i

CHOLESTEROL ABSORPTION AND TRANSINTESTINAL CHOLESTE ROL

EFFLUX (TICE) RATES ARE UNALTERED IN MICE OVEREXPRE SSING

INTESTINAL SR-BI.

By

KANWARDEEP SINGH BURA

A Thesis 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

MASTER OF SCIENCE

in the Molecular Pathology Program

December 2011

Winston-Salem, North Carolina

Approved by:

J. Mark Brown, Ph.D., Advisor

Paul A. Dawson, Ph.D., Committee Chairman

John S. Parks, Ph.D.

Lawrence L. Rudel, Ph.D.

John S. Parks, Ph.D.

Ryan E. Temel, Ph.D.

ii

ACKNOWLEDGMENTS

My work at Wake Forest University would not have been possible without

the help of the following individuals. My advisor, J. Mark Brown, was a central

figure in my success. He provided wonderful direction in the progression of my

project. His consistent guidance was instrumental in my completing this thesis

project. I am grateful for his gentle encouragement whenever I went to him

frazzled.

Furthermore, my committee members, Dr. Paul A. Dawson, Dr. Lawrence

L. Rudel, Dr. John S. Parks, and Dr. Ryan E. Temel were instrumental in

providing direction during my time here. I acknowledge that my time with them

was rather tough, but I am sure they had my best interests in mind. A special

thanks needs to be given to Dr. Temel for his consistent help throughout the

study.

Other members of the lab have made my time here memorable. I was very

fortunate that there was good lab environment here at Wake Forest. I have been

extremely fortunate to be allowed to work with Caleb, Allison, Jun, Gwyn, John,

Stephanie, Ramesh, Kathryn, Carol, Martha and Matt. They were always present

there to help me whenever I needed it.

None of these accomplishments would have been possible without my

family. My parents Mani Ram and Pushpa Bura have been wonderful role models

in my life. They have molded me into who I am and have always taught me to

look at the big picture. My brother, Yugdeep, has become a true friend these last

several years as he entered college.

iii

TABLE OF CONTENTS

LIST OF FIGURES……………………………………………………………………. iv

LIST OF ABBREVIATIONS………………………………………………………..... v

ABSTRACT……………………………………………………………………………. vii

CHAPTER

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

II. MATERIALS AND METHODS……………………………………………. 11

III. RESULTS………………………………………………………………….... 18

IV. DISCUSSION……………………………………………………………….. 28

REFRENCE LIST…………………………………………………………………….. 33

CURRICULUM VITAE……………………………………………………………….. 48

iv

LIST OF FIGURES

FIGURE

1. Working Model for the classic “biliary” view of RCT in mice. 2. Working model for the role of intestinal SR-BI in “non-biliary” fecal sterol loss

though the proximal small intestine.

3. Intestinal overexpression of SR-BI decreases plasma cholesterol, but does not alter intestinal cholesterol absorption.

4. Treatment with the NPC1L1 inhibitor ezetimibe does not unmask a role for

intestinal SR-BI in cholesterol absorption.

5. Hepatic and Biliary cholesterol levels in intestinal SR-BI and hepatic NPC1L1 double transgenic mice.

6. Intestinal overexpression of SR-BI does not augment non-biliary reverse

cholesterol transport.

PAGE

4

19

5

22

25

26

v

LIST OF ABBREVIATIONS

ACAT

Apo

CE

CHD

EDTA

ER

EZE

FC

HDL

ILDL

LDL

LRP

MW

MTP

PAGE

PBS

PCR

PEG

Acyl-coenzyme A: cholesterol acyltransferase

Apolipoprotein

Cholesteryl ester

Coronary heart disease

Ethylenediaminetetraacetic acid

Endoplasmic reticulum

Ezetimibe

Free Cholesterol

High Density Lipoprotein

Intermediate-size low-density lipoprotein

Low density lipoprotein

LDL receptor related protein

Molecular weight

Microsomal triglyceride transfer protein

Polyacrylamide gel electrophoresis

Phosphate buffer saline

Polymerase chain reaction

Polyethylene glycol

vi

LIST OF ABBREVIATIONS

PL

RCT

SRBI

SDS

TICE

TG

VLDL

WT

SR-BIInt-Tg

Phospholipid

Reverse Cholesterol Transport

Scavenger Receptor Class B type I

Sodium dodecyl sulfate

Trans Intestinal Cholesterol Efflux

Triglyceride

Very low-density lipoprotein

Wild type

SR-BI intestine transgenic mouse

vii

ABSTRACT

It has recently been shown that reverse cholesterol transport (RCT) does

not rely solely on biliary secretion (37), but also involves the direct excretion of

cholesterol by the proximal small intestine through a process known as trans-

intestinal cholesterol efflux (TICE). There is almost no information in literature

regarding molecular mechanisms regulating TICE. It has long been known that

scavenger receptor class B type I (SR-BI) plays a crucial role in RCT in the liver

by facilitating selective uptake of high density lipoprotein (HDL) cholesterol.

Interestingly, SR-BI is also expressed in the small intestine. Previously it has

been reported that mice overexpressing SR-BI specifically in the proximal small

intestine have accelerated cholesterol absorption (83). In contrast, we

hypothesized that over expression of SR-B1 would augment the TICE pathway

by facilitating intestinal clearance of plasma cholesterol. To test this hypothesis a

series of experiments were conducted by using mice transgenically

overexpressing SR-BI in the proximal small intestine (SR-BIInt.-Tg) in the presence

or absence of biliary cholesterol contributions either on a low (0.015%) or high

(0.2%) (wt/wt) cholesterol diet. As previously reported, SR-BIInt.-Tg mice had

significantly lower plasma cholesterol levels. However, in contrast to previous

reports, SR-B1Int.-Tg mice had unaltered fractional cholesterol absorption and

fecal neutral sterol excretion rates. Our results suggest that SR-BI is not rate-

limiting for intestinal cholesterol absorption or fecal neutral sterol loss through the

TICE pathway in mice. Therefore, additional studies are required to identify

viii

intestinal lipoprotein receptors involved the delivery of plasma cholesterol to the

intestine for TICE pathway.

