doi:10.1111/j.1440-1746.2008.05733.

G A S T R O E N T E R O L O G Y jgh_5733 830..840

Differential effect of oleic and palmitic acid on lipid

accumulation and apoptosis in cultured hepatocytes
Matteo Ricchi,* Maria Rosaria Odoardi,* Lucia Carulli,* Claudia Anzivino,* Stefano Ballestri,*
Adriano Pinetti,† Luca Isaia Fantoni,‡ Fabio Marra,§ Marco Bertolotti,* Sebastiano Banni,¶
Amedeo Lonardo,* Nicola Carulli* and Paola Loria*
*Dipartimento Integrato di Medicina Interna, Endocrinologia, Metabolismo & Geriatria – Università degli Studi di Modena e Reggio Emilia,
†
Dipartimento di Chimica, Università di Modena e Reggio Emilia, ‡Dipartimento di Scienze Biomediche, Sezione di Chimica Biologica, Università
degli Studi di Modena e Reggio Emilia, Modena, §Dipartimento di Medicina Interna and Center for Research, Transfer, and High Education
DENOTHE, Università di Firenze, Firenze, and ¶Dipartimento di Biologia Sperimentale, Università degli Studi di Cagliari, Cagliari, Italy

Key words Abstract

apoptosis, cell culture techniques, fatty acids,
saturated and insaturated, fatty liver, insulin
resistance.
Accepted for publication 21 October 2008.
Correspondence
Nicola Carulli and Paola Loria, Università degli
Studi di Modena, Dipartimento di Medicina
Interna, Endocrinologia, Metabolismo e
Geriatria, Nuovo Ospedale Civile
Sant’Agostino Estense di Baggiovara, Via
Giardini Baggiovara, Modena 41100, Italy.
Email: paola.loria@unimore.it
Partly supported by grants from MIUR
Ministero Istruzione Università e Ricerca
Scientifica -PRIN 2004061213_001.
Abbreviations:
DAPI, 4′,6-diamidino-2-phenylindole
dihydrochloride; FA, Fatty Acid; FFA, Free
Fatty Acids; IR, Insulin Resistance; NEFA,
Non esterified fatty acids; OA, Oleic Acid;
PA, Palmitic Acid; T2D, Type 2 Diabetes;
TG, Triglycerides; VLDL, Very low density
lipoproteins
.
Background and Aim: Studies have shown monounsaturated oleic acid to be less toxic
than palmitic acid and to prevent/attenuate palmitic acid hepatocites toxicity in steatosis
models in vitro. However, to what degree these effects are mediated by steatosis extent is
unknown.
Methods: We evaluated whether steatosis per se is associated with hepatocytes apoptosis
and determined the role of oleic and palmitic acid, the most abundant fatty acids in western
diets, on triglyceride accumulation and apoptosis in an in vitro model of steatosis induced
in three hepatocytic cell lines (HepG2, HuH7, WRL68). The impact of incubation for 24 h
with oleic (0.66 and 1.32 mM) and palmitic acid (0.33 and 0.66 mM), alone or combined
(molar ratio 2 : 1) on steatosis, apoptosis, and insulin signalling, was evaluated.
Results: Concurrent with PPARg and SREBP-1 gene activation, steatosis extent was larger
when cells were treated with oleic than with palmitic acid; the latter fatty acid was
associated with increased PPARa expression. Cell apoptosis was inversely proportional to
steatosis deposition. Moreover, palmitic, but not oleic acid, impaired insulin signalling.
Despite the higher amount of fat resulting from incubation of the two fatty acids combined,
the apoptosis rate and impaired insulin signalling were lower than in cells treated with
palmitic acid alone, indicating a protective effect of oleic acid.
Conclusions: Oleic acid is more steatogenic but less apoptotic than palmitic acid in
hepatocityc cell cultures. These data may provide a biological basis for clinical findings on
dietary patterns and pathogenetic models of nonalcoholic fatty liver disease.

Introduction
Fatty acids (FAs) are major components of biological cell mem-
branes that play important roles in intracellular signalling and as
precursors for ligands that bind to nuclear receptors.1,2 FAs repre-
sent vital energy stores, but high-fat diets are associated with the
development of obesity and type 2 diabetes (T2D).3,4 Several lines
of evidence indicate the importance of both quantitative and quali-
tative (e.g. saturated vs unsaturated) changes in dietary FAs as
relevant mechanisms for the development of nonalcoholic fatty
liver disease (NAFLD) both in rodent models and in humans.5–9
Finally, in obese NAFLD patients, the decreased unsaturated/
saturated fatty acid ratio in serum, fat and liver tissue might have a
pathogenetic role in the disease.10,11 Increased free fatty acids (FFA)
levels are linked with the pathogenesis of insulin resistance (IR),
which is considered a major determinant in the pathogenesis of
NAFLD. Peripheral IR results in increased concentrations of circu-
lating FFAs due to unopposed antilipolytic insulin action. Addition-
ally, accumulation of FFA results in an impaired post-receptor
insulin signalling12 contributing to IR and causing a further increase
in FFA.
The hepatocytes are not a physiological site of lipid storage and
development of steatosis is associated with cellular dysfunction
and apoptosis.13,14 This phenomenon, which also occurs in the

