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Tài liệu Báo cáo khoa học: Interruption of triacylglycerol synthesis in the endoplasmic reticulum is the initiating event for saturated fatty acid-induced lipotoxicity in liver cells pdf

Interruption of triacylglycerol synthesis in the
endoplasmic reticulum is the initiating event for saturated
fatty acid-induced lipotoxicity in liver cells
Michalis D. Mantzaris
1
, Epameinondas V. Tsianos
2
and Dimitrios Galaris
1
1 Laboratory of Biological Chemistry, University of Ioannina Medical School, Greece
2 First Division of Internal Medicine and Hepato-gastroenterology Unit, University of Ioannina Medical School, Greece
Introduction
Dietary habits in the Western world have changed dras-
tically during the last few decades, and this change cor-
relates with increasing levels of obesity, implying that
diet may be associated with the development of insulin
resistance, type 2 diabetes, cardiovascular disease and
other pathologies in the general population [1]. Con-
sumption of food rich in fat causes qualitative and
quantitative changes in serum free fatty acid (FFA) lev-
els, and increases the rate of uptake and accumulation

of lipids in nonadipose tissues such as the liver, which
is the main lipid-metabolizing organ. Inappropriate
accumulation of excess lipids in liver cells in the form
of lipid droplets has been proposed to lead to dysfunc-
tion of hepatocytes and, consequently, to serious path-
ological complications [2,3]. Nonalcoholic fatty liver
disease (NAFLD) is a term used to characterize a spec-
trum of pathological changes ranging from simple fatty
infiltration (steatosis) to hepatic steatosis accompanied
Keywords
endoplasmic reticulum stress; lipoapoptosis;
nonalcoholic fatty liver disease (NAFLD);
oleate; stearate
Correspondence
D. Galaris, Laboratory of Biological
Chemistry, University of Ioannina Medical
School, 451 10 Ioannina, Greece
Fax: +30 26510 07868
Tel: +30 26510 07562
E-mail: dgalaris@uoi.gr
(Received 14 October 2010, revised 16
November 2010, accepted 24 November
2010)
doi:10.1111/j.1742-4658.2010.07972.x
The aim of the present study was to investigate in detail the molecular mecha-
nisms by which free fatty acids induce liver toxicity in liver cells. HepG2 and
Huh7 human liver cell lines were exposed to varying concentrations of stea-
rate (18:0), oleate (18:1), or mixtures of the two fatty acids, and the effects on
cell proliferation, lipid droplet accumulation and induction of endoplasmic
reticulum stress and apoptosis were evaluated. It was observed that: (a) stea-
rate, but not oleate, inhibited cell proliferation and induced cell death; (b)
stearate-induced cell death had the characteristics of endoplasmic reticulum
stress-mediated and mitochondrial-mediated apoptosis; (c) the activation of
stearate in the form of stearoyl-CoA was a necessary step for the lipotoxic
effect; (d) the capacity of cells to produce and accumulate triacylglycerols in
the form of lipid droplets was interrupted following exposure to stearate,
whereas it proceeded normally in oleate-treated cells; and (e) the presence of
relatively low amounts of oleate protected cells from stearate-induced toxicity
and restored the ability of the cells to accumulate triacylglycerols. Our data
suggest that interruption of triacylglycerol synthesis in the endoplasmic retic-


ulum, apparently because of the formation of a pool of oversaturated inter-
mediates, represents the key initiating event in the mechanism of saturated
fatty acid-induced lipotoxicity.
Abbreviations
ACS, long-chain acyl-CoA synthetase; ATF4, activating transcription factor 4; BrdU, bromodeoxyuridine; CHOP, CCAAT ⁄ enhancer-binding
protein homologous protein; DAG, diacylglycerol; ER, endoplasmic reticulum; eIF2a, eukaryotic translation initiation factor 2a;
FITC, fluorescein isothiocyanate; FFA, free fatty acid; JNK, c-Jun N-terminal kinase; NAFLD, nonalcoholic fatty liver disease; PERK,
RNA-dependent protein kinase-like endoplasmic reticulum eukaryotic initiation factor-2a kinase; PI, propidium iodide; SD, standard deviation;
SFA, saturated fatty acid; TAG, triacylglycerol; TrC, triacsin C; UFA, unsaturated fatty acid.
FEBS Journal 278 (2011) 519–530 ª 2010 The Authors Journal compilation ª 2010 FEBS 519
by inflammation, fibrosis, and cirrhosis (nonalcoholic
steatohepatitis) [4,5]. Despite the high prevalence of
NAFLD and its potential for serious complications,
the underlying molecular mechanisms that determine
the progression to liver damage remain poorly under-
stood and need further investigation.