1

INTRODUCTION

According to American Heart association’s latest estimates one in every

three deaths in United States is due to one or more forms of coronary heart

disease (CHD). CHD events have costs more lives in this country over the past

century then the next four causes combined (1). According to the latest

estimates, in 2010 direct or indirect costs related to CHD have accounted for

$503.2 billion in United States alone which is a significant increase from $393.5

billion in 2005 (1). Results from numerous research studies done over the last

few decades support the concept that the most accurate predictor of CHD

incidence is the plasma concentrations of low-density lipoprotein cholesterol

(LDLc). Plasma LDLc concentrations shows a strong positive correlation with the

CHD related events. For the transport of cholesterol in the arteries, LDL particles

are retained by arterial proteoglycans which in turn attracts macrophages.

Macrophages engulf the LDL particles and start the formation of atherosclerotic

plaques, which if severe enough results in various CHD related events. Hence,

lowering LDLc has been the primary therapeutic goal for decades, and statins

have been the main drug used to accomplish this. However, even with the

substantial LDLc lowering achieved with statin treatment, these drugs have

reduced CHD-associated mortality and morbidity by barely 30% (2). , Since the

landmark Framingham Heart Study’s results showed a strong inverse correlation

between the HDLc levels and CHD (3), a number of therapeutic strategies are

being developed by many drug companies to elevate high-density lipoprotein

2

cholesterol (HDLc) .There have been many mechanisms proposed as to how

HDLc influences the development of CHD (4-6), but the most widely accepted is

that HDL facilitates the process of reverse cholesterol transport (RCT).

HDL is believed to be the major cholesterol transport particle in RCT

pathway. HDL is thought to facilitate the net movement of cholesterol from

peripheral tissues to the liver for excretion into the feces through bile. (7). Known

proteins involved in RCT in humans include LCAT, CETP, HL, and SR-BI. Along

with HDLc concentration, functionality of all these proteins can reflect the activity

of RCT pathway. Hepatic SR-BI mediates the majority of hepatic uptake of HDL

CE. Once in the liver, SR-BI-derived CEs are converted into FC by a lysosomal

cholesteryl esterase, which can either be excreted in bile as FC or bile acids (8,

9). A fraction of the bile acid and cholesterol excreted into bile is lost in the feces,

and this is the major route of disposal for cholesterol and its metabolites from the

body (8, 9). Research have shown that excess of FC is cytotoxic for the cells, so

it is generally accepted that the process of RCT has primarily evolved to protect

body from excess accumulation of unesterified cholesterol (10,11). However, in

the context of atherosclerosis, RCT is thought to involve HDL-mediated efflux of

cholesterol from the arterial wall, specifically from cholesterol-laden

macrophages (12).

As discussed on the previous page, in the classic model of RCT (Figure

1), cholesterol in peripheral tissues is returned to the liver for excretion into bile

3

(8, 9, 13-15). If this was true, plasma HDLc levels would predict both biliary and

fecal cholesterol levels. However, studies carried out in mouse model which have

extremely low HDLc levels (e.g. ABCA1-/-), the biliary and fecal sterol loss is

similar to wild type mouse (16, 17). In addition, various mouse models of

genetically altered hepatic cholesterol metabolism have shown a disconnect

between biliary and fecal sterol (18, 19). This has led us to believe that fecal

sterol loss likely involves two distinct excretory routes: 1) the classic hepatobiliary

pathway (Figure 1) , and 2) a non-biliary pathway (Figure 2) . As per the non-

biliary RCT pathway cholesterol effluxed from peripheral cells or macrophages is

delivered to the liver but instead of transporting it further to the bile, it re-enters

the plasma compartment where it can be directly excreted through the

enterocytes (gut wall). One of the reasons for studying and understanding non-

biliary route is that excessive augmentation of biliary cholesterol concentrations

can promote cholesterol gallstone formation in humans (20-22). Importantly, the

major mechanisms regulating cholesterol secretion into bile have been recently

defined (19, 23-27), but almost no information exists regarding mediators of non-

biliary cholesterol secretion by the intestine.

4

FIGURE 1. Working Model for the classic “biliary” view of RCT in a wild type mouse. In this model, free cholesterol is effluxed from peripheral tissues, and delivered to the liver via HDL-driven presentation to hepatic SR-BI. This free cholesterol is immediately shunted into the bile through the actions of hepatic ABCG5/G8. A portion of this biliary cholesterol can be reabsorbed by the intestine through the actions of intestinal NPC1L1, whereas intestinal ABCG5/G8 can efflux newly absorbed cholesterol back into the lumen. Taking into account dietary sterol, it is predicted that ~55-65% of fecal sterol loss can be attributed to biliary sterol loss in mice and humans (35, 36).

5

FIGURE 2. Working model for the role of intestinal SR-BI in “n on-biliary” fecal sterol loss though the proximal small intestine. We have previously demonstrated that by overexpressing NPC1L1 in the liver, biliary cholesterol secretion in blunted by >90%, likely resulting from NPC1L1’s ability to recapture newly secreted cholesterol (19). This unique model of biliary insufficiency will be the focus of many of the proposed studies. In the case of biliary insufficiency, we believe that peripheral tissue cholesterol efflux and HDL-mediated delivery back to the liver occurs in a similar fashion to the wild-type condition. However, once hepatic SR-BI accepts peripheral cholesterol into the liver, this free cholesterol burden cannot be disposed of through biliary secretion. Therefore, the liver initiates the secretion of nascent HDL particles, which acquire high levels of apoE likely from non-hepatic sources, and these apoE-HDLc particles accumulate in the plasma. These apoE-HDLc particles interact with SR-BI at the basolateral surface of proximal intestinal enterocytes, to deliver cholesterol into the cell. This free cholesterol is then effluxed from the apical membrane of the enterocyte into the lumen of the small intestine through the actions of ABCG5/G8 for fecal excretion. The sum of “biliary” and “non-biliary” pathways represents the total flux of peripheral cholesterol into the feces

