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i ' LIBRARY
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THE EFFECT OF SATURATED AND UNSATURATED FATTY
ACIDS ON HEPGZ CELLS AND
THE TREHALOSE PROTECTION OF HEGZ CELLS ON
PALMITATE INDUCED TOXICITY

presented by
YIFEI WU

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5108 K IPro;/Acc&Pres/ClRC/DateDue Indd

THE EFFECT OF SATURATED AND UNSATURATED FATTY ACIDS ON HEPGZ
CELLS AND THE TREHALOSE PROTECTION OF HEPGZ CELLS ON
PALMITATE INDUCED TOXICITY
By

Yifei Wu

A THESIS

Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of

MASTER OF SCIENCE

Chemical Engineering and Material Science

2008

ABSTRACT

THE EFFECT OF SATURATED AND UNSATURATED FATTY ACIDS ON HEPG2
CELLS AND THE TREHALOSE PROTECTION OF HEPGZ CELLS ON
PALMITATE INDUCED TOXICITY

By

Yifei Wu

Understanding the mechanism of saturated fatty acid-induced hepatocyte toxicity may
provide insight into cures for diseases such as obesity-associated cirrhosis. Trehalose,
a nonreducing disaccharide Shown to protect proteins and cellular membranes from
inactivation or denaturation caused by different stress conditions, also protects
hepatocytes from palmitate-induced toxicity. Our results suggest that trehalose serves
as a free radical scavenger and alleviates damage from hydrogen peroxide secreted by
the compromised cells. We also observe that trehalose protects HepGZ cells by
interacting with the plasma membrane to counteract the changes in membrane fluidity
induced by palmitate. Unsaturated fatty acids such as oleate and Iinoleate are not toxic
to HepGZ cells and do not induce significant biophysical changes to cell membrane.
The experimental results are supported by molecular dynamics simulations Of model

cell membranes that closely reflect the experimental conditions.

Acknowledgment

My utmost gratitude goes to my advisor, Dr. Christina Chan, for her expertise,
dedication, kindness, and most of all, for her patience. This thesis would not be
possible without her guidance and support. I would also like to thank our
collaborators Dr. Amadeu K. Sum and Sukit Leekumjom from Virginia Polytechnic
Institute and State University for their dedicated work and substantial contribution in
making the publication possible. My thanks and appreciation goes to Dr. Patrick
Walton and all members of Cellular & Biomolecular Lab, for their important inputs
and helpful comments in this project. I am grateful to Dr. Neil Wright in the
Department of Mechanical Engineering and Dr. John L. McCracken in the

Department of Chemistry, for gracefully providing the DSC and EPR measurements.

iii

TABLE OF CONTENTS

List Of Figures .................................................................................. V
Chapter 1 Saturated fatty acid toxicity on HepGZ cells and the trehalose protection of
toxicity .......................................................................................... 1
Introduction ................................................................................ 1
Materials and methods ..................................................................... 4
Cell culture ............................................................................ 4
Cytotoxicity assay ..................................................................... 5
Membrane fluidity .................................................................... 5
Results ....................................................................................... 7
Discussion .................................................................................. 12
Chapter 2 Unsaturated fatty acid effect on HepG2 Cells ................................. 13
Introduction ................................................................................. 13
Materials and methods .................................................................... 16
Results ....................................................................................... I7
Discussion .................................................................................. 22
Chapter 3 Molecular dynamics Simulation ................................................. 24
Discussion .................................................................................. 30
Conclusion and future work .............................................................. 31
References ....................................................................................... 34

iv

List Of Figures

FIGURE 1 Effect of trehalose on the HepG2 cells cytotoxicity in response to

palmitate ........................................................................................ 8
FIGURE 2 Effects Oftrehalose and palmitate on H202 release ....................... 9
FIGURE 3 Effect of palmitate exposure on cellular membrane fluidity ............ l I
FIGURE 4 HepG2 cells cytotoxicity in response to FF As .............................. 18
FIGURE 5 Effects of FFAS on H202 release ............................................. 19
FIGURE 6 Effect of FFAS exposure on cellular membrane fluidity .................. 20

FIGURE 7 Effect of F FAS on phase transition temperature of DOPC Iiposome ...... 21
FIGURE 8 Starting structure of POPC/POPE bilayer with one palmitate inserted into

the aqueous phase ............................................................................... 22
FIGURE 9 Snapshot of palmitate aggregation in the aqueous phase .................. 27
FIGURE 10 Snapshots of bilayer structures with and without palmitate ............. 28

FIGURE 1 1 Average number of hydrogen bonds per lipid for bilayers with FFAS... 29

Chapter 1

Saturated fatty acid toxicity on HepGZ cells and the trehalose protection of toxicity

Introduction

Non-esterified long-chain free fatty acids (FFAS) are major sources of cellular
energy (1) and essential components in triglycerides, cholesteryl esters,
prostaglandins, and phospholipid syntheses (2, 3). There have been numerous
reports on the toxic effects of fatty acids on model cells in vitro. Andrade et al.
showed that both saturated and unsaturated fatty acids exert toxic effects on
melanoma cells through the loss of membrane integrity or DNA fragmentation (4).
Lima et a1. evaluated the toxicity of various fatty acids on J urkat (T-lymphocytes)
and Raji (B-lymphocytes) cells (5) and found a positive correlation between the
toxicity and the chain length and number of double bonds in the fatty acids. Their
experiments identified palmitate among the most toxic of the fatty acids. FFAS,
especially saturated fatty acids, can cause cell death in many types Of cells,

including pancreatic ,B-cell (6, 7), cardiomyocytes (8, 9), and hepatocytes (10-12).

