Effect of chronic alcohol exposure on folate uptake by liver mitochondria

Material

[³H]Folic acid (specific activity 25.4 Ci/mmol; radiochemical purity > 98%) was obtained from Moravek Biochemicals (Brea, CA). TRIzol reagent was purchased from Life Technologies (Invitrogen, Carlsbad, CA). DNA oligonucleotide primers were from Sigma Genosys (The Woodlands, TX). All other chemicals were of analytical or molecular biology grade and were obtained from commercial vendors (Fisher Scientific and Sigma).

Chronic Alcohol Feeding of Rats

Male Wistar rats (Charles River, Wilmington, MA) weighing ∼120 g (∼14 wk old) were housed at the Animal Core of the NIAA-funded Southern California Research Center for Alcoholic Liver and Pancreatic Disease (ALPD) and Cirrhosis at the University of Southern California. The experimental protocol was approved by animal use committee of the Veterans Affairs at Long Beach and the University of Southern California. Rats were fed Lieber-Decarli (22) ethanol liquid diet (ethanol provided 36% of total calories) (Bio Serv, Frenchtown, NJ) as described by us (33, 34, 37, 38); control rats were pair-fed with the same liquid diet but without ethanol (maltose-dextrin replaced ethanol isocalorically). After 2, 4, and 6 wk of alcohol feeding, rats were euthanized, and the liver was removed for isolation of mitochondria (for uptake and protein study). A portion of the liver was also collected and stored at −80°C in TRIzol for subsequent determination of mRNA and heterogenous nuclear RNA (hnRNA) levels of mitochondrial folate transporter.

Cell Culture

Human-derived liver HepG2 cells (a hepatocellular carcinoma cell line) (passages 10–20; American Type Tissue Collection; ATCC, Manassas, VA) were used in this study. Cells were grown to confluence on collagenized plates in DMEM growth medium (Sigma, St. Louis, MO) containing 10% FBS, 100 U/ml penicillin, and 100 μg/ml streptomycin at 37°C. In examining the effect of chronic alcohol exposure on physiological and molecular parameters of mitochondrial folate uptake, confluent monolayers of HepG2 cells were exposed to 50 mM alcohol for 96 h as described by us and others recently (37, 38, 41). This concentration of ethanol was chosen because it is similar to the concentration of alcohol reported in the blood of chronic alcoholic subjects (28). Control cells were treated in the same manner but without added ethanol.

Isolation of Mitochondria

Liver mitochondria were isolated from rats by an established procedure, as described by us previously (32). Briefly, rats were anesthetized, and the liver was perfused with ice-cold saline solution in situ. The liver was then removed, connective tissue and fat was removed and washed with ice-cold H-media (70 mM sucrose, 220 mM mannitol, 0.05% fat-free bovine serum albumin, and 2 mM HEPES, pH 7.4). Approximately 2 g of liver tissue was minced, suspended in H-media (2 ml/g), and homogenized using a Teflon-glass homogenizer (800 revolutions/min). The homogenate was filtered through a double layer of gauze and centrifuged at 600 g for 15 min, and the supernatant was collected. The supernatant was then spun at 6,430 g for 15 min and the resulting pellet suspended in ice-cold H-media. The suspension was centrifuged at 9,680 g for 15 min and the fluffy layer on top of the pellet was washed away. The remaining pellet was suspended in H-media and centrifuged again at 9,680 g for 15 min. The pellet, containing the mitochondria, was finally suspended in suspension buffer [in mM: 140 KCl, 0.3 EDTA, 5 MgCl2, 10 2-N-morpholino ethanesulfonic acid (MES), and 10 mM HEPES, pH 7.4] to achieve a protein concentration of l0–15 μg/μl.

For isolating mitochondria from human liver HepG2 cells, again an established procedure was used (13) in which cells were washed twice with phosphate buffer, scraped off gently, centrifuged for 15 min, then resuspended in ice-cold buffer A (10 mM NaCl, 1.5 mM MgCl₂ and 10 mM Tris, pH 7.5), and disrupted by homogenization using a dounce glass homogenizer. The suspension was homogenized and suspended immediately with uptake buffer B (70 mM sucrose, 1 mM KH₂PO₄, 5 mM sodium succinate, 5 mM HEPES, 220 mm mannitol, 0.1 mm EDTA, pH 7.4), and the homogenate was centrifuged twice at 500 g for 5 min at 4°C. The resulting supernatant was centrifuged again at 13,000 g for 20 min. The obtained mitochondrial pellet was resuspended in uptake buffer B, sonicated (on ice), and remaining membrane/intact cells eliminated by centrifugation at 60,000 g for 90 min at 4°C. Freshly prepared mitochondria were used for uptake studies.

