Chronic ethanol feeding and folate deficiency activate hepatic endoplasmic reticulum stress pathway in micropigs
Farah Esfandiari,1
Jesus A. Villanueva,1
Donna H. Wong,1
Samuel W. French,2 and
Charles H. Halsted1
1Department of Internal Medicine, University of California-Davis, Davis; and 2Department of Pathology, Harbor-University of California Los Angeles Medical Center, Torrance, California
Submitted 9 December 2004
; accepted in final form 2 February 2005
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ABSTRACT
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Previously, we showed that feeding micropigs ethanol with a folate-deficient diet promoted the development of hepatic injury while increasing hepatic levels of homocysteine and S-adenosylhomocysteine (SAH) and reducing the level of S-adenosylmethionine (SAM) and the SAM-to-SAH ratio. Our present goals were to evaluate mechanisms for hepatic injury using liver specimens from the same micropigs. The effects of ethanol feeding or folate-deficient diets, singly or in combination, on cytochrome P-450 2E1 (CYP2E1) and signal pathways for apoptosis and steatosis were analyzed using microarray, real-time PCR, and immunoblotting techniques. Apoptosis was increased maximally by the combination of ethanol feeding and folate deficiency and was correlated positively to liver homocysteine and SAH. Liver CYP2E1 and the endoplasmic reticulum stress signals glucose-regulated protein 78 (GRP78), caspase 12, and sterol regulatory element binding protein-1c (SREBP-1c) were each activated in pigs fed folate-deficient or ethanol diets singly or in combination. Liver mRNA levels of CYP2E1, GRP78, and SREBP-1c, and protein levels of CYP2E1, GRP78, nuclear SREBP, and activated caspase 12 each correlated positively to liver levels of SAH and/or homocysteine and negatively to the SAM-to-SAH ratio. The transcripts of the lipogenic enzymes fatty acid synthase, acetyl-CoA carboxylase, and stearoyl-CoA desaturase were elevated in the ethanol-fed groups, and each was positively correlated to liver homocysteine levels. The induction of abnormal hepatic methionine metabolism through the combination of ethanol feeding with folate deficiency is associated with the activation of CYP2E1 and enhances endoplasmic reticulum stress signals that promote steatosis and apoptosis.
methionine metabolism; liver injury
CHRONIC ETHANOL FEEDING INDUCES functional and structural changes in the liver that include hepatocellular steatosis and apoptosis (36). The chronic exposure to ethanol alters hepatic methionine metabolism. Findings in alcoholic patients, and in several animal models of ethanol feeding, have included elevations in serum homocysteine and liver S-adenosylhomocysteine (SAH), with reductions in liver S-adenosylmethionine (SAM) and the SAM-to-SAH ratio (3, 12, 18, 28). At the same time, alcoholic liver disease (ALD) is commonly associated with deficiency of folate, a vitamin that is central to methionine metabolism by providing substrate for the transmethylation of homocysteine (16). These observations contribute to the hypothesis that abnormal hepatic methionine metabolism plays a role in the pathogenesis of ALD (19, 29, 30, 36). To test this hypothesis, we induced alcoholic liver injury in a micropig model by promoting abnormal methionine metabolism by feeding folate-deficient diets with ethanol at 40% of kcal. After 14 wk of feeding, the combined diet reduced liver folate, the SAM-to-SAH ratio, and GSH while increasing plasma levels of homocysteine and liver SAH, DNA strand breaks, and plasma aspartate transaminase by eightfold, together with histopathological features typical of steatohepatitis (19). Selected findings from this study are summarized in Table 1. This demonstration of rapid promotion of alcoholic liver injury through induction of abnormal methionine metabolism in the micropig contrasts with an earlier study in the same animal model in which induction of the histopathology of liver injury required 1 yr of ethanol feeding with an otherwise nutritionally adequate diet (17).