Most of the research up until now on the mechanism of cell death focused on the
production of potential or toxic intermediates, such as stearoyl-COA desaturase 1
(13-15), acids from omega oxidation (16,17 ), reactive oxygen Species (ROS),
ceramide (18,19), reduced mitochondrial potential (8), and reduction of
mitochondrial Bcl-2/Bax ratio (11). Recent studies in our lab suggest that palmitate

can cause Iipotoxicity in liver cells through increased production Of hydrogen

peroxide (H202) and hydroxyl (*OH) radicals (12). Measurements indicated that the
cytotoxicity was not completely prevented upon treatment with mitochondrial
complex inhibitors or free radical scavengers. This suggests that mechanisms other
than ROS production in the mitochondria may be contributing to the toxicity of
palmitate. Therefore, we investigated the possibility that palmitate-induced toxicity
may be due to hydrophobic effects on the cellular membrane. Fatty acids are known
to have toxic and fusogenic effects on cells (20, 21). The mechanism by which fatty
acids exert cytotoxicity has been identified as the detergent-effect (22). According to
this hypothesis, ionized fatty acid micelles solubilize membrane lipids or proteins

and disrupt the physical and functional integrity of cell membranes (20, 21).

Identifying chemical agents to prevent or reverse the effect Of fatty acid induced
cellular toxicity has been a major focus of research in the past decades. Studies have
suggested that saturated and unsaturated fatty acids have different effects on toxicity.
For example, there has been evidence indicating that dietary oleic acid can protect
endothelial cells against hydrogen peroxide-induced oxidative stress and reduce the
susceptibility of LDLs to oxidative modifications (23-25). Similarly, we found that
oleic acid does not induce the same level of cytotoxicity as palmitic acid in HepG2
cells at similar concentrations (12) and the addition of oleic acid reduces the
cytotoxicity induced by palmitate (26). In another related study, Kinter et al.
investigated the protective role of unsaturated FFA in oxygen-induced toxicity of
hamster fibroblasts and found that monounsaturated F FA increased cell survival as

compared to saturated and polyunsaturated F PAS (27). Furthermore, it has been

shown that unsaturated FFAS rescued palmitate-induced apoptosis by converting
palmitate into triglycerides (13). Recently, Natali et a1. investigated the effects Of
various types of FFAS in glial cells and found that oleic acid was apotent inhibitor
of fatty acid and cholesterol synthesis (28). In recent years, it has been established
experimentally that trehalose has a stabilizing effect on biological membranes (29)
by protecting cells from dehydration, heat, and cold (30-32). Moreover, evidence is
mounting suggesting that trehalose acts as an antioxidant, possibly serves as a free
radical scavenger (33-35), and inhibits the peroxidation of unsaturated fatty acids by
heat or oxygen radicals (35,36). In addition, trehalose has been found to protect
yeast cells and cellular proteins from damage by oxygen radicals during oxidative
stress (37). In light of the role trehalose plays in the stabilization of cells, we
investigated whether trehalose is protective in the presence of palmitate, and if so,
we aimed at understanding how trehalose protects against palmitate-induced

toxicity.

Therefore, to gain insight into how trehalose interacts with liver cells (human
hepatocellular carcinoma cell line, HepG2 cells) in the presence of palmitate, we
performed a series of experimental and computational measurements. The
experimental measurements focused on determining the influence of palmitate and
trehalose on the fate of HepG2 cells, and the computational part aimed at interpreting
and understanding the experimental results, shedding light into the role of palmitate
and trehalose in the toxicity of HepG2 cells. Insight into these mechanisms will add to

our understanding Of processes (i.e., metabolic, signaling, and biophysical) that are

altered by palmitate. We found that palmitate decreased the cellular membrane
fluidity of HepG2 cells. The addition of trehalose to palmitate cultures prevented this
lowering in membrane fluidity. Thus, we found that trehalose protects also against
palmitate-induced toxicity in liver cells. This study represents the first attempt to
obtain a comprehensive understanding of the biochemical and biophysical processes

leading to and resulting from the toxicity of palmitate on cells.

Materials and methods

Cell culture

Human hepatocellular carcinoma cell line, HepGZ (American Type Culture
Collection, Manassas, VA), was cultured in Dulbecco's modified Eagle's medium
(DMEM, lnvitrogen, Carlsbad, CA) containing 10% fetal bovine serum (American
Type Culture Collection) and 2% penicillin-streptomycin (Invitrogen). They were
seeded in six-well plates and incubated at 37°C in humidified atmosphere containing
10% C02. After cells reached confluence, the media were replaced with 2 ml control
medium (4% fatty-acid free bovine serum albumin, or BSA) or fatty acid (0.7 mM
with 4% BSA) and changed every 24 h. The BSA level used was close to
physiological conditions (38). A quantity of 0.7 mM F FAS was employed in this study
because the plasma FFA levels in the obese and type 2 diabetic patients have been
reported to be approximately this level (39-42). Experiments were conducted after 48
h of treatment. To determine the Optimal amount of trehalose to add, we performed a

dose-response in HepGZ cells with trehalose ranging from O to 0.2 mM.

Cytotoxicity assay

Cell viability was assessed by lactate dehydrogenase (LDH) leakage through the
membrane into the medium. The cell culture supernatant from control and
palmitate-treated cultures were tested after 48 h of incubation for the presence of LDH
[LDH(medium)] usingan LDH assay kit (Roche Applied Science, Indianapolis, IN).
Cells were washed with phosphate-buffered saline (PBS) and lysed with 1% triton-X
100 for 12 h at 37°C. The cell Iysate was then centrifuged at 5000 g for 10 min and
tested for LDH activity [LDH(trtoin)]. The LDH released was normalized to the total

LDH, given by

LDH (medium)

%LDH = x
LDH (medium) + LDH(trit0n)

 

Membrane fluidity

Two different stearic-acid derivatives were used to detect changes in the membrane
fluidity, S-n-doxylstearic acid (S-n-SASL) and l6-n-doxylstearic acid (I6-n-SASL)
(lnvitrogen, Carlsbad, CA). The S-n-SASL probe monitors the portion of the
membrane closest to the lipid headgroups, while the 16-n-SASL reflects changes in

the middle/end of the lipid hydrocarbon chains (43).