830 Journal of Gastroenterology and Hepatology 24 (2009) 830–840 © 2009 The Authors
Journal compilation © 2009 Journal of Gastroenterology and Hepatology Foundation and Blackwell Publishing Asia Pty Ltd
M Ricchi et al.
kidney, pancreas and heart, is referred to as lipotoxicity and is
believed to play a significant role in the pathogenesis of tissue
damage.15 In fact, the extent of apoptosis correlates with the sever-
ity of steatohepatitis and the stage of fibrosis in human NAFLD.16
Lipotoxicity also contributes to decreased insulin sensitivity,
perhaps by promoting the accumulation of fat-derived metabolites
that inhibit insulin signalling and action.3,17
Fatty acids are chemically classified as saturated and unsatur-
ated (monounsaturated and polyunsaturated) and their structure
affects their biological effects. This is of particular relevance for
the most abundant FAs present in the diet and in serum, namely
palmitic acid (PA), a saturated FA, and oleic acid (OA), a monoun-
saturated FA.18 In spite of the amount of information available for
the pathogenesis of triglyceride accumulation, little is known on
the metabolic effects of PA and OA as determinants of the patho-
physiology of NAFLD.
Studies on the effect of FAs-induced steatosis on cellular apo-
ptosis have demonstrated that PA and OA mixtures-induced ste-
atosis is associated with apoptosis in hepatocyte cell cultures.19
Moreover, similar to other cell lines, monounsaturated FAs were
less toxic also in hepatocytes20 and were able to prevent/attenuate
PA toxicity.21 However, it remains unknown to what extent these
effects were mediated by degree/type of lipid accumulated in the
hepatocytes.
Aim of the present study was to evaluate the role of OA and PA,
the major FA present in western diets, on steatogenesis, cell sur-
vival, FAs composition, gene expression and insulin signalling in
an in vitro model of steatosis. Given that a priori it could not be
ruled out that specific hepatocytic cell lines might differ in their
susceptibility to steatosis/apoptosis, we carried out experiments in
three different cell lines.
Methods
Hepatocyte cell cultures
Three cell lines with different characteristics were used: (i) HepG2
cells are derived from a well differentiated human hepatoblastoma
cell line that retain many characteristics of normal differentiated
quiescent hepatocytes, and are p53 wild-type;22,23 (ii) WRL-68
cells, a fetal liver cell line;24 and (iii) p53-mutated HuH7 cells,
derived from a differentiated hepatocellular carcinoma.25 HepG2
and WRL68 cells were purchased from Istituto Zooprofilattico
Sperimentale (Brescia, Italy) and HuH7 cells from Japanese
Cancer Research Resources Bank (Osaka, Japan).
Long-chain FAs, palmitic (16:0) and oleic (18:1) were provided
as sodium salts (Sigma-Aldrich, Milan, Italy). Palmitic acid and
OA were dissolved in MetOH 99% (stock solution 100 mM).
Stock solutions were kept at -20°C before the experiments. Solu-
tions and reagents used for cell cultures were from GIBCO Life
Technologies Ltd (Grand Island, NY, USA).
Protocol of the study—induction and
evaluation of steatosis
Steatosis was induced by a slight modification of previously
described methods.26,27 HepG2, WRL-68 and HuH-7 cell cultures
were incubated with phenol red-free medium containing 10% of
charcoal stripped fetal bovine serum (FBS; Cambrex, East
Journal of Gastroenterology and Hepatology 24 (2009) 830–840 © 2009 The Authors
Journal compilation © 2009 Journal of Gastroenterology and Hepatology Foundation and Blackwell Publishing Asia Pty Ltd
Fatty acids and apoptosis in hepatocytes
Rutherford, NJ, USA), 1% bovine serum albumin (BSA), supple-
mented with FFA (oleic and palmitic acid alone or in association)
at the following final concentrations: a) PA: 0.33 mM and
0.66 mM; b) OA: 0.66 mM and 1.32 mM and c) mixtures of OA
and PA (ratio 2:1) at two different final concentrations: PA
0.33 mM + OA 0.66 mM (final fatty acids concentration 1 mM)
and PA 0.66 mM + OA 1.32 mM (final fatty acids concentration
2 mM). Control cell cultures were incubated with plain medium or
with medium added with the vehicle in which fatty acids were
dissolved. After 24 h of incubation with PA and OA alone or in
association, the extent of steatosis, apoptosis and gene expression
were evaluated as detailed above. In agreement with previous
studies,19,27 we found 24 h to be optimal incubation time. At 12 h
or less the TG accumulation was low and no detectable variations
in gene expression could be shown (data not shown). A more
prolonged incubation time (e.g. 48 h and 72 h) while not providing
a significant advantage in terms of intracellular TG accumulation
was associated with a significant decrease in cell viability at higher
FFA concentrations in our and others’ experience (data not
shown,28).
After fixation with formaldehyde, neutral lipids were stained
using 0.5% Oil-Red-O (Sigma-Aldrich) in isopropanol for 30 min
and nuclei were stained with hematoxylin.
For the electron microscopy pictures cells were detached from
the substrate with trypsin, fixed in 2.5% glutaraldeyde for 1 h and
than post-fixed in tyrode 1% OsO4 (osmium tetraoxyde) for 1 h.
Cells were then dehydrated in progressive concentrations of
ethanol, dried in propylene oxide and embedded in Durcupan’s
resin. Sections 50 nm were cut on an Ultratatome (Reichert-Jung,
Wetzlar, Germany), placed on mesh copper grids (S162, ASSING,
Rome, Italy) and stained with 7% uranyl acetate and 2.66% lead
citrate. Micro-photographs were taken by an electron microscope
(JEOL, model 2011, Peabody, MA, USA) at 200 kV.
Intracellular triglyceride content was evaluated after lysis of the
cells with NaOH 0.3 N. Triglyceride concentration (mg/dL) was
determined by standard technique with an automatic analyzer
(Roche, Milan, Italy) and normalised by protein content (mg/mL).
Total intracellular lipid content was evaluated by Nile Red stain-
ing (Adipored, Cambrex); briefly, cells were grown in 96 black-
plates and treated with FAs. At the end of incubation cells were
washed twice with phosphate buffered saline (PBS) and incubated
with Adipored for 10 min. Fluorescence was evaluated as previ-
ously described.29
Evaluation of apoptosis
Apoptosis, assessed with DAPI (4’,6-diamidino-2-phenylindole
dihydrochloride, Sigma-Aldrich) staining and caspases 3/7 activ-
ity (Promega, Milan, Italy) were evaluated as previously
described.29
Measurement of the fatty acid composition
Lipids were extracted from cells using the method of Folch et al.30
Aliquots were mildly saponified as previously described31 in order
to obtain free fatty acids for high pressure liquid chromatography
(HPLC) analysis. Separation of FAs was carried out with a Agilent
1100 HPLC system (Agilent, Palo Alto, CA, USA) equipped with
a diode array detector. A C-18 Inertsil 5 ODS-2 Chrompack
831
Fatty acids and apoptosis in hepatocytes
column (Chrompack International BV, Middleburg, The Nether-
lands), 5 mm particle size, 150 ¥ 4.6 mm, was used with a mobile
phase of CH 3 CN/H2O/CH 3 COOH (70/30/0.12, v/v/v) at a flow
rate of 1.5 mL/min. Unsaturated fatty acids were detected at
200 nm. Spectra (195–315 nm) of the eluate were obtained every
1.28 s and were electronically stored.32 These spectra were taken
to confirm the identification of the HPLC peaks.
Analysis of saturated FAs and further confirmation of unsatur-
ated fatty acids was carried out by GC assay of FA methyl esters.
Free fatty acids obtained as described above were methylated by
the addition of 14% BF3/CH3OH at room temperature and imme-
diately extracted into a solvent consisting of n-hexane and water
(4:3 ratio). After centrifugation to separate the two phases, the
hexane phase was saved and the aqueous phase was further
extracted by another round of hexane. The two hexane collections
were combined, dried, and redissolved in 500 mL of n-hexane.33
The gas chromatograph (Model 6890, Agilent) was equipped
with split ratio of 20:1 injection port, a flame ionization detector
(FID), an autosampler (Model 7673, Agilent), a 100 m HP-88
fused capillary column (Agilent), and an Agilent ChemStation
software system. The injector and detector temperatures were set
at 250°C and 280°C respectively. Hydrogen served as carrier gas
(1 mL/min), and the FID gases were H2 (30 mL/min), N2 (30 mL/
min), and purified air (300 mL/min). The temperature program
was as follows: initial temperature was 120°C, programmed at
10°C/min to 210°C and 5°C/min to 230°C, then programmed at
25°C/min to 250°C and held for 2 min.
Determination of cellular mRNA level
In HepG2 cell line total RNA was isolated using the RNeasy lipid
tissue kit (Qiagen, Milan, Italy) and quantified/checked with RNA
Nano LabChip (Agilent, Milan, Italy). About 1 mg of total RNA
was reverse-transcribed with the high capacity cDNA Archive Kit
(Applied Biosystems, Monza Italy). TaqMan polymerase chain
reactions (PCR) were performed on cDNA samples using the
TaqMan Universal PCR Master Mix (Applied Biosystems)
according to PRISM 7900 HT Sequence Detection Systems.
The TaqMan strategies for each gene have been developed as
Assay-on-Demand by Applied Biosystems. Gene expression pro-
filing was achieved using the comparative cycle threshold (CT)
method of relative quantification (the calibrator samples were non-
treated cells, with 18S RNA used as endogenous control). Data are
expressed as log2 of the relative quantity (RQ) defined also as ‘fold
induction versus the controls’.
Western blot analysis of cellular proteins
Confluent, serum-starved (12 h) HepG2 were treated with fatty
acid, after stimulation with 100 nM of insulin for 15′, the cells
were quickly placed on ice, and washed with ice-cold PBS. The
mono-layer was lysed in RIPA buffer (20 mM Tris·HCl, pH 7.4,
150 mM NaCl, 5 mM EDTA, 1% Nonidet P-40, 1 mM Na3VO4,
1 mM phenyl methyl sulfonyl fluoride, 0.01% protease inhibitor
cocktail [Protease Inhibitor Cocktail, Sigma-Aldrich]) and trans-
ferred to micro-centrifuge tubes. Insoluble proteins were discarded
by centrifugation at 12 000 rpm at 4°C. Protein concentration in
the supernatant was measured in duplicate using a commercially
available assay (Pierce, Rockford, IL, USA). Equal amounts of
832
Journal compilation © 2009 Journal of Gastroenterology and Hepatology Foundation and Blackwell Publishing Asia Pty Ltd
M Ricchi et al.
total cellular proteins were separated by sodium dodecyl sulphate-
polyacrylamide gel electrophoresis (SDS-PAGE) and analyzed by
Western blot using primary antibodies as indicated. Detection was
performed using a chemiluminescent substrate (ECL, Amersham,
IL, USA). Primary antibodies were rabbit Phospho-Akt (Ser-473)
Antibody (Cell Signalling Technology, Danvers, MA, USA) and
mouse Anti-b-Actin antibody (Sigma-Aldrich).
Statistical analysis
Results were expressed as mean ⫾ standard error (SE). All data
represent a minimum of three experiments conducted in triplicate,
unless otherwise specified. The significance of differences was
assessed by Student’s t-test for independent data. Linear regres-
sion analysis was performed by the least square method. The
differences between slope values were evaluated by the analysis of
variance. Significance was accepted at the P < 0.05 level. Statisti-
cal analysis was performed with the aid of SPSS statistical soft-
ware (version 14.0 for Windows, SPSS Inc., Chicago, IL, USA).
Results
OA and PA differentially affect triglyceride
accumulation in hepatocytic cell lines
We first analyzed the development of lipid accumulation in an in
vitro model of hepatic steatosis. HepG2 were exposed to increas-
ing concentrations of OA or PA, or to a combination of the two
fatty acids at a 2:1 ratio in favour of OA. After 24 h, lipid accu-
mulation was evident in all cells exposed to FA, as indicated by
staining with Nile Red staining (Fig. 1). The degree of fat accu-
mulation was roughly proportional to the concentration of FA to
Figure 1 Effect of different fatty acids on lipid accumulation in HepG2
cells. After 24 h of incubation (for details see materials and methods)
intracellular lipid droplets were measured by Nile Red. Data are
expressed as means ⫾ standard error of Relative Fluorescence Units
(RFLU) per mg of protein. Three experiments conducted in duplicate.
*P < 0.01 vs control; **P < 0.01 vs palmitic acid (PA) 0.33 mM;
***P < 0.01 vs PA 0.66 mM; ****P < 0.01 vs oleic acid (OA) 0.66 mM;
*****P < 0.01 vs PO1 mM.
Journal of Gastroenterology and Hepatology 24 (2009) 830–840 © 2009 The Authors
M Ricchi et al. Fatty acids and apoptosis in hepatocytes

