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Alcoholic fatty liver is the earliest and most common response of the liver to alcohol and may be a precursor of more severe forms of liver injury. The mechanism by which ethanol causes fatty liver and liver injury is complex. We found that in both rat H4IIEC3 and McA-RH7777 hepatoma cell lines, ethanol induced transcription of a sterol regulatory element-binding protein (SREBP)-regulated promoter via increased levels of mature SREBP-1 protein. This effect of ethanol was blocked by addition of sterols. This effect is likely mediated by acetaldehyde, because the effect was only seen in cell lines expressing alcohol dehydrogenase, and inhibition of ethanol oxidation by 4-methylpyrazole blocked the effect in the hepatoma cells. Furthermore, the aldehyde dehydrogenase inhibitor cyanamide enhanced the effect of ethanol in the hepatoma cells. Consistent with these in vitro findings, feeding mice a low fat diet with ethanol for 4 weeks resulted in a significant increase in steady-state levels of the mature (active) form of SREBP-1. Activation of SREBP-1 by ethanol feeding was associated with increased expression of hepatic lipogenic genes as well as the accumulation of triglyceride in the livers. These finding suggest that metabolism of ethanol increased hepatic lipogenesis by activating SREBP-1 and that this effect of ethanol may contribute to the development of alcoholic fatty liver. Alcoholic fatty liver is the earliest and most common response of the liver to alcohol and may be a precursor of more severe forms of liver injury. The mechanism by which ethanol causes fatty liver and liver injury is complex. We found that in both rat H4IIEC3 and McA-RH7777 hepatoma cell lines, ethanol induced transcription of a sterol regulatory element-binding protein (SREBP)-regulated promoter via increased levels of mature SREBP-1 protein. This effect of ethanol was blocked by addition of sterols. This effect is likely mediated by acetaldehyde, because the effect was only seen in cell lines expressing alcohol dehydrogenase, and inhibition of ethanol oxidation by 4-methylpyrazole blocked the effect in the hepatoma cells. Furthermore, the aldehyde dehydrogenase inhibitor cyanamide enhanced the effect of ethanol in the hepatoma cells. Consistent with these in vitro findings, feeding mice a low fat diet with ethanol for 4 weeks resulted in a significant increase in steady-state levels of the mature (active) form of SREBP-1. Activation of SREBP-1 by ethanol feeding was associated with increased expression of hepatic lipogenic genes as well as the accumulation of triglyceride in the livers. These finding suggest that metabolism of ethanol increased hepatic lipogenesis by activating SREBP-1 and that this effect of ethanol may contribute to the development of alcoholic fatty liver. fatty acid synthase sterol regulatory element-binding protein stearoyl-CoA desaturase ATP citrate lyase malic enzyme acetyl-CoA carboxylase 3-hydroxy-3-methylglutaryl modified Eagle's medium phosphate-buffered saline fetal bovine serum SREBP cleavage-activating protein mitogen-activated protein kinase alcohol dehydrogenase aldehyde dehydrogenase Fatty liver, characterized by accumulation of lipid droplets, and of triglyceride and cholesterol in the liver, is a uniform response of the liver to alcohol (1Nanji A.A. Zakim D. Zakim D. Boyer T.D. Hepatology: A Textbook of Liver Disease. 3rd. Ed. W. B. Saunders Co., Philadelphia1996: 911-913Google Scholar). Although previously considered to be a benign consequence of alcohol use, it is now known that fatty livers are unusually susceptible to the effects of endotoxin, which has been implicated in the pathogenesis of alcoholic hepatitis and fibrosis (2Yang S.Q. Lin H.Z. Lane M.D. Clemens M. Diehl A.M. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 2557-2562Crossref PubMed Scopus (692) Google Scholar, 3Yang S. Lin H. Diehl A.M. Am. J. Physiol. 2001; 281: G382-G392Crossref PubMed Google Scholar, 4Diehl A.M. Alcohol. Clin. Exp. Res. 2001; 25: 8S-14SCrossref PubMed Scopus (41) Google Scholar). Furthermore, obesity, a well-known factor predisposing to fatty liver, has also been noted to be an independent risk factor for the development of cirrhosis in alcoholics (5Naveau S. Giraud V. Borotto E. Aubert A. Capron F. Chaput J.C. Hepatology. 