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 ALD3165
induced hepatic steatosis and insulin resistance. J. Biol. Chem. 282 :
22678 – 22688 .
9 . Liu , Y. , J. S. Millar , D. A. Cromley , M. Graham , R. Crooke ,
J. T. Billheimer , and D. J. Rader . 2008 . Knockdown of acyl-
CoA:diacylglycerol acyltransferase 2 with antisense oligonucleotide
reduces VLDL TG and ApoB secretion in mice. Biochim. Biophys.
Acta . 1781 : 97 – 104 .
10 . Yu , X. X. , S. F. Murray , S. K. Pandey , S. L. Booten , D. Bao , X. Z.
Song , S. Kelly , S. Chen , R. McKay , B. P. Monia , et al . 2005 . Antisense
oligonucleotide reduction of DGAT2 expression improves he-
patic steatosis and hyperlipidemia in obese mice. Hepatology . 42 :
362 – 371 .
11 . Halsted , C. H. , J. Villanueva , C. J. Chandler , S. P. Stabler , R. H. Allen ,
L. Muskhelishvili , S. J. James , and L. Poirier . 1996 . Ethanol feeding
of micropigs alters methionine metabolism and increases hepato-
cellular apoptosis and proliferation. Hepatology . 23 : 497 – 505 .
12 . Esfandiari , F. , M. You , J. A. Villanueva , D. H. Wong , S. W. French ,
and C. H. Halsted . 2007 . S-adenosylmethionine attenuates hepatic
lipid synthesis in micropigs fed ethanol with a folate-defi cient diet.
Alcohol. Clin. Exp. Res. 31 : 1231 – 1239 .
13 . Lee , T. D. , M. R. Sadda , M. H. Mendler , T. Bottiglieri , G. Kanel ,
J. M. Mato , and S. C. Lu . 2004 . Abnormal hepatic methionine and
glutathione metabolism in patients with alcoholic hepatitis. Alcohol.
Clin. Exp. Res. 28 : 173 – 181 .
14 . Song , Z. , Z. Zhou , S. Uriarte , L. Wang , Y. J. Kang , T. Chen , S. Barve ,
and C. J. McClain . 2004 . S-adenosylhomocysteine sensitizes to TNF-
alpha hepatotoxicity in mice and liver cells: a possible etiological
factor in alcoholic liver disease. Hepatology . 40 : 989 – 997 .
15 . Song , Z. , Z. Zhou , I. Deaciuc , T. Chen , and C. J. McClain . 2008 .
Inhibition of adiponectin production by homocysteine: a potential
mechanism for alcoholic liver disease. Hepatology . 47 : 867 – 879 .
16 . Pearson , G. , F. Robinson , T. Beers Gibson , B. E. Xu , M. Karandikar ,
K. Berman , and M. H. Cobb . 2001 . Mitogen-activated protein
(MAP) kinase pathways: regulation and physiological functions.
Endocr. Rev. 22 : 153 – 183 .
17 . Willumsen , B. M. , A. Christensen , N. L. Hubbert , A. G. Papageorge ,
and D. R. Lowy . 1984 . The p21 ras C-terminus is required for trans-
formation and membrane association. Nature . 310 : 583 – 586 .
18 . Chiu , V. K. , J. Silletti , V. Dinsell , H. Wiener , K. Loukeris , G. Ou ,
M. R. Philips , and M. H. Pillinger . 2004 . Carboxyl methylation
of Ras regulates membrane targeting and effector engagement.
J. Biol. Chem. 279 : 7346 – 7352 .
19 . Wang , Z. , T. Yao , and Z. Song . 2010 . Chronic alcohol consumption
disrupted cholesterol homeostasis in rats: down-regulation of low-
density lipoprotein receptor and enhancement of cholesterol bio-
synthesis pathway in the liver. Alcohol. Clin. Exp. Res. 34 : 471 – 478 .
20 . Lieber , C. S. , and L. M. De Carli . 1973 . Ethanol dependence and
tolerance: a nutritionally controlled experimental model in the rat.
Res. Commun. Chem. Pathol. Pharmacol. 6 : 983 – 991 .
21 . Ganji , S. H. , S. Tavintharan , D. Zhu , Y. Xing , V. S. Kamanna , and
M. L. Kashyap . 2004 . Niacin noncompetitively inhibits DGAT2 but
not DGAT1 activity in HepG2 cells. J. Lipid Res. 45 : 1835 – 1845 .
22 . Shinohara , M. , C. Ji , and N. Kaplowitz . 2010 . Differences in
betaine-homocysteine methyltransferase expression, endoplasmic
reticulum stress response, and liver injury between alcohol-fed
mice and rats. Hepatology . 51 : 796 – 805 .
23 . You , M. , X. Liang , J. M. Ajmo , and G. C. Ness . 2008 . Involvement
of mammalian sirtuin 1 in the action of ethanol in the liver. Am. J.
Physiol. Gastrointest. Liver Physiol. 294 : G892 – G898 .