a) b)

Figure 2 Representative electron microscopy photograph. HepG2

cells in the absence (Panel a) or presence (Panel b) of fatty acids 1 mM
(palmitic acid [PA] 0.33 mM + oleic acid [OA] 0.66 mM) for 24 h.

which cells were exposed. Similar findings were obtained for

WRL68 and HuH7 cell lines (data not shown). Finally, electron
microscopy pictures displayed a massive increase in the number
and size of lipid droplets into the cytosol of HepG2 cells incubated
with FAs as compared to controls (Fig. 2).
To evaluate quantitatively the relation between the type of FA
and the degree of triglyceride accumulation, we measured triglyc-
eride (TG) concentration in cells lysates (Fig. 3). All treatments
with FA caused a significant increase in triglyceride content of all
three cell lines tested in comparison to control cells. In addition,
there was a clear dose-dependency for all conditions tested.
However, OA induced a significantly higher triglyceride accumu-
lation than PA when used at equimolar concentrations (Fig. 3).
This effect was evident in all three lines, and was statistically
significant for HepG2 and WRL-68. Incubation of all cell cultures
with mixtures of both FAs showed a dose dependent increase in the
amount of triglycerides which was almost equivalent to the sum of
triglyceride levels induced by separate incubation with the two
different FA. Finally, the concentration of triglyceride in HepG2
cells was always greater than that found in WRL-68 cells but lower
than in HuH-7 treated with the same experimental schedule. To
sum up, the three different cell lines display a more pronounced
steatosis response in the order PA > PA + OA > OA; for each FA a
dose-response relationship was observed. However, the absolute Figure 3 Effect of different fatty acids on triglycerides accumulation.
ability to accumulate triglycerides was decreasing in the order HepG2 (Panel a), WRL-68 (Panel b) and HuH-7 (Panel c) cell cultures
HuH-7 > HepG2 > WRL-68. were incubated with palmitic acid (PA) (0.33 and 0.66 mM), oleic acid
(OA) (0.66 and 1.32 mM) or mixtures of the two fatty acids (1 mM or
2 mM) for 24 h. Triglyceride (TG) accumulation was evaluated as the
concentration of TGs in cell lysates after NaOH lysis. Columns represent
PA is a stronger apoptotic stimulus than OA
mean values ⫾ standard error of three different experiments conducted
We next analyzed the effects of the different FAs treatments on in triplicate. *P < 0.05 vs control; **P < 0.05 vs PA 0.66 mM.
programmed cell death in the three different lines of hepatocytic
cells, using the percentage of cells showing nuclear condensation
when stained with DAPI29 (Fig. 4). Palmitic acid at a concentration
of 0.33 mM increased significantly (P < 0.05) the percentage of indicate that PA, but not OA induces apoptosis in cultured
apoptotic cells only in HuH7 cell cultures while at 0.66 mM apo- hepatocytes.
ptosis was significantly increased in all cell lines tested. In con- Co-incubation with the two FAs at the lower concentrations
trast, OA, used at concentrations as high as 1.32 mM did not (1 mM) did not modify the rate of apoptosis. At the higher con-
induce any increase in the number of apoptotic cells as compared centrations (0.66 mM PA together with 1.32 mM OA) an increase
to untreated controls. Remarkably, comparison of the effects of the in the percentage of apoptotic cells was observed in all cell lines.
two FA used at equimolar concentrations showed a significantly However, in two out of three lines, the increase in apoptosis was
higher ability of PA to induce apoptosis in all cell lines. These data significantly lower in the presence of OA than when PA at a