1997; 25: 108-111Crossref PubMed Scopus (547) Google Scholar). In addition, fatty liver occurring in the absence of significant alcohol consumption may be associated with inflammation and fibrosis, a condition designated non-alcoholic steatohepatitis (6Teli M.R. James O.F. Burt A.D. Bennett M.K. Day C.P. Hepatology. 1995; 22: 1714-1719Crossref PubMed Google Scholar). Thus, there is increased interest in understanding the pathogenesis of fatty liver, with the hope that effective therapies against alcohol-induced liver injury might be targeted against the factors that maintain the steatosis. The mechanisms by which ethanol causes fatty liver appear to be complex. Historically, the main mechanism proposed was that reducing equivalents (NADH) generated during ethanol oxidation inhibit the NAD+-requiring steps of the tricarboxylic acid cycle and β-oxidation and, thereby, inhibit fatty acid oxidation (7Crabb D.W. . 1993; 11: 207-230Google Scholar, 8Grunnet N. Kondrup J. Alcohol. Clin. Exp. Res. 1986; 10: 64S-68SCrossref PubMed Scopus (64) Google Scholar). However, it was reported that, although the reductive stress on the liver abates over time in the ethanol-fed baboon model (reflected by a normalization of the ratio of lactate to pyruvate in the hepatic venous blood), fatty infiltration persists (9Salaspuro M.P. Shaw S. Jayatilleke E. Ross W.A. Lieber C.S. Hepatology. 1981; 1: 33-38Crossref PubMed Scopus (88) Google Scholar). Alternative explanations for the persistence of fatty liver include inhibition of lipoprotein export (possibly via formation of acetaldehyde protein adducts with tubulin) and oxidative stress leading to lipid peroxidation (10Grunnet N. Kondrup J. Dich J. Alcohol Alcohol. 1987; 1: 257-261Google Scholar, 11Reinke L.A. Lai E.K. DuBose C.M. McCay P.B. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 9223-9227Crossref PubMed Scopus (219) Google Scholar, 12Kurose I. Higuchi H. Kato S. Miura S. Ishii H. Alcohol. Clin. Exp. Res. 1996; 20: 77A-85ACrossref PubMed Scopus (92) Google Scholar). Although these mechanisms may contribute to the development of fatty liver, additional regulatory systems for fat metabolism have recently been elucidated, and may represent targets for ethanol toxicity. For example, we have demonstrated that ethanol blocks the ability of peroxisome proliferator-activated receptor α to activate transcription, in part due to impairment of its ability to bind target DNA sequences (13Galli A. Pinaire J. Fischer M. Dorris R. Crabb D.W. J. Biol. Chem. 2001; 276: 68-75Abstract Full Text Full Text PDF PubMed Scopus (185) Google Scholar, 14Crabb D.W. Keio. J. Med. 1999; 48: 184-188Crossref PubMed Scopus (72) Google Scholar). Peroxisome proliferator-activated receptor α is a fatty acid receptor that coordinates a number of metabolic pathways that may serve to dispose of excess fatty acids (e.g. by inducing fatty acid oxidizing systems in the mitochondrion and peroxisomes, fatty acid transporters, and binding proteins as well as several apolipoproteins) (15Gearing K.L. Gottlicher M. Widmark E. Banner C.D. Tollet P. Stromstedt M. Rafter J.J. Berge R.K. Gustafsson J.A. J. Nutr. 1994; 124: 1284S-1288SAbstract Full Text PDF PubMed Google Scholar). An additional mechanism that may underlie the development of alcoholic fatty liver is enhanced lipogenesis. Several studies have demonstrated that a significant increase in hepatic lipogenesis occurs in chronically ethanol-treated animals (16Lieber C.S. Spritz N. DeCarli L.M. J. Clin. Invest. 1966; 45: 51-62Crossref PubMed Scopus (129) Google Scholar, 17Carrasco M.P. Marco C. Segovia J.L. Life Sci. 2001; 68: 1295-1304Crossref PubMed Scopus (23) Google Scholar, 18Muramatsu M. Kuriyama K. Yuki T. Ohkuma S. Jpn. J. Pharmacol. 1981; 31: 931-940Crossref PubMed Scopus (10) Google Scholar) and is associated with a significant increase of the activities of hepaticl-α-glycerophosphate acyltransferase, fatty acid synthase (FAS),1 and malic enzyme (ME) (18Muramatsu M. Kuriyama K. Yuki T. Ohkuma S. Jpn. J. Pharmacol. 