A number of recent in vitro and in vivo studies have
shown that different forms of fatty acids exert remark-
ably different effects. Exposure of a variety of cell
types, including hepatocytes, to long-chain saturated
fatty acids (SFAs) led to increased expression of proin-
flammatory cytokines, inhibition of insulin signaling,
induction of endoplasmic reticulum (ER) stress, and
promotion of cell death, mainly by apoptosis [6–12].
On the other hand, unsaturated fatty acids (UFAs)
were not toxic at the same concentrations and, in addi-
tion, their presence protected cells from SFA-induced
effects [6,13–16]. A protective role for endogenously
generated UFAs was also indicated by in vivo experi-
ments using genetically modified mice bearing an inacti-
vating mutation in the gene encoding the enzyme
stearoyl-CoA desaturase 1. This enzyme is responsible
for the introduction of a double bound into long-chain
SFAs [17]. However, the exact molecular mechanisms
underlying these events remain partially understood,
and the data obtained, as well as the explanations pro-
vided, are often controversial. For instance, it has been
suggested that SFAs can influence important cellular
signaling pathways either directly or indirectly through
the generation of reactive oxygen species [18], ceramides
[19], or accumulation of saturated triacylglycerols
(TAGs) [20], leading to cellular dysfunction and ulti-
mately to cell death. The precise mechanisms lying
beneath these processes remain elusive, and the key ele-
ments that determine the induction of toxicity have not
been identified yet.
The aim of the present investigation was to perform
a detailed evaluation of several aspects concerning
SFA-induced lipotoxicity in order to define the key
event(s) involved in this mechanism. For this purpose,
human hepatoblastoma cells were exposed to varying
combinations of saturated (stearate, 18:0) and unsatu-
rated (oleate, 18:1 cis) fatty acids for various time peri-
ods, and cell proliferation, toxicity, induction of ER
stress and apoptosis and lipid droplet accumulation
were evaluated.
Results
SFAs inhibit proliferation and induce cell death
Exposure of HepG2 cells to 0.3 mm of the SFA stea-
rate (18:0), but not to the monounsaturated fatty acid
oleate (18:1, cis), induced a transient inhibition of cell
proliferation during the first 24 h (Fig. 1A). This
observation was also confirmed by analysis of bromo-
deoxyuridine (BrdU) incorporation into DNA, which
decreased by more than 50% after 24 h of stearate
treatment. However, cells regained their normal prolif-
eration capacity at longer incubation periods, whereas
coadministration of oleate (0.3 mm) prevented the
AB
CD
EF
Fig. 1. Oleate prevents (SA) stearate-induced cytotoxicity. (A)
HepG2 cells (1.0 · 10
5
) were seeded in 24-well plates and cultured
for 24 h before being treated with vehicle (
), 300 lM stearate ( ),
300 l
M oleate (OA) ( ), or a combination of the two ( ). At the
indicated time points, cells were harvested, and viable cells were
counted (Trypan blue exclusion). (B) Cells were treated as above,
except that for the last 8 h of treatment they were supplemented
with BrdU (100 l
M). BrdU incorporation into DNA was detected by
using an antibody against BrdU as described in Experimental proce-
dures. Results are expressed as percentages of the respective con-
trols (Ctrl) (*P < 0.05). (C, D) The conditions were exactly as in (A)
and (B), respectively, except that 600 l
M of each fatty acid was
used. (E) Cells were treated with stearate (600 l
M), and cell num-
bers were counted at the indicated time points (solid line). Oleate
(600 l
M) was added at 0, 12, 24 or 36 h following addition of stea-
rate, and cell numbers were counted at the indicated time points
(dashed lines). (F) Cells were supplemented with 600 l
M stearate
in the presence of increasing concentrations of oleate (0, 20, 60,
100 and 300 l
M). After 48 h, cells were harvested and cell num-
bers were counted. Data are expressed as mean ± SD of triplicate
measurements.
Saturated fatty acid-induced lipotoxicity M. D. Mantzaris et al.
520 FEBS Journal 278 (2011) 519–530 ª 2010 The Authors Journal compilation ª 2010 FEBS
transient inhibition of proliferation induced by stearate
alone (Fig. 1A,B).
When higher concentrations of FFA (0.6 mm) were
used, cells could not recover following stearate treat-
ment, and toxic effects were apparent (Fig. 1C). Again,
coadministration of oleate protected cells from stea-
rate-induced toxicity and restored the capacity of cells
to proliferate (Fig. 1C,D). The protection offered by
oleate was apparent even when it was administered 12,
24 or 36 h following stearate administration (Fig. 1E)
or in ratios of oleate to stearate lower than 1 : 1
(Fig. 1F). Essentially the same results were obtained
when another human hepatocellular carcinoma cell line
(Huh7) or other unsaturated (linoleic acid, 18:2 cis)or
saturated (palmitate) fatty acids were used instead of
HepG2 cells, oleate, and stearate, respectively (results
not shown).
