt protein expression. Indeed,

a small but significant reduction of total Fxr mRNA expression (25%) was observed in

Ostα-/- mice (Chapter II, Table 2). However, Asbt mRNA and particularly protein

expression was significantly reduced in the Ostα-/- mice.

To further test the relative contribution of down-regulation of Asbt expression

versus changes in bile acid pool on ileal Fxr activation, WT and Ostα-/- mice were fed

diets containing 0.2% (w/w) CA. This supplementation, based on the food intake,

provides a daily intestinal CA input that is approximately four times the amount of the

endogenous bile acid pool, effectively normalizing the differences in pool size and

composition between the genotypes. Chemical analysis of ileal tissue-associated BA (as

intracellular BA levels) in these mice showed a trend toward decreased levels in ileum of

Ostα-/- mice, and correlated with decreased ileal Asbt mRNA expression in Ostα-/- versus

WT mice. Also consistent with the decreased Asbt expression, ileal expression of Fxr

target genes remained low in Ostα-/-Fxr-/- mice, and no increase in inflammatory gene

expression was observed.

A surprising finding in the Ostα/Fxr study is that despite little change in ileal

Fgf15 mRNA expression, ileal tissue FGF15 protein expression were increased almost

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9-fold in Ostα-/- mice. Taking into account the increased total ileal RNA and protein in

Ostα-/- mice, ileal FGF15 protein levels were increased almost 20-fold. The underlying

mechanism responsible for the disproportionate increase in ileal FGF15 protein levels in

Ostα-/- mice is not known. Possible mechanisms include post-transcriptional regulation of

FGF15 protein expression. Post-transcriptional regulation was reported for another

member of the FGF family, FGF9 (7). In this mechanism, the Far Upstream Element

Binding Protein 3 (FUBP3) binds to the 3’ untranslated region of Fgf9 mRNA and

stimulates its translation. Co-expression of FGF9 and FUBP3 in HEK293 cells

significantly increased FGF9 protein levels, while knockdown of FUBP3 significantly

decreased FGF9 protein. In both cases, mRNA levels of Fgf9 were unchanged. Whether

such a post-transcriptional mechanism would be operative for FGF19 in ileum remained

to be determined. Our lab attempted to investigate de novo FGF15 protein synthesis

using [35S]methionine pulse-chase labeling with ileal explants. However, those efforts

were impeded by the lack of a precipitating antibody for mouse FGF15 (data not shown).

In addition to effects on translation, the enrichment of FGF15 in ileum may also result

from immediate retention after secretion. Indeed, although FGF15/19 has only a weak

affinity for heparin sulfate proteoglycans, heparin dose enhances FGF19-FGFR4

interaction in the presence of β-Klotho in a cell-free pull down assays (8). The

morphological changes of the villi in Ostα-/- mice may alter the properties of the

extracellular matrix leading to trapping of newly secreted FGF15. Further studies are

necessary to understand the underlying mechanism(s) responsible for this increased

ileal-associated FGF15 protein.

As mentioned above, another surprising finding in Ostα-/- mice is the

morphological changes of the ileal villi. These changes are not Fxr or Fgf15-dependent

since they were also observed in Ostα-/-Fxr-/- mice. Nor do the morphological changes

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appear to be due to chronic inflammation as the expression of inflammatory cytokines

such as Tnfα and Il-1β was not increased in the ileum of Ostα-/- mice. One possible

mechanism for this phenotype is intracellular accumulation of an OSTα/β substrate that

stimulates cell proliferation. Among the OSTα/β substrates identified to date,

prostaglandin E2 (PGE2) is a candidate (9). PGE2 is synthesized from arachidonic acid

(AA) by both isoforms of cyclooxygenase (COX-1 and COX-2). COX-1 is constitutively

expressed while COX-2 expression is induced by inflammatory cytokines or

lipopolysaccharides. Several clues point to the involvement of PGE2 in the regulation of

intestinal cell proliferation: 1) There is abundant supply of arachidonic acid supply in the

small intestine. Sources of arachidonic acid include direct dietary supply, de-novo

synthesis from dietary linolic acid (LA) (10), and bile (11). Biliary diversion significantly

reduces the amount of radiolabeled PGE2 associated with chylomicrons and albumins in

the circulation. 2) PGE2 is actively synthesized in the small intestine. Both COX-1 and

