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J. Biol. Chem., Vol. 277, Issue 32, 29342-29347, August 9, 2002

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Ethanol Induces Fatty Acid Synthesis Pathways by Activation of Sterol Regulatory Element-binding Protein (SREBP)^*

Min You, Monika Fischer, Mark A. Deeg, and David W. Crabb

From the Departments of Medicine, Indiana University School of Medicine and Richard Roudebush Veteran's Affairs Medical Center, Indianapolis, Indiana 46202

Received for publication, March 12, 2002, and in revised form, May 23, 2002

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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 (1). 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 (2-4). 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 (5). 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 (6). 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 -oxidation and, thereby, inhibit fatty acid oxidation (7, 8). 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 (9). 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 (10-12). 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  to activate transcription, in part due to impairment of its ability to bind target DNA sequences (13, 14). Peroxisome proliferator-activated receptor  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) (15).

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 (16-18) and is associated with a significant increase of the activities of hepatic L--glycerophosphate acyltransferase, fatty acid synthase (FAS),¹ and malic enzyme (ME) (18, 19). A study using KK-A^y 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 (20). 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 (21). Increased fatty acid synthesis could even be observed in non-hepatic cells overexpressing ADH and exposed to ethanol (22). 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 (23).

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 (24). 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 (25, 26). 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 (SCD), ACL, ME, and L--glycerophosphate acyltransferase), whereas SREBP-2 is more involved in regulation of genes involved in cholesterol metabolism (such as the low density lipoprotein receptor, HMG-CoA synthase, HMG-CoA reductase, and squalene synthase). The livers from mice overexpressing SREBP-1 have massive fatty livers due to increased accumulation of cholesteryl esters and triglycerides (25). More interestingly, studies have shown that increased hepatic levels of nuclear SREBP-1c contribute to the development of fatty liver in two mouse models of diabetes (27).

Despite the overlap between genes induced by ethanol feeding and those regulated by SREBPs, there have been no studies that examine the effect of ethanol on SREBP activity. Therefore, in the present study, we examined the effect of ethanol on the SREBP activation and transcriptional function in cultured hepatoma cells as well as in mice fed ethanol-containing diets.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Materials-- Most chemicals were purchased from Sigma Chemical Co. Trypsin and tissue culture media were purchased from Invitrogen. Delipidated fetal bovine serum was purchased from Sigma. CV-1 (African green monkey kidney) and rat hepatoma (H4IIEC3 and McA-RH7777) cell lines were purchased from the American Type Culture Collection. Cholesterol and 25-hydroxycholesterol were obtained from Sigma. All radioisotopes were purchase from PerkinElmer Life Sciences. The pSyn SRE plasmid, containing a generic TATA and three SRE elements (representing those found at 325 to 225 bp of the hamster HMG-CoA synthase promoter fused into the luciferase pGL2 Basic vector), was a kind gift of Dr. Timothy F. Osborne (University of California, Irvine, CA). The JS-15 plasmid (identical to pSyn SRE, except for a double-point mutation resulting in loss of sterol-regulated transcription) was a kind gift of Dr. Richard J. Deckelbaum (Columbia University, New York, NY). The cDNA probes for SREBP-1, FAS, SCD, ACL, ME, and glyceraldehyde-3-phosphate dehydrogenase were kind gifts of Dr. Jay D. Horton (University of Texas Southwestern Medical Center, Dallas, TX).

Transfection of Tissue Culture Cells-- All cells were grown in modified Eagle's medium (MEM) supplemented with 10% fetal bovine serum, 100 µg/ml streptomycin, and 63 µg/ml penicillin G. On the day for transfection, the cells were washed with PBS and switched to MEM supplemented 10% delipidated FBS serum. Ten µg of the reporter plasmid and 5 µg of pSV2CAT (as an internal control for transfection efficiency) were transfected by calcium phosphate precipitation. Four hours later the cells were exposed to PBS containing 15% glycerol for 3 min. The cells were rinsed twice with PBS, and fresh medium was added. When ethanol was present, the cells were incubated in a chamber containing a beaker of 1 liter of water containing ethanol at the same concentration to reduce the loss of ethanol from the cultures due to evaporation. Forty-eight hours after transfection, cells were washed twice with PBS and lysed in 150 µl of a buffer containing 25 mM Tris, pH 7.8, 2 mM EDTA, 20 mM dithiothreitol, 10% glycerol, and 1% Triton X-100. Fifty microliters of cell extracts was incubated with luciferase assay reagent based on the original protocol of de Wet et al. (28). Chloramphenicol acetyltransferase activity was measured as described previously (29), and quantified on a PhosphorImager (Amersham Biosciences).