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Table 1. Selected findings in micropig livers on metabolic effects of ethanol feeding with and without folate deficiency
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The most studied interactive processes of alcoholic liver injury include oxidative damage through activation of the cytochrome P-450 2E1 enzyme (CYP2E1) and the induction of apoptosis by extrinsic (death receptor) and intrinsic (mitochondrial) pathways, which differ in signaling mechanisms. The extrinsic pathway involves TNF-
stimulation of its TNF receptor-1 (TNFR-1) that activates an apoptotic pathway with stimulation of caspase 8 and then caspase 3. In the intrinsic or mitochondrial pathway, caspase 8 activates bcl-2 family members, which translocate to mitochondria and induce the mitochondrial permeability transition that results in membrane depolarization and rupture with release of proapoptotic cytochrome c and subsequent activation of caspases 9 and 3 (1, 23). Recent studies point to the endoplasmic reticulum (ER) as a third subcellular organelle involved in alcoholic liver injury. The ER is the main organelle for protein synthesis and modifications, calcium signaling, and the biosynthesis of lipids (5, 32). The ER stress pathway is activated by conditions such as glucose deprivation, calcium depletion, and oxidative stress, which can interfere with proper folding and maturation of proteins (33). The accumulation of unfolded proteins in the ER initiates an intracellular signaling pathway known as the unfolded protein response (UPR), which promotes either a cell survival or cell death pathway (6, 32, 33). The survival response activates genes that encode ER-resident chaperones such as glucose-regulated protein 78 (GRP78)/Bip, which uses energy from ATP hydrolysis to prevent aggregation of ER proteins and is considered the classical marker for UPR activation (42). If the adaptive response is inadequate, the UPR promotes apoptosis through nuclear transcription factor growth arrest and DNA damage-(GADD) inducible and ER-localized caspase 12. The UPR also initiates cellular lipid synthesis by activation of sterol regulatory element binding proteins (SREBPs) that regulate the transcription of lipid synthesis enzymes, including fatty acid synthase (FAS), acetyl-CoA carboxylase (ACC), and stearoyl-CoA desaturase (SCD) (2, 5, 32, 41). SREBP-1c and SREBP-2 are synthesized as
125-kDa precursors; upon activation, the NH2-terminal segments are translocated to the nuclear membrane as proteins (
68 kDa) that activate the transcription of the target genes for lipid synthesis. SREBP-1c plays an active role in the transcription of genes involved in hepatic triglyceride production, whereas SREBP-2 is involved in regulation of genes in cholesterol synthesis (7, 22).
Using frozen liver samples from our recent study (19), the goal of the present study was to identify signaling pathways and mechanisms for alcoholic liver injury in the micropig model of abnormal methionine metabolism induced by ethanol feeding and folate deficiency. In addition to selective enhancement of the ER stress pathway, we found significant correlations of the levels of activated CYP2E1 and ER stress signals to elevations in liver homocysteine and SAH. Our findings suggest an integral role for abnormal methionine metabolism in the pathogenesis of ALD that include activation of both CYP2E1 and the ER stress pathways.
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MATERIALS AND METHODS
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Animals and diets.
As previously described, 24 juvenile Yucatan micropigs that were obtained from Sinclair Farms, Columbia, MO, were pair fed diets that provided 90 kcal/kg body wt, vitamin-free casein at 15%, 30% of kcal as corn oil, and 55% as carbohydrate (control), or diets in which carbohydrate was reduced to 15% and ethanol was substituted to 40% of kcal (19). Folic acid was omitted or was added to diets at 14.5 µg/kg body wt, and each diet was supplemented with a vitamin and mineral mix that included levels that were in excess of pig requirements for choline at 60.3 mg and methionine at 675 mg/kg body wt (35) (Dyets, Bethlehem, PA). Thus there were four feeding groups of six animals each: folate sufficient control (FS), folate deficient only (FD), folate sufficient with ethanol (FSE), and folate deficient with ethanol (FDE). The micropigs were housed in facilities approved by the National Institutes of Health and were cared for following standards and procedures outlined in the National Academy of Sciences "Guide for the Care and Use of Laboratory Animals." All procedures were reviewed and approved by the Animal Welfare Committee of University of California Davis. After 14-wk feeding, each pig was fasted overnight, anesthetized with isofluorane, and subjected to surgical laparotomy. Liver tissues were removed, and inner segments of liver were placed in formalin for subsequent blocking in paraffin, or freeze-clamped in liquid nitrogen and frozen at 70°C for further analysis.
TUNEL assay.
