3158Journal of Lipid Research Volume 51, 2010
Copyright © 2010 by the American Society for Biochemistry and Molecular Biology, Inc.
This article is available online at http://www.jlr.org
Alcoholic liver disease (ALD) continues to be an impor-
tant health problem in the United States. Although much
progress has been made over the past two decades, the
mechanisms involved in its initiation and progression re-
main to be fully understood. The disease is characterized
by early steatosis, subsequent steatohepatitis (steatosis with
infl ammatory cell infi ltration and necrosis), and, in some
instances, progression to fi brosis and/or cirrhosis ( 1, 2 ).
Excessive neutral fat accumulation in hepatocytes (steato-
sis) is the most common and earliest response of the liver
to chronic alcohol consumption ( 3 ) and plays a critical
role in disease progression. Hepatic steatosis results from
an imbalance between intrahepatic triglyceride (TG) pro-
duction and removal. Both uptake of free fatty acids (FAs)
to the liver and de novo synthesis contribute to hepatic TG
production, whereas FA ? -oxidation and formation of very
low density lipoprotein (VLDL) particles contribute to he-
patic TG removal.
The fi nal step and rate-limiting reaction in TG synthesis
is catalyzed by acyl CoA:diacylglycerol acyltransferase
(DGAT), which covalently joins a fatty acyl-CoA and a
diacylglycerol (DG) molecule to form TG. In mammals,
DGAT occurs in two isoforms, DGAT1 and DGAT2, from
distinct gene families ( 4, 5 ). Although both isoforms
are widely expressed and present at high levels in white
adipose tissue, DGAT1 is most highly expressed in the
small intestine, whereas DGAT2 is primarily expressed in
the liver ( 4, 6 ). Evidence suggest that the two enzymes play
different roles in TG metabolism, with DGAT2 participat-
ing in steatosis and DGAT1 in VLDL synthesis. Overex-
pression of liver-specifi c DGAT2 in mice results in hepatic
Abstract The mechanisms involved in the development of
alcoholic liver disease (ALD) are not well established. We
investigated the involvement of acyl-CoA: diacylglycerol
acyltransferase 2 (DGAT2) upregulation in mediating he-
patic fat accumulation induced by chronic alcohol consump-
tion. Chronic alcohol feeding caused fatty liver and increased
hepatic DGAT2 gene and protein expression, concomitant
with a signifi cant suppression of hepatic MAPK/ERK ki-
nase/extracellular regulated kinase 1/2 (MEK/ERK1/2) ac-
tivation. In vitro studies demonstrated that specifi c inhibitors
of the MEK/ERK1/2 pathway increased DGAT2 gene ex-
pression and triglyceride (TG) contents in HepG2 cells,
whereas epidermal growth factor, a strong ERK1/2 activa-
tor, had the opposite effect. Moreover, chronic alcohol
feeding decreased hepatic S-adenosylmethionine (SAM):
S-adenosylhomocysteine (SAH) ratio, an indicator of dis-
rupted transmethylation reactions. Mechanistic investigations
revealed that N-acetyl-S-farnesyl- L -cysteine, a potent inhibi-
tor of isoprenylcysteine carboxyl methyltransferase, sup-
pressed ERK1/2 activation, followed by an enhanced DGAT2
expression and an elevated TG content in HepG2 cells.
Lastly, we demonstrated that the benefi cial effects of be-
taine supplementation in ALD were associated with im-
proved SAM/SAH ratio, alleviated ERK1/2 inhibition, and
attenuated DGAT2 upregulation. In conclusion, our data
suggest that upregulation of DGAT2 plays an important role
in the pathogenesis of ALD, and that abnormal methionine
metabolism contributes, at least partially, to DGAT2 upreg-
ulation via suppression of MEK/ERK1/2 activation. —Wang,
Z., T. Yao, and Z. Song. Involvement and mechanism of
DGAT2 upregulation in the pathogenesis of alcoholic fatty
liver disease. J. Lipid Res . 2010. 51: 3158–3165.
Supplementary key words alcohol • ERK1/2 • DGAT2 • betaine
This work was supported by the National Institutes of Health grants K01
AA015344 and R01 AA017442 (Z. S.), and the National Natural Science
Foundation of China 81000168 and the China Postdoctoral Science Founda-
tion 20100471022 (Z. W.). Its contents are solely the responsibility of the au-
thors and do not necessarily represent the offi cial views of the National Institutes
Manuscript received 23 April 2010 and in revised form 25 August 2010.