Stearate treatment induces ER stress and
mitochondrial-mediated apoptosis
As shown in Fig. 2, treatment of HepG2 cells with
0.6 mm stearate led to the appearance of condensed
and fragmented nuclei (Fig. 2A,B), hypodiploid sub-
G
1
DNA (Fig. 2C), DNA internucleolar fragmentation
(Fig. 2D), caspase-3 cleavage (Fig. 2E), and the
appearance of cytochrome c in the cytosol (Fig. 2F),
which are clear characteristics of mitochondrial-medi-
ated apoptotic cell death. In all cases, oleate was not
toxic by itself, and its coadministration with stearate
prevented the appearance of these apoptotic markers.
Different Bcl-2 family members serve as proapop-
totic and antiapoptotic mitochondrial regulators under
certain circumstances [21,22]. As shown in Fig. 3A, the
relative amount of the antiapoptotic protein Bcl-2 was
gradually increased in HepG2 cells during the initial
22 h of stearate treatment, but this increase was inter-
rupted thereafter, and the Bcl-2 concentration stabi-
lized at a somewhat lower level. On the other hand,
the proapoptotic Bcl-2-like protein Bax was activated,
as indicated by its translocation from the cytosolic
fraction to the mitochondrial fraction following stea-
rate administration (Fig. 3B). The presence of oleate
inhibited this translocation, indicating the involvement
of Bax activation in stearate-induced mitochondrial
destabilization and apoptosis.
In order to further evaluate specific molecular mech-
anisms contributing to induction of mitochondrial
destabilization, we analyzed changes in specific mark-
ers of ER stress, which has been proposed to be
involved in SFA-induced lipotoxicity [8,13,16,23]. As
shown in Fig. 4A, increased phosphorylation of
eukaryotic translation initiation factor 2a (eIF2a) was
apparent after 16 h of stearate treatment, reached a
peak at 22 h, and declined thereafter. Concomitantly
with eIF2a phosphorylation, dramatic elevations in the
expression of activating transcription factor 4 (ATF4)
and of CCAAT⁄ enhancer-binding protein homologous
protein (CHOP) proteins downstream of eIF2a were
observed (Fig. 4A), indicating the initiation of ER
stress-induced apoptosis. Oleate was unable to induce
the expression of the proapoptotic protein CHOP, and
its coadministration with stearate inhibited CHOP
expression induced by stearate alone (Fig. 4B). These
observations indicate that activation of the RNA-
dependent protein kinase-like ER eukaryotic initiation
factor-2a kinase (PERK) branch of ER stress is trig-
gered following exposure of HepG2 cells to stearate,
and the presence of oleate prevented this activation.
Along with PERK branch activation, the phosphor-
ylation of c-Jun N-terminal kinase (JNK) was also
increased, displaying a strong peak after 22 h of stea-
rate treatment (Fig. 4C). In oleate-treated cells, JNK
phosphorylation increased slightly after 16 h of treat-
ment, but oleate, in contrast to stearate, did not exhi-
bit the sharp increase at 22 h. In addition, oleate
prevented stearate-induced phosphorylation of JNK
when the two agents were coadministered (Fig. 4C).
Taken together, these results indicate that the location
of the protective action of oleate was upstream of ER
stress activation.
Whether the protection offered by oleate was specific
for SFA-induced ER stress or represented a more gen-
eral phenomenon was also investigated. The presence
of oleate was unable to prevent the toxic effects
induced by thapsigargin or tunicamycin (two classical
ER stress inducers), indicating that oleate is not a gen-
eral inhibitor of the ER stress response (results not
shown).
Stearate treatment interrupts TAG synthesis and
lipid droplet accumulation
In addition to its role in proper protein folding, the
ER is responsible for lipid synthesis. In particular,
excess availability of FFAs, as is the case in the pres-
ent experimental model, leads to increased formation
of TAGs, which are either released from the cells as
very low density lipoprotein or stored in the cytosol as
lipid droplets. It was observed that the accumulation
of lipid droplets was efficient in oleate-treated cells,
whereas stearate-treated cells contained fewer and
smaller lipid droplets after 24 h of treatment (Fig. 5A).
Moreover, coadministration of oleate restored the
capacity of stearate-treated cells to accumulate lipid
droplets. This observation was further confirmed by
M. D. Mantzaris et al. Saturated fatty acid-induced lipotoxicity
FEBS Journal 278 (2011) 519–530 ª 2010 The Authors Journal compilation ª 2010 FEBS 521
using TLC analysis, which showed that stearate-treated
cells contained much lower amounts of TAG than ole-
ate-treated and oleate plus stearate-treated cells (results
not shown). These observations indicate that the mag-
nitude of cellular steatosis as such is not responsible
for lipotoxicity. Rather, it is likely that diversion of
fatty acids into inert TAG stores contributes to cell
survival and preserves cellular functions.