To quantify the effects of the different diets on hepatocellular apoptosis, the terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling (TUNEL) assay was performed on liver slices from each micropig, in which apoptotic bodies were detected by DNA fragmentation, as described previously (31). For morphometric quantification of TUNEL-positive hepatocytes, at least 10 fields per liver section were examined in each experiment. The x10 objective was used, and counts were blinded as to the treatment groups. Only hepatocytes were counted, and the units were numbers of TUNEL-positive cells per millimeter squared. Liver sections of six pigs on each experimental group were evaluated.
Microarray.
To explore genomewide expression changes associated with ethanol feeding and folate deficiency, liver mRNA levels were examined in cRNA preparations from two liver specimens from each group. cRNAs were hybridized with HumU133A oligo arrays (Affymetrix, Santa Clara, CA), which contain 22,283 probe sets. Because pig-specific array is not commercially available, we used the human array system. All Affymetrix data were filtered for those genes with fluorescent intensity value of <100. Genes whose expressions in livers from the folate-deficient and ethanol-fed groups were either increased or decreased by twofold or more (P < 0.05) compared with those in livers from the control group were considered significantly changed. GeneChip cRNA probes used in GeneChip expressions were obtained by following a protocol from the manufacturer. First-strand cDNA was synthesized by reverse transcription of 8-µg total RNA with SuperScript II reverse transcriptase (Invitrogen, Carlsbad, CA) and T7 oligo(dT) 24 primer (Genset Oligos, Boulder, CO), followed by second-strand cDNA synthesis. Biotin-labeled cRNA probes were generated by reverse transcription of double-stranded cDNA using BioArray high-yield RNA transcript labeling kit (ENZO Life Sciences, Farmingdale, NY). The cRNA probes were purified from unincorporated nucleotides by RNeasy mini column (QIAGEN, Valencia, CA), fragmented, and hybridized overnight at 68°C to the Human GeneChip array (HG U133A, Affymetrix). The hybridized probe array was stained with streptavidin phycoerythrin conjugate and scanned by the GeneArray scanner. To confirm the reproducibility of microarray data, selected cRNA samples were analyzed twice. Data for the microarray experiments used in this study can be found in the NCBI Gene Expression Omnibus (GEO), with series accession number GSE2099.
RNA isolation, cDNA synthesis, and cloning of nucleic acids.
Total RNA was isolated from liver tissue using an RNeasy mini kit (QIAGEN) followed by DNase treatment. Reverse transcription was carried out with oligo(dT) primer and SuperScript II reverse transcriptase from 2 µg of total RNA following the protocol provided in the first-strand cDNA synthesis kit (Invitrogen). Each amplified cDNA fragment was ligated to TA-pCR-2.1 TOPO vector (TOPO TA Cloning kit, Invitrogen), transformed into E. coli cells, sequenced, and gel purified. Primers from the authenticated pig cDNA sequence were designed using a Primer Express program (version 2, Applied Biosystems, Foster City, CA) to generate PCR products of 50. Primer sequences were identified through GenBank for transcripts of genes of interest. All of the primers were designed from pig-specific sequences obtained from GenBank, and specificity of PCR products was verified by agarose gel purification and sequencing. The primer pairs included, for CYP2E1: 5'-CTACATCATCCCCAAGGGCA-3' and 5'-AAGACGGAGTCCAGTGTCGG-3'; for GRP78: 5'-GTGGAGATCATCGCCAACG-3' and 5'-CACATATGACGGCGTGATGC-3'; for SREBP-1c: 5'-GGCGAAGCTGAATAAATCCG-3' and 5'-CGGATGTAGTCGATGGCCTT-3'; for FAS: 5'-GACGGGTATACGCCACCATC-3' and 5'-TGGAACCGTCTGTGTTCGTG-3'; for ACC: 5'-TCCACTCAAGCATACCTCCCA-3' and 5'-CGTCAGCATGTCAGAAGGCA-3'; for SCD: 5'-CGGTATCCTGTTGATGTGCTTC-3' and 5'-CAATACCAGGGCACACGATCGT-3'; for TNFR-1: 5'-TCTTTCCCTGGCATTCTTCC-3' and 5'-CGTTGGTAGCGGCAAGCTAA-3'; and for
-actin: 5'-TCGATCATGAAGTGCGACGT-3' and 5'-CGTGTTGGCGTAGAGGTCCT-3'.
Real-time PCR.
Quantitative RT-PCR analysis was used to confirm microarray results for selected response genes and to analyze other genes of interest in all 24 liver samples.