6

RCT is a multi-step process resulting in the net movement of cholesterol

from peripheral tissues back to the liver. The model of “classical RCT pathway”

was proposed by Glomset in 1968 (7). Long before Glomset and colleagues

proposed the “biliary” model for RCT (7), the existence of a “non-biliary” pathway

of RCT (TICE) was proposed by Warren Sperry in 1927 (28). Sperry

demonstrated that as compared to control group (bile intact dogs), fecal neutral

sterol loss was paradoxically increased in the dogs that had undergone chronic

biliary fistula surgery (28). Since Sperry’s experiments, a handful of studies have

been done showing the importance of TICE pathway, one such study by

Pertsemlidis and colleagues showed that even in the surgical absence of biliary

cholesterol contributions to the small lintestine there was about 7-fold increase in

the fecal neutral sterol loss (29). Similar results have been seen under conditions

of biliary diversion in rats (30) and familial hypercholesterolemic humans (31). In

patients with complete obstruction of biliary and pancreatic ducts cholesterol,

Stanley and Cheng suggested that non-dietary fecal sterol loss in humans

consists of two distinct fractions: 1) a fraction coming through biliary secretion,

and 2) and a second fraction directly secreted by the intestine (32). Over the past

few decades a number of studies supporting the idea of TICE pathway can be

found scattered through the literature, but for the major part of last century these

studies have always been ignored. Unfortunately, a major drawback of these

biliary diversion or obstruction animal models is the interruption of enterohepatic

circulation of bile acids and other non-lipid biliary components. So the absence of

bile salts will compromise intestinal sterol absorption, which will upregulate the

7

endogenous cholesterol synthesis in intestine (33). However, strong evidence for

non-biliary fecal sterol loss continues to come to light in the age of genetically

modified mice. In fact, non-biliary RCT has now been demonstrated in several

mouse models with genetic or surgical deficiency of biliary cholesterol (34-38).

Hepatic clearance of HDL cholesterol facilitated by Scavenger receptor BI

(SR-BI) is critical for RCT. SR-BI is a membrane-associated glycoprotein, which

was first identified by its close homology to the lipoprotein scavenger receptor

CD36 (39, 40). SR-BI is conserved in evolution and is expressed in many

mammalian tissues and cell types including brain, intestine, liver, macrophages,

endothelial cells, smooth muscle cells, keratinocytes, adipocytes, platelets, and

human placenta. However, SR-BI is most highly expressed in tissues that are

known to be dependent on HDL FC and CE for bile acid synthesis (liver) and

steroidogenesis (adrenal, ovary and testis) (41-43). SR-BI delivers HDL CE to

the liver and steroidogenic tissues by a process known as “selective uptake

pathway”. Contrary to the classic LDL receptor endocytic pathway, in which the

entire lipoprotein is internalized in clathrin coated pits and degraded (84), in the

selective uptake pathway, SR-BI binds HDL and the core CE are delivered to the

plasma membrane without the concomitant uptake and degradation of the entire

HDL particle. The process though which SR-BI transfer lipids to the plasma

membrane is not well understood. The fact that the isolated plasma membranes

from a number of different sources exhibit HDL CE selective uptake in vitro

suggests that the initial phase involves bulk flow of the hydrophobic lipids without

8

the assistance of other cytoplasmic components (85, 86). Indeed, Krieger and

colleagues (87) showed that SR-BI was able to mediate selective uptake of HDL

CE into multilamellar vesicles without expression of other proteins or specialized

cellular structures. A recent thorough study (88) took a careful look at the

localization of different cell types supporting the idea that most SR-BI- mediated

HDL CE selective uptake occurs at the cell surface and not during endocytic

trafficking.

In the context of RCT, these studies suggest that SR-BI is not only crucial for

cholesterol delivery to the liver but is also important for cholesterol efflux at the

vessel wall. Therefore, the receptor acts at both ends of the RCT pathway.

It has been a matter of debate, but SR-BI seems to localize to both apical and

basolateral membranes in polarized cells (44-49), and likely undergoes sterol-

dependent basolateral to apical transcytosis (44-49). The precise molecular

mechanism by which SR-BI facilitates selective uptake remains elusive, but

purified SR-BI reconstituted into liposomes can recapitulate high-affinity HDL

binding and selective uptake, indicating that these processes are intrinsic to the

receptor itself (51). Direct evidence that SR-BI plays a role in RCT has come

from studies in mice. In support of this, SR-BI overexpression results in

diminished HDLc levels, increased biliary cholesterol secretion, and marked

protection against atherosclerosis (52-55). Furthermore, mice lacking SR-BI

accumulate large apoE-rich HDL particles in plasma, have decreased biliary

sterol excretion, and develop severe atherosclerosis (56-60). In fact, mice lacking

9

SR-BI in the apoE-deficient background develop occlusive coronary artery

atherosclerosis, myocardial infarction, and die at a very early age (61-63). The

hyperlipidemic and proatherogenic effects seen with SR-BI deficiency are

thought to be primarily due to SR-BI’s critical role in selective uptake of HDL

cholesteryl esters into the liver, thereby promoting biliary and fecal sterol loss. In

line with this, Zhang and colleagues (64) reported that hepatic overexpression of

SR-BI specifically promoted macrophage to feces RCT, and SR-BI-/- mice had

reduced RCT. However, these authors also recognized that intestinal SR-BI may

also play a role in RCT.

The role of SR-BI in the intestine is a matter of intense debate, and begs

further investigation. Several investigators have speculated that SR-BI facilitates

cholesterol absorption, instead of promoting basolateral to apical movement as

we propose (Figure 2) . The first evidence for this possibility was demonstrated

by Hauser et al. (65), who showed that SR-BI was present in brush border

membrane vesicles (BBMV), and SR-BI antibody reduced cholesterol binding to

BBMVs. Others have also demonstrated that SR-BI can be localized to the apical

membrane of the enterocyte (66), and is a high-affinity cholesterol receptor in

intestinal BBMV (67). However these in vitro findings have not been

substantiated in mice with targeted disruption of SR-BI. In fact, mice lacking SR-

BI have normal or enhanced intestinal cholesterol absorption (68-70). The most

thorough of these studies revealed that SR-BI deficiency significantly increased

fractional cholesterol absorption, under several conditions of dietary cholesterol

challenge (68). Further, the cholesterol absorption inhibitor ezetimibe potently

10

inhibits cholesterol absorption in SR-BI deficient mice, indicating that SR-BI in not

likely involved in ezetimibe-sensitive cholesterol transport (69). In contrast to SR-

BI deficiency, one study has demonstrated that intestine-specific overexpression

of SR-BI did result in modest increases in movement of a gavaged cholesterol

trace into the plasma, but fractional cholesterol absorption and mass fecal sterol

loss was not determined (71). In our studies we have chosen to study these

intestine-specific SR-BI transgenic mice, given that SR-BI knockout mice lack

any phenotypic abnormalities in intestinal cholesterol absorption (68).