Published, JLR Papers in Press, August 25, 2010
Involvement and mechanism of DGAT2 upregulation in
the pathogenesis of alcoholic fatty liver disease
Zhigang Wang, * ,† Tong Yao, * and Zhenyuan Song 1, *
Department of Kinesiology and Nutrition,* University of Illinois at Chicago , Chicago, IL 60612; and College
of Pharmacy, † Harbin Medical University , Harbin, Heilongjiang, China
Abbreviations: AF, alcohol-fed ; AFC, N-acetyl-S-farnesyl-L-cysteine;
ALD, alcoholic liver disease; ALT, alanine aminotransferases; DG, di-
acylglycerol; DGAT, acyl-CoA:diacylglycerol acyltransferase; EGF, epi-
dermal growth factor; ERK1/2, extracellular signal-regulated kinases 1
and 2; GSH, glutathione; ICMT, isoprenylcysteine carboxyl methyl-
transferase; MAPK, mitogen-activated protein kinase; MEK, MAPK/
ERK kinase; PF, pair-fed; SAH, S-adenosylhomocysteine; SAM, S-adeno-
sylmethionine; SREBP-1c, sterol regulatory element binding protein-
1c; TG, triglyceride.
1 To whom correspondence should be addressed.
by guest, on October 20, 2015
DGAT2 upregulation in ALD3159
or an isocaloric maltose-dextrin mixture (pair-fed, PF), accord-
ing to Lieber and De Carli ( 20 ). Food intake and body weight
were recorded daily and weekly, respectively. For betaine supple-
mentation, betaine (anhydrous; Sigma, St. Louis, MO) was sup-
plemented in the alcohol-containing liquid diet (1%, w/v) and
started simultaneously with AF diet. Mice were euthanized
and plasma and liver tissue samples harvested at the end of the
Cells and culture conditions
HepG2 cells, a human hepatoma cell line, were obtained from
the American Type Culture Collection (ATCC, Manassas, VA)
and cultured in DMEM containing 10% (v/v) fetal bovine serum,
2 mM glutamine, 5U/ml penicillin, and 50 ? g/ml streptomycin
at 37°C in a humidifi ed O 2 /CO 2 (19:1) atmosphere.
Plasma biochemical assays
Plasma levels of alanine aminotransferases (ALT) and TG
were assayed using commercially available kits (Infi nity, Thermo
Electron, Melbourne, Australia).
Liver TG measurements
For intrahepatic TG measurement, liver tissues ( ? 80 mg) were
homogenized in 1.0ml NaCl (50 mM) solution and hepatic total
lipids were extracted overnight in 10 ml heptane:isopropanol
(3:2) mixture at 4°C. Hepatic TG content was determined by
enzymatic colorimetric methods using a commercially available
kit (Infi nity, Thermo Electron, Melbourne, Australia).
Measurement of methionine metabolites
Liver tissue was homogenized and deproteinized in 4% meta-
phosphoric acid (1:8, m/v). The homogenates were centrifuged
at 15,000 g for 10 min. SAM and SAH were determined via a high-
performance liquid chromatography (HPLC) method using a
5-mm Hypersil C-18 column (250 × 4.6 mm). The mobile phase
consisted of 40 mM ammonium phosphate, 8 mM heptane sul-
fonic acid [ion-pairing reagent (pH 5.0)], and 6% acetonitrile,
and was delivered at a fl ow rate of 1.0ml/minute. SAM, SAH,
betaine, and GSH were detected using a Waters 740 UV detector
at 254nm. An internal standard, S-adenosylethionine, was added
to all samples and standard solutions to a concentration of
Measurement of intracellular TG content
To determine the intracellular TG content, HepG2 cells
seeded in 24-well plates were washed twice with phosphate
buffered saline (PBS) and cellular lipids were extracted by
1ml hexane:isopropanol (3:2) mixture. TG content was measured
using a TG assay kit (Infi nity, Thermo Electron, Melbourne, Aus-
tralia). Cells undergoing the same treatment conditions were
lysed in RIPA buffer for protein concentration determination
and data normalization.