-Actin was used as an internal control in all mRNA expression analyses, and each reaction was performed in triplicate using the ABI Prism 7900 sequence detection system (Applied Biosystems). Separate standard-curve cDNA dilutions were included in each PCR run, and liver transcripts were expressed as a ratio normalized to
-actin levels.
Protein isolation and Western blot analysis.
For total protein isolation, 100 mg of each liver tissue sample were homogenized in ice-cold lysis buffer (50 mM Tris·HCl, pH 7.4, 150 mM NaCl, 2 mM EDTA and 1% Nonidet P-40) containing protease inhibitor cocktail (Sigma, St. Louis, MO). After 30-min incubation on ice, the tissue suspensions were centrifuged at 15,000 g for 15 min at 4°C. The supernatants were collected, and protein concentrations were measured by the Bio Rad protein assay (Bio Rad, Hercules, CA). For nuclear extract preparation, 100 mg of liver tissues were homogenized in ice-cold homogenization buffer A (10 mM HEPES pH 7.9, 10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 1 mM DTT) containing 1% Nonidet P-40 and protease inhibitors. After 10-min incubation on ice, the tissue suspensions were centrifuged for 1 min at 4°C. The nuclear pellets were dissolved in buffer B (20 mM HEPES, pH 7.9, 400 mM NaCl, and 1 mM each of DTT, EDTA, and EGTA) containing protease inhibitors and placed on a rotatory shaker for 15 min, followed by centrifugation at 11,000 g for 15 min at 4°C. The supernatants (nuclear extracts) were stored at 70°C. Microsomal fractions were obtained by homogenizing 500 mg of liver tissue in 6 ml of ice-cold 0.1 M sodium phosphate buffer, pH 7.6, containing protease inhibitor cocktail followed by centrifugation at 40,000 g for 30 min at 4°C. The supernatants were collected and centrifuged at 100,000 g for 60 min to give microsomal pellets. The pellets were dissolved in 0.1 M sodium phosphate buffer and stored at 70°C. Total liver homogenates (100 µg), microsomes (8 µg), and/or nuclear extracts (20 µg) were separated on SDS-PAGE, electroblotted onto 0.2-µm nitrocellulose membranes, and blocked in 5% milk in TBST (Tris-buffered saline at pH 7.5 containing 0.1% Tween 20) for 1 h at room temperature. Immunoblot analyses were carried out using the following multispecies antibodies: anti-GRP78 (1:1,000), anti-caspase 9 (1:1,000) (StressGen, Victoria, BC), anti-caspase 12 (1:700), anti-fas-associated protein with death domain (FADD) (1:1,000) (Chemicon, Temecula, CA), anti-SREBP-1 (1:500), anti-cytochrome c (1:500) (Santa Cruz), and anti-
-actin (1:10,000) (Sigma). Antibody to purified liver human CYP2E1 was a gift from J. M. Lasker. Dilutions (1:5,000) of horseradish peroxidase-conjugated anti-rabbit IgG (Pierce, Rockford, IL) were used as the secondary antibody (1:10,000 dilution of anti-mouse IgG for
-actin). After incubation with primary and secondary antibodies, blots were developed with the horseradish peroxidase SuperSignal chemiluminescent detection system (Pierce). Band intensities were quantified using ImageQuant software (Molecular Dynamics) and standardized against
-actin.
Statistical analyses.
All values obtained from each group are expressed as means ± SE. Significant differences among the four feeding groups were determined by two-way ANOVA. For these analyses, the effect of folate deficiency was determined by changes in FD and FDE groups, the effect of ethanol by changes in FSE and FDE groups, and the interactive or additive effects of both factors were determined by changes in FDE according to two-way ANOVA. One-way ANOVA was used to qualify selective results. Data from the present study were correlated to previously published values for SAH, the SAM-to-SAH ratio, and/or homocysteine (19) by linear regression analysis by using SPSS 10 for windows (SPSS).
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RESULTS
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Induction of apoptosis by ethanol feeding and folate deficiency.
Hepatocellular apoptosis was analyzed according to the appearance of TUNEL-positive hepatocytes that exhibited the morphological features of decreased size with highly condensed and fragmented nuclei (Fig. 1A) and was induced by ethanol feeding and enhanced by the interaction of ethanol feeding with folate deficiency (Fig. 1B). According to linear regression analysis using data from all 24 micropigs, the numbers of apoptotic nuclei were correlated separately to hepatic SAH and homocysteine levels (see Table 3).