Collectively, these studies have revealed that SR-BI is expressed at very low

levels in the intestine, but its role in intestinal cholesterol absorption has not been

definitively established (68-71). In contrast, we wanted to test the hypothesis that

intestinal SR-BI plays a critical role in the non-biliary TICE pathway for RCT

(Figure 2) . The results from our studies are detailed below.

11

EXPERIMENTAL PROCEDURES

Animals

Mice overexpressing SR-BI specifically in the small intestine were generated

as previously described (71). Briefly, the murine cDNA for SR-BI was cloned

downstream of the apolipoprotein C-III enhancer (-500/-890 bp)-apolipoprotein A-

IV promoter (-700 bp), and the resulting plasmid was linearized via SalI and AvrI

restriction digestion, and used to generate transgenic mice by pronuclear

microinjection into the B6D2 background. Resulting SR-BI intestine transgenic

mice were subsequently backcrossed to the C57BL/6 background for over 12

generations, and were kindly provided by Dr. Xavier Collet (INSERM, France).

These mice were intercrossed with mice transgenically overexpressing NPC1L1

specifically in the liver (NPC1L1LiverTg ). Creation of NPC1L1LiverTg mice has been

described previously (72). All experiments were done in the high overexpressing

line NPC1L1-Tg112 (72), which had been backcrossed to C57BL/6 background

for over 9 generations. Therefore, all mice had been backcrossed to C57BL/6

background for greater than 9 generations before study, and male sibling

controls were used to minimize effects of genetic heterogeneity and sex.

Genotyping was confirmed by PCR analysis on genomic DNA isolated from tail

snip as previously described (71, 72). At 6-8 weeks of age, mice were switched

from a diet of rodent chow to a low fat (10% of energy as palm oil-enriched fat)

and either low (0.015%, w/w) or high cholesterol (0.2%, w/w) for a period of 2-8

weeks. For ezetimibe treatment experiments, male wild type (WT) and intestine-

12

specific SR-BI transgenic (SR-BI) mice littermates were fed a diet containing

either 0.015% cholesterol (w/w) for 4 weeks. After 4 weeks on diet, mice were

orally gavaged daily for 3 consecutive days with either vehicle or 10mg/kg body

weight (BW) ezetimibe (EZE) as previously described (72). All mice were

maintained in an American Association for Accreditation of Laboratory Animal

Care-approved animal facility, and all experimental protocols were approved by

the institutional animal care and use committee at the Wake Forest University

School of Medicine.

Plasma Lipid and Lipoprotein Analyses

Total plasma cholesterol (TPC) and plasma triglyceride (TG) concentrations

were measured using colorimetric assay as previously described (73, 74).

Cholesterol concentrations in very low-density lipoproteins (VLDL), intermediate-

density lipoproteins (IDL), low-density lipoproteins (LDL), and high density

lipoproteins (HDL) were determined after separation of plasma samples by high

performance liquid chromatography (HPLC) with a Pharmacia Superose-6

column run at a flow rate of 0.5 ml/min. as previously described (73,74).

Immunoblotting of Hepatic and Intestinal Proteins

Total liver and jejunal homogenates were made in a modified RIPA buffer

containing PBS (pH 7.5), 1% Nonidet P-40, 0.1% SDS, 0.5% sodium

deoxycholate, 2 mM Na3VO4, 20 mM -glycerophosphate, 10 mM NaF, and a

protease inhibitor mixture (Calbiochem) including 500 µM AEBSF, 1 µg/ml

13

aprotinin, 1 µM E-64, 500 µM EDTA, and 1 µM leupeptin. Homogenates (25 µg

per lane) were separated by 4-12% SDS-PAGE, transferred to polyvinylidene

difluoride membrane, and incubated with the following antibodies: 1) rabbit anti-

SR-BI IgG (abcam®, #ab396) and 2) mouse anti-β actin antibody (Sigma,

#A1978).

Immunolocalization of intestinal SR-BI.

Jejunum of Wild type (WT) and SR-BIInt-tg mice were taken out and

flushed with (0.9%) saline to get rid of food. The segments were next cut opened

along the length of intestine, rolled with the help of a toothpick, and were

embedded in OCT. Tissues in OCT were then frozen with dry ice and sections

were obtained on the glass slide with the help of a cryostat (Lecia, #CM1510 S).

The tissues were fixed with 10% formaldehyde for 10 minutes and air dried for 30

minutes. Next the slides were washed in PBS (pH=7.4) for 3 times, 5 minutes

each. PBS was carefully removed by blotting and then a circle was drawn around

each section with a hydrophobic pen (Vector Labs, ImmEdge cat#H-4000). Next

the sections were blocked for 30 minutes with 4% blocking buffer (2% goat

serum and 2% BSA in PBS at RT in a humid chamber. Blocking solution was

removed by tapping. SR-BI primary antibody (rabbit anti-SR-BI IgG, abcam®,

#ab396) was diluted 1:1000 in 2% blocking buffer ( 1 ml PBS + 20 µl 50% BSA)

and incubated for 1 hr at RT. Sections were again washed in PBS for three times

for five minutes each. Goat anti rabbit secondary antibody (FITC anti-rabbit IgG

H+L, Vector Cat # BA-4000) was diluted 1:5000 in 2% blocking buffer and added

14

dropwise on the tissue sections for 30 minutes. The sections are again washed in

PBS as described above and then the slides were stained with DAPI to identify

nuclei, Briefly, 2ul of the stock solution (5 mg/ml) of DAPI dihydrochloride (Sigma

32670) in Dimethyl formamide was added to 50 ml of PBS for making a working

solution) for 10 minutes after the final wash with PBS, coverslips were carefully

installed on the sections. Next fluorescence microscopy was conducted, and for

visualization the following parameter were used: used UV filter (340-380 nm) for

DAPI and blue filter (465-495) for FITC).