COX-2 are expressed throughout the gastrointestinal tract in rat (12), and PGE2

synthesis is detected down the length of the intestine in rabbits (13). 3) Cancer is a form

of uncontrolled cell proliferation, and activation of COX-2 and subsequent PGE2

synthesis augments cancer development in colon and ileal-cecal valve (14). 4) Under

non-cancerous conditions, PGE2 and arachidonic acid supply plays a critical role in

maintenance of intestinal tissue renewal. While subcutaneous administration of

indomethacin, an inhibitor of COX-1 and COX-2, significantly decreased villus cell

number in rats, methyl-PGE2 (PGE2 analogue) reversed the phenotype and induced

villus hyperplasia in the distal small intestine (15). In a recent study of an AA deficient

mouse model, genetic inactivation of Fads1, an enzyme critical for LA conversion to AA,

reduced PGE2 levels along the GI tract and decreased cell proliferation in the colon (16).

These studies suggest that PGE2 concentrations in the intestinal mucosa impact cell

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proliferation. It is possible that PGE2 concentrations are elevated in distal intestine of

Ostα-/- mice due to impaired lipid absorption in proximal intestine, secondary to the

decreased bile acid pool size and altered bile acid pool. Since the relative contribution of

Ostα/β and other basolateral PGE2 transporters, such as MRP4, to PGE2 metabolism is

unknown, further study is necessary to test this hypothesis. Possible experiments

include measurements of tissue-associated PGE2 levels and examining the effects of

blocking PGE2 synthesis using low-doses of NSAID in ileum morphology.

Analysis of a subset of genes important for bile acid and cholesterol metabolism

was performed in the liver and ileum of Ostα-/- mice (Table 2, in Chapter II). This

experiment was performed to find potential changes in cholesterol homeostasis following

Ostα deficiency. Among these genes, changes in Bsep, Cyp8b1, Mrp4, Mrp2, Pxr and

SR-BI mRNA may all be explained by Fxr-mediated regulation. Cyp8b1 is a well-

characterized target for Fxr-mediated repression, and its expression was increased in

both Fxr-/- and Ostα-/-Fxr-/- mice (17). FXR inhibits Mrp4 expression by a mechanism

involving the CAR binding site (18). This likely explains the significant increase of Mrp4

expression in Fxr-/- and Ostα-/-Fxr-/- mice. On the other hand, as a direct target gene of

FXR, expression of Bsep and Pxr are both significantly decreased in Fxr-/- and Ostα-/-Fxr-

/- mice (19, 20). This may explain the decrease in expression of Mrp2, a well-

characterized PXR target gene. The significant decrease of SR-BI expression in all the

knockout models (Ostα-/-, Fxr-/- and Ostα-/-Fxr-/- mice) may result from a reduction of

hepatic Fxr activation. In addition to being a direct target gene for Fxr, SR-BI expression

is induced indirectly by FXR enhancing the activity of HNF1α, an important activator of

SR-BI expression (2). In Ostα-/- mice, a block of intestinal bile acid return and repression

of Cyp7a1 may significantly decrease the concentration of hepatic bile acid and reduce

the activation of FXR. The significant increases of mRNA expression for Hmgcs, Hmgcr

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and Fgfr4 in Ostα-/-Fxr-/- mice can all be explained by the rescue of bile acid homeostasis.

As discussed earlier, the phenotype of Ostα-/-Fxr-/- mice was similar to that induced by

administration of bile acid sequestrants (BAS). The increased hepatic cholesterol

turnover results in activation of SREBP2 target genes, including Hmgcs and Hmgcr. The

increase of Fgfr4 in Ostα-/-Fxr-/- mice may involve a more complicated mechanism.

Hepatic Fgfr4 expression is positively regulated by hepatic insulin signaling (21). Similar

to a recently discovered pathway for bile acid sequestrant treated mice, the increased

synthesis of bile acids and their passage into the colon of Ostα-/-Fxr-/- mice may activate

TGR5 and increase GLP-1 release, an incretin hormone that improves hepatic insulin

sensitivity (22). However, whether this pathway contributes to upregulation of Fgfr4

mRNA levels in Ostα-/-Fxr-/- mice requires further studies, such as measurement of

systemic GLP-1 levels and hepatic insulin signaling. In general, hepatic gene expression

changes between the four genotypes were consistent with the changes in bile acid and

cholesterol homeostasis and the level of FXR activation.