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Fig. 1. Effects of diets on hepatocyte apoptosis. Liver sections from animals fed folate-deficient (FD), ethanol [folate sufficient with ethanol (FSE)], or combined diets [folate-deficient with ethanol (FDE)] were processed for the detection of DNA fragmentation by the terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling (TUNEL) assay. A: note the presence of several nuclei positively stained for fragmented DNA (arrows) in the FSE and more significantly in the FDE animals. B: for quantification, apoptotic nuclei were counted in 10 fields in each liver sample to obtain average values for each specimen. Values are means ± SE. +Ethanol effect, P < 0.002; interaction with folate deficiency, P < 0.04.
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Microarray analysis of effects of folate deficiency and ethanol feeding on gene expression.
Microarray analyses of liver specimens from each group showed that more than 200 genes were differentially expressed in the livers of animals fed the folate-deficient, ethanol-containing, and combined diets by greater or lesser than twofold (P < 0.05) compared with control group results. Activated genes in the FD, FSE, and FDE groups included CYP2E1 and GRP78, whereas acyl-coenzyme A dehydrogenase, which promotes lipid oxidation, was downregulated in each group (Table 2). Comparing results with microarrays from other species, hydroxyacyl-coenzyme A dehydrogenase type II was downregulated in ethanol-fed mice (14) and regucalcin senescence marker protein-30 was downregulated in ethanol-fed rats (13), contrary to upregulation of each gene in the present study (Table 2).
Effects of diets on hepatic CYP2E1 levels.
Confirming the microarray data, CYP2E1 transcripts were activated by folate deficiency and ethanol separately and by an interaction of the two diets (Fig. 2A). CYP2E1 protein levels were increased independently by folate deficiency and ethanol feeding (Fig. 2, B and C). According to linear regression analyses, the hepatic levels of CYP2E1 transcripts were correlated positively to levels of SAH and negatively to the SAM-to-SAH ratio, whereas protein levels of CYP2E1 correlated positively to both SAH and homocysteine and negatively to the SAM-to-SAH ratio (Table 3).

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Fig. 2. Effects of diets on the hepatic expressions of cytochrome P-450 2E1 (CYP2E1). A: CYP2E1 transcript levels were quantified by real-time RT-PCR. *Folate effect, P < 0.01; +ethanol effect, P < 0.02; interaction, P < 0.04. B: immunoblot analysis of CYP2E1 in microsomal extracts from livers of four experimental groups. Aliquots of liver microsomal fractions (8 µg) from each animal were resolved in 14% SDS-polyacrylamide gel, transferred to nitrocellulose membrane, and probed with anti-human CYP2E1. C: quantification of immunoblot results using ImageQuant software. *Folate effect, P < 0.02; +ethanol effect, P < 0.04. Values are means ± SE. FS, folate sufficient control.
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Effects of diets on ER stress-response genes.
The mRNA expression of the UPR response marker GRP78 was increased in ethanol groups, with an interaction of both factors in the combined feeding group (Fig. 3A). GRP78 protein was induced independently by folate deficiency and ethanol feeding (Fig. 3, B and C). According to linear regression analyses, levels of both GRP78 transcripts and protein were correlated positively to liver SAH and homocysteine levels and negatively to the SAM-to-SAH ratio (Table 3).

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Fig. 3. Effects of diets on expression of glucose-regulated protein 78 (GRP78) in liver. A: GRP78 transcript levels by real-time RT-PCR. +Ethanol effect, P < 0.001; interaction, P < 0.04. B: immunoblots of GRP78 (100 µg of protein) with anti-GRP78 polyclonal antibody. C: quantification of GRP78 protein bands using ImageQuant. *Folate effect, P < 0.03; +ethanol effect, P < 0.001. Values are means ± SE.
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The ER signaling of apoptosis was analyzed as the ratio of the active cleaved form to its uncleaved precursor form of caspase 12. Accordingly, caspase 12 activation was increased independently by both folate deficiency and ethanol feeding (Fig. 4, A and B). The ratio of the cleaved to uncleaved forms of caspase 12 was correlated positively to liver homocysteine levels and negatively to the SAM-to-SAH ratio (Table 3).