24 . Ji , C. , C. Chan , and N. Kaplowitz . 2006 . Predominant role of sterol
response element binding proteins (SREBP) lipogenic pathways in
hepatic steatosis in the murine intragastric ethanol feeding model.
J. Hepatol. 45 : 717 – 724 .
25 . Kong , W. , J. Wei , P. Abidi , M. Lin , S. Inaba , C. Li , Y. Wang , Z. Wang ,
S. Si , H. Pan , et al . 2004 . Berberine is a novel cholesterol-lowering
drug working through a unique mechanism distinct from statins.
Nat. Med. 10 : 1344 – 1351 .
26 . Tsai , J. , W. Qiu , R. Kohen-Avramoglu , and K. Adeli . 2007 . MEK-
ERK inhibition corrects the defect in VLDL assembly in HepG2
cells: potential role of ERK in VLDL-ApoB100 particle assembly.
Arterioscler. Thromb. Vasc. Biol. 27 : 211 – 218 .
27 . Sampey , B. P. , B. J. Stewart , and D. R. Petersen . 2007 . Ethanol-
induced modulation of hepatocellular extracellular signal-regu-
lated kinase-1/2 activity via 4-hydroxynonenal. J. Biol. Chem. 282 :
1925 – 1937 .
28 . Yeon , J. E. , S. Califano , J. Xu , J. R. Wands , and S. M. De La Monte .
2003 . Potential role of PTEN phosphatase in ethanol-impaired sur-
vival signaling in the liver. Hepatology . 38 : 703 – 714 .
29 . Richards , C. A. , S. A. Short , S. S. Thorgeirsson , and B. E. Huber .
1990 . Characterization of a transforming N-ras gene in the human
hepatoma cell line HepG2: additional evidence for the importance
of c-myc and ras cooperation in hepatocarcinogenesis. Cancer Res.
50 : 1521 – 1527 .
30 . Deaciuc , I. V. , N. B. D’Souza , R. Burikhanov , E. Y. Lee , C. N.
Tarba , C. J. McClain , and W. J. de Villiers . 2002 . Epidermal growth
factor protects the liver against alcohol-induced injury and sensi-
tization to bacterial lipopolysaccharide. Alcohol. Clin. Exp. Res. 26 :
864 – 874 .
31 . Mandrekar , P. , and G. Szabo . 2009 . Signalling pathways in alcohol-
induced liver infl ammation. J. Hepatol. 50 : 1258 – 1266 .
32 . Thakur , V. , M. T. Pritchard , M. R. McMullen , Q. Wang , and L.
E. Nagy . 2006 . Chronic ethanol feeding increases activation of
NADPH oxidase by lipopolysaccharide in rat Kupffer cells: role
of increased reactive oxygen in LPS-stimulated ERK1/2 activation
and TNF-alpha production. J. Leukoc. Biol. 79 : 1348 – 1356 .
33 . Lee , Y. J. , A. R. Aroor , and S. D. Shukla . 2002 . Temporal activation
of p42/44 mitogen-activated protein kinase and c-Jun N-terminal
kinase by acetaldehyde in rat hepatocytes and its loss after chronic
ethanol exposure. J. Pharmacol. Exp. Ther. 301 : 908 – 914 .
34 . Kramer , K. , E. O. Harrington , Q. Lu , R. Bellas , J. Newton , K. L.
Sheahan , and S. Rounds . 2003 . Isoprenylcysteine carboxyl methyl-
transferase activity modulates endothelial cell apoptosis. Mol. Biol.
Cell . 14 : 848 – 857 .
35 . Winter-Vann , A. M. , B. A. Kamen , M. O. Bergo , S. G. Young ,
S. Melnyk , S. J. James , and P. J. Casey . 2003 . Targeting Ras sig-
naling through inhibition of carboxyl methylation: an unex-
pected property of methotrexate. Proc. Natl. Acad. Sci. USA . 100 :
6529 – 6534 .
36 . Sedding , D. G. , M. Tröbs , F. Reich , G. Walker , L. Fink , W.
Haberbosch , W. Rau , H. Tillmanns , K. T. Preissner , R. M. Bohle ,
et al . 2009 . 3-Deazaadenosine prevents smooth muscle cell prolif-
eration and neointima formation by interfering with Ras signaling.
Circ. Res. 104 : 1192 – 1200 .
37 . Kharbanda , K. K. , M. E. Mailliard , C. R. Baldwin , H. C. Beckenhauer ,
M. F. Sorrell , and D. J. Tuma . 2007 . Betaine attenuates alcoholic
steatosis by restoring phosphatidylcholine generation via the phos-
phatidylethanolamine methyltransferase pathway. J. Hepatol. 46 :
314 – 321 .
38 . Yamazaki , T. , E. Sasaki , C. Kakinuma , T. Yano , S. Miura , and O.
Ezaki . 2005 . Increased very low density lipoprotein secretion and
gonadal fat mass in mice overexpressing liver DGAT1. J. Biol. Chem.
280 : 21506 – 21514 .
by guest, on October 20, 2015