Immunoblots-- Nuclear protein extracts from cells or animal livers were prepared as described previously (30, 31). Measurement of SREBP protein was performed using 100 µg of crude cell or liver nuclear protein, separated by electrophoresis in an 8% SDS-polyacrylamide gel and transferred to nitrocellulose filters. SREBP-1 and SREBP-2 were visualized using antibodies obtained from Santa Cruz Biotechnology. Detection of the protein bands was performed using the Amersham Biosciences ECL kit.

Northern Blots-- Total RNA was prepared from mouse liver using an RNeasy Total RNA kit (Qiagen Inc.). Equal aliquots of total RNA (40 µg) from each mouse liver was denatured with formaldehyde and formamide, subjected to electrophoresis in a 1.2% agarose gel, and transferred to Hybond N+ membrane (Amersham Biosciences) for hybridization. The cDNA probes were labeled with [-³²P]dCTP using the Megaprime DNA labeling system kit (Amersham Biosciences). The filters were hybridized with the indicated ³²P-labeled probes (~1 × 10⁶ cpm/ml) for 2 h at 65 °C using Rapid-hyb buffer (Amersham Biosciences), washed with 0.1% (w/v) SDS/0.1× SSC at 70 °C for 60 min, and exposed at 80 °C to Reflection NFE496 film (PerkinElmer Life Sciences Inc.) with intensifying screens for 3-36 h at room temperature. The resulting bands were quantified by exposure of the filter to a PhosphorImager screen, and the results were normalized to the signal generated from glyceraldehyde-3-phosphate dehydrogenase mRNA.

Total Cholesterol and Triglyceride Levels-- Mouse livers were homogenized in radioimmune precipitation lysis buffer. Lipids were extracted using chloroform/methanol (2:1 v/v), evaporated under dry nitrogen, and dissolved in 5% fatty acid free bovine serum albumin. Protein was assayed using a Protein Assay kit (Bio-Rad Laboratories). Colorimetric cholesterol and triglyceride assays were carried out using the Sigma Diagnostics Cholesterol and Triglyceride Reagents (Sigma Chemical Co.).

Animals and Diets-- 6- to 8-week-old male C57BL/6J mice were obtained from The Jackson Laboratory (Bar Harbor, ME). Animals were housed individually in a room with controlled temperature (20-22 °C), humidity (55-65%), and lighting (on at 6 a.m. and off at 6 p.m.). For animals on an ethanol-containing diet, animal cages were placed on heating pads to maintain body temperature because ethanol consumption can induce hypothermia. Liquid diets provide 1 kcal/ml (prepared by Dyets, Inc., Bethlehem, PA) and were based upon the Lieber-DeCarli formulation (32). Protein content was constant at 18% of calories, and each diet had identical mineral and vitamin content. The animals were divided into two dietary groups: (a) control diet (fat comprising 10% of total calories, 6% from cocoa butter, and 4% from safflower oil, 72% of calories as carbohydrate) and (b) ethanol-containing diet (identical to the control diet but with ethanol added to account for 27.5% of total calories and the caloric equivalent of carbohydrate (maltose-dextrin) removed). The animals were pair-fed for 4 weeks then sacrificed. The experimental protocols were approved by the Indiana University School of Medicine Animal Care and Use Committee.

Histology of Liver-- Frozen sections of the liver were stained with Oil Red O.