Journal of Gastroenterology and Hepatology 24 (2009) 830–840 © 2009 The Authors 833
Journal compilation © 2009 Journal of Gastroenterology and Hepatology Foundation and Blackwell Publishing Asia Pty Ltd
Fatty acids and apoptosis in hepatocytes
Figure 4 Effect of fatty acids on apoptosis rate. In HepG2 (Panel a),
WRL-68 (Panel b) and HuH-7 (Panel c) apoptosis was quantified by
assessing the characteristic nuclear changes of apoptosis using the
nuclear binding dye 4’,6-diamidino-2-phenylindole dihydrochloride
(DAPI) and counting cell numbers under a fluorescent microscopy.
Columns represent mean values ⫾ standard error of three different
experiments conducted in triplicate. *P < 0.05 vs control; **P < 0.05 vs
palmitic acid 0.66 mM.
concentration of 0.66 mM was used alone. These data are strongly
suggestive of a protective effect of OA on apoptosis induced
by PA.
To add further evidence to the differential effects of the two fatty
acids on apoptosis, we measured the activity of caspases 3/7, that
belong to the group of the execution caspases, activated once the
cell death program is fully operative (Fig. 5). In cells exposed to
PA, the activity of caspases 3/7 increased in a dose-dependent
fashion. In contrast, OA did not have any effects on caspase
834
Journal compilation © 2009 Journal of Gastroenterology and Hepatology Foundation and Blackwell Publishing Asia Pty Ltd
M Ricchi et al.
Figure 5 Effect of fatty acids on caspases 3/7 activity. In HepG2 (Panel
a), WRL-68 (Panel b) and HuH-7 (Panel c) caspases 3/7 activity was
quantified measuring the fluorescence from Z-DEVD-R110 cleavage and
normalised by protein content in parallel plates. Columns represent
mean values ⫾ standard error of three different experiments conducted
in triplicate. *P < 0.05 vs control; **P < 0.05 vs palmitic acid (PA)
0.66 mM; ***P < 0.05 vs PA 0.33 mM.
activity except in HepG2, where a significant increase was
observed. However, it should be stressed that the increase in
response to OA was only 1–2 fold as compared to control, while
PA induced an 8–16 fold increase. As a result, caspases 3/7 activity
in HepG2 exposed to OA was always significantly lower than in
cells treated with PA.
In co-incubation experiments, the addition of OA to PA
decreased caspases activity in comparison to cells treated with PA
alone in HepG2 and in WRL68 but not in HuH7 cell line. Thus,
data on caspases activity are closely similar to those obtained with
DAPI staining.
Journal of Gastroenterology and Hepatology 24 (2009) 830–840 © 2009 The Authors
M Ricchi et al. Fatty acids and apoptosis in hepatocytes

PA and OA are associated with different FA

584.360*

578.460*

991.330*

662.510*

1058.980*
355.990
58.228
440.570
36.410

69.171

28.104

64.352

90.473

92.724
Total FAs
profiles in HepG2 cells
We hypothesized that the mechanisms of cell toxicity due to palm-
itic acid could be ascribed to different fatty acid profiles and to the
esterified/unesterified ratio. To test this hypothesis, we compared

1.674
0.018
1.646
0.388
1.786
0.289
1.772
0.195
2.303
0.172
1.806
0.209
2.154
0.202
22:0
individual and total fatty acid composition obtained by HPLC with
the results of TG content assessed enzymatically.
The absolute FAs composition in steatotic HepG2 cultures after

1.500
0.213
1.771
0.885
1.814
0.173
1.616
0.120
2.120
0.238
1.676
0.154
1.480
0.319
20:0
exposure to different treatments is shown in Table 1. All treatments
induced significant changes in the concentration of various FAs.
PA and OA concentration increased after incubation with the

37.363*

33.767*
23.949
4.812
25.694
4.244
28.533
3.536
22.165
1.211

1.287
27.514
3.065

4.272
respective FA. In addition, while PA incubation was associated

18:0
with an increase 16:1, 18:1c11 and 14:0, the incubation with OA
induced an increase of 18:0, 16:0 and 14:0. As expected, the

150.288*

223.582*

152.017*

169.168*

280.682*
incubation with the two FAs induced a combined pattern and was

99.095
20.159

20.891

40.311
104.335
8.484

5.296

18.811

27.117
also associated with a reduction in the concentration of 16:1 as

16:0
compared to PA alone. The putative metabolic fate of FAs added to
the medium based on results shown in Table 1 is depicted in

2.654
0.082
2.791
0.653
3.405
0.431
3.404
0.581
3.447
0.300
3.084
0.363
3.601
0.019
Figure 6.

15:0
Total FA content was proportional to the moles of fatty acids
added to the medium (data not shown). Contrasting with data on

15.323*

17.634*

16.687*
TG content, we found that OA and PA at equimolar doses induced

10.308
2.169
12.053
0.067

1.112
12.083
1.790

2.344
13.505
3.052

1.022
a comparable accumulation of total fatty acids. This strongly sug-
14:0
Fatty acid profile of HepG2 cells incubated with palmitic acid (PA), oleic acid (OA) or PA and OA combined
gests that after incubation with PA the concentration of TG is lower
(Fig. 1) than total FA concentration due to the presence of a con-

2.659*
sistent FFA not being detected in the total TG assay. It could be 1.567
0.158
1.584
0.178
1.859
0.441
1.314
0.504
1.542
0.181
1.625
0.070

0.154
12:0

indirectly estimated that the percentage of FFAs following incu-

bation with PA is in the order of magnitude of 15% (vs 0–1%
following incubation with OA).
18:1 c11

97.634*

86.541*
63.996
10.617
74.485
0.586

5.627
66.224
3.265
92.485
2.594
70.854
21.176

5.623
FAs structure and not TG content is the key
determinant of apoptosis
291.330*

594.991*

293.478*

532.038*
80.847
15.220
85.252
5.729
99.727
9.266

3.547

29.215

35.181

35.333
To better define the relationship between the degree of steatosis
18:1

and induction of apoptosis, we plotted the intracellular TG con-

centration with the activity of caspases 3/7 (Fig. 7). While these
7.276
0.345
6.696
0.036
8.037
0.353
8.441
1.174
11.683
3.459
8.433
1.808
9.594
0.610
parameters were significantly correlated for both FA, the slope of
22:4

the curve obtained with PA was significantly steeper than the one
observed for OA. It is of relevance that the regression for com-
bined treatments fall in between PA and OA, again suggesting a
16.400
0.207
17.143
0.207
21.924
0.833
18.484
3.109
20.588
3.826
19.636
2.014
23.122
6.802
18:2

protective effect of OA on PA induced cytotoxicity. Similar rela-

tionships were observed also plotting TG versus percentage of
apoptosis evaluated by DAPI staining (data not shown). These data
8.966
0.392
8.180
0.291
10.138
1.054
10.061
1.151
11.745
1.948
9.466
0.433
10.301
0.662

confirm that for a given amount of intracellular triglycerides, the

20:4

Data are expressed as nmoles/mg of proteins.

degree of apoptosis depends on the FA used for the induction of

steatosis being far higher for PA, followed by their association and
47.159*

68.936*

49.472*

by OA.
31.346
3.168

1.939

3.297
30.651
2.227
35.756
12.192
36.038
3.719

10.301
16:1

Gene expression
5.139
0.603
4.653
0.274
5.336
0.415
5.218
0.539
6.240
1.246
5.061
0.319
5.445
0.263
22:6

The expression of PPARalpha, gamma and SREBP-1 constantly

increased following exposure to FAs. However, as shown in
*P < 0.05 vs control.