In order to further investigate these fundamen-
tally different effects of different FFAs, we performed
A
C
DE
F
B
Fig. 2. Stearate (SA) promotes cell death via mitochondria-mediated apoptosis. HepG2 cells were treated with vehicle (Ctrl), stearate, oleate
(OA) or a combination of the two (SA ⁄ OA) at 600 l
M each, and different markers related to mitochondria-mediated apoptosis were evalu-
ated. (A) Micrographs showing the morphology of cell nuclei after 48 h of treatment and subsequent chromatin staining with Hoechst 33342.
(B) Quantification of the percentage of apoptotic nuclei (condensed and fragmented) by random counting of 200 nuclei in each sample. (C)
Cells were treated for 24 and 48 h before analysis of cellular DNA content by flow cytometry as described in Experimental procedures. Sub-
G
1
region indicates hypodiploid DNA content. (D) Cells were treated as in (C), and DNA was isolated from each cell population. The charac-
teristic ladder pattern of DNA appeared after electrophoresis in 1.4% agarose gel. (E) Cells were supplemented with fatty acids for 48 h,
and cleavage of procaspase-3 was evaluated in total cell extracts by western blotting. (F) The appearance of cytochrome c in the cytosolic
fractions (S-100) of fatty acid-treated cells for 48 h was evaluated by western blotting.
Saturated fatty acid-induced lipotoxicity M. D. Mantzaris et al.
522 FEBS Journal 278 (2011) 519–530 ª 2010 The Authors Journal compilation ª 2010 FEBS
long-scale time-course experiments. Cells were treated
with FFAs for time periods of 3, 6, 12, 24 and 36 h,
before staining of the accumulated neutral lipids with
Nile red and analysis of the fluorescence intensity of
individual cells by flow cytometry. Cell fluorescence
increased progressively in oleate-treated and oleate
plus stearate-treated cells, whereas it was significantly
lower in stearate-treated cells during the first 3 and 6 h
of treatment (Fig. 5B). Interestingly, the fluorescence
in stearate-treated cells started to decrease gradually at
exposure times longer than 6 h, giving rise to a distinct
cell population with basal levels of fluorescence
(Fig. 5B). After 36 h, almost the entire cell population
was devoid of lipid droplets.
It is obvious from these results that TAG synthesis
was initially hindered following stearate administration
and was completely interrupted at longer incubation
periods. Interestingly, the interruption of TAG synthe-
sis preceded the appearance of toxic effects, supporting
the notion that it constitutes the initiating event in the
process of lipotoxicity.
Stearate has to be activated in order to be toxic
The first enzyme involved in metabolism of FFAs after
their uptake into liver cells is the long-chain acyl-CoA
synthetase (ACS), which activates fatty acids by link-
ing them to coenzyme A. As shown in Fig. 6, triac-
sin C (TrC), a specific competitive inhibitor of ACS
[24,25], was not toxic by itself, whereas it inhibited the
accumulation of lipid droplets following exposure of
cells to either stearate or oleate (Fig. 6A,B). At the
same time, TrC protected cells from stearate-induced
death, as indicated by estimation of cell viability
(Fig. 6C), annexin-V plus propidium iodide (PI) stain-
ing (Fig. 6D), and other ER stress markers (results not
shown). This protective effect could not be attributed
to a nonspecific inhibitory effect on ER stress, as TrC
was not able to prevent CHOP induction by thapsigar-
gin or tunicamycin, two classic ER stressors (Fig. 6E).
These observations show that it is not stearate as such
that is responsible for inducing cell toxicity, but one or
more of its metabolic intermediates in the pathway of
A
B
Fig. 3. Stearate (SA) treatment alters Bcl-2 protein levels and pro-
motes Bax translocation to mitochondria. (A) HepG2 cells were
treated with 600 l
M stearate for the indicated times. Total protein
extract was prepared from each sample, and the expression of Bcl-
2 was examined by western blotting. (B) Cells were incubated with
vehicle (Ctrl) or 600 l
M stearate, 600 lM oleate (OA) or a combina-
tion of the two (SA ⁄ OA) for 48 h. Mitochondrial (M-10) and cyto-
solic (S-100) fractions were prepared, and the presence of Bax
protein in these fractions was analyzed by western blotting.
A
B
C
Fig. 4. Stearate (SA) treatment promotes the induction of the ER
stress response. (A) HepG2 cells were treated with 600 l
M stea-
rate for the indicated times. Total cell extracts were prepared, and
the phosphorylation of eIF2a and the expression of ATF4 and
CHOP proteins were examined by western blotting with specific
antibodies. (B) Western blot analysis of the expression of CHOP in
total cell extracts prepared from cells treated with 600 l
M stearate,
600 l
M oleate (OA) or a combination of the two (SA ⁄ OA) for 36 h.