Data Analysis-- The paired t test was used to evaluate statistical differences in values between ethanol- and pair-fed mice. Both t tests and analysis of variance were used to analyze the in vitro data. All data are presented as the mean ± S.E.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Effects of Ethanol on Transcription of SRE-containing Promoters in Vitro-- Effects of ethanol on cellular metabolism may be due directly to ethanol, or to the products of its oxidation, including NADH, acetaldehyde, and acetate. We chose two rat hepatoma cell lines and a non-hepatic cell line for study. Previous studies from our laboratory have shown that H4IIEC3 expresses moderately high levels of class I (low K_m) ADH and ALDH2 protein and enzyme activity (33). This line also expresses SREBP-1 and -2 (data not shown). Studies by others demonstrated that the amount of SREBP-1c mRNA was nearly 3-fold greater than that of SREBP-1a in the McA-RH7777 cells, the ratio that approaches that of normal rat liver (9:1) (34). Furthermore, Western blot analysis showed that McA-RH7777 cells have substantial protein expression of both ADH and ALDH2 (data not shown). CV-1 cells were chosen as a cell type known not to express ADH. We tested the cells for responsiveness to the effects of sterols on a SRE reporter gene, pSyn SRE-luciferase. pSV2-CAT was used as internal control. In each of three cell lines, the reporter activity was markedly reduced by incubation with sterol (10 µg/ml cholesterol plus 1 µg/ml 25-hydroxycholesterol) to 10-35% of control activity. Thus, each cell appears to express functional SREBP whose activity can be suppressed by sterol supplementation.

The effect of ethanol on pSyn SRE expression was then tested in the three cell lines. The cells were transfected with the reporter, pSyn SRE-luciferase, and the internal control plasmid (pSV2CAT) and exposed to various concentrations of ethanol for 48 h then harvested for assay of reporter enzymes. As shown in Fig. 1, ethanol markedly increased the activity of the SREBP reporter in both hepatoma cell lines but not in the CV-1 cells. Ethanol had no effect on the expression of luciferase from the parent plasmid, pGL2 Basic (data not shown). To confirm that the effect of ethanol was mediated by SREBPs, the hepatoma cells were also transiently transfected with JS-15, a pSyn SRE plasmid mutated in the SRE regions, resulting in insensitivity to sterol. Incubation of these cells with sterols did not down-regulate SRE expression, and ethanol had no effect on the activity of the reporter (data not shown).

View larger version (30K): [in this window] [in a new window]   Fig. 1.   Ethanol enhances the expression of pSyn SRE in hepatoma cells. CV-1, H4IIEC3, and McA-RH7777 cells were plated in MEM (low glucose) supplemented with 10% delipidated FBS and transfected with pSyn SRE and internal control (pSV2CAT) for 5 h, and the cells were exposed to PBS containing 15% glycerol for 3 min. Then the cells were incubated with varying concentrations of ethanol for 48 h. The concentration of ethanol in the medium was maintained by culturing the cells in an incubator containing a reservoir of ethanol as indicated in water. The cells then were harvested for assay of the reports. Luciferase and CAT activity were determined as described under "Experimental Procedures." The data are expressed as percentages of control (mean ± S.E.) from at least five experiments performed in duplicate. *, p < 0.05; **, p < 0.01 by t test, compared with control.

We further tested the effect of sterols on the ability of ethanol to activate the pSyn SRE-luciferase reporter. If the effect of ethanol were mediated through activation of SREBPs, it would be predicted to be blocked by supplemental sterols. In both H4IIEC3 and McA-RH7777 cells, addition of sterols completely blocked the effect of ethanol on activation of the pSyn SRE reporter activity (Fig. 2). These data provide strong evidence that ethanol induced expression of the reporter by way of activation of one or more of the SREBP forms, which interacts with a sterol response element of the reporter.

View larger version (15K): [in this window] [in a new window]   Fig. 2.   Sterols block the effect of ethanol on activation of pSyn SRE reporter activity. H4IIEC3 and McA-RH7777 cells were plated in MEM (low glucose) supplemented with 10% delipidated FBS and transfected with pSyn SRE and internal control (pSV2CAT) for 5 h. Cells were then incubated with increasing concentrations of ethanol and increasing amounts of sterol (indicated as micrograms/ml cholesterol/25-hydroxycholesterol) for 48 h. The cells then were harvested for assay of the reporter enzymes. Luciferase and CAT activity were determined as described under "Experimental Procedures." The data are expressed as percentages of control (mean ± S.E.) from at least three experiments performed in duplicate. *, p < 0.05; **, p < 0.01 by t test, compared with control.