Figure 8, PA but not OA induced a significant increase in PPARa

1.274
0.065
1.176
0.043
1.327
0.033
1.362
0.208
1.415
0.054
1.167
0.100
1.436
0.025

expression vs controls (P < 0.05). In contrast, incubation of

20:5

HepG2 with OA but not with PA was associated, at both concen-

trations used, with an increase in the expression of PPARg vs
Table 1

control

controls (P < 0.05). In addition OA at the highest concentration

O 0,6

O 1,3
P 0,3

P 0,6

PO 1

PO 2
SD

SD

SD

SD

SD

SD

SD

increased the expression of SREBP-1 vs controls (P < 0.05). When

Journal of Gastroenterology and Hepatology 24 (2009) 830–840 © 2009 The Authors 835
Journal compilation © 2009 Journal of Gastroenterology and Hepatology Foundation and Blackwell Publishing Asia Pty Ltd
Fatty acids and apoptosis in hepatocytes M Ricchi et al.

Figure 6 Putative metabolic pathway compatible with changes in fatty

acid profile observed after adding palmitic acid (PA) and oleic acid (OA)
to cell cultures base on findings shown in Table 1. PA (16:0) increases
the concentration of 16:1, 18:1c11 presumably by stimulating delta 9
desaturation of PA yielding 16:1, further elongation (18:1c11). The
increase of 14:0 may be ascribed to peroxisomal beta oxidation of PA.
OA (18:1c9) increases 18:0, 16:0 and 14:0. 18:0 increase might be due
to a partial inhibition of SCD1 with a consequent increase of 16:0 and
14:0 as products of peroxisomal beta oxidation of 18:0.

Figure 7 Relationship between extent of triglyceride (TG) accumula-

tion and caspases 3/7 activity. Regression lines represent the relation-
ship between the two parameters after incubation of HepG2 cells
respectively with palmitic acid (PA; 䉲), PA + oleic acid (OA; 䊉) and OA
(䊏). Single points are the mean of three experiments that have in
parallel evaluated TGs content and caspases activity. The slope values
of PA vs PA + OA (t = 6.805; P < 0.0001)and OA vs PA + OA (t = -5.353;
P < 0.0001) regression lines are significantly different.

the effect of two equimolar doses of PA and OA were compared,

the difference did not reach statistical significance.

PA inhibits signaling downstream of the
insulin receptor
Peripheral IR, is caused, at least in part, by blunted transmission
of the signal along the enzymatic cascade downstream of the
insulin receptor. A critical step in this pathway is represented by
Figure 8 Effect of palmitic acid (PA) and oleic acid (OA) on PPARg,
SREBP-1 and PPARa gene expression. Expression was evaluated by
real time-polymerase chain reaction (PCR; for details see materials and
methods). Data are reported as mean ⫾ standard error of log2(RQ) nor-
malized to the controls. *P < 0.05 vs control.