(C) The intensity of phosphorylation of JNK (p-JNK) and the amount
of the total protein was examined by western blot analysis in total
cell extracts derived from cells treated as in (A). Where indicated
thapsigargin (Thap)-treated cells (2 l
M for 24 h) were used as posi-
tive controls (Ctrl).
M. D. Mantzaris et al. Saturated fatty acid-induced lipotoxicity
FEBS Journal 278 (2011) 519–530 ª 2010 The Authors Journal compilation ª 2010 FEBS 523
TAG synthesis. In addition, these results show that the
properties of the metabolic intermediates of stearate
and oleate must be fundamentally different.
Discussion
The results presented in this investigation, in agree-
ment with previously reported observations, revealed
fundamentally different effects of SFAs and UFAs on
liver cells [8,11–13]. In an attempt to identify the key
event(s) responsible for these differences, we examined
the main steps involved, following the uptake of satu-
rated and unsaturated FFAs into the cells. After their
internalization, FFAs are converted to fatty acyl-CoA,
a reaction catalyzed by ACS. Fatty acyl-CoAs are acti-
vated forms of fatty acids that can be either oxidized
in mitochondria or utilized in the ER as substrates for
the synthesis of phospholipids, cholesterol esters, and
TAGs [26–28]. The observation in this investigation
that inhibition of ACS by TrC abolished both FFA-
induced lipid droplet accumulation and SFA-induced
toxicity (Fig. 6) indicates that SFA activation is essen-
tial for the manifestation of toxicity. It has to be noted
that TrC was not able to inhibit thapsigargin-induced
Ctrl
3 h
12 h
24 h 36 h
6 h
OA
OA
SA/OA
SA/OA
SA/OA
SA/OA
SA/OA
SA
OA
FL1-Log
Counts
OA
OA
SA
SA
SA
SA
Ctrl
Ctrl
Ctrl
Ctrl
Ctrl
SA OA SA/OA
A
B
Fig. 5. Stearate (SA) supplementation interrupts lipid droplet accumulation. HepG2 cells were exposed to 600 lM stearate, 600 lM oleate
(OA) or a combination of the two at 600 l
M each. (A) To identify lipid droplets, cells treated with FFA media for 24 h were stained with Nile
red and analyzed by confocal microscopy. (B) Graphs showing the distribution of Nile red fluorescence intensity of individual cells at 3, 6, 12,
24 and 36 h were obtained by flow cytometric analysis in the FL1 channel (logarithmic scale). Control (Ctrl) cells (blue line), stearate-treated
cells (green line), oleate (OA)-treated cells (black line) and cells supplemented with both fatty acids (red line) are shown. These experiments
were repeated two more times, with essentially the same results.
Fig. 6. Acyl-CoA formation is necessary for stearate-induced toxicity. HepG2 cells were exposed for 48 h to vehicle (Ctrl), 600 lM of stearate
(SA), 600 l
M oleate (OA) or a combination of the two (SA ⁄ OA) in the absence or presence of 0.5 lM TrC, a specific inhibitor of ACS. (A)
Cells were stained with Nile red and analyzed by confocal microscopy. Representative photographs show inhibition of FFA-induced lipid drop-
let formation by TrC. (B) Quantitation of Nile red fluorescence by flow cytometry. Bars represent the mean fluorescence value of each distri-
bution ± SD of duplicate measurements from two independent experiments (*P < 0.05 versus control;
#
P < 0.05 versus TrC-untreated
cells). (C) Cells were harvested, and cell numbers were assessed by Trypan blue exclusion. Each bar represents the mean ± sd from tripli-
cate measurements (*P < 0.05). (D) Annexin V–FITC binding and PI staining were performed in order to assess cell death. Fluorescence
was analyzed by flow cytometry in 10
4
cells per sample. (E) Cells were exposed to typical ER stressors, thapsigargin (Tg, 2 lM) or tunicamy-
cin (Tm, 3 l
M), for 24 h, in the presence or absence of 0.5 lM TrC. Total cell extracts were isolated, and CHOP expression was examined
by western blot analysis.
Saturated fatty acid-induced lipotoxicity M. D. Mantzaris et al.