A stock solution of the spin labeled stearic acids at 10’3 M was prepared in
dimethyl-sulfoxide and the aliquots stored at —20°C. Immediately before use, the

stock solution was thawed and diluted 50 times with PBS. Preliminary experiments

were conducted to confirm that the spin-label solution did not affect the cell viability.
Cell suspensions collected after Trypsin-EDTA (GIBCO, Billings, MT) exposure
were centrifuged and the pellets were resuspended in spin label solution and kept on
ice. The labeled cell suspensions were then transferred to a flat cell and placed in the
cavity of the electron paramagnetic resonance (EPR) spectrometer (model NO.
ESP-300E X-band; Bruker AXS, Madison, WI). The microwave power was set at
15.8 mW, the modulation frequency at 100 kHz, and the modulation amplitude at
2.53 G. For indexes of membrane fluidity, we evaluated the values of the outer and
inner hyperfine splitting (2 T .L and 2T” in Gauss, respectively) in the EPR spectra for

5-n-SASL. The order parameter was calculated from 2T .L and 27‘" by

§_ TH—(Ti +C)

__ X 1.66
'l‘ll + (2'1" + C)

 

where C = 1.4 — 0.053(T” — T .L)- In the EPR spectra for l6-n-SASL, we used the
peak height ratio (ho/h — I) for an index of the membrane fluidity (44,45), where ho
and h — 1 are the heights of the central and high-field peaks, respectively. The
greater the values of the order parameter and peak height ratio, the lower the
freedom of motion of the spin labels in the membrane bilayers, indicating lower

membrane fluidity (46).

Results

Palmitate cytotoxicity and trehalose protective role

We previously found that palmitate-induced toxicity led to ROS production in
HepG2 cells (12), but its effect was not prevented upon treating with ROS
scavengers. During oxidative stress, yeast cells produce trehalose to protect
themselves from damage by oxygen radicals (37). Therefore, we evaluated the effect
of trehalose on palmitate-induced toxicity in HepG2 cells. The level of cytotoxicity
was measured by the relative amount ofLDI-I released in the medium. The control
consisted of HepG2 cells exposed to DMEM with 4% BSA. From Fig 1, our
measurements indicate that palmitate significantly increased the amount of LDH
released, relative to the control. As the concentration of trehalose increased, the
LDH released reduced significantly. This protective effect reached a maximum at a
trehalose concentration of 0.13 mM, whereupon further increase in the trehalose
concentration was detrimental to the l-IepGZ cells. Although the mechanism of
trehalose-induced toxicity is not a focus Of this study, we infer from previous studies,
including our own, that trehalose preferentially binds to the membrane and possibly
causes surface modifications that may affect cell activity (47-52). As it will be
demonstrated fiom our computational studies, we found that trehalose can induce
local hydrophobic/hydrophilic domains along the bilayer interface, a membrane
reorganization process which may have potentially detrimental effects. Based on
these findings, atrehalose concentration of 0.13 mM was optimum for alleviating

the palmitate-induced toxicity in HepGZ cells and it was used in all subsequent

experiments.

75( ‘ - --—-— — - » —- - — —.__- -__._______ __ _-

41.1 #

% LDH

 

 

 

 

 

 

 

 

0.00 0.10 0.13 0.16 0.20
Trehalose concentrations (le0

FIGURE 1 Effect of trehalose on the HepGZ cells cytotoxicity in response to palmitate.
Confluent HepG2 cells in bovine serum albumin (BSA) medium were exposed to 0.7 mM
palmitate in the presence of different concentrations of trehalose. The LDH released was
measured after 48 h. Open and shaded bars represented the effect of trehalose alone or the
mixtures of trehalose/palmitate, respectively. Note that the first shaded bar shows the effect
of palmitate alone. Error bars are standard deviation of three independent experiments. The
symbols * and # indicate statistical difference from control and palmitate, respectively (p <
0.05).

Trehalose on H202 release

We previously identified H202 as one of the ROS species involved in the
palmitate-induced toxicity of hepatoma cells. To determine whether trehalose
protected HepG2 cells by scavenging H202, the measured H202 released into the
medium was normalized to total cellular protein. As shown in Fig 2, 48 h of
palmitate exposure enhanced H202 release into the medium, while trehalose
Significantly reduced the H202 release in the presence of palmitate. The results

suggest that trehalose protects the cells in part by reducing H202 release.

 

* ,#
24.8

10
0.77 0.52

o——-+ L

BSA BSA+Treh BSA+ Palm BSA+Treh+Pa|m

H202 Released (nmol/mg protein)

   

 

 

 

 

FIGURE 2 Effects of trehalose and palmitate on H202 release. Confluent HepGZ cells in
BSA medium were treated with 0.7 mM palmitate (Palm) with or without 0.13 mM
trehalose (Treh) for 48 h. The H202 released into the medium was measured and normalized
to total cellular protein. Error bars are standard deviation of three independent experiments.
The symbols * and # indicate statistical difference from control and palmitate, respectively
(p < 0.05).

Membrane fluidity for HepG2 cells

Since palmitate is hydrophobic, there may be nonspecific cytotoxic effects due to its
hydrophobicity. It has been established experimentally that trehalose has a
stabilizing effect on biological membranes (53), therefore we investigated the
changes in cellular membrane structure upon exposure to palmitate in the presence
and absence of trehalose. The cellular membrane fluidity of HepG2 cells in the
presence of palmitate or trehalose was measured by EPR. The EPR spectra of the
spin-label agents were used to detect changes in the freedom of motion of the lipids
in the cell membrane, thus providing a measure of the membrane fluidity. The

membrane fluidity of HepG2 cells were measured after 48 h of exposure to palmitate,

trehalose, and combinations thereof. The control was HepG2 cells exposed to
DMEM with 4% BSA. Using l6-n-SASL as a probe to monitor the ordering of the
lipid tails near the center of the bilayer core, we observed a greater peak height ratio
for the palmitate-treated cells as compared to the control, which correlated with
reduced freedom of motion of the spin labels in the membrane. This indicates a
decrease in membrane fluidity of the cells treated with palmitate for 48 h, as shown
in Fig. 3a. The exposure of HepG2 cells to trehalose had an insignificant effect on
the bilayer core region since trehalose is excluded from the bilayer. The interaction
of trehalose with the membrane is only at the surface of the bilayer. Treating HepG2
cells with trehalose and palmitate increased the peak height ratio. This suggests that
a complex interaction exists between the cellular membrane, palmitate, and
trehalose. Similarly, using 5-n-SASL as a probe to monitor the lipid carbons nearthe
lipid headgroups, we Observed that the presence of trehalose in the palmitate-treated

cells increased the fluidity near the surface of the membrane (see Fig. 3b).

