Chronic Ethanol Feeding and Folate Deficiency Activate Hepatic Endoplasmic Reticulum Stress Pathway...

FINAL ACCEPTED VERSION MANUSCRIPT NUMBER (G-00542-2004.R1)

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Chronic Ethanol Feeding and Folate Deficiency Activate Hepatic Endoplasmic Reticulum

Stress Pathway in Micropigs

Farah Esfandiari1, Jesus A. Villanueva1, Donna H. Wong1, Samuel W. French2 and Charles H.

Halsted1*

1. Department of Internal Medicine, University of California Davis, Davis, CA,

2. Department of Pathology, Harbor-UCLA Medical Center, Torrance, CA

Running head: Ethanol and folate deficiency activate liver ER stress

*Corresponding author:

Charles H. Halsted, MD

School of Medicine

University of California, Davis

Davis, CA 95616

Phone 530-752-6778; Fax: 530-752-3470; email:[email protected]

Articles in PresS. Am J Physiol Gastrointest Liver Physiol (February 10, 2005). doi:10.1152/ajpgi.00542.2004

Copyright © 2005 by the American Physiological Society.


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ABSTRACT

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 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 ER stress signals glucose-

regulated protein 78 (GRP78), caspase 12, and sterol regulatory element binding protein 1-c

(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 1-c, and protein levels of

CYP2E1, GRP78, nSREBP, 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 lipogenic

enzymes fatty acid synthase (FAS), acetyl-CoA carboxylase (ACC) and stearoyl-CoA desaturase

(SCD) 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 ER stress signals that promote steatosis and apoptosis.

Key Words: Methionine metabolism and liver injury


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INTRODUCTION

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 SAH with reductions in liver

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 weeks 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 (AST) by 8-fold together with

histopathologic 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 one year

of ethanol feeding with an otherwise nutritionally adequate diet (17).

The most studied interactive processes of alcoholic liver injury include oxidative damage

through activation of the cytochrome P450 enzyme CYP2E1 and the induction of apoptosis by

extrinsic (death receptor) and intrinsic (mitochondrial) pathways, which differ in signaling

mechanisms. The extrinsic pathway involves TNFα stimulation of its TNFR-1 receptor that


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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 pro-apoptotic cytochrome C and subsequent activation

of caspases 9 and 3 (1, 23). Recent studies point to the endoplasmic reticulum 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 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 GADD 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 FAS, ACC and SCD (2, 5, 32, 41). SREBP-1c and SREBP-2 are synthesized

as ~125 kDa precursors; upon activation, the N-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).


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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

Animals and diets- As previously described, 24 juvenile Yucatan micropigs that were

obtained from Sinclair Farms, Columbia, MO, USA were pair fed diets that provided 90 kcal/kg

body weight, 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 weight, and each diet

was supplemented with a vitamin and mineral mix that include levels that were in excess of pig

requirements for choline at 60.3 mg, and methionine at 675 mg/kg body weight (35)(Dyets, Inc.

Bethlehem, Pennsylvania, USA). Thus, there were four feeding groups of 6 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 weeks 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 -

70º 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 sections were examined in each


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experiment. The 10x 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 mm2. Liver

sections of six pigs on each experimental group were evaluated.

Microarray- To explore genome-wide 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. Since pig-specific array is not commercially

available, we use the human array system. All Affymetrix data were filtered for those genes with

fluorescent intensity value of less than 100. Genes whose expressions in livers from the folate

deficient and ethanol fed groups were either increased or decreased by 2-fold or more (p<0.05)

compared to 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, Inc., Farmingdale, NY). The cRNA

probes were purified from unincorporated nucleotides by RNeasy mini column (QIAGEN,

Valencia, CA), fragmented and hybridized overnight at 680 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.