524 FEBS Journal 278 (2011) 519–530 ª 2010 The Authors Journal compilation ª 2010 FEBS
Gate: R1 Gate: R1 Gate: R1 Gate: R1
Gate: R1 Gate: R1 Gate: R1
Q1: 0.27%
Q3: 92.26% Q3: 37.71% Q3: 91.27% Q3: 84.98%Q4: 3.71% Q4: 27.00% Q4: 3.82% Q4: 9.37%
Q3: 93.40%
Q3: 85.00%
Q3: 90.48%
Q3: 92.44%
Q4: 2.89%
Q4: 9.94%
Q4: 3.90%
Q4: 2.81%
Q1: 3.80% Q1: 0.34% Q1: 0.48%Q2: 3.76%
Gate: R1
Q1: 0.54%
Q2: 3.17%
Q2: 31.49%
Q1: 0.43%
Q2: 4.64%
Q2: 4.57% Q2: 5.17%
Q1: 0.49%
Q1: 0.44%
Q2: 5.13%
Q2: 4.32%
A
B
D
E
C
M. D. Mantzaris et al. Saturated fatty acid-induced lipotoxicity
FEBS Journal 278 (2011) 519–530 ª 2010 The Authors Journal compilation ª 2010 FEBS 525
or tunicamycin-induced ER stress (Fig. 6E), thus
excluding the possibility of nonspecific inhibition of
ER stress. When the available acyl-CoAs are in excess,
they are channeled towards TAG synthesis.
The main findings of the present investigation were
the observations that TAG synthesis in liver cells
exposed to excess stearate was interrupted, and that
this interruption preceded the appearance of toxic
effects (Fig. 5A,B). In sharp contrast, oleate-treated
cells, which continued to proliferate normally, were
able to produce TAGs continuously and accumulate
them in the form of lipid droplets. Moreover, coad-
ministration of oleate restored the ability of stearate-
treated cells to synthesize TAGs and prevented cell
toxicity. These findings are in agreement with previous
observations from the Schaffer group, indicating
increased incorporation of palmitate (16:0) into the
TAG pool only in the presence of oleate [18].
The above results raise two main questions: (a) what
is the cause of TAG synthesis inhibition, and (b) what
is the exact nature of the events that ultimately lead to
cell toxicity?
Regarding the first question, it is obvious that one
or more steps (following acyl-CoA formation) in the
cascade of TAG formation that take place in ER
membranes are defective in SFA-treated but not in
UFA-treated cells. It has been previously shown that
the degree of saturation of fatty acyl chains in TAG
synthesis intermediates, such as phosphatidic acid and
diacylglycerol (DAG), can influence their physicochem-
ical properties, and in this way modulate their interac-
tions with specific proteins [29–32]. Oversaturated
DAGs, for example, were unable to interact with
protein kinase C, and this effect was attributed to the
formation of gel-like domains (instead of liquid-
crystalline domains) in the membranes, making these
molecules unavailable for the required interactions
[30,33]. Addition of UFAs could restore the liquid-
crystalline phase, making DAG molecules accessible to
the interacting proteins [33]. We propose that a similar
mechanism can satisfactorily explain the results
reported in this work, as well as the majority of previ-
ous observations from other laboratories.
Regarding the second question, the induction of ER
stress and apoptosis observed in SFA-treated cells can
be explained by modulation of the physicochemical
properties of ER membranes by saturated lipid inter-
mediates, such as PA and DAG. Excessive saturation
accompanied by the formation of gel-like domains can
influence the rigidity and fluidity of ER membranes,
thus compromising the functional integrity of these
organelles. It has to be stressed here that the ER is
especially vulnerable, as its membranes require higher
concentrations of UFAs in order to be functional [34].
In support of this notion, previous investigations have
shown major irregularities in the morphology of the
ER in SFA-treated but not UFA-treated cells
[9,20,35]. Moffitt et al. [20] suggested that accumula-
tion in the ER lumen of oversaturated TAGs, which
cannot be further processed, because of their inappro-
priate physicochemical properties (high melting point),
is the main cause of toxicity. This proposal, however,
is not consistent with the disappearance of lipid drop-
lets from stearate-treated cells, as observed in this
investigation (Fig. 5B).
The ER is the site of synthesis of all secretory pro-
teins and resident proteins of the membrane system,
and any perturbation that compromises the protein-
folding capacity of the organelle can lead to ER stress
[36–38]. ER stress is a general, integrated stress
response displayed by mammalian cells. This response
can be divided in two phases according to the intensity
and the duration of the stress. An initial adaptive
response culminates in the temporary inhibition of
protein synthesis, providing cells with the opportunity
to recover and restore normal homeostasis. The data
presented in this work demonstrate that cells exposed
to stearate are moved initially towards such an adap-
tive state, as indicated by the transient inhibition of
cell proliferation (Fig. 1A,B) and the early phosphory-
lation of eIF2a (Fig. 4A). When the stress is more
intensive and prolonged, secondary events, such as
ATF4 and CHOP protein expression and JNK activa-
tion, were induced, leading ultimately to cell death by
apoptosis (Fig. 4A,C). Prolonged ER stress and JNK
activation, as observed in this study, usually stimulate
apoptosis by several pathways, including the transloca-
tion of Bax to mitochondria, and CHOP-regulated
inhibition of the expression of antiapoptotic proteins,
such as Bcl-2 [23,39]. A schematic representation of
the events observed in FFA-supplemented cells is pre-
sented in Fig. 7.