10

 

 

 

 

 

 

 

 

 

 

 

 

a)
4.5 ~ *
4.29
*3;
,1." 4.11
E42-
6
5 ,_
.2 379 3.84
E 3.9 — °
.c a
.9 .. l
(D , r.
.c 1'
.é .
a) 3.6 ~ i l E
0.
li '.
3'3 ” '- I - ; f I
BSA BSA+Treh BSA+Palm BSA+Treh+Palmy
b)
0.72 —
*
0.702
_ 0.70
e
0
E
e
8 0.68
5
E
O
(I)
0.66
0.64
BSA BSA+Treh BSA+PaIm BSA+Treh+Palm

 

FIGURE 3 Effect of palmitate exposure on cellular membrane fluidity. Cells were treated
with 0.7 mM palmitate in the presence or absence of 0.13 mM trehalose for 48 h. The
cellular membrane fluidity was measured using EPR. (a) Values are peak height ratio for
16-n-SASL-labeled HepGZ cells. (b) Values are order parameter for 5-n-SASL-labeled
HepGZ cells. Error bars are standard deviation of four independent experiments. The
symbols * and # indicate statistical difference from control and palmitate, respectively (p <
0.05).

11

Discussion:

FFAS are known to play important roles in the development of many hepatic
disorders. A number of studies have shown that elevated levels of fatty acids are
important mediators of Iipotoxicity and can impair cellular functions and/or cause
cell death (54). Others have found that the negative effect of FFA-induced toxicity
may be reduced or alleviated by the addition of unsaturated fatty acids, antioxidants,
or, as of more recently, disaccharides. To evaluate whether palmitate-induced
toxicity can be reduced by adding trehalose, we exposed HepGZ cells to palmitate
alone or a combination of trehalose and palmitate. As demonstrated from our
experiment (Fig 1 and 2), cells exposed to palmitate resulted in a significant amount
of LDH and H202 released into the medium, indicating cell death or compromised
cellular membrane. With increasing trehalose concentration, reduced amount of
LDH released is observed in the presence of palmitate, up to ~13 mM, an optimal
concentration for HepGZ cells. Furthermore, a significant reduction in H202 released
is observed for these cells. To provide insight into the biochemical and biophysical
processes altered by the presence of palmitate in HepG2 cells and to interpret these
results from a molecular level, we collaborated with Dr. Amadeu K. Sum and Sukit
Leekumjom from Virginia Polytechnic Institute and State University, and they
studied the effect of palmitate and trehalose on model cell membranes (lipid bilayers)
using molecular dynamic simulations. The simulations results reveal the early stages
of how palmitate induces biophysical changes to the cellular membrane and the role

of trehalose in protecting the membrane structure. (Chapter 3)

12

Chapter 2

Unsaturated Fatty Acid Effect on HepG2 cells

Introduction
Unbound free fatty acids (FFAS), derived from dietary triglycerides (TGs) and
phospholipids, are aliphatic monocarboxylic acids and among important energy
sources for cells and tissues (55, 56). Typically containing a lipid chain of 4 to 28
carbons, they are classified according to the degree of unsaturation: saturated,
monounsaturated, and polyunsaturated (57-60). The optimal FF A concentrations in
the plasma stream are regulated by plasma protein albumin that leave about less than
0.01% unbounded (61, 62). Recently, in vitro studies confirm that saturated FFAS
induced significant toxic effects on various cells types, however, unsaturated F FAs
have been shown to induce less toxic effects or reduce and prevent the toxic effects by
saturated FFAS (63-66).
It was suggested that saturated F F As-induced toxicity may be due to hydrophobic
effects on the cellular membrane rather than ROS, mainly because the cytotoxicity
was not completely prevented upon treatment with mitochondrial complex inhibitors
or free radical scavengers (66). Currently, there is very limited knowledge on how
unsaturated F FAS play a role in preventing or reducing the toxicity.
There have been numerous studies that examined the effects of unsaturated FAS on
lipid bilayers or liposomes at various conditions. For example, Sunamoto et a1.

investigated the autoxidation of phosphatidylcholine (PC) Iiposome containing

13

arachidonic and linoleic acids (67). Using 1,1- diphenyl-Z-picrylhydrazyl as radical,
they found that the oxidation rate of unsaturated FAS or lipids became faster in
liposomes compared to the reaction in solution. Their results suggested that the
reactions are preferential within the bilayer core regions. In another related

studies, Lee et al. exposed liposomes containing different amounts of oleic, linoleic,
and arachidonic acid to oxidizing medium and found that liposomes containing
linoleic and arachidonic acid were less susceptible to oxidation than oleic acid (68).
After exposing the fragments of lipid peroxidation to endothelial cells and found that
the amount of monocyte chemotaxis and monocyte adhesion were significantly
increased, they concluded that oxidation products of linoleic and arachidonic acid can
triggered cellular immune response. Furthermore, Samuni et al. investigated the
oxidative damage of egg phosphatidylcholine (EPC) Iiposome containing arachidonic
acid (C20z4), cis- 7,10,13,16,19-docosapentaenoic acid (C225), and
ciS-4,7,10,13,16,19-docosahexaenoic acid (C22z6) in the presences Of vitamin E,
antioxidant (Tempo), and cholesterol (69). Their results showed that all
polyunsaturated FFAS are highly sensitive to oxidation and hydrolytic degradation.
Based on the residual fragment of FFAS collected overtime, cholesterol demonstrated
some protective effect and Tempo were better antioxidant than vitamin E.
Furthermore, Hyv"onen et al. investigated the membrane properties at the final stage
of the phospholipases A2 enzyme process where phospholipids within the bilayer are
hydrolyzed to fatty acids (70). Using PLPC, lyso-PC molecules (a PC headgroup,

glycerol backbone, and palmitic acid chain), and Iinoleate/linoleic acid in their

14

models, they found that the bilayers become unstable, as a result of water penetrating
into the bilayer core region. Recently, Watabe et a1. examined the decomposition rate
of unsaturated FAS in DPPC Iiposome containing photoporphyrin IX (PpIX) from
light irradiations and determined the oxidation rate from fast to slow: arachidonic acid
< oleic acid < linoleic acid (71). Although the oleic acid contains less number of
double-bond than linoleic derivatives, they have a greater oxidation rate because the
locations of double-bonds are in close range of PpIX molecules embedded within the
bilayer. In summary, experimental studies mentioned here are based on the resulting
products of lipid oxidation, however, none have addressed the interactions between
lipid constituent and FA at the initial stage of this process.