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RNA Isolation, cDNA synthesis and cloning of nucleic acids- Total RNA was isolated from

liver tissue using an RNeasy mini kit (QIAGEN, Valencia, CA) 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, Calsbad, CA). Each amplified cDNA fragment was ligated to TA-pCR-2.1 TOPO

vector (TOPO TA Cloning kit, Invitrogen, Calsbad, CA), 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 the primers were designed from pig specific sequences obtained from GenBank and

specificity of PCR products were 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 TNFR1:5’-TCTTTCCCTGGCATTCTTCC-3’ and

5’-CGTTGGTAGCGGCAAGCTAA-3’;and for β-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. β-actin was

used as an internal control in all mRNA expression analysis, and each reaction was performed in

triplicate using the ABI Prism 7900 sequence detection system (Applied Biosystems, Foster City,


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CA). Separate standard curve cDNA dilutions were included in each PCR run and liver transcripts

were expressed as a ratio normalized to β-actin levels.

Protein Isolation and Western Blot Analysis- For total protein isolation, 100 mg of each

liver tissue sample was homogenized in ice-cold lysis buffer (50 mM Tris-HCl, pH 7.4, 150 mM

NaCl, 2 mM EDTA and 1% NP-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

40 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% NP-40 and protease inhibitors. After 10

min incubation on ice, the tissue suspensions were centrifuged for 1 min at 40 C. The nuclear

pellets were dissolved in buffer B (20mM 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 40 C. The supernatants (nuclear extracts) were

stored at -700 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 40 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 -700 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 one hour at room temperature. Immunoblot analyses were carried

out using the following multispecies antibodies: anti-GRP78 (1:1000), anti-caspase 9 (1:1000)

(StressGen, Victoria, CA), anti-Caspase12 (1:700), anti-FADD (1:1000) (Chemicon, Temecula,


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CA), anti-SREBP-1 (1:500), anti-cytochrome C (1:500) (Santa Cruz, CA) and anti-β-actin

(1:10,000) (Sigma, St. Louis, Mo). Antibody to purified liver human CYP2E1 was a gift from

J.M. Lasker. 1:5000 dilutions of horseradish peroxidase-conjugated anti-rabbit IgG (Pierce,

Rockford, IL) was used as the secondary antibody (1:10,000 dilution of anti-mouse IgG for β-

actin). After incubation with primary and secondary antibodies, blots were developed with the

HRP SuperSignal chemiluminescent detection system (Pierce, Rockford, IL). Band intensities

were quantified using ImageQuant software (Molecular Dynamics) and standardized against β-

actin.

Statistical Analyses-All values obtained from each group are expressed as mean + SEM.

Significant differences between 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 using SPSS 10 for windows (SPSS Inc.).


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RESULTS

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

(Table 3).

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 2-fold (p<0.05) compared to control group results.

Activated genes in the FD, FSE, and FDE groups included CYP2E1 and GRP 78, whereas acyl-

Coenzyme A dehydrogenase, which promotes lipid oxidation, was down-regulated in each group

(Table 2). Comparing results with microarrays from other species, hydroxyacyl-Coenzyme A

dehydrogenase type II was down-regulated 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. 2b, 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,


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while protein levels of CYP2E1 correlated positively to both SAH and homocysteine and

negatively to the SAM to SAH ratio (Table 3).

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. 3b, c). According to linear regression analyses, levels of both GRP78

transcripts and protein were correlated positively to liver SAH and homocysteine levels and


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. 4a, b). The ratio of the cleaved

to uncleaved forms of caspase 12 was correlated positively to liver homocysteine levels and


The ER signaling of steatosis was analyzed according to the activation of SREBP- 1c and

the expression of its nuclear protein nSREBP-1c.The mRNA transcripts of SREBP-1c were

increased independently by folate deficiency according to one-way ANOVA and by ethanol

according to 2-way ANOVA (Fig. 5a). The protein levels of nSREBP-1c protein were increased

independently by both folate deficiency and ethanol feeding (Fig. 5b, c). SREBP-1c mRNA was

correlated to SAH and homocysteine and negatively to the SAM to SAH ratio, while nuclear

SREBP-1c protein levels were correlated with liver SAH and negatively to the SAM to SAH ratio

(Table 3).