Although the conditions prevailing in this cellular
model are quite different from those prevailing in vivo,
previous experiments with rats fed a diet enriched in
SFAs demonstrated similar characteristics of ER stress
activation and apoptosis in the liver [10]. Moreover, in
accordance with the results presented in this article,
the above characteristics were not apparent in animals
fed a control diet or a diet containing UFAs, although
steatosis developed. In addition, phosphorylation of
eIF2a, which is characteristic of PERK branch activa-
tion of ER stress, has been demonstrated in humans
with NAFLD and nonalcoholic steatohepatitis [40].
In conclusion, it is proposed that the key event deter-
mining SFA-induced lipotoxicity is the interruption of
Saturated fatty acid-induced lipotoxicity M. D. Mantzaris et al.
526 FEBS Journal 278 (2011) 519–530 ª 2010 The Authors Journal compilation ª 2010 FEBS
TAG synthesis. It is suggested that the creation of a
pool of oversaturated lipid intermediates makes these
molecules inaccessible to the enzymes of TAG synthe-
sis, whereas a certain degree of unsaturation can
restore normal TAG formation. Excessive saturation
compromises the functional integrity of the ER, lead-
ing ultimately to ER stress and apoptosis. Unraveling
the exact molecular mechanism(s) of lipotoxicity may
lead to new strategies for the management of NAFLD.
Experimental procedures
Cell culture and treatment
Human hepatocellular HepG2 (ATCC, HB-8065) and
Huh7 (Health Science Research Resources Bank,
JCRB0403, Osaka, Japan) carcinoma cells were grown in
DMEM containing 10% heat-inactivated fetal bovine
serum, 2 mm glutamine, 100 UÆmL
)1
penicillin, and
100 ngÆmL
)1
streptomycin, at 37 °C in air with 5% CO
2
.
Cells were seeded and left under normal conditions for 24 h
before any further treatment. Stock solutions of FFAs
(100 mm) were prepared in isopropanol by heating to
70 °C, and the desired concentrations were prepared in
growth medium supplemented with BSA, as described pre-
viously [41]. Briefly, a 5% (w ⁄ v) BSA solution in DMEM
was filtered and mixed with the fatty acid stock solution,
giving a concentration of 5 mm FFA (FFA ⁄ BSA molar
ratio of 6.6 : 1). The solution was left for 30 min at 50 ° C,
and diluted with DMEM, giving the desired concentrations.
Estimation of cell viability
Following FFA treatment, cell numbers were assessed by
Trypan blue exclusion. Floating and attached cells were
Fig. 7. Schematic representation of the molecular events that take place following exposure of liver cells to FFAs. Interruption of TAG syn-
thesis in conditions of excess availability of SFAs is the key point in the molecular mechanism of SFA-induced lipotoxicity. It is suggested
that creation of a pool of oversaturated lipid intermediates determines whether TAG formation will proceed normally or whether the process
will be diverted towards induction of ER stress and apoptosis. SAT, saturated intermediates; UNSAT, unsaturated intermediates; DGAT,
acyl-CoA:diacylglycerol acyltransferase; LD, lipid droplets; VLDL, very low density lipoprotein; IRE1, inositol-requiring enzyme 1.
M. D. Mantzaris et al. Saturated fatty acid-induced lipotoxicity
FEBS Journal 278 (2011) 519–530 ª 2010 The Authors Journal compilation ª 2010 FEBS 527
collected, centrifuged at 200 g for 5 min, and resuspended
in DMEM plus Trypan blue (0.4% in NaCl ⁄ P
i
) at a ratio
of 1 : 1. Viable cells were counted with a hemocytometer,
and cell numbers were expressed as percentages of the
respective control, unless otherwise indicated.
Estimation of DNA synthesis
Cells were seeded in 24-well plates onto 11-mm glass cover-
slips at a density of 5 · 10
4
cells per well. After 24 h, cells
were supplemented with media containing the indicated
concentrations of FFA for 24 and 48 h. In the final 8 h,
cells were supplemented with 100 lm BrdU (Sigma,
St. Louis, MO, USA) and analyzed by indirect immunoflu-
orescence. Briefly, cells were fixed with 3.7% paraformal-
dehyde, quenched with 50 mm ammonium chloride for
15 min, and permeabilized with 0.1% Triton X-100 for
4 min, before being treated with 1.5 m hydrochloric acid
for 10 min. Incorporation of BrdU into newly synthesized
DNA was detected with an antibody against BrdU (Sigma),
and analysis was performed with a Leica TCS-SP scan-
ning confocal microscope. Cell nuclei were detected by
Hoechst 33342 staining (Sigma). More than 300 cells per
sample were counted, and BrdU-positive cells were
expressed as a percentage of the total cell number.