In Chapl, We found that trehalose, a non-reducing disaccharide widely used as

a stabilizer and preservant, has a protective role in palmitate-induced toxicity. Unlike
trehalose, which is impermeable to membranes (72), unsaturated FFAS can be
transported through the membrane into cells through passive and active transport.
Once inside the cells, FA can modify the membrane lipid compositions by altering the
membrane fluidity and in turn affect cellular function (73,74). Based on this fact that
saturated F A decreased membrane fluidity in Chapl, we speculated that the presence
of unsaturated FA may help maintain or restore membrane fluidity to its normal state.
TO compare the effect of saturated and unsaturated FAS on cell membranes, we
conducted the cytotoxicity experiments using HepGZ cells exposure to palmitate,
oleate, and Iinoleate. Computational investigations through MD simulations were used

to confirm the experimental results and determine the role of palmitate, oleate, and

15

Iinoleate on model DOPE bilayers. To relate experimental and computational results,
the phase transition study of DOPC Iiposome containing various concentrations Of
saturated and unsaturated FAS was used. We try to interpret and understand the
interactions of F As embedded inside the lipid bilayers and identify the role

of unsaturated FAS in preventing the changes in membrane fluidity. Insight into these
mechanism will add to our understanding of processes (i.e., metabolic, signaling, and

biophysical) that are induced by FFAS.

Experimental Materials and Methods

Cell culture

Human hepatocellular carcinoma cell line HepGZ was cultured in DMEM containing
10% fetal bovine serum and 2% penicillin-streptomycin. They were seeded in six-well
plates and incubated at 37 °C in humidified atmosphere containing 10% C02. Afier
cells reached confluence, the media were replaced with 2 ml control medium (4%
fatty-acid free bovine serum albumin, or BSA) or fatty acids (0.7 mM palmitate or
oleate or Iinoleate with 4% BSA) and changed every 24 h. Experiments were
conducted after 48 h of treatment.

Cytotoxicity assay

Same as in Chap 1.

Membrane fluidity

Same as in Chap 1.

Liposome preparation and DSC measurement

l6

To correlate the fluidity measurements to our computational studies, a Simpler
model cell membrane also was used. Liposomes (multilamellar vesicles) were
prepared by the thin film method according to the protocol fi'om Avanti Polar Lipids.
The lipid 1,2-dioleoyl-sn-glycerO-3-phosphocholine (DOPC, Avanti Polar Lipids,
Alabaster, AL) and fatty acid (palmitic acid, oleric acid or linolenic acid ,
Sigma-Aldrich, St. Louis, MO), obtained in chloroform stock solutions, were mixed
in appropriate amounts in a glass tube. After vortexing, the solvent was dried under
nitrogen. This formed a thin lipid film on the inside wall of the glass tube. The film
was dried in a freeze-dryer to ensure complete evaporation of chloroform. Deionized
water was added into the tube before it was placed in a bath sonicator for 10 min.
Differential scanning calorimetry (DSC) analysis were performed on the liposomes
samples at a scan rate of 1°C/min. Samples containing 20 mg/ml of lipid and 10 ,uL
of liposome suspensions were used. DOPC was used in these experiments because
its phase transition temperature (~—19°C) allowed us to obtain clean and clear DSC
scans, whereas POPC (one of the lipids used in the simulations studies) has a phase
transition temperature near the normal melting point of water (—2°C for POPC),

which causes severe interference in Obtaining reliable data.

Results

The experimental results are divided into four sections: cytotoxicity, peroxide (H202),
membrane fluidity, DSC measurements. Cytotoxicity and peroxide measurements are
used to determine HepG2 cells viability after exposure to palmitate, oleate, or

Iinoleate. Membrane fluidity is measured by EPR using stearic acid probes, 5-n-SASL

l7

and 16-n-SASL embedded inside HepG2 cells. Lastly, phase transition study of
DOPC Iiposome containing various concentrations of palmitate, oleate, and Iinoleate
are measured by DSC.

Cytotoxicity Measurements

The level of cytotoxicity was measured by the relative amount of LDH released in the
medium after exposure Of HepG2 cells with palmitate, oleate, or Iinoleate. The control
consisted of HepGZ cells exposed to DMEM with 4% BSA alone. From Fig. 4, our
measurements indicate that palmitate significantly increased the amount of LDH
released, relative to the control. Oleate and Iinoleate did not induce toxic effect on

HepG2 cells.

 

I

60

50.2

 

 

 

 

 

BSA BSA+Palm BSA+OIea BSA+Lino

FIGURE 4. HepG2 cells cytotoxicity in response to FFAS. Confluent HepGZ cells in Bovine
serum albumin (BSA) medium were exposed to 0.7 mM palmitate, oleate, or Iinoleate. The
LDH released was measured after 48 hrs. Error bars are standard deviation of three
independent experiments.

Peroxide Measurements

18

Relative to the amount of LDH released, we found a direct correlation between the
amounts of H202 release into the medium. The amount H202 release after 48 hrs
exposure of HepG2 to palmitate, oleate, or Iinoleate are shown in Fig. 5. The results

suggested palmitate induced cell death by triggering cellular immune response.

 

 

 

 

 

 

 

60
*
45.3
.E 50 ”
0:
‘5?
o. 40 —
o:
E
S
E 30 ‘
E,
r zo—
m
2
c
a: 10 - 0.77 2.24 1,41
0:
I 0 I
BSA BSA+Palm BSA+OIea BSA+Lino

 

FIGURE 5. Effects of FFAS on H202 release. Confluent HepG2 cells in Bovine serum
albumin (BSA) medium were treated with 0.7 mM palmitate (Palm), oleate (Olea), or
Iinoleate (Lino) for 48 hrs. The H202 released into the medium was measured and
normalized to total cellular protein. Error bars are standard deviation of three independent
experiments. The symbols * indicates statistical difference from control (p < 0.01).