1981; 31: 931-940Crossref PubMed Scopus (10) Google Scholar, 19Joly J.G. Feinman L. Ishii H. Lieber C.S. J. Lipid Res. 1973; 14: 337-343Abstract Full Text PDF PubMed Google Scholar). A study using KK-Ay mice, a model for obesity and overt diabetes, indicated that acetyl-CoA carboxylase (ACC), ATP citrate lyase (ACL), ME, and 6-phosphogluconate dehydrogenase were markedly increased following administration of ethanol (20Arakawa M. Taketomi S. Furuno K. Matsuo T. Iwatsuka H. J. Nutr. 1975; 105: 1500-1508Crossref PubMed Scopus (14) Google Scholar). Others reported that the mRNA for the low density lipoprotein receptor and 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase was significantly increased in ethanol-fed rats (21Seitz H.K. Kuhn B. von Hodenberg E. Fiehn W. Conradt C. Simanowski U.A. Hepatology. 1994; 20: 487-493PubMed Google Scholar). Increased fatty acid synthesis could even be observed in non-hepatic cells overexpressing ADH and exposed to ethanol (22Galli A. Price D. Crabb D. Hepatology. 1999; 29: 1164-1170Crossref PubMed Scopus (67) Google Scholar). More recently, it was shown that the hepatic lipogenic pathway is activated after consumption of a mere 24 g of ethanol per day in humans (23Siler S.Q. Neese R.A. Hellerstein M.K. Am. J. Clin. Nutr. 1999; 70: 928-936Crossref PubMed Scopus (179) Google Scholar). The battery of enzymes and proteins reported to be induced in liver by ethanol feeding are a subset of those regulated by sterol regulatory element-binding proteins (SREBP-1a, -1c, and -2). SREBPs are synthesized as precursors (∼125 kDa) bound to the endoplasmic reticulum and nuclear envelope. Upon activation, SREBPs are released from the membrane into the nucleus as a mature protein (∼ 68 kDa) by a sequential two-step cleavage process (24Brown M.S. Goldstein J.L. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 11041-11048Crossref PubMed Scopus (1110) Google Scholar). A key role of SREBPs in regulating fatty acid and cholesterol synthesis in liver was suggested by studies of transgenic mice overexpressing the constitutively active mature forms of SREBPs (25Shimano H. Horton J.D. Hammer R.E. Shimomura I. Brown M.S. Goldstein J.L. J. Clin. Invest. 1996; 98: 1575-1584Crossref PubMed Scopus (699) Google Scholar, 26Horton J.D. Shimomura I. Brown M.S. Hammer R.E. Goldstein J.L Shimano H. J. Clin. Invest. 1998; 101: 2331-2339Crossref PubMed Google Scholar). These transgenic mouse studies have suggested that, broadly speaking, SREBP-1 plays an active role in regulating the transcription of genes involved in hepatic triglyceride synthesis (including ACC, FAS, stearoyl-CoA desaturase-1 ME, and is more involved in of genes involved in cholesterol metabolism as the low density lipoprotein and The livers from mice overexpressing SREBP-1 have fatty livers due to increased accumulation of and (25Shimano H. Horton J.D. Hammer R.E. Shimomura I. Brown M.S. Goldstein J.L. J. Clin. Invest. 1996; 98: 1575-1584Crossref PubMed Scopus (699) Google Scholar). More studies have shown that increased hepatic levels of nuclear contribute to the development of fatty liver in mouse of I. Horton J.D. J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar). the genes induced by ethanol feeding and those regulated by there have been studies that the effect of ethanol on SREBP in the we the effect of ethanol on the SREBP and in hepatoma cells as well as in mice were from and were from fetal bovine serum was from and rat hepatoma and cell lines were from the and were from were from Life The a and those found to of the synthase promoter into the was a of F. of The to for a in of was a of J. The for FAS, ME, and dehydrogenase were of D. Horton of cells were in modified Eagle's medium with fetal bovine and the day for the cells were with and to of the and of an for were by the cells were exposed to for The cells were with and medium was ethanol was the cells were in a a of of ethanol the to the of ethanol from the due to after cells were with and in of a and of cell was with on the of M. S. Biol. 1987; PubMed Scopus Google Scholar). was as previously D.W. 1987; PubMed Scopus