Ileal gene expression changes also follow the extent of FXR activation. FXR

target genes including Slc13a1, Pxr, Ibabp and Ostβ were downregulated in Ostα-/- mice,

consistent with the mRNA levels of Fgf15 and ileal bile acid content in the CA diet study.

Like Asbt, a number of cholesterol transporters (Abca1, Abcg1 and Npc1l1) were down-

regulated in both Ostα-/- and Ostα-/-Fxr-/- mice. The simultaneous down-regulation of

these genes may indicate a partial de-differentiation of the enterocytes secondary to the

effects on villus morphology.

To summarize, in Chapter II we demonstrated clearly that intestinal FXR

signaling plays a critical role in the unique bile acid metabolism phenotype in Ostα-/- mice.

In response to the blocking of ileal basolateral bile acid transporter, adaptive responses

in the ileum of Ostα-/- mice (downregulation of Asbt, ileal thickening of villi and increases

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in ileal FGF15 levels) function to limit bile acid uptake and down-regulate de novo

synthesis to prevent bile overload. Further studies are needed to identify the

mechanism involved in the Fxr-independent responses in Ostα-/- mice.

There is significant crosstalk between cholesterol homeostasis and bile acid

metabolism. In Ostα-/- mice, hepatic turnover of cholesterol is predicted to be either

reduced by repression of Cyp7a1 expression or increased by inhibition of intestinal

cholesterol absorption. Under chow-fed conditions, WT and Ostα-/- mice had similar

levels of plasma cholesterol, with Ostα-/- mice exhibiting a 2-fold increase of hepatic

cholesteryl esters. Fxr deficiency has been introduced into atherogenic mouse models in

order to demonstrate its atheroprotective effect (23). In the same manner, we introduced

either Asbt or Ostα deficiency into atherogenic models to compare their differential

effects on the metabolism of atherogenic lipoproteins.

Asbt deficiency was introduced into the two commonly used atherogenic mouse

models (ApoE-/- and LDLr-/-) and showed similar responses as the administration of BAS,

ASBT inhibitors, or ileal bypass. Genetic inactivation of Asbt significantly increased

hepatic bile acid synthesis, reduced hepatic cholesterol levels, plasma cholesterol levels,

and decreased the development of atherosclerosis after 16 weeks on diet. In contrast,

deficiency of the basolateral bile acid transporter (Ostα-/-) showed a very different

phenotype when introduced into an ApoE-/- background. Despite a lower starting point

for plasma cholesterol levels at 6 weeks of age and an increased fecal neutral sterol

excretion both before and after being put on an atherogenic diet, TPC levels and hepatic

total cholesterol levels in Ostα-/-ApoE-/- mice were similar to the ApoE-/- mice after 16

weeks on diet.

Inhibition of intestinal cholesterol absorption has been shown to lower plasma

concentration of ApoB-containing lipoproteins (24). Many studies were performed using

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either genetic ablation of the intestinal cholesterol transporter Npc1l1 or its inhibitor

ezetimibe. In all cases, plasma cholesterol levels, especially the ApoB-containing

lipoproteins, were significantly decreased after Npc1l1 inhibition. In contrast, our study

suggests that benefits of a mild decrease in intestinal cholesterol absorption can be

overcome by repression of hepatic bile acid synthesis. A comparison between our Ostα-/-

ApoE-/- and the Npc1l1-/-ApoE-/- study by Davis et al. (25) gives insights to the difference

between the two models.

The cholesterol content of the diets were comparable in both studies and

sufficient to initiate hypercholesterolemia (0.1% for Ostα-/-ApoE-/- and 0.15% for Npc1l1-/-

ApoE-/-). At the end of the feeding period (16 weeks for Ostα-/-ApoE-/- and 24 weeks for

Npc1l1-/-ApoE-/-), total plasma cholesterol is significantly decreased in Npc1l1-/-ApoE-/-

mice (250 mg/dl vs 900 mg/dl in Npc1l1+/+ApoE-/-), whereas the levels in Ostα-/-ApoE-/-

mice remained high (975 mg/dl vs 872 mg/dl in Ostα+/+ApoE-/- mice).