836 Journal of Gastroenterology and Hepatology 24 (2009) 830–840 © 2009 The Authors
Journal compilation © 2009 Journal of Gastroenterology and Hepatology Foundation and Blackwell Publishing Asia Pty Ltd
M Ricchi et al.
Figure 9 Effect of fatty acids (FAs) on Akt phosphorylation. Akt phos-
phorylation was evaluated in confluent, serum-starved (12 h) HepG2
were treated with FAs, after stimulation with 100 nM of insulin for 15′
and analyzed by Western blot using Phospho-Akt (Ser-473) Antibody.
phosphorylation and activation of the serine/threonine kinase
Akt, a central regulator of glucose uptake, anabolic metabolism
and antiapoptotic signals.34 We compared the effects of steatosis
induced by the different FAs, alone or in association, on insulin-
induced phosphorylation of Akt (Fig. 9). Insulin caused a rapid
and robust phosphorylation of Akt on Ser473, an activation-
specific residue, in HepG2 cells. Steatosis induced by PA was
associated with a marked decrease of insulin-induced Akt phos-
phorylation, while in cells treated with OA Akt phosphorylation
was maintained. Interestingly, in cells exposed to both FA, an
intermediate level of phosphorylation was detected. No differ-
ences were found among different fatty acid treatments in the
activity of ERK1-2 (data not shown). Taken together, these data
demonstrate that PA and OA differentially affect the early signal-
ing pathways downstream of the insulin receptor.
Discussion
In this in vitro model of steatogenesis, three different lines of
hepatocytes were exposed to increasing doses of FAs for
comparison purposes. The effects of administering OA, PA or
their combination on TG accumulation, cell survival, FAs
composition, gene expression and insulin signalling were
explored. Data showed that while the extent of steatosis was more
severe in cells treated with OA than in those exposed to PA,
opposite effects of the two FAs were found on cell apoptosis. The
greater steatosis extent observed following exposure to OA was
associated with prominent PPARg and SREBP-1 activation. In
contrast, PA supplementation was associated with PPARa activa-
tion. Moreover, PA, but not OA, impaired insulin signalling.
Despite the higher steatosis extent resulting from incubation with
the two FAs combined, apoptosis rate and insulin signalling
impairment were lower than in cells treated with PA alone, indi-
cating a protective effect of OA.
As expected, in our study the accumulation of TGs was propor-
tional to the concentration of FAs in the culture medium. However,
OA was a more powerful trigger for TG accumulation than PA
irrespective of the cell line we used. This is a novel finding for
hepatocytes and may represent a general property of OA, which
has been previously reported to induce a more marked fat
Journal of Gastroenterology and Hepatology 24 (2009) 830–840 © 2009 The Authors
Journal compilation © 2009 Journal of Gastroenterology and Hepatology Foundation and Blackwell Publishing Asia Pty Ltd
Fatty acids and apoptosis in hepatocytes
accumulation also in other cell types, such as islet b cells.35 It is
tempting to associate the more effective steatogenic property of
OA with the specific pattern of gene expression (PPAR-gamma
and SREBP-1) shown in the present study. In contrast, the lesser
steatosis extent and increased apoptosis are due to PPAR-alpha
activation which results in enhanced b-oxidation and thus oxida-
tive stress.36
The most intriguing finding of the present study is the dissocia-
tion between the effects on steatosis and apoptosis of the different
FAs. Steatosis per se induces cell apoptosis.14,15 Other studies have
indicated a greater pro-apoptotic effect of PA as compared to OA,
but few studies have examined the relationship between steatosis
extent and apoptosis in hepatic cell cultures.20,28 The finding that
OA is less efficient in the induction of apoptosis is in agreement
with the recent work by Mahli et al. who, however, at variance
with our study, found no differences in the steatogenic effect of
OA and PA.20 This discrepancy might probably be accounted for
by differences in the experimental conditions notably including the
lower (0.2 mM in Mahli’s vs 0.66 mM in our study) FA concen-
trations used.19 In this study, we have shown a correlation between
TG accumulation and apoptosis with both OA and PA. However,
the slope of this function was markedly different comparing the
two FAs indicating that, for a given amount of TG accumulation,
the effects on apoptosis were dramatically enhanced when cells
were treated with PA (Fig. 6).
A large number of molecular mechanisms have been implicated
in PA-associated apoptosis: ceramide production, NO synthesis,
suppression of antiapoptotic factors such as Bcl-2,15 reactive
oxygen species generation,37 endoplasmic reticulum stress;37,38
nuclear factor-kB activation39 and decreased synthesis of cardio-
lipin.40 In hepatocytes, steatosis may enhance the expression of
inflammatory cytokines, such as TNF-a, and increase apoptosis
via Fas receptor activation.16,19,27 A JNK stimulated mitochon-
drial20 and a reticulum stress-mediated9,21 apoptotic pathways have
been also reported. Death receptors are cell surface proteins, which
can trigger apoptosis when bound by their ligands. Ribeiro &
Cortez-Pinto41 observed enhanced expression of the death receptor
Fas in alcoholic hepatitis and tumor necrosis factor (TNF)
receptor-1 in NASH. Others have identified enhanced Fas expres-
sion in alcoholic and nonalcoholic steatohepatitis16,42 Thus steato-
hepatitis may sensitize hepatocytes to extracellular death ligands
(e.g. Fas ligand, tumor necrosis factor-alpha) promoting an extrin-
sic pathway to apoptosis.19,27 NF-kB is a transcription factor that
can upregulate both death receptors and ligands.43,44 NF-kB acti-
vation in Kupffer cells or infiltrating monocytes is proinflamma-
tory and induces the expression of death ligands such as TNFa.
FFAs can directly activate the IKK-k/ NF-kB pathway in hepa-
tocytes via a lysosomal, cathepsin B– dependent mechanism.27
This pathway involves the translocation of Bax to lysosomes with
subsequent lysosomal destabilization and release of the cysteine
protease, cathepsin B, into the cytosol. This subsequently leads to
the activation of NF-kB, via IKK-k, and a subsequent increase in
the expression of TNFa apoptosis has increasingly been linked to
inflammation and hepatic fibrogenesis.45–47
More recently, Gores’ group48 reported OA to sensitise hepato-
cytes in vitro to to the death ligand TRAIL. Oleic acid led to
upregulation of the cognate TRAIL receptor death receptor 5
whose expression was enhanced in steatotic human liver samples.
DR5 was responsible for FFA sensitisation to TRAIL killing medi-
837
Fatty acids and apoptosis in hepatocytes
ated by a JNK dependent mechanism. Moreover, as previously
reported, if Kupffer cells are activated they produce death ligands,
including Fas ligand, and TNFa which, in turn, induce apoptosis
in hepatocytes expressing death receptors,49 this might be also a
condition in NASH patients. Given that inhibiting apoptosis may
well prevent the feared consequences of chronic liver disease,
cirrhosis, portal hypertension, and liver failure thus drugs are
being developed to block apoptosis.50
The cells’ ability to readily incorporate FAs into cytoplasmic
TGs might serve as a protection against their pro-apoptotic
effects.51 The finding that OA resulted in a greater steatosis extent
implies that this FA is more readily incorporated into TG and
therefore is associated with less apoptosis than PA which was less
steatogenic. Along these lines, the cytoprotective effect of OA in
vitro could thus be explained by this FA’s ability to promote
channelling of PA into TG synthesis, as demonstrated in cultured
fibroblasts.51 The toxic effect of PA on hepatocytic cell cultures
could be, at least, in part related to the amount of FFA associated