The lack of effect of ethanol on pSyn SRE-luciferase in CV-1 cells suggested that ethanol metabolism was required for this effect. This was further substantiated by the use of inhibitors of ethanol metabolism. We used the class I alcohol dehydrogenase inhibitor 4-methylpyrazole and the aldehyde dehydrogenase inhibitor cyanamide. Fig. 3 shows that neither inhibitor significantly affected pSyn SRE-luciferase activity in McA-RH7777 cells in control experiments. However, 4-methylpyrazole nearly abolished the effect of ethanol on pSyn SRE-luciferase expression in McA-RH7777 cells, whereas cyanamide augmented the effect markedly. The results suggest that acetaldehyde generated from ethanol may be responsible for the ability of ethanol to activate pSyn SRE-luciferase.

View larger version (20K): [in this window] [in a new window]   Fig. 3.   Effect of inhibitors of alcohol and aldehyde dehydrogenase on the action of ethanol on pSyn SRE reporter activity in hepatoma McA-RH7777 cells. McA-RH7777 cells were transfected as described in Fig. 1 with the pSyn SRE reporter plasmid and internal control (pSV2CAT); the inhibitors were added 24 h later, and the cells were exposed to ethanol as indicated, beginning at the same time. The cells then were harvested 24 h later for assay of the reports. Luciferase and CAT activities were determined as described under "Experimental Procedures." The data are expressed as percentages of control (mean ± S.E.) from at least three experiments performed in duplicate. *, p < 0.05; **, p < 0.01 by paired t test, compared with control.

Effect of Ethanol and Acetaldehyde on Levels of Mature SREBP Protein-- We further examined the effect of ethanol and acetaldehyde on the levels of the mature SREBP-1 protein. As shown in Fig. 4, Western analysis of nuclear extracts from hepatoma H4IIEC3 cell incubated with ethanol or acetaldehyde showed a substantial but transient increase in the amount of mature SREBP-1. The mass of the mature form of SREBP-1 was found to increase ~2.5-fold after 30 min of exposure of the hepatoma cells to ethanol (Fig. 4A). The amount of mature SREBP-1 returned to control levels within 1 h. Similarly, acetaldehyde treatment resulted in increase the mature form of SREBP-1 ~2-fold after 30 or 45 min (Fig. 4B), and the amount of mature SREBP-1 returned to control levels within 1 h. There was no discernable change in the mass of the precursor SREBP-1 protein. The amount of mature SREBP-2 protein was not altered by ethanol treatment (data not shown).

View larger version (21K): [in this window] [in a new window]   Fig. 4.   Immunoblot analysis of SREBP-1 in nuclear extracts of rat hepatoma H4IIEC3 cells. H4IIEC3 cells were incubated in MEM (low glucose) supplemented with 10% delipidated FBS medium with or without 50 mM ethanol (A) or 200 µM acetaldehyde (B) for the indicated times. Aliquots of nuclear extracts (100 µg of protein) were electrophoresed on 10% SDS-polyacrylamide gels under reducing conditions, transferred to nitrocellulose membranes, and immunostained with anti-SREBP-1 antibody.

Increase in Hepatic Lipids after Ethanol Feeding-- To determine the effect of ethanol on SREBP in vivo, we first established the effects of ethanol feeding of mice using the usual liquid diet pair feeding protocol. The mice were fed a low fat, high carbohydrate diet (4% safflower oil, 6% cocoa butter, and 72% carbohydrate) or the low fat diet with ethanol substituted for 27.5% of the carbohydrate calories for 4 weeks. Ethanol intake had no apparent effect on the health status of the animals. A steady average 2- to 3-g increase in the body weight was observed in both groups during the entire 4-week study. As shown in Table I, no significant differences in plasma triglycerides and cholesterol were detected between ethanol- and pair-fed animals. Similar levels of hepatic cholesterol (esterified plus unesterified) levels were also found in both groups. However, hepatic triglycerides were significantly elevated by about 3.5-fold by ethanol feeding compared with pair-fed controls. The body weights did not vary between these groups, but the liver weights were significantly increased in the low fat diet plus ethanol group. Consistent with this observation, livers from ethanol-fed mice were significantly larger than livers from control group. Histological analysis showed prominent accumulation of lipid droplets in the livers of ethanol-fed mice, whereas lipid droplets were rare in the livers of control groups (Fig. 5). Our data clearly demonstrate that ingestion of an ethanol-containing low fat diet for 4 weeks led to the development of fatty liver.