Detection of lipid accumulation
Lipid droplet accumulation was detected by Nile red stain-
ing as previously described [42]. Cell imaging for Nile red
staining was performed by confocal microscopy. Quantifica-
tion of lipid droplets was performed by flow cytometric
analysis of the distribution of Nile red fluorescence in indi-
vidual cells. Briefly, cells were seeded in 24-well plates onto
11-mm glass coverslips for confocal microscopy, or in six-
well plates for flow cytometry. After FFA treatment, cells
were fixed with 3.7% paraformaldehyde for 10 min, washed
twice, and stained with Nile red (Sigma) solution (final con-
centration, 2 lgÆmL
)1
). Samples were kept for 45 min in
the dark at 37 °C to allow equilibrium with the dye. Cover-
slips were mounted in Mowiol, and viewed with a
Leica TCS-SP scanning confocal microscope, equipped with
an argon ⁄ krypton laser and Leica TCS software. Flow
cytometric analysis (15 000 events per sample) was carried
out with a CyFlow ML (Partec) equipped with an argon
laser, in the FL1 channel (logarithmic scale).
Determination of TAG levels
Cells seeded in six-well plates were treated with FFA med-
ium for 24 h. Cells were harvested, and lipids were
extracted twice with CHCl
3
⁄ MeOH ⁄ ddH
2
O (1 : 1 : 0.9).
Lipid extracts were dried under a nitrogen stream, redis-
solved in chloroform ⁄ methanol (2 : 1), and separated by
TLC (silica gel ⁄ TLC; Fluka), with hexane ⁄ diethyl
ether ⁄ acetic acid (70 : 30 : 1). For visualization of lipids,
the TLC plates were immersed in a solution of cupric sul-
fate (10%, w ⁄ v) in aqueous phosphoric acid (8%, v ⁄ v),
allowed to dry at room temperature, and charred at 150 °C
for 10 min. TAGs were identified with glyceryl tripalmitate
standard (Sigma).
Estimation of apoptosis
For nuclear morphological observations, cells were fixed with
3.7% paraformaldehyde, neutralized with 50 mm NH
4
Cl,
and stained with Hoechst 33342. Nuclear morphology was
observed under a fluorescence microscope (Axiovert S 100;
Zeiss, Ontario, NY, USA) equipped with a UV filter.
Flow cytometric analysis of cellular hypodiploid sub-G
1
DNA content was performed according to [43]. Cells were
harvested, fixed with 70% ethanol, resuspended in NaCl ⁄ P
i
for 20 min at 37 ° C, and stained with Hoechst 33342
(30 min, 37 °C). Annexin V–fluorescein isothiocyanate
(FITC) (BD Pharmigen, San Diego, CA, USA) binding and
PI staining were performed according to [44]. Following
incubation with 1.0 lgÆmL
)1
annexin V and 4.0 lgÆmL
)1
PI
for 20 min, cells were analyzed by flow cytometry.
Preparation and analysis of DNA and protein
extracts
After the appropriate treatment, cellular DNA was isolated
from 3 · 10
6
cells per sample, and analyzed for internucleo-
lar fragmentation by agarose gel electrophoresis. Prepara-
tion of mitochondrial and cytosolic fractions was achieved
by differential centrifugation, as described previously [45].
For western blot analysis, cell lysates (40–50 lg of protein)
were subjected to SDS ⁄ PAGE, and the separated proteins
were demonstrated by immunoblotting after being trans-
ferred to nitrocellulose membranes. Antibodies against
cytochrome c (sc-13156), Bax (2D2, sc-20067), Bcl-2(100)
(sc-509), ATF4 (sc-200) and CHOP ⁄ GADD153 (R-20,
sc-793) were from Santa Cruz Biotechnology. Antibodies
against phospho-JNK (#9251), total JNK (#9252) and
phospho-eIF2a (#9721) were from Cell Signaling. Horse-
radish peroxidase-conjugated antibody against caspase-3
(#610325) was from BD Pharmigen, and antibody against
b-actin (A5441) was from Sigma.
Statistical analysis
All data are expressed as the mean ± standard deviation
(SD). Differences between groups were compared by one-
way ANOVA followed by a post hoc Bonferroni correction
test for multiple comparisons, using originpro 8 software
(OriginLab). Differences were considered to be statistically
significant at P < 0.05.
Saturated fatty acid-induced lipotoxicity M. D. Mantzaris et al.
528 FEBS Journal 278 (2011) 519–530 ª 2010 The Authors Journal compilation ª 2010 FEBS
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