with incubation with this FA. However, given that this explanation
is based on in vitro indirect data, our hypothesis needs to be
verified by further studies given that a recent report disproves the
paradigm that FFAs are increased in human NAFLD.52 In addition
to PA channelling into TG synthesis, changes in the composition of
the intracellular FAs pool may represent an alternative strategy to
protect cells from PA-dependent apoptosis. The addition of PA to
the medium resulted in an increased proportion of the less dam-
aging palmitoleic acid indicating the attempt to ‘detoxify’ PA. This
detoxification might be achieved through increased steroyl-CoA
desaturase 1 (D9) activity (Fig. 8), which protects from
PA-induced apoptosis.53
Our data provide in vitro evidence for the principle that TG
accumulation might be a defence mechanism against the toxicity
of excess FFAs. Yamaguchi et al. recently reached the same con-
clusion in the obese mouse model, wherein inhibited triglyceride
synthesis was associated with improved hepatic steatosis though
to the expenses of worsened hepatic inflammatory and fibrotic
changes.54 Observations that simple steatosis is rarely progressive
in humans55–57 further confirm that FAs stored as inert triglycerides
are harmless unless associated to another injuring factor, such as
IR, or oxidative stress, that may switch on the inflammatory
cascade. Familial heterozygous hypobetalipoproteinemia repre-
sents a naturally occurring model of human simple steatosis due to
genetically impaired capacity to export VLDL from the hepato-
cytes into the bloodstream. Of interest, in the absence of IR,
steatosis in these individuals, although massive, does not appear to
be progressive.58 Studies have shown that PA impairs insulin sig-
nalling via increased JNK activity leading, in turn, to phosphory-
lation of insulin receptor substrate-1 and 2 at the inhibitory sites
eventually leading to IR.59 Increased circulating FFAs concentra-
tions represent an early correlate of peripheral IR, which may be
demonstrated to occur before the development of obesity in
experimental condition.12 In this connection, our data have shown
that PA administration, in itself, impaired insulin signalling while
OA partially restored it. This finding implies that the steatosis
extent is not the only, nor perhaps the major, determinant of
impaired insulin action. Indeed, rat models7,9 and human stud-
ies5,6,8 consistently support the concentrations and the chemical
structure of FAs to be fundamental players in the development
and progression of NAFLD. In human NAFLD, FAs that are
838
Journal compilation © 2009 Journal of Gastroenterology and Hepatology Foundation and Blackwell Publishing Asia Pty Ltd
M Ricchi et al.
incorporated in hepatic TG are mostly derived from the circulating
NEFA pool and thus reflect the adipose tissue FAs composition.60
Individuals consuming western-type high-fat diets show a serum/
tissue predominance of PA with a relative reduction in OA.6,10,11 In
principle, characterization of FAs profile in the serum/liver may
assist in predicting those individuals with progressive/severe
NAFLD.20 Our data also highlight that variations in the desaturase
activity might have a role in the type of FAs stored in hepatic TG
and thus in the pathogenesis of human NAFLD. Conversely, PA
deprivation and OA enrichment in diet might be a goal to prevent/
treat the disease.
In conclusion, while a limitation of the current study is the use
of three hepatocyte cultures of different origin but no data on
primary hepatocyte culture, our study showing that OA is more
steatogenic but less damaging than PA in hepatocyte cell cultures
may provide a biological clue useful to a better understanding of
animal models of NAFLD. As far as human NAFLD is concerned,
our data might suggest two major clinical implications/research
hypothesis. First, for the diagnosis of NASH, steatosis extent
evaluated histologically might not be as relevant as its chemical
composition. Second, in agreement with recent studies envisaging
for saturated FAs a role of ‘second hit’,61 the chemical composition
of steatosis might help differentiate those non-progressive (‘inert’)
forms of NAFLD from those that, due to their enrichment in
saturated FAs, are at a substantial risk of progression. These
hypotheses are worthy being tested in specific studies.
Acknowledgments
We thank Dorval Ganazzi for the assistance in statistical analysis.
We are also grateful to Dr. Cristiana Bertolani and Nadia Navari,
coworkers of Prof. Marra, for their contribution.
Part of these data have been presented in abstract form at the
European Association for the Study of the Liver, Wien, April 2006.
References
1 Chawla A, Repa JJ, Evans RM, Mangelsdorf DJ. Nuclear receptors
and lipid physiology: opening the x-files. Science 2001; 294:
1866–70.
2 Clarke SD. The multi-dimensional regulation of gene expression by
fatty acids: polyunsaturated fats as nutrient sensors. Curr. Opin.
Lipidol. 2004; 15: 13–18.
3 McGarry JD. Banting lecture 2001: dysregulation of fatty acid
metabolism in the etiology of type 2 diabetes. Diabetes 2002; 51:
7–18.
4 Haslam DW, James WP. Obesity. Lancet 2005; 366: 1197–209.
5 Nehra V, Angulo P, Buchman AL, Lindor KD. Nutritional and
metabolic considerations in the etiology of non-alcoholic
steatohepatitis. Dig. Dis. Sci. 2001; 46: 2347–52.
6 Musso G, Gambino R, De Michieli F et al. Dietary habits and their
relations to insulin resistance and postprandial lipemia in
nonalcoholic steatohepatitis. Hepatology 2003; 37: 909–16.
7 Hernandez R, Martinez-Lara E, Canuelo A et al. Steatosis recovery
after treatment with a balanced sunflower or olive oil-based diet:
involvement of perisinusoidal stellate cells. World J. Gastroenterol.
2005; 11: 7480–5.
8 Cortez-Pinto H, Jesus L, Barros H, Lopes C, Moura MC,
Camilo ME. How different is the dietary pattern in non-alcoholic
steatohepatitis patients? Clin. Nutr. 2006; 25: 816–23.
Journal of Gastroenterology and Hepatology 24 (2009) 830–840 © 2009 The Authors
M Ricchi et al.
9 Wang D, Wei Y, Pagliassotti MJ. Saturated fatty acids promote
endoplasmic reticulum stress and liver injury in rats with hepatic
steatosis. Endocrinology 2006; 147: 943–51.
10 De Almeida IT, Cortez-Pinto H, Fidalgo G, Rodrigues D,
Camilo ME. Plasma total and free fatty acids composition in human
non-alcoholic steatohepatitis. Clin. Nutr. 2002; 21: 219–23.
11 Videla LA, Rodrigo R, Araya J, Poniachik J. Oxidative stress and
depletion of hepatic long-chain polyunsaturated fatty acids may
contribute to nonalcoholic fatty liver disease. Free Radic. Biol. Med.
2004; 37: 1499–507.
12 Samuel VT, Liu ZX, Qu X et al. Mechanism of hepatic insulin
resistance in non-alcoholic fatty liver disease. J. Biol. Chem. 2004;
279: 32345–53.
13 Browning JD, Horton JD. Molecular mediators of hepatic steatosis
and liver injury. J. Clin. Invest. 2004; 114: 147–52.
14 Canbay A, Gieseler RK, Gores GJ, Gerken G. The relationship
between apoptosis and non-alcoholic fatty liver disease: an
evolutionary cornerstone turned pathogenic. Z. Gastroenterol. 2005;
43: 211–17.
15 Unger RH, Orci L. Lipoapoptosis: its mechanism and its diseases.
Biochim. Biophys. Acta 2002; 1585: 202–12.
16 Feldstein AE, Canbay A, Angulo P et al. Hepatocyte apoptosis and
fas expression are prominent features of human nonalcoholic
steatohepatitis. Gastroenterology 2003; 125: 437–43.
17 Schmitz-Peiffer C, Craig DL, Biden TJ. Ceramide generation is
sufficient to account for the inhibition of the insulin-stimulated PKB
pathway in C2C12 skeletal muscle cells pretreated with palmitate. J.
Biol. Chem. 1999; 274: 24202–10.
18 Baylin A, Kabagambe EK, Siles X, Campos H. Adipose tissue
biomarkers of fatty acid intake. Am. J. Clin. Nutr. 2002; 76:
750–7.
19 Feldstein AE, Canbay A, Guicciardi ME, Higuchi H, Bronk SF,
Gores GJ. Diet associated hepatic steatosis sensitizes to Fas
mediated liver injury in mice. J. Hepatol. 2003; 39: 978–83.