Cholesterol Absorption and Fecal Neutral Sterol Excretion Measurements

Fractional cholesterol absorption was measured using the fecal dual isotope

method, and fecal neutral sterol excretion was measured by gas-liquid

chromatography as previously described (73). Briefly, for fractional cholesterol

absorption experimental mice were gavaged with 0.1 µCi of [4-14C] cholesterol

(Amersham Pharmacia Biotech) and 0.2 µCi of [22, 23-3H] sitosterol (New

England Nuclear) dissolved in ~50 µl of corn oil. Each mouse was individually

housed in a cage with a wire bottom and was allowed free access to diet and

water for a total of 72 hours. The feces were collected and the feces were

collected, dried in a 70°C vacuum oven, weighed, and cr ushed into a fine

powder. A measured mass (50–100 mg) of feces was placed into a glass tube,

saponified by adding 50% KOH to a final concentration of 5% (w/v) and heating

at 65°C for 2 h. Neutral lipids were extracted by add ing hexane and deionized

water, vortexing, and centrifuging at 2,000 g for 10 min. The hexane phase was

15

transferred to a scintillation vial and dried at 65°C under N2. In addition, aliquots

of the initial [14C] cholesterol/ [3H] sitosterol dose were placed into scintillation

vials to determine dose ration. Following complete removal of organic solvent by

evaporation, Bio-Safe II scintillation fluid (Research Products International) was

added directly to the scintillation vials, and the [14C] cholesterol and [3H] sitosterol

counts were measured in a scintillation spectrometer. Percentage cholesterol

absorption was calculated using the following equation:

For mass fecal neutral sterol quantification, mice were singly housed as

described above on wire bottom cages. After a 3 day fecal collection, the mice

were weighed, and the feces were collected, dried in a 70°C vacuum oven,

weighed, and crushed into a fine powder. A measured mass (50–100 mg) of

feces was placed into a glass tube containing 103 ug of 5α-cholestane as an

internal standard. The feces were saponified and the neutral lipids were

extracted into hexane as described above. Mass analysis of the extracted neutral

sterols was conducted by gas-liquid chromatography as described previously

(72, 73). Fecal neutral sterol mass represents the sum of cholesterol,

coprostanol, and coprostanone in each sample. Fecal neutral sterol excretion

was expressed as mg sterol/day/100 g body weight.

16

Hepatic Lipid Quantification

Liver lipid extracts were made and total cholesterol (TC), cholesterol ester

(CE), free cholesterol (FC), triglyceride (TG), or phospholipids (PL) mass was

measured using detergent-solubilized enzymatic assays as previously (75).

Briefly, �100 mg of liver was thawed, minced, and weighed in a glass tube.

Lipids were extracted in 2:1 CHCl3/methanol at room temperature overnight. The

protein was quantitatively separated from the lipid extract, which was then dried

down under N2 and redissolved in a measured volume of 2:1 CHCl3/methanol.

Dilute H2SO4 was added to the sample, which was then vortexed and centrifuged

to split the phases. The aqueous upper phase was aspirated and discarded, and

an aliquot of the bottom phase was removed and dried down; 1% Triton X-100 in

CHCl3 was then added, and the solvent was evaporated (75). Deionized water

was then added to each tube and vortexed until the solution was clear. Lipids

were then quantified using the Triglycerides/GB kit (Roche) plus the enzymatic

cholesterol assays described above.

Biliary Lipid Collection and Analysis

Determination of lipid concentrations in gall bladder bile lipid was conducted

as previously described (72, 73). Briefly, bile was collected by directly puncturing

the gall bladder with a 30G1/2 needle. A measured volume (10 µl) of bile was

placed into a glass tube and the neutral lipids were extracted and analyzed using

enzymatic methods as described for liver lipids. Aliquots of the aqueous phase of

17

the extraction were analyzed for bile acid content using an enzymatic assay

using hydroxysteroid dehydrogenase (76).

Statistical analyses

Data are expressed as the mean ± standard error of the mean (SEM). All

data were analyzed using either a one-way or two-way analysis of variance

(ANOVA) followed by Student’s t tests for post hoc analysis. Differences were

considered significant at p <0.05. All analyses were performed using JMP version

5.0.12 (SAS Institute; Cary, NC) software.

18

RESULTS

Intestinal overexpression of SR-BI decreases plasma cholesterol, but does not

alter intestinal cholesterol absorption.

To determine the relative protein abundance and subcellular localization of

SR-BI we utilized immunoblotting and immunofluorescent techniques to detect

endogenous and ectopically expressed SR-BI in the liver and small intestine. As

previously described SR-BIInt-Tg mice have 2-3 fold more SR-BI in the liver, when

compared to wild type mice (Figure 3A) , likely due to the fact that the apo-

CIII/apoA-IV enhancer region can drive modest hepatic expression. Interestingly,

in the jejunum, we could barely detect endogenous SR-BI in WT mice (Figure

3A), whereas SR-BIInt-Tg had SR-BI protein levels similar to that found in the liver

of wild type mice (Figure 3A) . In agreement with Western blotting,

immunofluorescence detection of endogenous SR-BI in the jejunum was barely

detectable, yet the SR-BIInt-Tg mice had readily detectable levels of protein that

localized to both the apical and basolateral membranes of enterocytes (Figure

3B). In previous studies, mice overexpressing SR-BI specifically in the small

intestine were shown to have dramatic reduction in plasma cholesterol levels

when maintained on rodent chow (71).

19

FIGURE 3. Intestinal overexpression of SR-BI decreases plasma cholesterol, but does not alter intestinal cholesterol absorption. Male wild type (WT) and intestine-specific SR-BI transgenic (SR-BI) mice littermates were fed diets containing either 0.015% or 0.2% cholesterol (w/w) for a period of 8 weeks. (A) Western blot analysis of liver and jejunal homogenates (n=3 per group) from mice fed 0.015% cholesterol for 2 weeks. (B) Immunolocalization of SR-BI in the mouse jejunum. Sections (6 µm) of mouse jejunum were fixed with 3.7% formaldehyde/PBS, permeabilized with 0.05% Tween 20, and stained with a polyclonal antibody raised against SR-BI to determine tissue and subcellular localization; Blue = nucleus, Red = SR-BI. (C) Total plasma cholesterol (TPC) and high density lipoprotein cholesterol (HDL) in 6 week old chow fed mice (Chow) or mice fed 0.015% or 0.2% cholesterol for 4 weeks. (D) Represents fractional cholesterol absorption measured by fecal duel-isotope method, and mass fecal neutral sterol loss determined by gas liquid chromatography measured after 6 weeks on diet. Data in panels (C,D) represent the mean ± S.E.M. from 6-10 mice per group, * = significantly different from WT mice within each diet group (p<0.05).