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


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by ethanol feeding according to two-way ANOVA (Fig. 6a, 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.6 c), but were

correlated to liver homocysteine levels (Table 3).

Extrinsic and intrinsic apoptotic pathway responses- Ethanol feeding alone increased

transcript levels of TNFR1 (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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DISCUSSION

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 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 down-regulation of acyl-coenzyme

A dehydrogenase, in keeping with other studies that showed reduced mRNA levels of this

peroxisome proliferator-activated receptor alpha (PPARα) - 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 folate deficient diet (FD), an interactive effect of folate deficient diet

combined with ethanol feeding (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


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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. 3, 4, and 5, Table 3). These effects appeared to be specific for the ER stress pathway, since

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.

Since all pig liver samples were collected after an 18 hour 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 up-regulated according to real time PCR

transcript analyses (Fig. 2-6) 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) is 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


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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 over expressing 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 (MCD) diet (25). Others found that the MCD diet

enhances the TNFα 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 TNFα in cultured murine macrophages (38).

Most recently, elevated SAH levels were induced together with TNFα in ethanol-fed mice, and in

vitro induction of SAH enhanced the apoptosis response to TNFα in HepG2 cells (34).

Potential mechanisms to relate abnormal methionine metabolism to the activation of

CYP2E1 are speculative. Since 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


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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; Table 2) is consistent with prior evidence that SAH and/or homocysteine are

activators of the ER survival pathway (2, 33). At the same time, we found up-regulation 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 1-c transcripts and the translocation of its mature protein to

the nuclear membrane were up regulated by both folate deficiency and ethanol feeding, while the

levels of both transcripts and nuclear protein correlated to levels of SAH (Fig. 5, Table 3).

Furthermore, two SREBP 1-c- regulated lipid synthesis genes, SCD and ACC, were activated both

by folate deficiency and ethanol (Fig. 6), while 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.


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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

weeks developed fatty liver with 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, since 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 (ADH-1), so that a

decrease in SREBP allows an increase in the expression of ADH-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).

Acknowledgments-We are grateful to Dr. Jerome M. Lasker, Senior Scientist, Bell Atlantic

Laboratories, for providing the antibody to human CYP2E1.

Grants- This work was supported by a grant from the National Institutes of Health, number AA

14145-02 to CHH and AA 08116-12 to SWF.


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Disclosures- None of the authors have any intellectual or financial conflict of interest with this

work.


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FIGURE LEGENDS

Figure 1. Effects of diets on hepatocyte apoptosis. Liver sections from animals fed folate

deficient, ethanol or combined diets were processed for the detection of DNA fragmentation by

the TUNEL assay. a) Note the presence of several nuclei positively stained for fragmented DNA

(arrows) in the ethanol fed and more significantly in the ethanol fed/folate deficient animals. For

quantification, b) apoptotic nuclei were counted in 10 fields in each liver sample to obtain average

values for each specimen. + Ethanol effect; p<0.002; § Interaction with folate deficiency (p<0.04).

Figure 2. Effects of diets on the hepatic expressions of 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 �g) 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)

Figure 3. Effects of diets on expression of 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).

Figure 4. Effects of diets on Caspase 12 activation. (a) Analysis of caspase 12 activation by

western blotting using 100 ug of liver cytosolic extracts. P, precursor; C, cleaved form of Caspase


26

12. (b) Quantification of caspase 12 activation as ratio of cleaved to precursor form. * Folate

effect (p<0.04); + Ethanol effect (p<0.001).

Figure 5. Effects of diets on expression of hepatic SREBP-1c. (a) SREBP-1c transcript levels by

real-time RT-PCR. * Folate effect by one-way ANOVA; + Ethanol effect (p<0.006). (b) SREBP-

1c activation by immunoblot (20 �g of nuclear extract). (c) Quantification of nSREBP-1c protein

levels by ImageQuant. * Folate effect (p<0.005); + Ethanol effect (p<0.01).