Suppression of DGAT2 expression by siRNA
RNA targeting the human DGAT2 gene and a control small
interfering (si)RNA containing a scrambled sequence (Ambion,
Austin, TX) were transfected by siPORT TM NeoFX TM Transfection
Agent (Ambion, Austin, TX), according to the manufacturer’s
Measurement of DGAT2 activity
The microsomal fraction from HepG2 cells was prepared as
described previously ( 21 ). Because DGAT2 activity is inhibited
at high concentrations of MgCl 2 (100 mM), the DGAT assay
was carried out in the presence of 8 mM and 100 mM MgCl 2
steatosis ( 7 ). Conversely, inhibition of DGAT2 with anti-
sense oligonucleotides reverses hepatic steatosis in ob/ob
mice and in mice challenged with high-fat diet ( 8–10 ),
suggesting that this enzyme plays a critical role in the
development of fatty liver disease. Although strong evi-
dence supports that fatty liver development induced by
chronic alcohol consumption is secondary to increased de
novo synthesis, the effect of alcohol exposure on DGAT
regulation remains to be clarifi ed.
Disrupted methionine metabolism plays a pathological
role in ALD ( 11–15 ). S-adenosylmethionine (SAM) is the
fi rst product in the methionine metabolic pathway and a
universal methyl donor. After donating its methyl group
for transmethylation reaction, SAM is converted into
S-adenosylhomocysteine (SAH), which is subsequently hy-
drolyzed to homocysteine and adenosine. In turn, SAH
is a potent inhibitor of most known methyltransferases.
Alcohol-induced abnormal methionine metabolism is char-
acterized by disrupted transmethylation reactions due to
decreased SAM and increased SAH levels and affects
numerous signaling pathways involved in apoptosis, regen-
eration, and lipid metabolism. Among these is the Ras/
ERK1/2 pathway. The extracellular signal-regulated ki-
nases 1 and 2 (ERK1/2) are members of the mitogen-
activated protein kinase (MAPK) family, whose activation
results in cell growth, proliferation, survival, and infl am-
mation ( 16 ). The activation of ERK1/2 is strongly depen-
dent on the GTPase Ras, whose activity requires association
with cellular membranes ( 17, 18 ). Ras plasma membrane
association requires a series of posttranslational modifi -
cations of its carboxyl terminus, including farnesylation,
proteolysis, and methylation of its CAAX sequence. The
methylation reactions require SAM as a methyl group do-
nor to produce methylated Ras. In this context, ERK1/2
activation is closely related to methionine metabolism.
We previously reported that ERK1/2 phosphorylation
was reduced in the liver of alcohol-fed rats, and this is as-
sociated with hypercholesterolemia and reduced hepatic
low density lipoprotein (LDL) receptor expression, sug-
gesting that the ERK1/2 pathway participates in the regu-
lation of hepatic lipid metabolism ( 19 ). In the present
study, we conducted in vivo and in vitro experiments to
investigate the role of DGAT2 upregulation in the devel-
opment of hepatic steatosis in alcohol-fed mice and the
potential mechanistic involvement of suppression of the
MEK/ERK1/2 pathway in DGAT2 upregulation.
MATERIALS AND METHODS
Animals and treatments
Male C57BL/6 mice weighing 25 ± 0.5 g (means ± SD) were
obtained from the Jackson Laboratory (Bar Harbor, ME). Stud-
ies were approved by the Institutional Animal Care and Use
Committee, which is certifi ed by the American Association of
Accreditation of Laboratory Animal Care. Eight mice were ran-
domly assigned to two groups and fed for 4 weeks with liquid di-
ets containing (in percent of energy intake) 18% protein, 35%
fat, 12% carbohydrate, and 35% either ethanol (alcohol-fed, AF)
by guest, on October 20, 2015
3160Journal of Lipid Research Volume 51, 2010
Chronic alcohol exposure resulted in ERK1/2
suppression in the liver
To examine the effect of AF on MAPK activation in the
liver, we conducted immunoblotting analysis using total
liver tissue extracts from both PF and AF mice. As shown in
Fig. 2 , AF had no effect on c-Jun N-terminal kinases (JNK)
activation ( Fig. 2A ), whereas the activation of p38 was min-
imally enhanced ( Fig. 2B ). However, AF resulted in a sig-
nifi cant reduction in the phosphorylation of ERK1/2 ( Fig.