Membrane Fluidity Measurements

Since typical FFAS are hydrophobic in nature, we investigated the non-specific
cytotoxic effects due to their hydrophobicity. For this study, we investigated the
changes in cellular membrane structure upon exposure to saturated and unsaturated
FAS using EPR. The membrane fluidity of HepGZ cells were measured afier 48 hrs of

exposure to palmitate, oleate, or Iinoleate. The control was HepG2 cells exposed to

19

DMEM with 4% BSA. Using both 5-n-SASL and 16-n-SASL as probes to monitor
the ordering of the lipid tails near the lipid headgroups and the center of the bilayer
core, we observed an increase in the S order parameter and peak height ratio for
HepG2 cells exposed to palmitate, as shown in Fig. 6. No significant changes are
observed for HepG2 cells exposed to either oleate or Iinoleate, in comparison to

the control. The results suggested a grater reduction of membrane fluidity due to the

hydrophobic effect of saturated FA than unsaturated FAS.

 

      

 

 

0.72 - a)
*
0.702

5 0.70 "-
‘65
E
‘3
80%—
E
O
“9 0.66 —

0.64 —

BSA+Palm BSA+OIea BSA+Lino

 

20

 

P
01
I

4.29

:5
N
I

Peak height ratio (h0/h4)
co 00
CD (0
I I

 

.0)
CD
I

  

 

 

 

BSA BSA+Palm BSA+OIea BSA+Lin0

 

FIGURE 6. Effect of FFAS exposure on cellular membrane fluidity. Cells were treated with
0.7 mM palmitate (Palm), oleate (Olea), or Iinoleate (Lino) for 48 hrs. The cellular membrane
fluidity was measured using EPR. a) Values are order parameter for 5-n-SASL labeled HepG2
cells. b) Values are peak height ratio for 16-n-SASL labeled HepG2 cells. Error bars are
standard deviation of three independent experiments. The symbols "' indicates statistical
difference from control (p < 0.05)

DSC Measurements

To corroborate the membrane fluidity results, we measured the phase transition
temperature of DOPC Iiposomes by DSC measurements. The DSC therrnographs for
DOPC Iiposomes with varying mole fractions of palmitate, oleate, or Iinoleate are
shown in Fig.7. The figure demonstrated a significant increase in the phase transition
temperature of the DOPC Iiposomes with increasing concentration of palmitate.
However, slight changes are Observed for DOPC Iiposome containing the same
concentration of Oleate and Iinoleate. This suggests that only palmitate increase

the ordering of the phospholipids in the Iiposomes, which correlates with the decrease

in membrane fluidity measured by EPR.

21

-140 ~

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

A '15.0 7

9

E -16.0 4

a)

j.

c

,3 -170 -

0)

C

e

l- -

m -18.0

m

to

Z

“L -190 ~

-20.0
0% 1% 5% 10% 15% 20%

!_Palmtiate -_1_9,0 -18.7 ,, -18.1 -17.4 -16.7 '13.;‘L_.
gggggb -19.1 -19.1 _ -19.2 -19.3 M -19.5 Mfi-1M9.§__
ILinoleate -18.9 -190 -19.1 -19.1 -19.2 -19.3

 

 

 

 

 

 

 

 

Mole Fraction of F FAs in DOPC Iiposome
FIGURE 7 Effect of FFAs on phase transition temperature of DOPC Iiposome. Phase
transition temperature of DOPC Iiposomes containing various concentrations of palmitate,
oleate and linoleate(grey, white and black bar). The phase transition was measured with DSC.

Error bars are standard deviation of four independent experiments. The symbols “*” indicates
statistical difference from control (p < 0.01)

Discussion

Unlike most FFAS, palmitate has been shown to be very toxic to HepG2 cells at 0.7
mM. Unsaturated FFAS, on the other hand, have been shown to have both positive and
negative effects among various cell types. We found from the cytotoxicity and
peroxide measurements that Oleate and Iinoleate are not toxic to HepGZ cells at same
concentration considered for palmitate. Our EPR measurements indicated that there is
a significant change in membrane fluidity in the presence of palmitate, compared to
Oleate and Iinoleate systems. This change is mainly associated with hydrophobic

effect of saturated FA which resulted in reducing membrane fluidity. To compare the

22

biochemical and biophysical processes associate with the change in membrane

fluidity in the presence of palmitate, oleate, or Iinoleate at a molecular level, we
collaborate with Dr. Amadeu K. Sum and Sukit Leekumjom from Virginia
Polytechnic Institute and State University, and they studied the effect of these FFAS

on model cell membranes (DOPC lipid bilayers) using molecular dynamic simulations.

(Chapter 3)

23

Chapter 3

Molecular dynamics Simulation

(Dr. Amadeu K. Sum and Sukit Leekumjom from Virginia Polytechnic Institute and

State University performed all the simulations discussed in this chapter)

To provide insight into the biochemical and biophysical processes altered by the
presence of palmitate, Oleate, or Iinoleate in HepG2 cells and to interpret these results
from a molecular level, we collaborated with Dr. Amadeu K. Sum and Sukit
Leekumjom from Virginia Polytechnic Institute and State University, and they studied
the effect of FFAS and trehalose on model cell membranes (lipid bilayers) using
molecular dynamic simulations.

The lipid bilayers used here are equimolar
l -palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine and
l-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPC/POPE) bilayers with
a total of 288 lipid molecules evenly distributed in each leaflet. This mixed bilayer
was chosen for these studies because it represents the main phospholipid constituents
of HepG2 cells (75). All simulations were performed at 310 K, by Dr. Amadeu K.
Sum and Sukit Leekumjom with the GROMACS 3.3.1 software package in parallel
using Virginia Tech's System X (dual 2.3 GHz Apple Xserve GS). Images in this
chapter are presented in color.