View larger version (130K): [in this window] [in a new window]   Fig. 5.   Ethanol effects on liver. Oil red O staining of liver sections of mice fed a low fat diet alone (a) or the low fat diet with ethanol (b).

The Effect of Ethanol Feeding on Levels of Mature SREBP-1 Protein in Mouse Livers-- To determine the effect of ethanol feeding on SREBPs in mouse livers, immunoblot analysis of liver nuclear extracts from these mice was performed. Fig. 6 showed that addition of ethanol to the low fat diet resulted in a substantial increase in the amount of mature SREBP-1 protein. The amount of mature SREBP-2 protein was not altered by addition of ethanol to the diet. In contrast to the level of the mature SREBP-1 protein, which was enhanced by ethanol feeding, no significant increase was observed in either the membrane-bound precursor protein levels of SREBP-1 or the corresponding mRNA (data not shown). This suggested that ethanol regulates the abundance of mature SREBP-1 protein mainly at a post-translational level. This is similar to the results observed with the hepatoma cells.

View larger version (28K): [in this window] [in a new window]   Fig. 6.   Immunoblot analysis of SREBP-1 and -2 in nuclear extracts from livers of mice fed a low fat diet with or without ethanol. Eight male C57BL/6J mice were fed a low fat diet with (lanes 3 and 4) or without ethanol (lanes 1 and 2) for 4 weeks. Aliquots of nuclear extracts (80 µg of protein) from pooled livers from four pooled livers of each group were electrophoresed on 10% SDS-polyacrylamide gels under reducing conditions, transferred to nitrocellulose membranes, and immunostained with anti-SREBP-1 or anti-SREBP-2 antibody. P and N denote the precursor and cleaved nuclear forms of SREBP, respectively. In the left lower panel, an intense cross-reacting protein is indicated by an asterisk. The positions to which prestained protein markers migrated to in the polyacrylamide gel are indicated on the left of the lumigrams.

Effect of Ethanol Feeding on Expression of Genes Regulated by SREBPs-- To determine whether the ethanol-mediated induction of SREBP-1 maturation was associated with a corresponding increase in expression of genes known to be regulated by SREBPs, a series of Northern blot analyses were performed using total RNA from the livers of mice described in Table I. As shown in Fig. 7, ethanol feeding increased the expression of mRNAs from several SREBP target genes by ~2-fold (normalized to the level of glyceraldehyde-3-phosphate dehydrogenase). These targets included the major enzyme of fatty acid synthesis, FAS, the enzymes that supply NADPH and acetyl-CoA for fatty acid synthesis (ME and ACL, respectively), and the enzyme responsible for conversion of palmitate and stearate to palmitoleate and oleate, SCD. Two isoforms of SCD (SCD1 and SCD2) are currently known. SCD1 is the only SCD isoform found in liver (35). Thus, our data mainly reflect SCD1 expression. We attempted to measure the abundance of ACC in the liver RNA samples, but the signal was too low to quantify. These findings suggest that the increase in the mature form of SREBP-1 by ethanol was associated with increased transcriptional activity of this factor, reflected in the increased mRNA levels of a battery of lipogenic genes.