2C, D ), which was in line with our previous observation in
rats ( 19 ). No changes in protein levels of the three mem-
bers of the MAPK family were observed in the liver of AF
animals when compared with PF controls.
and DGAT2 activity was calculated by subtracting the value ob-
tained at 100 mM from the value obtained at 8 mM. Brief,
200µl of the reaction mixture containing 200µM sn-1,2-dio-
leoylglycerol (Sigma-Aldrich, Saint Louis, MO) in acetone,
30µM [ 14 C]oleoyl-CoA (American Radiolabeled Chemicals,
Saint Louis, MO), 8 mM MgCl 2 , 200µg BSA, and 10µg mi-
crosomal protein in 175 mM Tris-HCl buffer, pH 8.0 was incu-
bated for 30 min at 25°C. After incubation, the reaction was
stopped by adding 1.5ml stop solution [2-propanol-heptane-
water (80:20:2, v/v)]. The top heptane phase was collected and
washed with alkaline ethanol. An aliquot of the top heptane
phase was used for radioactivity measurements using a Beck-
man LS 5801 counter.
Quantitative real-time RT-PCR
Total RNA from either frozen liver tissue or cultured cells
was isolated with a phenol-chloroform extraction. For each sam-
ple, 1.0 ? g total RNA was reverse transcribed using a high-
capacity cDNA reverse transcription kit (Applied Biosystems,
Foster City, CA). The cDNA was amplifi ed in MicroAmp Optical
96-well reaction plates with a SYBR Green PCR Master Mix
(Applied Biosystems) on an Applied Biosystems Prism 7000
sequence detection system. Relative gene expression was calcu-
lated after nomalization by a house-keeping gene (mouse or hu-
man 18S rRNA).
Liver tissues were homogenized and HepG2 cells were lysed in
RIPA buffer and the isolated proteins were separated by SDS
polyacrylamide gel electrophoresis and transferred to 0.45 ? m
polyvinylidene difl uoride membrane. After transfer, membranes
were blocked in 5% BSA in PBS with 0.1% Tween 20 and probed
with anti-DGAT2 (Novus Biologicals, Littleton, CO), anti-SREBP-
1c (Santa Cruz Biotechnology, Santa Cruz, CA), anti-phospho-
ERK1/2 or anti-ERK1/2 (Cell Signaling Technology, Danvers,
MA) antibodies. Horseradish peroxidase-conjugated secondary
antibodies and enhanced chemiluminescence substrate kit were
used in detection of specifi c proteins.
All data are expressed as means ± SD. Statistical analysis was
performed using a one-way ANOVA and further analyzed by
Newman-Keuls test for statistical difference. Differences be-
tween treatments were considered to be statistically signifi cant
at P < 0.05.
Chronic alcohol exposure increased hepatic DGAT2 gene
expression and protein production
Chronic alcohol consumption for 4 weeks caused fatty
liver and liver injury as evidenced by signifi cantly increased
plasma ALT levels, increased liver weight versus body
weight ratio, and elevated liver TG content in the alcohol-
fed group (data not shown). Long-term AF increased
DGAT2 gene ( Fig. 1A ) and protein expression ( Fig. 1B, C )
in the liver when compared with the PF group. We also
examined the effect of AF on hepatic expression of sterol
regulatory element binding protein-1c (SREBP-1c), the
master regulator of de novo FA synthesis. In line with pre-
vious studies ( 22–24 ), AF signifi cantly elevated SREBP-1c
protein in the liver ( Fig. 1D, E ).