20

We examined total plasma cholesterol and HDL cholesterol levels in mice fed

either a standard rodent chow, or semi-synthetic diets containing either 0.015%

or 0.2% cholesterol (wt/wt). Under all dietary conditions the SR-BIInt-Tg mice

exhibited significantly reduced TPC and HDLc levels, when compared to wild

type littermates (Figure 3C). Interestingly, Bietrix and colleagues reported

modest increases in cholesterol absorption rates as measured by the

appearance of gavaged 14C-cholesterol into the plasma over a four hour time

course in SR-BIInt-Tg mice (71). However, measuring the appearance of a tracer

amount of 14C-cholesterol into the plasma compartment reflects not only

intestinal cholesterol absorption, but also reflects plasma lipoprotein clearance

rates as well as hepatic lipoprotein secretion. To more definitely measure

cholesterol absorption rates without these other confounding factors, we utilized

both the dual fecal isotope method as well as quantifying mass fecal cholesterol

loss by gas chromatography. Importantly, on both the low and high cholesterol

diets there was similar intestinal cholesterol absorption and fecal neutral sterol

loss in wild type and SR-BIInt-Tg littermates (Figure 3D).

Treatment with the NPC1L1 inhibitor ezetimibe does not unmask a role for

intestinal SR-BI in cholesterol absorption of fecal sterol loss.

Given that fractional cholesterol absorption rates were particularly high in

our initial studies (Figure 3D) , we were concerned that relative contributions of

the TICE pathway to fecal cholesterol loss may have been masked since most of

the lumenal cholesterol was being recaptured by the intestinal cholesterol

21

transporter Niemann Pick-C1-Like 1 (NPC1L1). To overcome this concern we

treated mice with ezetimibe to block intestinal reuptake of lumenal cholesterol by

NPC1L1. Ezetimibe treatment for 2 days significantly reduced intestinal

cholesterol absorption by ~40% in both wild type and SR-BIInt-Tg mice (Figure

4A). In this cohort of mice, before ezetimibe treatment was initiated fecal neutral

sterol loss no different between wild type and SR-BIInt-Tg mice (Figure 4B) .

Twenty four hours after ezetimibe treatment both wild type and SR-BIInt-Tg mice

increased fecal neutral sterol loss by ~5-fold (Figure 4C) . This ezetimibe-induced

increase in fecal neutral sterol loss was still apparent after 48 hours of ezetimibe

treatment (Figure 4D) . Collectively, these studies suggest that regardless of

whether intestinal cholesterol uptake was intact or blocked with ezetimibe,

intestinal SR-BI overexpression has no impact on the amount of cholesterol

excreted in the feces.

22

FIGURE 4. Treatment with the NPC1L1 inhibitor ezetimibe does not unmask a role for intestinal SR-BI in cholesterol absorption. Male wild type (WT) and intestine-specific SR-BI transgenic (SR-BI) mice littermates were fed a diet containing either 0.015% cholesterol (w/w) for 4 weeks. After 4 weeks on diet, mice were orally gavaged daily for 3 consecuative days with either vehicle or 10mg/kg Body Weight (BW) ezetimibe (EZE). (A) Represents fractional cholesterol absorption measured by fecal duel-isotope method for feces collected during the first 48 hours of experiment. (B-C) Represent mass fecal neutral sterol loss determined by gas liquid chromatography measured after 4 weeks on diet prior to ezetimibe treatment at baseline (B), and 1 day (C) or 2 days (D) post ezetimibe treatment. Data represent the mean ± S.E.M. from 6-10 mice per group, and values not sharing the same superscript differ significantly(p<0.05).

23

Intestinal overexpression of SR-BI does not augment non-biliary reverse

cholesterol transport.

The main goal of these studies was to determine the role of intestinal SR-

BI in non-biliary RCT or TICE. The rationale for studying SR-BI stems from the

well known ability of SR-BI to traffic cholesterol across hepatocytes in a

unidirectional basolateral to apical manner (43-46, 88). We hypothesized that

intestinal SR-BI may likewise traffic plasma cholesterol across the enterocyte in a

basolateral to apical manner to contribute to TICE. To study the role of intestinal

SR-BI in TICE we crossed SR-BIInt-Tg mice with the TICE-predominant

NPC1L1LiverTg mouse model, which have been previously described to

specifically lack biliary cholesterol secretion (37,72), resulting in four

experimental genotypes for comparisons: wild type (WT), Intestine SR-BI

transgenics (SR-BIInt-Tg ), Liver NPC1L1 transgenics (NPC1L1LiverTg ), and

SRBI/NPC1L1 double transgenic mice. As expected, regardless of dietary

cholesterol burden, NPC1L1LiverTg and SRBI/NPC1L1 double transgenic mice

had severely reduced biliary cholesterol levels, when compared to either wild

type or SR-BIInt-Tg (Figure 5E ). Likely due to the 2-3 fold overexpression of SR-BI

in the liver, SR-BIInt-Tg mice exhibit a small increase in biliary cholesterol levels

when fed a 0.2% cholesterol diet (Figure 5E ). As previously reported,

NPC1L1LiverTg mice have specific depletion of only biliary cholesterol (Figure 5E) ,

without affecting biliary bile acid (Figure 5F) , or biliary phospholipid (Figure 5G) .

Despite alterations in biliary cholesterol levels, NPC1L1LiverTg mice do not

accumulate cholesterol or triglyceride in the liver (Figure 5A, 5B, 5C) .

24

Interestingly, SR-BIInt-Tg mice have modest reductions in hepatic free and

esterified cholesterol, compared to wild type mice (Figure 5B,5D) , but this is only

seen when the mice are fed 0.2% cholesterol in the diet. Collectively, hepatic and

biliary lipid levels in this experiment mimic what was anticipated for dietary

cholesterol feeding in these mouse models (72).