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Fig. 4. Effects of diets on caspase 12 activation. A: analysis of caspase 12 activation by Western blotting using 100 µg of liver cytosolic extracts. P, precursor; C, cleaved form of caspase 12. B: quantification of caspase 12 activation as ratio of cleaved to precursor form. Values are means ± SE. *Folate effect, P < 0.04; +ethanol effect, P < 0.001.
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The ER signaling of steatosis was analyzed according to the activation of SREBP-1c and the expression of its nuclear protein SREBP-1c (nSREBP-1c). The mRNA transcripts of SREBP-1c were increased independently by folate deficiency according to one-way ANOVA and by ethanol according to two-way ANOVA (Fig. 5A). The protein levels of nSREBP-1c protein were increased independently by both folate deficiency and ethanol feeding (Fig. 5, B and C). SREBP-1c mRNA was correlated to SAH and homocysteine and negatively to the SAM-to-SAH ratio, whereas nSREBP-1c protein levels were correlated with liver SAH and negatively to the SAM-to-SAH ratio (Table 3).

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Fig. 5. Effects of diets on expression of hepatic sterol regulatory element binding protein (SREBP)-1c. A: SREBP-1c transcript levels by real-time RT-PCR. *Folate effect by one-way ANOVA: P < 0.05, +ethanol effect: P < 0.006. B: SREBP-1c activation by immunoblot (20 µg of nuclear extract). C: quantification of nuclear SREBP (nSREBP)-1c protein levels by ImageQuant. *Folate effect, P < 0.005; +ethanol effect, P < 0.01. Values are means ± SE.
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The physiological effects of SREBP-1c signaling were analyzed by measurements of the mRNA transcripts of the lipid synthesizing genes SCD, ACC, and FAS. The transcripts of SCD and ACC were each increased separately by folate deficiency, according to one-way ANOVA, and by ethanol feeding, according to two-way ANOVA (Fig. 6, A and B). The transcripts of each of these genes were correlated to levels of SAH and/or homocysteine and negatively to the SAM-to-SAH ratio (Table 3). The mRNA transcripts of FAS were increased only by ethanol (Fig. 6C), but were correlated to liver homocysteine levels (Table 3).

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Fig. 6. Effects of diets on transcripts of lipogenic enzymes. A: stearoyl-CoA desaturase (SCD). *Folate effect, P < 0.05 (one-way ANOVA); +ethanol effect, P < 0.03. B: acetyl-CoA carboxylase (ACC). *Folate effect, P < 0.05; +ethanol effect, P < 0.002 (two-way ANOVA). C: fatty acid synthase (FAS). +Ethanol effect, P < 0.002. Values are means ± SE.
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Extrinsic and intrinsic apoptotic pathway responses.
Ethanol feeding alone increased transcript levels of TNFR-1 (data not shown). The protein levels of FADD, cytosolic cytochrome c, and the cleaved form of caspase 9 were each elevated by ethanol feeding, with no additive effect of folate deficiency (Fig. 7).

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Fig. 7. Effects of diets on proteins of extrinsic and intrinsic (mitochondrial) apoptotic pathways. Immunoblots of representative proteins are shown. There were ethanol effects but no effect of folate deficiency on the levels of cytochrome c (P < 0.05), fas-associated protein with death domain (FADD) (P < 0.05), and the cleaved form of caspase 9 (P < 0.05).