Fig. 1. Chronic alcohol exposure increased hepatic DGAT2 gene
expression and protein production. Male C57BL/6 mice were pair-
fed liquid diets with or without ethanol for 4 weeks. Chronic alco-
hol exposure increased DGAT2 gene expression (A) and protein
abundance (B) in the liver. C: Quantitative analysis of DGAT2 pro-
tein in the liver, normalized by corresponding GAPDH expression
level. D: The protein abundance of SREBP-1c in the liver was aug-
mented signifi cantly in alcohol-fed mice; E: quantitative analysis of
SREBP-1c protein expression in the liver, normalized by corre-
sponding actin expression level. Data are means ± SD (n = 4). * P <
0.05. PF, pair-fed; AF, alcohol-fed.
by guest, on October 20, 2015
DGAT2 upregulation in ALD3161
Fig. 3. MEK/ERK1/2 inhibition leads to increased intracellular
TG accumulation in hepatocytes. HepG2 cells were pretreated with
MEK/ERK1/2 inhibitors or activator for 2 h, and then challenged
with 0.3 mM oleic acids for 20 h. A: MEK/ERK1/2 inhibitors,
U0126 (10µM) or PD98059 (10µM), elevated intracellular TG con-
tent; whereas (B) EGF (100ng/ml), an activator of ERK1/2 signal-
ing, reduced intracellular TG content in HepG2 cells. C: The time
course for the effect of EGF on ERK1/2 activation. HepG2 cells
were treated with EGF (100ng/ml) and cell lysates were collected
at different time points. ERK1/2 activation was measured by detect-
ing its phosphorylation. On the other hand, HepG2 cells were
treated with U0126 (10µM) or EGF (100ng/ml) for 18 h, then the
total RNA was isolated and DGAT2 gene expression was analyzed
by real time RT-PCR. D: U0126 enhanced gene expression of
DGAT2; conversely, (E) EGF inhibited the gene expression of
DGAT2 in HepG2 cells. Further, the microsomes in HepG2 cells
between untreated group and U0126-treated group were isolated
for determining the DGAT2 activity. F: Application of U0126 to in-
hibit ERK1/2 activation elevated the DGAT2 activity signifi cantly in
HepG2 cells. All values are denoted as means ± SD from three or
more independent batches of cells, * P < 0.05.
MEK/ERK1/2 inhibition leads to increased intracellular
TG accumulation in hepatocytes
To determine if ERK1/2 inhibition was associated with
increased TG accumulation in the liver, we treated HepG2
cells with two specifi c inhibitors of the MEK/ERK1/2 path-
way, U0126 (10µM) and PD98059 (10µM), before challeng-
ing cells with oleic acid. Intracellular TG levels were assayed
20 h later. As shown in Fig. 3 , both inhibitors signifi cantly
Chronic alcohol exposure caused abnormal methionine
metabolism in the liver
To determine the effects of AF on methionine metabo-
lism in the liver, we measured SAM, SAH, betaine, and glu-
tathione (GSH) concentrations in the liver via HPLC. In
line with our previous reports, AF signifi cantly decreased
hepatic SAM levels, whereas hepatic SAH levels were sig-
nifi cantly elevated, leading to a signifi cantly decreased
SAM/SAH ratio. Liver betaine levels decreased slightly in
AF, whereas GSH levels in the liver signifi cantly decreased
( Table 1 ).
Fig. 2. Chronic alcohol exposure resulted in prominent ERK1/2
suppression in the liver. Male C57BL/6 mice were pair-fed liquid
diets with or without ethanol for 4 weeks. Chronic alcohol expo-
sure had no effect on JNK activation (A) and minimally enhanced
p38 activation (B) in the liver. C: Chronic alcohol exposure de-
creased the phosphorylation levels of ERK1/2 in the liver; D: quan-
titative analysis of ERK1/2 phosphorylation in the liver, normalized
by corresponding total ERK1/2 expression level. Data are means ±
SD (n = 4). * P < 0.05. PF, pair-fed; AF, alcohol-fed.
TABLE 1. Changes in methionine metabolism in the liver
172.25 ± 15.95 115.93 ± 36.04 a 221.33 ± 36.69 b
69.75 ± 8.62219.33 ± 94.36 a 175.67 ± 51.40 a
2.48 ± 0.08
6.74 ± 1.11
0.63 ± 0.35 a
4.89 ± 2.05
1.30 ± 0.25 ab
18.87 ± 8.76 ab
4.66 ± 0.10 3.93 ± 0.22 a 4.49 ± 0.19 b
Data represent mean ± SD (N = 4). PF, pair-fed; AF, alcohol-fed;
a P < 0.05 compared with PF.
b P < 0.05 compared with AF.