20 Malhi H, Bronk SF, Werneburg NW, Gores GJ. Free fatty acids
induce JNK-dependent hepatocyte lipoapoptosis. J. Biol. Chem.
2006; 281: 12093–101.
21 Wei Y, Wang D, Topczewski F, Pagliassotti MJ. Saturated fatty acids
induce endoplasmic reticulum stress and apoptosis independently of
ceramide in liver cells. Am. J. Physiol. Endocrinol. Metab. 2006;
291: E275–81.
22 Javitt NB. Hep G2 cells as a resource for metabolic studies:
lipoprotein, cholesterol, and bile acids. FASEB J. 1990; 4:
161–8.
23 Dashti N, Wolfbauer G. Secretion of lipids, apolipoproteins, and
lipoproteins by human hepatoma cell line, HepG2: effects of oleic
acid and insulin. J. Lipid Res. 1987; 28: 423–36.
24 Gutierrez-Ruiz MC, Bucio L, Souza V, Gomez JJ, Campos C,
Carabez A. Expression of some hepatocyte-like functional properties
of WRL-68 cells in culture. In Vitro Cell. Dev. Biol. Anim. 1994;
30A: 366–71.
25 Nakabayashi H, Taketa K, Yamane T, Miyazaki M, Miyano K,
Sato J. Phenotypical stability of a human hepatoma cell line, HuH-7,
in long-term culture with chemically defined medium. Gann 1984;
75: 151–8.
26 Okamoto Y, Tanaka S, Haga Y. Enhanced GLUT2 gene expression
in an oleic acid-induced in vitro fatty liver model. Hepatol. Res.
2002; 23: 138–44.
27 Feldstein AE, Werneburg NW, Canbay A et al. Free fatty acids
promote hepatic lipotoxicity by stimulating TNF-alpha expression
via a lysosomal pathway. Hepatology 2004; 40: 185–94.
28 Gomez-Lechon MJ, Donato MT, Martinez-Romero A, Jimenez N,
Castell JV, O’Connor JE. A human hepatocellular in vitro model to
investigate steatosis. Chem. Biol. Interact. 2007; 165: 106–16.
Journal of Gastroenterology and Hepatology 24 (2009) 830–840 © 2009 The Authors
Journal compilation © 2009 Journal of Gastroenterology and Hepatology Foundation and Blackwell Publishing Asia Pty Ltd
Fatty acids and apoptosis in hepatocytes
29 Ricchi M, Bertolotti M, Anzivino C et al. 17Beta-estradiol prevents
cytotoxicity from hydrophobic bile acids in HepG2 and WRL-68 cell
cultures. J. Gastroenterol. Hepatol. 2006; 21: 894–901.
30 Folch J, Lees M, Sloane Stanley GH. A simple method for the
isolation and purification of total lipides from animal tissues. J. Biol.
Chem. 1957; 226: 497–509.
31 Banni S, Carta G, Contini MS et al. Characterization of conjugated
diene fatty acids in milk, dairy products, and lamb tissues. J. Nutr.
Biochem. 1996; 7: 150–5.
32 Melis MP, Angioni E, Carta G et al. Characterization of conjugated
linoleic acid and its metabolites by RP-HPLC with diode array
detector. Eur. J. Lipid Sci. Technol. 2001; 103: 617–21.
33 Ip C, Banni S, Angioni E et al. Conjugated linoleic acid-enriched
butter fat alters mammary gland morphogenesis and reduces cancer
risk in rats. J. Nutr. 1999; 129: 2135–42.
34 Whiteman EL, Cho H, Birnbaum MJ. Role of Akt/protein kinase B
in metabolism. Trends Endocrinol. Metab. 2002; 13: 444–51.
35 Cnop M, Hannaert JC, Hoorens A, Eizirik DL, Pipeleers DG. Inverse
relationship between cytotoxicity of free fatty acids in pancreatic
islet cells and cellular triglyceride accumulation. Diabetes 2001; 50:
1771–7.
36 Li Z, Berk M, McIntyre TM, Gores GJ, Feldstein AE. The
lysosomal-mitochondrial axis in free fatty acid-induced hepatic
lipotoxicity. Hepatology 2008; 47: 1495–503.
37 Listenberger LL, Ory DS, Schaffer JE. Palmitate-induced apoptosis
can occur through a ceramide-independent pathway. J. Biol. Chem.
2001; 276: 14890–5.
38 Karaskov E, Scott C, Zhang L, Teodoro T, Ravazzola M, Volchuk A.
Chronic palmitate but not oleate exposure induces endoplasmic
reticulum stress, which may contribute to INS-1 pancreatic beta-cell
apoptosis. Endocrinology 2006; 147: 3398–407.
39 Rakatzi I, Mueller H, Ritzeler O, Tennagels N, Eckel J.
Adiponectin counteracts cytokine- and fatty acid-induced apoptosis
in the pancreatic beta-cell line INS-1. Diabetologia 2004; 47:
249–58.
40 Ostrander DB, Sparagna GC, Amoscato AA, McMillin JB,
Dowhan W. Decreased cardiolipin synthesis corresponds with
cytochrome c release in palmitate-induced cardiomyocyte apoptosis.
J. Biol. Chem. 2001; 276: 38061–7.
41 Ribeiro PS, Cortez-Pinto H, Sola S et al. Hepatocyte apoptosis,
expression of death receptors and activation of NfkappaB in the liver
of non-alcoholic and alcoholic steatohepatitis patients. Am. J.
Gastroenterol. 2004; 99: 1718–19.
42 Natori S, Rust C, Stadheim LM et al. Hepatocyte apoptosis is a
pathologic feature of human alcoholic hepatitis. J. Hepatol. 2001;
34: 248–53.
43 Ghosh S, May MJ, Kopp EB. NF-kappa B and Rel proteins:
evolutionarily conserved mediators of immune responses. Annu. Rev.
Immunol. 1998; 16: 225–60.
44 Green DR. Death and NF-kappaB in T cell activation: life at the
edge. Mol. Cell 2003; 11: 551–2.
45 Faouzi S, Burckhardt BE, Hanson JC et al. Anti-Fas induces hepatic
chemokines and promotes inflammation by an Nfkappa
B-independent, caspase-3-dependent pathway. J. Biol. Chem. 2001;
276: 49077–82.
46 Canbay A, Guicciardi ME, Higuchi H et al. Cathepsin B inactivation
attenuates hepatic injury and fibrosis during cholestasis. J. Clin.
Invest. 2003; 112: 152–9.
47 Canbay A, Taimr P, Torok N et al. Apoptotic body engulfment by a
human stellate cell line is profibrogenic. Lab. Invest. 2003; 83:
655–63.
48 Malhi H, Barreyro FJ, Isomoto H, Bronk SF, Gores GJ. Free fatty
acids sensitise hepatocytes TRAIL mediated cytotoxicity. Gut 2007;
56: 1124–31.
839
Fatty acids and apoptosis in hepatocytes
49 Canbay A, Feldstein AE, Higuchi H et al. Kupffer cell engulfment
of apoptotic bodies stimulates death ligand and cytokine expression.
Hepatology 2003; 38: 1188–98.
50 Feldstein A, Gores GJ. Steatohepatitis and apoptosis: therapeutic
implications. Am. J. Gastroenterol. 2004; 99: 1718–19.
51 Listenberger LL, Han X, Lewis SE et al. Triglyceride accumulation
protects against fatty acid-induced lipotoxicity. Proc. Natl. Acad. Sci.
USA 2003; 100: 3077–82.
52 Puri P, Baillie RA, Wiest MM et al. A lipidomic analysis of
nonalcoholic fatty liver disease. Hepatology 2007; 46: 1081–90.
53 Warensjo E, Ohrvall M, Vessby B. Fatty acid composition and
estimated desaturase activities are associated with obesity and
lifestyle variables in men and women. Nutr. Metab. Cardiovasc. Dis.
2006; 16: 128–36.
54 Yamaguchi K, Yang L, McCall S et al. Inhibiting triglyceride
synthesis improves hepatic steatosis but exacerbates liver damage
and fibrosis in obese mice with nonalcoholic steatohepatitis.
Hepatology 2007; 45: 1366–74.
55 Matteoni CA, Younossi ZM, Gramlich T, Boparai N, Liu YC,
McCullough AJ. Nonalcoholic fatty liver disease: a spectrum of
clinical and pathological severity. Gastroenterology 1999; 116:
1413–19.
840
Journal compilation © 2009 Journal of Gastroenterology and Hepatology Foundation and Blackwell Publishing Asia Pty Ltd
M Ricchi et al.
56 Harrison SA, Neuschwander-Tetri BA. Nonalcoholic fatty liver
disease and nonalcoholic steatohepatitis. Clin. Liver Dis. 2004; 8:
861–79.
57 Adams LA, Lymp JF, St Sauver J et al. The natural history of
nonalcoholic fatty liver disease: a population-based cohort study.
Gastroenterology 2005; 129: 113–21.
58 Lonardo A, Lombardini S, Scaglioni F et al. Hepatic steatosis and
insulin resistance: does etiology make a difference? J. Hepatol.
2006; 44: 190–6.
59 Solinas G, Naugler W, Galimi F, Lee MS, Karin M. Saturated fatty
acids inhibit induction of insulin gene transcription by JNK-mediated
phosphorylation of insulin-receptor substrates. Proc. Natl. Acad. Sci.
USA 2006; 103: 16454–9.
60 Donnelly KL, Smith CI, Schwarzenberg SJ, Jessurun J, Boldt MD,
Parks EJ. Sources of fatty acids stored in liver and secreted via
lipoproteins in patients with nonalcoholic fatty liver disease. J. Clin.
Invest. 2005; 115: 1343–51.
61 Gentile CL, Pagliassotti MJ. The role of fatty acids in the
development and progression of nonalcoholic fatty liver disease.
J. Nutr. Biochem. 2008; 19: 567–76.
Journal of Gastroenterology and Hepatology 24 (2009) 830–840 © 2009 The Authors