25

FIGURE 5. Hepatic and B iliary cholesterol levels in intestinal SR-Bi and hepatic NPC1L1 double transgenic mice. Wild type (WT), Intestine SR-BI transgenics (SR-BI), Liver NPC1L1 transgenics (NPC1L1), and SRBI/NPC1L1 double transgenic male mice were fed either 0.015% or 0.2% cholesterol(w/w) for 2 weeks. Liver samples were extracted and enzymatically analyzed to quantify the mass of (A) total cholesterol (TC), (B) cholesteryl esters (CE), (C) triglycerides (TG), or (D) free cholesterol (FC). All hepatic lipid values were normalized to total tissue protein content. Panels E-G represent raw concentrations and molar ratios of gallbladder cholesterol (E), bile acids (F), and phospholipids (G). Data shown represent the mean ± S.E.M. from 4-8 mice per group; * = significantly different from WT mice within each diet group (p<0.05).

26

More importantly, when we measured fractional cholesterol absorption in the four

genotypes of mice, only the NPC1L1LiverTg mice fed 0.2% dietary cholesterol

were significantly different that the wild type controls on the same diet (Figure

6A). As seen in the previous two experiments (Figure 3D, 4A) , fractional

cholesterol absorption rates were unaltered in SR-BIInt-Tg regardless of NPC1L1

genotype (Figure 6A) .

27

FIGURE 6. Intestinal overexpression of SR-BI does not augment non-biliary reverse cholesterol transport. Wild type (WT), Intestine SR-BI transgenics (SR-BI), Liver NPC1L1 transgenics (NPC1L1), and SRBI/NPC1L1 double transgenic male mice were fed either 0.015% or 0.2% cholesterol(w/w) for 2 weeks. (A) Represents fractional cholesterol absorption measured by fecal duel-isotope method. (B) Represent mass fecal neutral sterol loss determined by gas liquid chromatography measured after 2 weeks on diet. Data shown represent the mean ± S.E.M. from 4-8 mice per group; * = significantly different from WT mice within each diet group (p<0.05).

28

In agreement with this, fecal neutral sterol loss was also unaltered across

the four genotypes (Figure 6B) . Only in the 0.2% dietary cholesterol group did

we see a modest increase in fecal neutral sterol loss in the SR-BIInt-Tg mice,

compared to all other groups (Figure 6B) . Collectively, these studies

demonstrate under several experimental conditions that intestinal overexpression

of SR-BI does not impact either cholesterol absorption or TICE rates in mice.

29

DISCUSSION

Although several groups have hypothesized a role for SR-BI in intestinal

cholesterol absorption (65-67), studies where cholesterol absorption was actually

quantified in mouse models of SR-BI deficiency (68) and now intestine-specific

overexpression (Figures 3,4,6) have not supported such a role. Instead there is

much stronger evidence that SR-BI does not play a rate-limiting role in intestinal

cholesterol absorption (68-70). The major findings of these studies are the

following: (1) endogenous SR-BI protein expression in the small intestine barely

detectable, when compared to very abundant levels in the liver, (2) endogenous

and ectopically expressed SR-BI localizes to both apical and basolateral

membranes in enterocytes, (3) intestine-specific overexpression of SR-BI does

not alter intestinal cholesterol absorption either in the absence or presence of the

cholesterol absorption inhibitor ezetimibe, (4) intestine-specific overexpression of

SR-BI does not alter fecal neutral sterol loss in the absence or presence of the

cholesterol absorption inhibitor ezetimibe, and (5) intestine-specific

overexpression of SR-BI does not increase TICE rates in mice genetically lacking

the biliary pathway for RCT (NPC1L1LiverTg ). Collectively, these findings support

the conclusion of many others (68-70) that intestinal SR-BI is not rate limiting for

the trafficking of cholesterol across the enterocyte either in an apical to

basolateral fashion (68-70), or through the basolateral to apical TICE pathway

(77).

30

Several laboratories have previously proposed a role for SR-BI as a high

affinity receptor for cholesterol at the brush border membrane (65, 67, and 78),

as well as a mechanism by which cholesterol is trafficked from the intestinal

lumen across the enterocyte in an apical to basolateral pattern for packaging into

chylomicrons (66-67, 78). However, all cell-based examining the subcellular

trafficking pattern of SR-BI has agreed that in polarized cells SR-BI preferentially

sorts HDL-derived cholesterol from basolateral membranes to apical membranes

(43-46), and the receptor itself has shown a similar trafficking pattern (43-46). In

agreement with this directional trafficking pattern in polarized cells, hepatic

overexpression or knockout of SR-BI either promotes or diminishes the

movement of HDL-derived cholesterol into bile, respectively (50, 52-55). In fact

all experimental evidence in the liver supports a rate-limiting role for SR-BI in

moving HDL-derived cholesterol preferentially into bile (50-64). Based on both

the in vitro and in vivo evidence supporting a role for SR-BI in unidirectional

transcytosis of cholesterol (43-64), and the lack of evidence that SR-BI promotes

intestinal cholesterol absorption (68-70,78), we hypothesized that intestinal SR-

BI may play a rate limiting role in TICE to move plasma cholesterol across the

enterocyte for efflux into to feces (Figure 2) .

Although it has been well documented that SR-BI knockout mice do not have

defects in intestinal cholesterol absorption (68-70), a previous study in intestine-

specific SR-BI transgenic mice indirectly demonstrated a potential role for

intestinal SR-BI in cholesterol absorption (71). In this study Bietrix and colleague

31

measured intestinal cholesterol absorption by gavaging WT and SR-BIInt-Tg mice

with 14C-cholesterol and followed the appearance of this tracer into the plasma

compartment over a four hour period (71). Unfortunately, examining plasma

appearance of a gavaged tracer reports not only on intestinal cholesterol

absorption, but also plasma lipoprotein clearance as well as hepatic repackaging

of intestinally derived cholesterol during the four hours period. Using the

standardized fecal dual isotope assay to measure cholesterol absorption in these

same mice, we were able to show in three separate experiments that SR-BIInt-Tg

mice do not have accelerated intestinal cholesterol absorption rates (Figure 3, 4,

6). In agreement with this, mass fecal loss of cholesterol is normal in SR-BIInt-Tg

mice (Figure 3, 4, 6) . Importantly, both cholesterol absorption and fecal neutral

sterol loss were normal in SR-BIInt-Tg mice fed different amounts of dietary

cholesterol as well as in mice treated with the intestinal cholesterol absorption

inhibitor ezetimibe (Figure 4). Collectively, in agreement with studies in SR-BI

knockout mice (68, 70), our results clearly demonstrate that intestinal SR-BI is

not rate limiting for intestinal cholesterol absorption.