by guest, on October 20, 2015
3162Journal of Lipid Research Volume 51, 2010
The benefi cial effect of betaine in ALD was associated
with improved methionine metabolism, alleviated
ERK1/2 inhibition, and attenuated DGAT2 induction in
Betaine exerts benefi cial effects on fatty liver diseases
by improving methionine metabolism. In an attempt to
elucidate the mechanisms for the benefi cial effect of be-
taine in ALD, betaine, at a dose of 1% (v/w), was supple-
mented in the alcohol-containing liquid diet. Four weeks
later, we found that betaine supplementation prevented
alcohol-induced fatty liver and liver injury, as evidenced
by lowered TG content in the liver ( Fig. 6A ) and ALT
levels in plasma ( Fig. 6B ), which were associated with
signifi cantly improved hepatic methionine metabolism
( Table 1 ). Data showed that the benefi cial effect of
betaine supplementation was associated with alleviated
ERK1/2 inhibition and attenuated DGAT2 induction
( Fig. 6C–E ) in the liver.
Neutral fat (mainly TG) deposition is the initial stage of
alcoholic fatty liver disease and plays a critical role in dis-
ease progression. Although the exact mechanisms remain
elusive, it is generally accepted that increased hepatic de
novo FA synthesis plays a critical role in the development
of fatty liver. FA synthesis is centrally regulated by SREBP-1c,
a nuclear transcription factor, via a subset of enzymes in-
volved in de novo FA synthesis, including acetyl-coA car-
boxylase, fatty acid synthase, and stearoyl-CoA desaturase.
increased intracellular TG content ( Fig. 3A ). To examine
whether TG accumulation could be prevented by enhanc-
ing ERK1/2 activation, we treated HepG2 cells with epi-
dermal growth factor (EGF) (100ng/ml), an activator of
ERK1/2 signaling, prior to oleic acid challenge. EGF treat-
ment reduced intracellular TG content ( Fig. 3B ), which was
associated with enhanced ERK1/2 activation ( Fig. 3C ).
MEK/ERK1/2 suppression upregulated DGAT2
expression and activity in HepG2 cells
To test whether hepatic ERK1/2 inhibition affected
DGAT2 expression, gene and protein expression and DGAT2
activity were measured. Exposure to the ERK1/2 inhibitor
U0126 (10µM) resulted in an ? 5-fold increase in DGAT2
mRNA expression in HepG2 cells compared with the un-
treated group ( Fig. 3D ). In contrast, enhanced ERK1/2 acti-
vation by EGF (100ng/ml) decreased DGAT2 mRNA levels
( Fig. 3E ). Moreover, exposure of cells to U0126 to inhibit
ERK1/2 activation signifi cantly increased DGAT2 enzymatic
activity ( Fig. 3F ). Furthermore, transfection of HepG2 cells
with DGAT2 siRNA, which caused an 80% decrease in DGAT2
mRNA expression, signifi cantly decreased intracellular TG
levels ( Fig. 4A , B ). Accordingly, treatment with DGAT2 siRNA
completely prevented U0126-induced intracellular TG in-
crease in HepG2 cells ( Fig. 4B ), implying that upregulation
of DGAT2 contributes to the increased hepatic TG content
by ERK1/2 inhibition.
Inhibition of methyltransferase suppressed MEK/
ERK1/2 activation, enhanced DGAT2 expression, and
elevated TG contents in HepG2 cells
In order to determine whether inhibition of transmethyl-
ation reactions was related with hepatic ERK1/2 inhibition
and DGAT2 induction, we applied N-acetyl-S-farnesyl-
L -cysteine (AFC), an inhibitor of SAM-dependent meth-
yltransferases (specifi c for isoprenylcysteine carboxyl meth-
yltransferase, ICMT), to HepG2 cells. Result showed that
treatment with AFC (from 10µM to 40µM) for 24 h de-
creased ERK1/2 phosphorylation in a dose-dependent
fashion ( Fig. 5A ). In addition, AFC signifi cantly augmented
intracellular TG levels ( Fig. 5B ) and DGAT2 gene expres-
sion ( Fig. 5C ) when compared with the untreated group.
Fig. 5. Inhibition of methyltransferases suppressed MEK/ERK
activation, enhanced DGAT2 expression, and elevated TG contents
in HepG2 cells. A: AFC suppressed the phosphorylation level of
ERK1/2. HepG2 cells were treated with methyltransferases inhibi-
tor AFC (10µM, 20µM or 40µM) for 24 h and cell lysates were col-
lected for detecting ERK1/2 phosphorylation. B: AFC increased
the intracellular TG content. HepG2 cells were pretreated with
AFC (40µM) for 2 h before challenged with 0.3 mM oleic acids.