Figure 6. Effects of diets on transcripts of lipogenic enzymes. (a) SCD, * Folate effect (p<0.05,

one-way ANOVA); + Ethanol effect (p<0.03,). (b) ACC, * Folate effect (p<0.05,); + Ethanol effect

(p<0.002, two-way ANOVA). (c) FAS, + Ethanol effect (p<0.002,).

Figure 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), FADD (p<0.05) and the

cleaved form caspase 9 (p<0.05).


27

Figure 1a


28

0

20

40

60

80

FS FD FSE FDE

§#

of A

popt

otic

live

r ce

lls/m

m2

+

+

Figure 1b

A


29

Figure 2

CYP2E1β-actin

FS FD FSE FDE

B

0.0

0.5

1.0

1.5

2.0

CY

P2E

1 tr

ansc

ript

/β-a

ctin

FS FD FSE FDE

*§+*+

0.0

0.5

1.0

1.5

2.0

2.5

3.0

CY

P2E

1 pr

otei

n/β-

actin

FS FD FSE FDE

*+ +*

A C


30

GRP78

β-actin

FS FD FSE FDE

Figure 3

B

0.0

0.3

0.6

0.9

FS FD FSE FDE

*

+

GR

P78

prot

ein/β-

actin

+*

AC

0

1

2

3

4

GR

P78

tra

nscr

ipt/β-

acti

n

FS FD FSE FDE

§+

+


31

FS FD FSE FDE

PC

Figure 4

A

B

0.0

0.1

0.2

0.3

0.4

Cas

pase

12 c

leav

ed/p

recu

rsor

form

s

FS FD FSE FDE

+

*

+ *


32

nSREBP-1c

β-actin

FS FD FSE FDE

Figure 5

B

A C

SRE

BP

-1c

tran

scri

pt/β

-act

in

0.0

0.5

1.0

1.5

2.0

2.5

3.0

FS FD FSE FDE

+

+

*

*

0

10

20

30

40

50

60

nSR

EB

P-1

c pr

otei

n/β-

acti

n

FS FD FSE FDE

+

+*

*


33

Figure 6

0

5

10

15

FS FD FSE FDE

+

+

FAS

tran

scri

pt/β

-act

inA

B

C

0

1

2

3

4

SCD

tra

nscr

ipt/β-

acti

n

FS FD FSE FDE

++

*

*

0

1

2

3

4

AC

C t

rans

crip

t/β-

acti

n

FS FD FSE FDE

++

*

*


34

Figure 7

Cytochrome C

Cleaved Caspase 9

FADD

FS FD FSE FDE

β-actin


35

Table 1. Selected findings in micropig livers on metabolic effects of ethanol feeding with and without folate deficiency (16)

Diet

Folate, µg/g

Homocysteine,

mnol/mg

protein

SAM, pmol/mg

protein

SAH, pmol/mg

protein

SAM/SAH Plasma AST,

units liter

FS 4.56 ± 1.23 0.77 ± 0.12 364 ± 30 136 ± 10 2.74 ± 0.21 42.0 ± 2.7

FD 2.30 ± 0.49* 0.93 ± 0.05 298 ± 10 243 ± 10* 1.23 ± 0.04* 47.7 ± 2.4

FSE 6.83 ± 0.66 1.10 ± 0.05† 261 ± 40† 218 ± 20† 1.18 ± 0.10† 108.6 ± 22.2†

FDE 2.52 ± 0.37* 1.13 ± 0.05† 246 ± 50† 291 ± 20*†‡ 0.83 ± 0.11*†‡ 350.8 ± 97.4†‡

P, folate

P, ethanol

P,FDE > FSE

p<0.001

p<0.003

p<0.03

p<0.001

p<0.001

p<0.02

p<0.001

p<0.001

p<0.04

p<0.01

p<0.04


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Table 2. Gene expression profile in ethanol-fed/folate-

deficient pig liver as compared with control *

GENE DESCRIPTION

FD FSE FDE

Oxidation Gene acc # FC

p value FC p value FC p value

Cytochrome P450, subfamily IIE (ethanol-inducible) AF182276.1 22.08 0.001 8.53 0.010 12.41 0.004 Cytochrome P450, subfamily IIA (phenobarbital-inducible)