View larger version (30K): [in this window] [in a new window]   Fig. 7.   Northern blot analysis of lipogenic enzymes from livers of mice fed a low fat diet with or without ethanol. A, total RNA (20 µg) from livers of mice fed low fat diet without (lanes 1 and 2) or with (lanes 3 and 4) ethanol was subjected to Northern blotting, followed by hybridization with the indicated cDNA probes. A cDNA probe for glyceraldehyde-3-phosphate dehydrogenase was used to confirm equal loading. Northern blots from two representative mouse livers are shown. B, results were quantified by phosphorimaging. All RNA levels are expressed relative to low fat pair-fed control (mean ± S.D., n = 4-5 animals). *, p < 0.05; **, p < 0.01 by paired t test, compared with low fat diet control. FAS, fatty acid synthase; SCD, stearoyl-CoA desaturase; ACL, ATP citrate lyase; ME, malic enzyme.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

In the present study, we demonstrated that, in both rat H4IIEC3 and McA-RH7777 hepatoma cell lines, ethanol induced transcription of SRE-regulated promoter via increased levels of mature SREBP-1 protein. Expression of promoters was blocked by the presence of sterols known to inhibit the activation of SREBPs. This effect of ethanol is likely mediated by acetaldehyde, because inhibition of ethanol oxidation by 4-methylpyrazole, an inhibitor of the class I ADH known to be expressed in these cells, blocked the effect. Conversely, the aldehyde dehydrogenase inhibitor cyanamide enhanced the effect of ethanol. Furthermore, ethanol did not have any effect on an SRE- regulated promoter in CV-1 cells, which do not express ADH. Consistent with our in vitro findings, feeding mice a low fat diet with ethanol for 4 weeks resulted in a significant increase in the abundance 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 controlled by SREBP-1 as well as the accumulation of triglyceride in the livers. It is interesting to note that increased activity of these enzymes has already been reported in livers of ethanol-fed animals (18-20). Our data indicate that the increased activity is the result of increased levels of the corresponding mRNAs. The increase in SCD activity in turn is reflected in the increased amounts of palmitoleic and oleic acid in the triglyceride stored in the liver of ethanol-fed animals (36).

The action of acetaldehyde could be due to increased rates of formation of the mature SREBP-1 or impairment of its further degradation. The current model of SREBP regulation holds that the 125-kDa precursors of SREBPs are anchored to intracellular membranes as a complex with SREBP cleavage-activating protein (SCAP), a membrane protein with a sterol-sensing domain (24). When cells are deprived of sterols, SCAP is activated and escorts SREBPs to the Golgi complex. In the Golgi, SREBPs are activated by sequential proteolytic cleavage by two proteases, site 1 protease and site 2 protease. Thus, one effect of acetaldehyde could be to reduce the concentration of sterols that influence SCAP activity. One enzyme that metabolizes sterols is known to be inhibited by acetaldehyde, namely ⁴-3-ketosteroid 5--reductase, an enzyme participating in the bile salt synthetic pathway (37-40). It is not known if other enzymes that participate in sterol metabolism aside from HMG-CoA reductase are affected by ethanol or acetaldehyde. When cells are overloaded with sterols, the SCAP·SREBP complex fails to move to the Golgi, and SREBPs are not processed. Our in vitro study showed that addition of sterols blocked the effect of ethanol on SRE-dependent gene expression in both hepatoma cell lines. This would be consistent with either an effect of acetaldehyde on sterol levels or with an effect mediated further downstream.

It is well known that the mature form of SREBP-1 is ultimately disposed via the proteasomal pathway (24). Exposure to ethanol could affect the level of the mature form of SREBP-1 via inhibition of proteasomal degradation. Ethanol has been shown to inhibit proteasomal protein degradation in rats fed ethanol chronically (41), although the mechanism for this action is unknown. Furthermore, the effect of ethanol appeared to be somewhat selective, because SREBP-1 but not SREBP-2 levels were increased in the cells and the mouse livers. An additional explanation could be the formation of acetaldehyde adducts with mature SREBP-1 and, subsequently, inhibition of SREBP-1 degradation, because acetaldehyde is known to form adducts with a number of proteins (36, 42, 43).

SREBPs are not only regulated by intracellular sterol levels but also by MAPK signaling (44), and the SREBPs are possibly substrates of MAPK (45). Several studies have shown that ethanol plays a positive role in the regulating MAPK signaling pathway (46-48). Therefore, ethanol could activate SREBPs indirectly via activation of MAPK. Clearly, additional study will be needed to understand the exact mechanism by which ethanol or acetaldehyde affects the SREBP pathway both in vitro and in vivo.