Alternatively, we hypothesized that intestinal SR-BI may play a key role in

removing plasma cholesterol for delivery to the TICE pathway of RCT. To more

definitely address a potential role for intestinal SR-BI in promoting TICE, we

crossed SR-BIInt-Tg mice to mice genetically lacking the biliary pathway for RCT

(NPC1L1LiverTg ). However, we found no role for intestinal SR-BI in promoting

TICE. This is in agreement with previous reports using an intestinal perfusion

32

system that demonstrated that small intestine segments from SR-BI deficient

mice exhibited functional TICE (77). This indicates that other basolateral

cholesterol transporters must be involved in clearance of plasma cholesterol for

the TICE pathway. The intestinal low density lipoprotein receptor (Ldlr) is an

obvious candidate, given that the small intestine has the second most Ldlr-

mediated lipoprotein clearance behind the liver (78). However, we have

previously shown functional TICE in Ldlr-deficient mice (18). Furthermore, it has

recently been shown that intestinal Ldlr is proteolytically degraded in mice treated

with a liver X receptor (LXR) agonist (79), which is a condition that substantially

augments TICE (37). Collectively, these studies do not support a rate-limiting role

for the Ldlr in the small intestine for mediating TICE. Given that the canonical

HDL and LDL receptors (SR-BI and Ldlr) are not likely involved in TICE, it will be

critical to determine the role of other Ldlr related proteins as well as novel

candidate receptors in the TICE pathway.

Although it seems quite clear that intestinal SR-BI is not rate limiting for

cholesterol absorption (68-70) or TICE (Figure 5,6) , it now becomes important to

gain better understanding into what role SR-BI does play in the small intestine.

There is strong support for SR-BI in the physical binding of cholesterol isolated

brush border membrane vesicles (BBMV) (65-67,). However, binding of

cholesterol to BBMV is not predictive of intestinal cholesterol absorption rates in

mice (80). These studies strongly suggest that ex vivo binding of cholesterol to

BBMV does not accurately predict the true rate of intestinal cholesterol

33

absorption, and results using the BBMV system need to be validated using

standard in vivo assays for cholesterol absorption such as the dual fecal isotope

assay or lymph duct cannulations. An alternative role for SR-BI in the small

intestine was recently postulated by Beaslas and colleagues (81). In this study it

was demonstrated that SR-BI can activate intracellular signaling pathways in

response to extracellular lipid stimuli (81). These results are not inconsistent with

a similar signaling role for SR-BI in eNOS activation in endothelial cells (82).

Further studies are needed to definitively address a primary signaling role for SR-

BI in the enterocyte in vivo. In summary, intestinal SR-B1 is not rate-limiting for

cholesterol absorption or fecal neutral sterol loss through the TICE pathway in

mice. Therefore, additional studies are required to identify intestinal lipoprotein

receptors involved the delivery of plasma cholesterol to the intestine for TICE

pathway, as well as further defining the true function of SR-BI in the enterocyte.

34

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FOOTNOTES

Acknowledgments -This work was supported by the National Heart, Lung, and

Blood Institute through a pathway to independence grant (1K99/R00- HL096166

to J.M.B.) and a program project grant (5P01HL049373 to L.L.R.).

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Kanwardeep Singh Bura

1606 Northwest Blvd. Apt # F, kbura@wakehealth.edu Winston-Salem, NC (336) 829 8039

Education

• Wake Forest University, Winston-Salem GPA: 3.38/4.0 MS in Molecular Pathology Dec, 2011

• Illinois Institute of Technology, Chicago, IL GPA: 3.5/4.0 MS in Biotechnology July, 2009

• Kurukshetra University, Kurukshetra, India 1St class (honors) B.Tech. in Biotechnology June, 2006

Industrial Experience

Orchid Biomedical Systems, Goa, India July 2006 - May 2007Trainee (Internship) Designed rapid Kit for pregnancy on the principle of Lateral Flow

• Learned the technique of binding antibodies to the gold particles of 10 nm. Manufactured dipstick, lateral flow and flow through rapid diagnostic test devices and an ELISA kit for hCG detection with complete sensitivity and stability studies.

Haryana Agriculture University , Hissar, India June 2005 – Nov 2005 Trainee (Internship)

• Plant tissue culture techniques • Worked on the micropropagation of the Bio-diesel producing plant Zetropa

Projects Undertaken

Illinois Institute of Technology, Chicago, USA August 2008-July 2009 Research Assistant Production of hybrid Spectrin Type Repeats(STR) by exon skipping in human Dystrophin gene. Insertion of the Dystrophin gene( ligated with tags on both ends) into pET vector system, then the expression of the gene in DH5α cell lines, purifying the protein and studying the properties of the particular STR.

Kurukshetra University Jan. 2006-May 2006 Research Assistant

Designing Lab Scale Fermentor Designing and construction of one liter glass jacket lab scale fermentor. During which instead of using mechanical seal and electric motor, we used a pair of magnets, on which the application of a strong magnetic field used to revolve the impellors.

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Relevant coursework:- Molecular Biology, Biochemistry, microbiology Immunology, Diagnostics, Industrial Microbiology, Biostatistics, Intellectual Property Rights, Animal and Plant Biotechnology, Bioinformatics.

Technical comptency

• Molecular biology Techniques – PCR, Southern Blots, Gene cloning, plasmid extraction and Western Blotting. Lateral Flow Device, Dialysis, FASTA, BLAST, NEBCUTTER, Swiss Pbd.

• Analytical Techniques – FPLC, Gel electrophoresis, basic microbiological techniques, Affinity chromatography, size exclusion chromatography, Gel chromatography)

• Software Skills:- Programming Language C, Operating Systems :- Windows Vista/XP/98, MS Office

Achievements:- • Won poster presentation competition at South East Lipid Research Conference, Pine

Mountain, GA,Oct 7-10, 2010. • Presented paper on Environmental Degradation in the National Seminar held at

Kurukshetra University, Kurukshetra. • Best poster award in National Symposium “Indian Biotechnology scenario: Challenges

and opportunities” at Kurukshetra University, Kurukshetra.