The initial stage of cell exposure to palmitate is modeled by introducing a single
palmitate molecule in the aqueous phase of previously equilibrated bilayers with and
without trehalose. Snapshots of the two systems at the start of the simulation are
shown in Fig 8. F ig 8 b and d, show representative trajectories of palmitate along the
simulation (trajectory is traced by the position of the central carbon atom in the
palmitate tail). As shown in Fig 8, palmitate can penetrate the bilayer within the
simulation time considered. Eight of the ten simulations resulted with palmitate in the

bilayer and palmitate remained in the aqueous phase for the duration of the

24

simulations. For the simulations with trehalose, similar results were obtained. The
observed penetration of palmitate in the bilayer is consistent with experimental studies
that demonstrated that palmitate can be readily incorporate into the hydrophobic
region of the bilayer (76,77).

f'o’xl ‘4 fry}

5“ 7, Riv" Re

 

C"
a.

'3 i ' l 411'.
x h,

a, --

T' .
17} it.
t _/ '0' T,

. Isa '

.“ 1"“. '
\u- '
\_\

  

8-

   

I
N
I

 

Center of aqueous phase (nm)
0

 

 

 

 

4 F 1 l 1 L 1
5 10 15 20
Time (n8)

 

‘0
“3
. '- 7:9???
- ‘F- ‘s
D.
&

 

 

 

Center of aqueous phase (nm)
0

 

 

 

 

FIGURE 8 Starting structure of POPC/POPE bilayer with one palmitate inserted into the
aqueous phase. Bilayers are modeled (a) without and (c) with trehalose. Colored molecules
are POPC/POPE headgroup (blue), lipid tails (red), water (gray), trehalose (green), and
palmitate (cyan). The dynamics of palmitate, represented by the position of central carbon
atom in its tail, are shown in panels b and (1. Blue horizontal lines correspond to the average
position of the phosphorus atoms of POPC and POPE along the interface and are used to
identify the interface. Gray area denotes the aqueous regions. The position 2 = 0 corresponds
to middle of the aqueous phase.

To investigate the protective role of trehalose on palmitate-induced toxicity, an

extensive hydrogen-bond analysis was carried out to investigate the interactions

25

between lipids, trehalose, and palmitate. [See previous publication (78) for details]
The observations led us to conclude that palmitate, a hydrophobic molecule, prefers to
penetrate the bilayer through hydrophobic regions. And trehalose has the ability to
modify the bilayer surface (79, 80), creating large hydrophobic regions exposed to the
aqueous phase. The results also demonstrates the dual role of trehalose: on the bilayer
surface, trehalose can alter the H-bond distribution, thus inducing hydrophobic
regions for palmitate to penetrate the bilayer, while in the aqueous phase, trehalose
can interact with palmitate and prevent it fi'om approaching the bilayer surface. These
two competing processes help us to understand why the experimental measurements
have shown that trehalose at high concentrations (>O.13 ‘mM) is detrimental to HepG2
cells. At high trehalose concentrations, the bilayer surface is significantly modified by
trehalose such that hydrophobic regions are more accessible for palmitate to penetrate
the bilayer.

From the Simulation, we found single palmitate can penetrate the bilayer. However,
attempts to model multiple palmitate molecule dynamics was not successful.
Palmitate molecules aggregate, as shown in Fig 9, due to the hydrophobic/hydrophilic
interactions. The aggregation reduces the driving force for palmitate to penetrate the
bilayer. To investigate effects of palmitate on bilayer, model bilayers with palmitate

embedded in them were created.

26

o
'
- 1'
I ' . ' «”‘Al ‘ .'
.’ ’ 7'." I 1""

- 1 .1 .‘ . ~ "I
‘ . r' 1‘ I . 4— _- f .3

-' "i 3:33-44 5“ (’I 7*, ' ' Jg’é‘ "4 A
- . «\“Ii’wcwfl‘sfgfi‘fi- it“

A

b

4.. ’\
,

 

Figure 9: Snapshot of palmitate aggregation in the aqueous phase. The colored groups
correspond to the DPPC headgroups (blue), lipid tails (light gray), palmitate (red/dark gray),
and water (pink).

Fig 10 shows the changes to the bilayer structure caused by the addition of palmitate
for the cases where the bilayer freely expands and remains constrained as palmitate is
embedded in the bilayer. To mimic the local effect of palmitate embedded in the
bilayer, the lateral expansion of the bilayer was constrained. As shown in Fig 100,
straight lipid tails with tilted arrangements are Observed at higher palmitate
concentrations. This is related to the ordered lipid phase, which has been shown to be
detrimental to cells by limiting their transport activities (81, 82), binding sites for
pathogens and toxins (83-85), and possibly the cause of palmitate-induced toxicity. As
the palmitate concentration decreases, the arrangement of the lipid tails become more

random as observed in Fig 10a, thus restoring the bilayer to its normal structure.

27

a) Normal bilayer . b) Membrane swelling (1 Lipid order phase

 

FIGURE 10 Snapshots of bilayer structures with (44 mol %) and without palmitate. (a) No
palmitate is embedded in the bilayer. (b) The bilayer is allowed to expand as more palmitate
molecules are embedded in the bilayer. (c) The bilayer is constraint in the lateral directions as
more palmitate molecules are embedded in the bilayer. Colored molecules are POPC
headgroup (blue), POPE headgroup (green), lipid tails (silver), water (red), and palmitate
(brown).

As seen from Fig 10, the increase in the ordering of the lipid tails is related to the
mixing of lipid and fatty acid components, where straight chain fatty acid exhibits
higher order parameters. Since the ordering Of the lipid tails is directly related to the
phase transition, the simulation results agree well with the DSC measurements for
DOPC Iiposomes containing palmitate, EPR measurements of HepG2 cells exposed to
palmitate (Chap 1&2), all of which Showed an increase in the phase-transition
temperature with increasing palmitate concentration.