Hepatic cholesteryl ester in the Ostα-/-/ApoE-/- study is low in both genotypes

(15.19 and 12.18 mg/liver in Ostα+/+ApoE-/- and Ostα-/-ApoE-/- mice, respectively) and

similar to the levels in Npc1l1-/-ApoE-/- mice on diet (15.59 mg/liver compared to 46.76

mg/liver in Npc1l1+/+ApoE-/- mice). The difference in hepatic cholesteryl ester levels

between the ApoE-/- mice in the two studies may result from differences in diet

composition or genetic background of the mice (pure C57BL/6 for Ostα-/-ApoE-/- study

while 75% C57BL/6 and 25%129Ola/Hsd for the Npc1l1-/-ApoE-/- mice). However, the

plasma cholesterol levels, the defining factor for atherosclerosis development, are

similar in ApoE-/- mice in both studies.

We should note that the extent and difference in fecal neutral sterols between

Ostα+/+ApoE-/- and Ostα-/-ApoE-/- mice is much greater on chow (5 and 40 mg/day/100g

BW respectively) than on the diet (1.9 and 8.7 mg/day/100g BW respectively). The high

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levels of fecal neutral sterol likely results from the cholesterol-binding feature of fiber in

chow versus the alpha-cellulose in the synthetic diet. This raises the question of whether

the difference in fecal neutral sterol excretion was too small to affect plasma cholesterol

levels. Fecal neutral sterols were not measured in the Npc1l1-/-/ApoE-/- study, but

blocking this cholesterol transporter should eliminate absorption completely. In a

separate study, maximal dose of ezetimibe (10mg/day/kg BW) reduced cholesterol

absorption from 70% to less than 10%, and hepatic Cyp7a1 expression is repressed

when these mice were put on cholesterol-containing diet (26). In the Npc1l1-/-/ApoE-/-

study, when the mice were put on chow, Npc1l1 deficiency in ApoE-/- mice reduces

plasma cholesterol to the similar levels as the Ostα-/-ApoE-/- mice on chow, but when put

on the western diet, plasma cholesterol levels in Npc1l1-/-ApoE-/- mice remained

significantly lower (25). Since Cyp7a1 expression may also remain low in Npc1l1-/-ApoE-

/- mice, the much more significant decrease of cholesterol absorption should account for

the atheroprotective effects in Npc1l1-/-ApoE-/- mice.

On the other hand, the comparatively smaller reduction in cholesterol absorption

in Ostα-/-ApoE-/- mice does not exclude the importance of Cyp7a1 in the differential

cholesterol metabolism in comparison to Asbt-/-ApoE-/- mice. Although cholesterol

absorption was not directly measured in Asbt-/-ApoE-/- mice, a previous study showed

inactivation of Asbt only reduces cholesterol absorption from 80% to 70% (27).

Consequently, the different effects on Cyp7a1 expression in the two models should be a

major contributor to the differential susceptibility to hypercholesterolemia and

atherosclerosis.

One interesting distinction between our studies is the response of Cyp7a1

expression changes following Ostα inactivation. As described in Chapter II, we have

demonstrated hepatic Cyp7a1 expression and presumably hepatic bile acid synthesis is

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significantly decreased in Ostα-/- mice on a chow diet. In the atherosclerosis study,

Cyp7a1 levels were not significantly decreased, but the ratio of TCA/TβMCA is lower in

Ostα-/-ApoE-/- mice, indicating a decrease of CYP7A1 activity. Alternative measurements

of hepatic bile acid synthesis are required to verify this observation. Potential assays

include measurements of hepatic CYP7A1 activity, or analysis of hepatic and plasma

7alpha-hydroxy-4-cholesten-3-one (C4) levels.