NM_000762.2 3.05 0.030 6.30 0.004 7.85 0.002

Peroxiredoxin 4 NM_006406.1 ---- ---- ---- ---- 2.10 0.003

Apoptosis Caspase 6, apoptosis-related cysteine protease BC000305.1 2.00 0.050 4.55 0.003 4.72 0.003 Programmed cell death 4 NM_005016.1 ---- ---- 2.00 0.038 4.54 0.004 Gelsolin (amyloidosis, Finnish type) BE675337 ---- ---- ---- ---- 2.24 0.008

Cell signaling Insulin-like growth factor binding protein 2, 36kDa NM_000597.1 ---- ---- 3.12 0.027 2.60 0.047 STAT induced STAT inhibitor-2 AB004903.1 ---- ---- ---- ---- 2.15 0.048 Regucalcin (senescence marker protein-30) D31815.1 ---- ---- 2.57 0.015 2.69 0.015 SOCS box-containing WD protein SWiP-1 NM_015626.1 ---- ---- ---- ---- 2.20 0.018 Follistatin NM_013409.1 ---- ---- ---- ---- 2.23 0.008 Guanine nucleotide binding protein (G protein) NM_005273.1 --- ---- ---- ---- 2.40 0.047 Thyroid hormone receptor interactor 12 NM_004238 ---- ---- ---- ---- -4.35 0.016 Brain abundant, membrane attached signal protein 1 NM_006317 ---- ---- ---- ---- -2.95 0.041 Annexin A2 NM_004039.1 -2.42 0.010 -2.00 0.014 -2.02 0.012 v-kit Hardy-Zuckerman 4 feline sarcoma viral oncogene NM_000222.1 ---- ---- ---- ---- 2.37 0.009

Cytoskeletal Gelsolin (amyloidosis, Finnish type) BE675337 ---- ---- ---- ---- 2.24 0.008 Adducin 2 (beta) NM_017482.1 ---- ---- ---- ---- 2.39 0.007 Ankyrin 3, node of Ranvier (ankyrin G) NM_020987.1 ---- ---- -2.18 0.041 -2.21 0.040

Transcription factor Transcription factor IIB NM_013242.1 ---- ---- ---- ---- 3.02 0.025 ZAP3 protein AI952986 ---- ---- ---- ---- 2.93 0.003 v-maf musculoaponeurotic fibrosarcoma oncogene NM_005461.1 -2.75 0.006 -2.66 0.008 -2.10 0.013 RAR-related orphan receptor A L14611.1 -5.68 0.028 -4.29 0.033 -4.75 0.031

Translation/post-translational Poly(rC) binding protein 2 NM_005016.1 ---- ---- 2.00 0.038 4.54 0.004 Ribosomal protein, large, P0 AA555113 ---- ---- 2.24 0.048 2.40 0.038 Eukaryotic translation initiation factor 3 BC000733.1 ---- ---- ---- ---- 2.12 0.015 Heat shock 70kDa protein 1A NM_005345.3 ---- ---- ---- ---- 2.21 0.020 Heat shock 70kDa protein 8 AB034951.1 ---- ---- ---- ---- 2.13 0.036 Ribosomal protein L7a NM_000972.1 ---- ---- ---- ---- 2.09 0.017 Glucose-regulated protein, 78kDa AF216292.1 2.06 0.043 4.30 0.030 2.00 0.030 Arginine-glutamic acid dipeptide (RE) repeats AB036737.1 -2.13 0.006 -2.00 0.009 -2.29 0.005