Google and on a protein from cells livers were as previously Res. PubMed Scopus Google Scholar, H. Brown M.S. Goldstein J.L. Natl. Acad. Sci. U. S. A. 1995; PubMed Scopus Google Scholar). of SREBP protein was using of cell liver nuclear by in an and to SREBP-1 and were using from of the protein was using the was from mouse liver using an of from mouse liver was with and to in a and to membrane for The were with using the DNA The were with the for using with for and exposed to Life with for The were by of the to a and the were to the generated from dehydrogenase livers were in were using and in fatty acid bovine serum was using a cholesterol and triglyceride were using the and to mice were from The were in a with and and For animals on an were on to maintain because ethanol consumption by and were the C.S. DeCarli L.M. Hepatology. 10: PubMed Scopus Google Scholar). was of and diet and The animals were into diet of from and from of as and diet to the diet with ethanol to for of and the of The animals were for 4 weeks The were by the of and of the liver were with The was to in and and of were to the in vitro are as the of ethanol on metabolism may be due to to the of its acetaldehyde, and We rat hepatoma cell lines and a non-hepatic cell for studies from have shown that H4IIEC3 levels of ADH and protein and enzyme D.W. Alcohol. Clin. Exp. Res. 1995; PubMed Scopus Google Scholar). This also SREBP-1 and by demonstrated that the of mRNA was that of in the McA-RH7777 the ratio that that of rat liver R.A. J. Goldstein J.L. Brown M.S. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: PubMed Scopus Google Scholar). Furthermore, that McA-RH7777 cells have protein expression of both ADH and cells were as a cell known to We the cells for to the effects of on a was as In of cell lines, the was markedly by with sterol cholesterol to of Thus, cell to SREBP be by sterol The effect of ethanol on expression was in the cell The cells were with the and the and exposed to of ethanol for for of shown in ethanol markedly increased the of the SREBP in both hepatoma cell lines in the cells. effect on the expression of from the that the effect of ethanol was mediated by the hepatoma cells were also with a in the in to of these cells with and ethanol effect on the of the We the effect of on the ability of ethanol to activate the the effect of ethanol were mediated of it be to be blocked by sterols. In both H4IIEC3 and McA-RH7777 addition of blocked the effect of ethanol on of the These that ethanol induced expression of the by of of more of the SREBP which with a sterol response of the The of effect of ethanol on in cells suggested that ethanol metabolism was for this This was by the of of ethanol We the alcohol dehydrogenase inhibitor 4-methylpyrazole and the aldehyde dehydrogenase inhibitor that inhibitor significantly in McA-RH7777 cells in However, 4-methylpyrazole the effect of ethanol on expression in McA-RH7777 cyanamide the effect The suggest that acetaldehyde generated from ethanol may be for the ability of ethanol to activate We the effect of ethanol and acetaldehyde on the levels of the mature SREBP-1 protein. shown in of nuclear from hepatoma H4IIEC3 cell with ethanol acetaldehyde a increase in the of mature SREBP-1. The of the mature form of SREBP-1 was found to increase after of of the hepatoma cells to ethanol 4 The of mature SREBP-1 to levels acetaldehyde resulted in increase the mature form of SREBP-1 after 4 and the of mature SREBP-1 to levels was in the of the precursor SREBP-1 protein. The of mature protein was by ethanol the effect of ethanol on SREBP in we the effects of ethanol feeding of mice using the diet feeding The mice were a low diet and the low fat diet with ethanol for of the for 4 effect on the of the A to increase in the was observed in both during the shown in significant in and cholesterol were and levels of hepatic cholesterol levels were also found in both However, hepatic were significantly by by ethanol feeding with The these the liver were significantly increased in the low fat diet ethanol Consistent with this livers from ethanol-fed mice were significantly livers from accumulation of lipid in the livers of ethanol-fed mice, lipid were in the livers of that of an low fat diet for 4 weeks to the development of fatty of mice low fat diet with fat significant to the low fat animals Liver cholesterol and triglyceride are to the protein of the significant to the low fat animals Liver cholesterol and triglyceride are to the protein of the cholesterol triglyceride significant to the low fat animals Liver cholesterol and triglyceride are to the protein of the cholesterol to mice were into fat of from and from of from the low fat diet and were the C.S. DeCarli L.M. Hepatology. 