Intracellular TG contents were measured 20 h later. C: AFC de-
creased the gene expression of DGAT2 in HepG2 cells. HepG2
cells were treated with AFC (40µM) for 18 h and total RNA was
isolate and DGAT2 gene expression was determined by real time
RT-PCR. All values were denoted as means ± SD from three or more
independent batches of cells, * P < 0.05.
Fig. 4. DGAT2 silencing prevented the elevation of TG content
caused by MEK/ERK1/2 suppression in HepG2 cells. Transfection
of siRNA for DGAT2 into HepG2 cells decreased DGAT2 mRNA
levels (A), which prevented U0126 (10µM)-induced intracellular
TG elevation (B). All values were denoted as means ± SD from
three or more independent batches of cells, * P < 0.05.
by guest, on October 20, 2015
DGAT2 upregulation in ALD3163
ERK1/2-regulated pathways play an important role in
controlling lipid metabolism in the liver ( 25, 26 ). We and
others previously reported that chronic alcohol exposure
causes signifi cant MEK/ERK1/2 suppression in the liver
( 19, 27, 28 ). In the present study, we demonstrate that
alcohol-induced hepatic MEK/ERK1/2 suppression is as-
sociated with increased DGAT2 expression and protein
production. In vitro studies reveal that specifi c inhibition
of MEK/ERK1/2 activation increases gene expression and
enzymatic activity of DGAT2 in HepG2 cells and causes
intracellular TG accumulation. Conversely, enhanced
ERK1/2 activation through EGF stimulation, slightly but
signifi cantly suppresses DGAT2 gene expression and pre-
vents intracellular TG accumulation. Furthermore, our
data show that elevation of intracellular TG content in-
duced by MEK/ERK1/2 is inhibited by DGAT2 siRNA in
HepG2 cells, indicating that DGAT2 upregulation contrib-
utes to increased TG synthesis by MEK/ERK1/2 inactiva-
tion. Because HepG2 cells have high N-ras activity, and
thus high ERK1/2 activity ( 26, 29 ) compared with primary
hepatocytes, it is conceivable that the decreased amplitude
in DGAT2 gene expression by EGF in HepG2 cells was
much less than the increased one triggered by ERK1/2 in-
hibitor U0126. Collectively, our data support the hypo-
thesis that suppression of ERK1/2 activation by chronic
alcohol exposure plays a critical role in upregulating
DGAT2 in the liver. Our study suggests that the hepatic
MEK/ERK1/2 pathway may be a therapeutic target for
treatment of ALD. Further support for this hypothesis de-
rived from a study showing that EGF, a strong ERK1/2 ac-
tivator, confers benefi cial effects on alcohol-induced fatty
liver and liver injury ( 30 ).
ERK1/2 is a member of the MAPK family, whose activa-
tion results in cell growth, proliferation, survival, and in-
fl ammation. The results with regard to the effect of alcohol
exposure on ERK1/2 activation are controversial, mainly
due to the cell type usage and exposure duration. For in-
stance, accumulating evidence indicates that chronic al-
cohol exposure activates the MEK/ERK1/2 pathway and
potentiates endotoxin-stimulated ERK1/2 activation in
Kupffer cells, resulting in increased synthesis of tumor ne-
crosis factor and augmented infl ammatory responses ( 31,
32 ). It has also been well documented that acute exposure
to alcohol results in modest activation of ERK1/2 in hepa-
tocytes ( 33 ), whereas chronic alcohol exposure suppresses
both constitutive and growth factor-stimulated ERK1/2 ac-
tivation in the liver ( 27, 28 ). The mechanisms involved in
MEK/ERK1/2 inhibition by chronic alcohol exposure are
not well understood and are probably multifactorial. The
ethanol-induced lipid peroxidation product 4-hydroxy-2-
nonenal has been reported to be an upstream modulator
of ERK1/2 activation ( 27 ). Inhibition of the insulin recep-
tor substrate 1 signaling pathway by chronic alcohol con-
sumption may also contribute to suppressed ERK1/2
activation in the liver ( 28 ). The Ras/Raf/MEK/ERK1/2
pathway is the major signal transduction pathway for
ERK1/2 activation, which is strongly dependent on the
GTPase Ras, whose activity requires its association with cel-
lular membranes ( 17, 18 ). Ras plasma membrane associa-
Enhanced gene expression and transactivation of SREBP-1c
in the liver and subsequently increased hepatic de novo
FA synthesis by AF have been documented by several
groups ( 22–24 ). However, the effects of alcohol consump-
tion on the downstream steps in TG synthesis remain
unclear. TG is synthesized from three FAs and glycerol
through ester bonds. Among the complex metabolism
network for TG synthesis in the liver, DGAT is a rate-limit-
ing enzyme catalyzing the fi nal step in TG synthesis by
facilitating the linkage of sn-1,2-diacylglycerol with a long-
chain fatty acyl-CoA. Overexpression of DGAT2 is associ-
ated with hepatic steatosis ( 7 ); suppression of DGAT2 with
antisense oligonucleotides reverses diet-induced hepatic
steatosis ( 8–10 ), suggesting that DGAT2 exerts an im-
portant role in the development of fatty liver diseases.