Palmitate has been Shown to be very toxic to HepG2 cells. Unsaturated FAS, on the
other hand, are not toxic. To obtain a better understanding on how unsaturated FA
interacts with the lipid bilayers in comparison to the effect induced by saturated FA,
hydrogen bonding was analyzed to characterize the effect of unsaturated FAS (oleate
and Iinoleate) on the properties of DOPC bilayers. The goal was to investigate the
effect Of lipid hydration with increasing FA concentrations. Using the hydrogen bond

analysis previously described by Brady and Schmidt (86) with the first hydration

28

cut-off from RDFs, Fig. 11 Shows the average number of hydrogen bonds per lipid
between lipid oxygen atoms and water for all systems considered. As seen in the
figure, the average number of hydrogen bonds reduces significantly with increasing
palmitate concentrations. This is because the increase in lipid packing resulted in the
removal of potential binding sites for water. On the other hand, the number of
hydrogen bonds for oleate systems remains relatively the same regardless of the oleate
or Iinoleate concentrations. This is related to fact that oleate and Iinoleate can reduce
the packing between lipids, thus maintaining the suitable area per lipid and the level
of hydration. From this analysis, we confirm that the role of saturated and unsaturated
fatty acid are very different in that unsaturated FA induced less change in the bilayer

structure and help maintain the level of hydration.

 

7.04 ~

F"

6.96 £ 3; 7 g,
r f r f i
6.88 .
6.80 i I I
0 5 10 15 20 25
FA concentration (mole%)

H-bond/ lipid

 

 

 

Figure 11. Average number of hydrogen bonds per lipid for bilayers with palmitate (circles),
oleate (square) and Iinoleate (triangle); water as H-donors. Solid, dotted, and dash lines are
drawn to guide the eye, respectively. Error bars represent standard deviations.

29

Discussion

Modification of the membrane lipid composition may alter the membrane fluidity and
in turn affect cellular function (87, 88). As palmitate molecules are embedded within
the membrane, it is observed from the EPR measurements that the membrane fluidity
is Significantly decreased. This phenomenon can be explained by many factors. First,
palmitate is hydrophobic in nature and by exposing it to cell membranes, palmitate is
most likely dissolving into the membrane, thus reducing the membrane fluidity.
Second, as palmitate can be metabolized into phospholipid components of cell
membranes, these additional components can cause an increase in the packing
between the lipids, consequently decreasing the membrane fluidity (87,88). Last, since
H202 and *OH are present in cultured cells exposed to palmitate, it has been reported
that unsaturated phospholipids are oxidized into fragment of saturated hydrocarbons
with various headgroup functionalities, some of which are highly toxic to cells (89-92).
Although, the oxidation of unsaturated lipids generally results in an increase in
fluidity and permeability Of the membrane (93-95), the remaining fi'agments inside the
membrane, which are hydrophobic in nature, can reduce the membrane fluidity. In this
study, we found that palmitate decreased the cellular membrane fluidity of HepG2
cells. This was expected, Since others have shown that fatty acids incorporated into the
membrane disrupted the bilayer structure and changed the lipid phase-transition
temperature (76,77). We have also observed increasing the concentration of palmitate

increases the phase-transition temperature of DOPC.

3O

Conclusions and Future work

We performed a series of experimental and computational measurements to gain
insight into how trehalose interacts with HepG2 cells in the presence of palmitate, and
the different effects Of saturated and unsaturated fatty acids on HpeG2 cells.
Experimentally, we found that healthy HepG2 cells exposed to palmitate resulted in a
significant amount of LDH and H202 released into the medium, indicating cell death
or compromised cellular membrane. However, cells exposed to Oleate and Iinoleate
did not Show cell damage. Furthermore, it is observed fi'om EPR measurements that
the membrane fluidity is significantly decreased in the presence of palmitate, while
fluidity is not significantly changed in the presence of oleate and Iinoleate. The

leading hypotheses for the observed results are:

l. Palmitate dissolves into the membrane, thus reducing the membrane fluidity.

2. Palmitate metabolizes into phospholipid components of cell membranes, thus
increasing the packing between the lipids.

3. The remaining fragments of oxidized lipids inside the membrane (oxidized by
H202 and *OH), which are hydrophobic in nature, reduce the membrane

fluidity.

The simulation is aimed at interpreting and understanding the experimental results,
providing knowledge at the molecular level into the role of fatty acids and trehalose in
the toxicity of HepG2 cells. As illustrated by the results, the computation analyses

confirmed that palmitate can dissolve into the bilayers within a short time. As the

31

palmitate concentration in the bilayer increased, it forces the surrounding lipid
molecules to become highly packed, resulting in a more ordered structure. The local
effect of palmitate embedded in the bilayer was also considered. The simulation
results yielded a highly order bilayer structure with the lipid tails in a tilted
arrangement. These results agreed well with DSC measurements for DOPC Iiposomes
containing palmitate and EPR measurements of HepG2 cells exposed to palmitate.
Furthermore, we verified that the direct interactions of trehalose and palmitate in the
medium through hydrogen bonding potentially hinder palmitate from dissolving into
the bilayer. The binding of palmitate to trehalose seems beneficial to cell membranes;
however, we have also discovered that trehalose can potentially modify the bilayer
surface by altering the surface hydrogen-bond distribution, thus inducing hydrophobic
regions for palmitate to penetrate the bilayer. We confirmed that the role of saturated
and unsaturated fatty acid are very different in that unsaturated FA induced less

change in the bilayer structure and help maintain the level of hydration.

We have demonstrated a potential mechanism by which palmitate incorporates into the
bilayer. We further hypothesize that palmitate can aggregate in lipid bilayer to form
small domains, and possibly pores. However, due to the limited time scale of
simulation, we were not able to Observe significant diffusion of palmitate in bilayer.
To get evidence for aggregation, we can use fluorescence resonate energy transfer
(FRET). Fatty acids and phospholipids would be tagged covalently with optical donor
and acceptor chromophores. A time correlated single photon counting system can be

used to measure the fluorescence lifetime of donor and acceptor incorporated in the

32

Iiposome. Aggregation would be seen as a deviation from the predictions of the
Forster model for a random distribution of the chromophores. Understanding the
mechanism Of saturated fatty acid-induced hepatocyte toxicity may provide insight

into cures for diseases such as obesity-associated cirrhosis.

33

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