10: PubMed Scopus Google Scholar). ethanol was a of was was to for of was of and and The animals were for 4 are as the significant to the low fat animals Liver cholesterol and triglyceride are to the protein of the in a to mice were into fat of from and from of from the low fat diet and were the C.S. DeCarli L.M. Hepatology. 10: PubMed Scopus Google Scholar). ethanol was a of was was to for of was of and and The animals were for 4 are as the the effect of ethanol feeding on SREBPs in mouse of liver nuclear from these mice was that addition of ethanol to the low fat diet resulted in a increase in the of mature SREBP-1 protein. The of mature protein was by addition of ethanol to the In to the of the mature SREBP-1 which was enhanced by ethanol significant increase was observed in the precursor protein levels of SREBP-1 the mRNA This suggested that ethanol the of mature SREBP-1 protein a This is to the observed with the hepatoma cells. the of SREBP-1 was associated with a increase in expression of genes known to be regulated by a of were using from the livers of mice in I. shown in ethanol feeding increased the expression of from several SREBP target genes by to the of These targets the enzyme of fatty acid FAS, the enzymes that and acetyl-CoA for fatty acid synthesis and and the enzyme for of and to and of and are is the only found in liver Lane M.D. J. Biol. Chem. Full Text PDF PubMed Google Scholar). Thus, We to the of in the liver the was low to These suggest that the increase in the mature form of SREBP-1 by ethanol was associated with increased of this in the increased mRNA levels of a battery of lipogenic In the we demonstrated that, in both rat H4IIEC3 and McA-RH7777 hepatoma cell lines, ethanol induced transcription of promoter via increased levels of mature SREBP-1 protein. of was blocked by the of known to inhibit the of This effect of ethanol is likely mediated by acetaldehyde, because inhibition of ethanol oxidation by an inhibitor of the ADH known to be in these blocked the the aldehyde dehydrogenase inhibitor cyanamide enhanced the effect of Furthermore, ethanol have effect on an regulated promoter in which Consistent with vitro findings, feeding mice a low fat diet with ethanol for 4 weeks resulted in a significant increase in the of the mature form of SREBP-1. Activation of SREBP-1 by ethanol feeding was associated with increased expression of several hepatic lipogenic genes known to be by SREBP-1 as well as the accumulation of triglyceride in the livers. is to that increased of these enzymes has been reported in livers of ethanol-fed animals (18Muramatsu M. Kuriyama K. Yuki T. Ohkuma S. Jpn. J. Pharmacol. 1981; 31: 931-940Crossref PubMed Scopus (10) Google Scholar, 19Joly J.G. Feinman L. Ishii H. Lieber C.S. J. Lipid Res. 1973; 14: 337-343Abstract Full Text PDF PubMed Google Scholar, M. Taketomi S. Furuno K. Matsuo T. Iwatsuka H. J. Nutr. 1975; 105: 1500-1508Crossref PubMed Scopus (14) Google Scholar). that the increased is the of increased levels of the The increase in in is in the increased of and acid in the triglyceride in the liver of ethanol-fed animals H. C. PubMed Scopus Google Scholar). The of acetaldehyde could be due to increased of formation of the mature SREBP-1 impairment of its The model of SREBP that the precursors of SREBPs are to as a with SREBP cleavage-activating protein a membrane protein with a (24Brown M.S. Goldstein J.L. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 11041-11048Crossref PubMed Scopus (1110) Google Scholar). cells are of is activated and SREBPs to the complex. In the SREBPs are activated by sequential cleavage by and Thus, effect of acetaldehyde could be to the of that enzyme that is known to be by acetaldehyde, an enzyme in the pathway Crabb D. L. Lin Hepatology. 1996; Google Scholar, L. Hepatology. 10: PubMed Scopus Google Scholar, A.A. Lin Hepatology. 1997; PubMed Scopus Google Scholar, Hepatology. 1998; PubMed Scopus Google Scholar). is known enzymes that in sterol metabolism from reductase are by ethanol cells are with the to to the and SREBPs are in vitro study that addition of blocked the effect of ethanol on expression in both hepatoma cell This be with an effect of acetaldehyde on sterol levels with an effect mediated is well known that the mature form of SREBP-1 is via the pathway (24Brown M.S. Goldstein J.L. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 11041-11048Crossref PubMed Scopus (1110) Google Scholar). to ethanol could the of the mature form of SREBP-1 via inhibition of has been shown to inhibit protein in rats ethanol chronically F. J. C. M. . although the mechanism for this is Furthermore, the effect of ethanol to be because SREBP-1 levels were increased in the cells and the mouse livers. An additional could be the formation of acetaldehyde adducts with mature SREBP-1 and, inhibition of SREBP-1 because acetaldehyde is known to form adducts with a number of proteins H. C. PubMed Scopus Google Scholar, J. Clin. Invest. 84: PubMed Scopus Google Scholar, D. Hepatology. 1996; PubMed Google Scholar). SREBPs are only regulated by sterol levels also by J. D. L. M. S. B. W. J. Lipid Res. Full Text Full Text PDF PubMed Google and the SREBPs are of J. L. S. C. W. D. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). Several studies have shown that ethanol plays a role in the regulating pathway Pharmacol. 1996; PubMed Scopus Google Scholar, Scopus Google Scholar, J. F. B. J. PubMed Scopus Google Scholar). ethanol could activate SREBPs via of additional study be to the mechanism by which ethanol acetaldehyde the SREBP pathway both in vitro and in The increased of mature SREBP-1 after ethanol feeding was associated with increased for lipogenic enzymes and triglyceride accumulation in liver. However, we found that nuclear levels were in livers from the ethanol feeding mice as with the mice, and hepatic levels of cholesterol were found in the and ethanol-fed These are with the vitro and in studies that SREBP-1 enzymes involved in is for the of genes involved in cholesterol (24Brown M.S. Goldstein J.L. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 11041-11048Crossref PubMed Scopus (1110) Google Scholar). However, suggest that ethanol feeding increased cholesterol levels in rat liver 20: PubMed Scopus Google it is that the to an increase in cholesterol in the mice is a effect is to in The of these is it was reported that, with alcohol the stress to the of by ADH and abates in the ethanol-fed However, fatty liver in these suggested by studies is that the may contribute to the of fat in the of alcohol use, effects on transcription factors as SREBP may be for of increased levels of fat synthesis and fatty liver. In of the lipogenic enzyme battery may increased of fatty acid synthesis even of alcohol the battery of enzymes include only for fatty acid synthesis by FAS, the is a inhibitor of I. This enzyme is the of fatty acid oxidation in the liver, with a of T.D. L. J. 1997; PubMed Scopus Google Scholar). the of in the livers of animals ethanol has been it is to with increased of This the that inhibition of fatty acid oxidation by to the accumulation of fat in the liver, and this effect may be independent of the effect of ethanol on the of the The is that the SREBP pathway is to by as sterols. Thus, it may be to the of fat synthesis and the of fatty liver by the diet of alcoholics with the sterols. Although the of is from there are in which liver injury might be by of fatty liver. Fatty livers are to endotoxin, which in is to an role in the pathogenesis of alcoholic hepatitis and of fatty liver might the of this to injury from We are to Ross for
You et al. (Thu,) studied this question.
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