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, li
Trang 1endoplasmic reticulum is the initiating event for saturated fatty acid-induced lipotoxicity in liver cells
Michalis D Mantzaris1, Epameinondas V Tsianos2and Dimitrios Galaris1
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.
Trang 2by 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 monounsatustea-rated 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
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 l M 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.
Trang 3transient 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-G1 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 antiapopproapop-totic 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 experimpres-ental 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
Trang 4using TLC analysis, which showed that stearate-treated
cells contained much lower amounts of TAG than
ole-ate-treated and oleate plus stearole-ate-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
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.
Trang 5long-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 l M 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).
Trang 6TAG 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 unsatusatu-rated 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-FFA-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
6 h
SA/OA
SA
OA
FL1-Log
SA
SA
Ctrl
Ctrl
Ctrl
A
B
Fig 5 Stearate (SA) supplementation interrupts lipid droplet accumulation HepG2 cells were exposed to 600 l M stearate, 600 l M 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 l M of stearate (SA), 600 l M oleate (OA) or a combination of the two (SA⁄ OA) in the absence or presence of 0.5 l M 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 l M ) or tunicamy-cin (Tm, 3 l M ), for 24 h, in the presence or absence of 0.5 l M TrC Total cell extracts were isolated, and CHOP expression was examined
by western blot analysis.
Trang 7Gate: R1 Gate: R1 Gate: R1 Gate: R1
Q1: 0.27%
Q3: 92.26% Q4: 3.71% Q3: 37.71% Q4: 27.00% Q3: 91.27% Q4: 3.82% Q3: 84.98% Q4: 9.37%
Q3: 93.40% Q4: 2.89% Q3: 85.00% Q4: 9.94% Q3: 90.48% Q4: 3.90% Q3: 92.44% Q4: 2.81%
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% Q2: 5.13% Q1: 0.44% Q2: 4.32%
A
B
D
E
C
Trang 8or 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 propro-teins 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
Trang 9TAG 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 Unravellead-ing
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
JCRB0403, Osaka, Japan) carcinoma cells were grown in
DMEM containing 10% heat-inactivated fetal bovine
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
growth medium supplemented with BSA, as described
was filtered and mixed with the fatty acid stock solution,
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.
Trang 10collected, centrifuged at 200 g for 5 min, and resuspended
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
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,
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
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 imagstain-ing 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
Cover-slips were mounted in Mowiol, and viewed with a
Leica TCS-SP scanning confocal microscope, equipped with
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
Lipid extracts were dried under a nitrogen stream,
the TLC plates were immersed in a solution of cupric
for 10 min TAGs were identified with glyceryl tripalmitate standard (Sigma)
Estimation of apoptosis For nuclear morphological observations, cells were fixed with
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
DNA content was performed according to [43] Cells were
(FITC) (BD Pharmigen, San Diego, CA, USA) binding and
PI staining were performed according to [44] Following
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
cells per sample, and analyzed for internucleo-lar fragmentation by agarose gel electrophoresis Prepara-tion of mitochondrial and cytosolic fracPrepara-tions was achieved
by differential centrifugation, as described previously [45] For western blot analysis, cell lysates (40–50 lg of protein)
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-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