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DISCUSSION
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The present study suggests that alcoholic liver injury is promoted in the micropig through alterations in hepatic methionine metabolism that also enhance the activation of CYP2E1 and induce the ER stress signaling pathway. Our previous evidence for involvement of abnormal methionine metabolism in the pathogenesis of ALD included the observation that the combination of dietary folate deficiency with chronic ethanol feeding accelerated the onset of alcoholic liver injury in the micropig, together with increases in serum homocysteine and hepatic levels of its SAH metabolite with reduction in hepatic SAM (Table 1) that correlated with reduced GSH (19), in contrast to our laboratorys prior studies in the micropig that showed a more delayed response to ethanol feeding with folate-sufficient diets (17, 18). Using liver samples from this recent study (19), we measured the effects of folate deficiency and ethanol feeding on CYP2E1 and signal pathways for apoptosis and steatosis. Analysis of the microarray data showed marked elevation in CYP2E1 and modest elevation of the ER stress marker GRP78 in all three experimental groups (Table 2). Of potential importance, the microarray also demonstrated modest downregulation of acyl-coenzyme A dehydrogenase, in keeping with other studies that showed reduced mRNA levels of this peroxisome proliferator-activated receptor-
-mediated gene in ethanol-fed mice (15). In analyzing the individual gene expression data, we considered that the effect of one or more abnormal methionine metabolites on CYP2E1 or an ER stress signal was suggested by a separate effect of the FD diet, an interactive effect of FDE, a positive correlation of an effect with elevated levels of either homocysteine or SAH, and/or a negative correlation with the ratio of SAM to SAH. Our data show that these criteria for the effect of abnormal methionine metabolism applied to the activations of CYP2E1 and GRP78, according to microarray analysis (Table 2), the numbers of TUNEL-positive apoptotic bodies in hepatocytes (Fig. 1 and Table 3), hepatic transcript and protein levels of CYP2E1 (Fig. 2 and Table 3), and several critical markers of ER stress pathways (Figs. 35 and Table 3). These effects appeared to be specific for the ER stress pathway, as there were no apparent effects of folate deficiency, either with or without ethanol feeding, to changes in selected signals in the extrinsic and intrinsic pathways for apoptosis. On the other hand, these signals were elevated, as anticipated, in the two ethanol-fed groups (Fig. 7).
While our data suggest that SAH and/or homocysteine act as mediators for the activation of CYP2E1 and ER stress signals, the experiments do not clearly establish cause and effect, since the data are associative and are based on results from tissues that were obtained at one point in time. Also, certain other experimental limitations could impact on the strength of our findings. Because all pig liver samples were collected after an 18-h fast, potential acute ethanol effects on CYP2E1 or ER signal pathways would not have been detected. Also, the levels of CYP2E1 might have been influenced by ethanol withdrawal (4) or overnight fasting (8). In the absence of pig-specific markers, we used the human Affimatrix chip in our microarray studies, which may account for the fact that many relevant genes that were upregulated according to real-time PCR transcript analyses (Fig. 26) were undetected by microarray (Table 2). Also, in the absence of pig-specific antibodies, we relied on human antibody to CYP2E1 or multispecies-specific antibodies in our other immunoblotting studies, which were effective in all experiments, except for the quantification of the three lipid synthesis enzymes.
Our findings on the CYP2E1 activation response pattern to the different diets with similar effects of folate deficiency and ethanol feeding (Fig. 2) are not consistent with the findings of progressive oxidative liver injury (19) and apoptosis (Fig. 1) that occurred with maximal effect of the combined diet (Table 1) (19). These comparisons suggest that the potential oxidative and apoptotic responses to CYP2E1 may differ in response to ethanol feeding and to folate deficiency, which, rather than adding to the ethanol effect, modulated the ethanol effect through its interaction (Fig. 2). Furthermore, CYP2E1 is but one of several mechanisms for apoptosis, as shown in one of our prior studies that demonstrated an apoptotic response to ethanol feeding with a folate-sufficient diet. Without invoking a CYP2E1 effect, this prior study related the apoptotic response to the effects of lowered SAM-to-SAH ratio on dysregulation of DNA synthesis (18).
Data from other studies indicate potential roles for methionine metabolites in the oxidative injury response to ethanol feeding. Studies in CYP2E1-overexpressing HepG2 cells have shown that the ethanol-induced activation of CYP2E1 produces hydroxyethyl radical, which promotes lipid peroxidation, mitochondrial damage, and decreased cell viability (9) that is aggravated by depletion of GSH (39). Others found that SAM plays an important role in the regulation of GSH levels and mitochondrial antioxidant defense (11, 26). While ours is the first study to show that CYP2E1 can be induced by folate deficiency alone (Fig. 2), others showed that CYP2E1 was activated by a methionine- and choline-deficient diet (25). Others found that the methionine- and choline-deficient diet enhances the TNF-
and liver injury response to LPS in association with low hepatic SAM, all of which were attenuated by the addition of SAM to the diet (10, 30). A subsequent study found that SAM attenuated the LPS-mediated transcription of TNF-
in cultured murine macrophages (38). Most recently, elevated SAH levels were induced together with TNF-
in ethanol-fed mice, and in vitro induction of SAH enhanced the apoptosis response to TNF-
in HepG2 cells (34).