The present study provides initial evidence showing
that chronic alcohol consumption induces expression of
DGAT2 at both the gene and protein level. Because FA for
TG synthesis in the liver derives not only from endoge-
nous de novo lipogenesis but also exogenous sources (e.g.,
dietary fat or white adipose tissue via lipolysis during fast-
ing), it is conceivable that upregulation of DGAT2 by
chronic alcohol consumption may play an important role
in the development of alcohol-induced hepatic steatosis.
Fig. 6. Betaine supplementation alleviated alcohol-induced
ERK1/2 inhibition and DGAT2 induction in the liver. Male
C57BL/6 mice were fed alcohol-containing liquid diets (AF) with
or without betaine [1% (wt/vol)] supplementation for 4 weeks. A:
Liver TG contents; B: Plasma ALT levels; C: Betaine supplementa-
tion attenuated alcohol-induced ERK1/2 inhibition and DGAT2
induction in the liver. Data are means ± SD (n = 4). D: Quantitative
analysis of ERK1/2 phosphorylation in the liver, normalized by cor-
responding total ERK1/2 expression level. E: Quantitative analysis
of DGAT2 expression in the liver, normalized by corresponding
actin expression level. Data are means ± SD (n = 4). * P < 0.05. AF,
alcohol-fed; BT, betaine.
by guest, on October 20, 2015
3164 Journal of Lipid Research Volume 51, 2010
to the intrahepatic FA pool for TG synthesis. Although in
the present study we did not directly measure the effect of
chronic alcohol exposure on VLDL secretion and mito-
chondrial ? -oxidation of FA, elevated plasma ketone bod-
ies and fasting TG concentrations in AF mice provide
indirect evidence of enhanced FA oxidation and VLDL se-
cretion in the early stages of ALD. The elevated plasma TG
content is in line with the observed hepatic DGAT1 ex-
pression (data not shown), which plays an important role
in VLDL secretion ( 38 ). These data altogether suggest
that both TG synthesis and disposal are enhanced in the
early stage of alcoholic liver disease and that fatty liver de-
velops when the capability of the liver to remove TG dose
not compensate for the increased synthesis.
In conclusion, we demonstrate that upregulation of he-
patic DGAT2 by chronic alcohol exposure contributes to
the development of ALD. Abnormal hepatic methionine
metabolism, specifi cally the suppression of transmethyla-
tion reactions, plays a mechanistic role in alcohol-induced
DGAT2 upregulation by suppressing MEK/ERK1/2 activa-
tion. Therefore, inhibition of ERK1/2 activation repre-
sents a critical link between methionine metabolism and
fatty liver development via upregulation of hepatic DGAT2
The authors thank Dr. Alan Diamond from the Department of
Pathology, Dr. P. V. Subbaiah from the Department of Medicine,
and Dr. Giamila Fantuzzi from the Department of Kinesiology
and Nutrition, University of Illinois at Chicago, for their
technical support, scientifi c advice, and generous editorial
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els suggest that FA from adipose tissue lipolysis contributes
by guest, on October 20, 2015
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