Lipid metabolism Hydroxyacyl-Coenzyme A dehydrogenase, type II NM_004493.1 ---- ---- ---- ---- 2.25 0.006 Acyl-Coenzyme A dehydrogenase, long chain AI367275 -2.10 0.022 -2.44 0.015 -2.50 0.016 Farnesyl diphosphate synthase NM_002004 -3.60 0.022 -4.80 0.027 -7.39 0.013 Solute carrier family 27 (fatty acid transporter), member 2 NM_003645.1 ----- ---- ---- ---- 2.83 0.002

Other metabolism Lactate dehydrogenase B NM_002300.1 ---- ---- 2.71 0.030 2.70 0.032 Carboxypeptidase A2 (pancreatic) NM_001869.1 ---- ---- ---- ---- 2.48 0.017


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Table 2 (continued)

GENE DESCRIPTION

FD FSE FDE

Oxidation Gene acc # FC

p value FC p value FC p value

Sulfotransferase family, cytosolic, 2A NM_003167.1 ---- ---- ---- ---- 2.50 0.009 Catechol-O-methyltransferase BC000419.1 ---- ---- ---- ---- 2.90 0.009 Alanine-glyoxylate aminotransferase D13368.1 ---- ---- ---- ---- 2.15 0.005 Nicotinamide N-methyltransferase NM_006169.1 2.41 0.049 2.48 0.046 3.20 0.022

Cell adhesion Cadherin, EGF LAG seven-pass G-type receptor 1 BC000059.1 ---- ---- ---- ---- 3.19 0.019 Putative N-acetyltransferase Camello 2 NM_016347.1 -6.42 0.043 -7.45 0.041 -6.71 0.042

Immune response Interferon-induced transmembrane protein 1 (9-27) AA749101 ---- ---- 2.64 0.005 2.55 0.011 Interferon-induced transmembrane protein 2 (1-8D) NM_006435.1 ---- ---- ---- ---- 3.67 0.008 Interferon induced transmembrane protein 3 (1-8U) BF338947 ---- ---- ---- ---- 3.13 0.001

Other Pecanex-like 1 AB018348.1 3.10 0.012 2.61 0.022 2.20 0.043 KIAA0916 protein NM_014873 ---- ---- -2.03 0.053 -2.11 0.049 B-cell translocation gene 1, anti-proliferative AL535380 ---- ---- ---- ---- 2.91 0.001 Chromosome 20 open reading frame 40 NM_014054.1 7.09 0.002 3.46 0.009 3.57 0.007

* Data are representative of two separate microarray hybridizations. The fold change (FC) of gene

expression represents increase or decrease of expression in each of the experimental groups vs the control group.


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Table 3. Correlations of findings with methionine metabolites

SAM/SAH SAH Homocysteine

r p r p r p

Apoptosis -0.4 0.056 0.530 0.008 0.425 0.038

CYP2E1

mRNA -0.57 0.003 0.604 0.002 0.351 0.09

Protein -0.631 0.002 0.548 0.008 0.534 0.01

GRP78

mRNA -0.44 0.032 0.666 0.001 0.434 0.034

Protein -0.68 0.001 0.536 0.007 0.571 0.004

Caspase 12

Cleaved/

precursor -0.6 0.002 0.39 0.06 0.662 0.001

SREBP-1c

mRNA -0.44 0.033 0.510 0.01 0.442 0.03

Protein -0.45 0.027 0.576 0.008 0.392 0.058

FAS

mRNA -0.367 0.078 0.16 0.46 0.548 0.006

SCD

mRNA -0.41 0.050 0.32 0.14 0.596 0.003

ACC

mRNA -0.52 0.013 0.54 0.01 0.492 0.002

Chronic Ethanol Feeding and Folate Deficiency Activate Hepatic Endoplasmic Reticulum Stress Pathway...

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