Potential mechanisms to relate abnormal methionine metabolism to the activation of CYP2E1 are speculative. Because SAM is the principal methyl donor for DNA, RNA, and proteins, and its effects are opposed by its product SAH, the SAM-to-SAH ratio is regarded as an index of methylation potential (21, 40). While the expression of rat CYP2E1 is developmentally regulated by hypomethylation in the 5' promoter region (37), others showed that overall cellular mRNA synthesis in HepG2 cells was enhanced by hypomethylation that was induced by SAH hydrolase inhibitors that decreased the SAM-to-SAH ratio (21). Although the promoter sequence of pig CYP2E1 is unknown, our finding that transcript levels of CYP2E1 were correlated negatively to the SAM-to-SAH ratio (Table 3) suggests a hypomethylation mechanism for its activation in our animal model. The same mechanism could apply to the activation of ER stress pathway observed in the present experiments.
Our data provide substantial evidence that both apoptosis and steatosis were mediated through the ER stress pathway in the micropig model and that activation of this pathway was mediated positively by homocysteine or SAH, or negatively by the SAM-to-SAH ratio. The finding that the UPR chaperone GRP78 was activated in response to ethanol feeding and folate deficiency (Fig. 3 and Table 2) is consistent with prior evidence that SAH and/or homocysteine is an activator of the ER survival pathway (2, 33). At the same time, we found upregulation of the proapoptotic pathway, as represented by enhanced production of the cleaved and active form of caspase 12 (Fig. 4). Our findings are also consistent with the SREBP activation mechanism for steatosis and suggest that this pathway is under regulatory control by abnormal methionine metabolism. The activation of SREBP-1c transcripts and the translocation of its mature protein to the nuclear membrane were upregulated by both folate deficiency and ethanol feeding, whereas the levels of both transcripts and nuclear protein correlated to levels of SAH (Fig. 5 and Table 3). Furthermore, two SREBP-1c-regulated lipid synthesis genes, SCD and ACC, were activated both by folate deficiency and ethanol (Fig. 6), whereas transcript levels of each of these genes, as well as FAS, were correlated to liver SAH or homocysteine (Table 3). The findings that levels of selected signals in the extrinsic and intrinsic pathways of apoptosis were only influenced by ethanol feeding (Fig. 7) underscores the specificity of the ER stress pathway for activation by abnormal methionine metabolites.
Others showed that SREBP-1c activation can be induced by ethanol feeding as a mechanism for alcoholic steatosis. Mice fed 27.5% of kcal as ethanol with a low-fat diet for 4 wk developed fatty liver with a 3.5-fold increase in triglyceride, together with increased levels of the mature nuclear form of SREBP-1c and increased transcripts of SREBP-1c-targeted triglyceride synthesis genes FAS, ACC, and SCD. Acetaldehyde may be central to the SREBP response to ethanol, because complementary experiments in rat hepatoma cells showed that the SREBP-1 promoter was activated and protein levels of nuclear SREBP-1 were increased by graded concentrations of ethanol but blocked by 4-methylpyrazole (41). Others showed that SREBP-1c is a negative inhibitor of the transcription of cytosolic alcohol dehydrogenase-1, so that a decrease in SREBP allows an increase in the expression of alcohol dehydrogenase-1 (20), whereas SREBP-1c was induced in HepG2 cells exposed to increasing concentrations of acetaldehyde (27). Another in vivo mouse study linked the effects of ethanol-induced ER stress to methionine metabolism, whereby elevated liver SREBP-1 and caspase 12 were associated with hepatic steatosis and hepatocellular apoptosis, all of which were attenuated by the concurrent feeding of betaine, a compound that is known to reduce homocysteine levels (24).
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GRANTS
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This work was supported by National Institutes of Health Grants AA-1414502 and DK 35747-19 to C. H. Halsted and AA-0811612 to S. W. French.
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ACKNOWLEDGMENTS
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We are grateful to Dr. Jerome M. Lasker, Senior Scientist, Bell Atlantic Laboratories, for providing the antibody to human CYP2E1.
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FOOTNOTES
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Address for reprint requests and other correspondence: C. H. Halsted, School of Medicine, Univ. of California, Davis, Davis, CA 95616 (E-mail: chhalsted{at}ucdavis.edu)
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.
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