intestinal sr-bi. by kanwardeep singh bura a thesis ...kanwardeep singh bura a thesis submitted to...
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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.
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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.
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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
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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
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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
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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).
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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
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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.
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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.).
49
Kanwardeep Singh Bura
1606 Northwest Blvd. Apt # F, [email protected] 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.
50
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.