The increased level of mature SREBP-1 after ethanol feeding was associated with increased mRNAs for multiple SREBP-regulated lipogenic enzymes and triglyceride accumulation in liver. However, we found that nuclear SREBP-2 levels were unchanged in livers from the ethanol feeding mice as compared with the pair-fed mice, and similar hepatic levels of total cholesterol were found in the control and ethanol-fed groups. These results are consistent with the previous in vitro and in vivo studies that SREBP-1 preferentially activates enzymes involved in lipogenesis, whereas SREBP-2 is primarily responsible for the transcriptional regulation of genes involved in cholesterol homeostasis (24). However, earlier reports suggest that ethanol feeding increased cholesterol ester levels in rat liver (49); it is possible that the failure to see an increase in cholesterol in the mice is a species-dependent effect or is related to other differences in diets.

The importance of these findings is 3-fold. First, it was reported many years ago that, with continued alcohol feeding, the redox stress attributable to the generation of NADH by ADH and ALDH abates in the ethanol-fed baboon. However, fatty liver persisted in these animals. One explanation suggested by our studies is that the redox perturbation may contribute to the retention of fat early in the course of heavy alcohol use, but effects on transcription factors such as SREBP may be responsible for maintenance of increased levels of fat synthesis and fatty liver. In fact, activation of the lipogenic enzyme battery may perpetuate increased rates of fatty acid synthesis even between periods of heavy alcohol consumption.

Second, the SREBP-regulated battery of enzymes include ACC. ACC not only supplies malonyl-CoA for fatty acid synthesis by FAS, but the malonyl-CoA is a potent inhibitor of carnitine palmitoyl transferase I. This enzyme is the major regulator of fatty acid oxidation in the liver, with a flux control coefficient of about 0.7-0.8 (50). Surprisingly, the content of malonyl-CoA in the livers of animals fed ethanol has not been reported, but it is expected to rise with increased activity of ACC. This raises the possibility that inhibition of fatty acid oxidation by malonyl-CoA contributes to the accumulation of fat in the liver, and this effect may be independent of the effect of ethanol on the redox state of the hepatocyte.

The third point is that the SREBP pathway is amenable to manipulation by exogenous compounds, such as sterols. Thus, it may be possible to reduce the rate of fat synthesis and the severity of fatty liver by supplementing the diet of alcoholics with the appropriate sterols. Although the long term goal of treatment is abstinence from alcohol, there are conditions in which liver injury might be reduced by rapid control of fatty liver. Fatty livers are exquisitely sensitive to endotoxin, which in turn is felt to play an important role in the pathogenesis of alcoholic hepatitis and cirrhosis. Resolution or control of fatty liver might reduce the sensitivity of this organ to ongoing injury from heavy drinking.

	ACKNOWLEDGEMENT

We are grateful to Ruth Ann Ross for her excellent technical assistance.

	FOOTNOTES

^* This work was supported by Grant AA06463 from the National Institute on Alcohol Abuse and Alcoholism (to D. W. C.) and Grant AA07611 from the Alcohol Research Center.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed: Veterans Administration Research 151, Third Floor D-3035, 1481 West Tenth St., Indianapolis, IN 46202. Tel.: 317-554-0000 (ext. 4544); Fax: 317-554-0116; E-mail: miyou@iupui.edu.

Published, JBC Papers in Press, May 29, 2002, DOI 10.1074/jbc.M202411200

	ABBREVIATIONS

The abbreviations used are: FAS, fatty acid synthase; SREBP, sterol regulatory element-binding protein; SCD, stearoyl-CoA desaturase; ACL, ATP citrate lyase; ME, malic enzyme; ACC, acetyl-CoA carboxylase; HMG, 3-hydroxy-3-methylglutaryl; MEM, modified Eagle's medium; PBS, phosphate-buffered saline; FBS, fetal bovine serum; SCAP, SREBP cleavage-activating protein; MARK, mitogen-activated protein kinase; ADH, alcohol dehydrogenase; ALDH, aldehyde dehydrogenase.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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