Our studies demonstrated ileal FXR and increased ileal FGF15 protein

expression is responsible for the unique bile acid metabolism phenotype in Ostα-/- mice

(Chapter II). The atherosclerosis study (Chapter III) further demonstrated the altered ileal

FXR-FGF15 signaling resulted in differential cholesterol homeostasis and related

diseases. The effect of Fxr deficiency on the development of atherosclerosis has been

studied using various models and experimental designs. Unfortunately, conflicting

results have been obtained and experimental design differences have made the studies

difficult to compare. For example, a study in LDLr-/- model showed an atheroprotective

effect of Fxr deficiency, but used a very high cholesterol-containing diet (1.25%), and the

decrease of atherosclerotic lesion was observed despite an absence of decreased

ApoB-containing lipoproteins (28). Another study showed benefits in Fxr deficient mice

fed a very high fat diet without cholesterol (29). In contrast, several other studies showed

that activation of FXR protected against the development of atherosclerosis and was

associated with a corresponding decrease of plasma ApoB-containing lipoproteins and

inflammatory markers in liver and macrophages (30-32).

To summarize, our studies demonstrated the critical involvement of ileal FXR-

FGF15 signaling pathway in the phenotype of Ostα-/- mice, and the possible effects of

ileal FXR signaling on cholesterol metabolism. These data emphasized the importance

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of bile acid signaling and bile acid transporters in the small intestine for the whole body

lipid homeostasis.

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C. L. Null, Y. Luo, and B. T. Keller. 2003. Inhibition of ileal bile acid transport and reduced atherosclerosis in apoE-/- mice by SC-435. J Lipid Res 44: 1614-1621.

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Intestinal sterol transporters and cholesterol absorption inhibition. Curr Opin Lipidol 22: 467-478.

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CURRICULUM VITAE Tian Lan

Contact Information

Department of Internal Medicine - Gastroenterology

Wake Forest School of Medicine

Medical Center Blvd, Winston-Salem, NC 27157

p. 336.716.2253; m. 336.529.1325

tlan@wakehealth.edu

Research Interest Intestinal physiology and regulation of metabolism; bile acid malabsorption; gastrointestinal disease; cardiovascular disease Education 2007-present Ph.D. Candidate of Molecular Pathology, Section in Lipid Sciences Wake Forest School of Medicine, Winston–Salem, NC Advisor: Paul A. Dawson, Ph. D. 2003-2007 B.S. in Biological Sciences Fudan University, Shanghai, China Honors and Awards

• Travel Award, Research Workshop: Bile Acid-Mediated Integration of Metabolism and Liver Disease, NIH, Bethesda, MD. June 2012

Poster Presentations • Wake Forest University Graduate Student Research Day. March 2012 • South East Lipid Research Conference, Pine Mountain, GA. October 2010 • Research Triangle Park Drug Metabolism Discussion Group, Chapel Hill, NC.

June 2009 Oral Presentations

• Research Workshop: Bile Acid-Mediated Integration of Metabolism and Liver Disease, NIH, Bethesda, MD. June 2012

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Original Articles 1. Lan T, Rao A, Haywood J, Davis CB, Han C, Garver E, Dawson PA. 2009.

Interaction of macrolide antibiotics with intestinally expressed human and rat organic anion-transporting polypeptides. Drug Metab Dispos 37: 2375-2382.

2. Yang Q, Lan T, Chen Y, Dawson PA. 2012. Dietary fish oil increases fat absorption and fecal bile acid content without altering bile acid synthesis in 20-day-old weanling rats following massive ileocecal resection. Pediatr Res 72: 38-42

3. Lan T, Rao A, Haywood J, Kock ND, Dawson PA. 2012. Mouse organic solute

transporter alpha deficiency alters FGF15 expression and bile acid metabolism. J Hepatol 57: 359-365

Reviews 1. Dawson PA, Lan T, Rao A. 2009. Bile acid transporters. J Lipid Res 50: 2340-2357.

2. Lan T, Haywood J, Rao A, Dawson PA. Molecular mechanisms of altered bile acid

homeostasis in organic solute transporter-alpha knockout mice. 2011. Dig Dis 29(1): 18-22

Abstracts 1. Lan T, Haywood J, Rao A, Dawson PA. 2010. Inactivation of organic solute

transporter (OST)-alpha alters bile acid homeostasis via the FXR-FGF15 pathway. Hepatology 52, 431A.