Uptake Study

Uptake studies were performed on freshly isolated mitochondrial preparations immediately following isolation utilizing a rapid-filtration technique as described previously (18). Incubation was performed in uptake buffer B in the presence of labeled (and unlabeled) folic acid at 37°C; the reaction was stopped by adding ice-cold buffer and the suspension was placed on nitrocellulose filters. Washing was then done with 5 ml of ice-cold buffer, the filter was dissolved in scintillation fluid, and radioactivity was measured in a liquid scintillation counter. Protein content of cell digests was measured with a Bio-Rad Dc protein assay kit (Bio-Rad).

Real-Time PCR Analysis

Total RNA (5 μg) was isolated from the liver of alcohol-fed rats and their pair-fed controls using TRIzol reagent following manufacturer's protocol. The RNA sample was treated with DNase I (Invitrogen) and reverse transcribed with oligo-dT primers to synthesize first-strand cDNA (Superscript First-Strand synthesis RT-PCR kit; Invitrogen). With the use of gene-specific primers against rat MFT (rMFT) and β-actin, the coding region was amplified following the conditions described before (29) in a CFX 96 real-time system (Bio-Rad). The primers used for rMFT and rβ-actin are rMFT: forward 5′-AGTAACCCCGAATGTGTG-3′, reverse 5′-TTCAGCAGCTGAGATGAG-3′ and β-actin: forward 5′-GTCAGGTCATCACTATCGGC-3′; reverse 5′-CATGGATGCCACAGGATTCC-3′. The data were normalized to β-actin and then quantified using a relative relationship method supplied by the vendor (Bio-Rad) (23). The real-time PCR analysis of hMFT expression in human liver HepG2 cells was carried out using gene-specific primers for the hMFT (forward, 5′-GCATAAGAGTCCTCTCGT-3′; reverse, 5′-TAGAGAGGGCCCACCACAC-3′) and β-actin (forward, 5′-CATCCTGCGTCTGGACCT-3′; reverse, 5′-TAATGTCACGCACGATTTCC-3′) as described by us previously (29, 37, 38).

hnRNA Analysis

To determine whether chronic alcohol feeding affects the transcription of the rat liver MFT gene, we determined the level of hnRNA in the liver of alcohol-fed rats and their pair-fed controls as described recently (2, 7, 12, 20, 31). Briefly, total RNA isolated from the liver of chronic alcohol-fed rats and their pair-fed controls were subjected to RT-PCR using gene specific primers. Primers used for amplification are MFT (forward 5′-TAGAGTTGAAGCAGCTGAC-3′, reverse-5′-CCATCGACTTTCCAAATGG-3′); and β-actin (forward 5′-CTGCTCTTTCCCAGATGAG-3′, reverse -5′CTCATAGATGGGCACAGTG-3′). To ensure specificity, the RNA samples were treated with DNase I (1 μg RNA/unit; Invitrogen), and the DNase I-treated RNA was reverse transcribed with the random hexamer (Invitrogen). Relative expression of the genes was measured by semiquantitative RT-PCR using gene-specific primers and visualized on 1% agarose gel. To establish specificity, a reaction without cDNA template was run in parallel as negative control. For quantification, the gel was scanned and the band intensity was measured using UN-SCAN-IT gel automated digitizing system, version 6.1 (Silk Scientific).

Western Blot Analysis

Western blot analysis was performed using equal amounts (150 μg) of mitochondrial proteins isolated from the liver of alcohol-fed and pair-fed rats. Proteins were resolved onto premade 4–12% Bis-Tris mini gel (Invitrogen) following standard procedure (37, 38). The protein gel was then electro-blotted onto nitrocellulose membrane (Bio-Rad). The blot was blocked overnight at 4°C with 5% dried milk and then incubated with specific rMFT polyclonal antibodies raised in rabbit (Santa Cruz Biotechnology, Santa Cruz, CA). rMFT on the blot was incubated with anti-rabbit IgG conjugated with horseradish peroxidase (Santa Cruz) and developed using an enhanced chemiluminescence detection system (Amersham, Arlington Heights, IL). Intensity of the specific MFT band was determined by UN-SCAN-IT gel automated digitizing system, version 6.1 (Silk Scientific) from scanned Western blot image.

5′-Rapid Amplification of the cDNA Ends

Transcription initiation site for SLC25A32 in HepG2 cells was identified by 5′-rapid amplification of the cDNA ends (5′RACE) technique using 5′-RACE kit (version 2.0, Life Technologies) as described by us previously (26). Sequence information of the human SLC25A32 cDNA deposited in GenBank (accession no. NM_030780.3) was utilized as a guide in the design of the primers. Total RNA (5 μg) isolated from HepG2 cells was used with gene-specific primer 1 (Gsp1) 5′-TATATGACTTGATGGCATTG-3′ followed by isolation and tailing of the first-strand cDNAs. PCR was then performed on tailed cDNAs using the gene-specific primer 2 (Gsp2) 5′-TCATAAAGTCCCCGTAGTCCATC-3′ and Abridged Anchor primer provided by the company. Nested amplification was then performed using Abridged Universal Amplification primer provided by the company and the gene-specific primer 5′-GCAGCGCAAGGTTGGATAAGAC-3′. Products of the PCR were then analyzed on agarose gel (2%), subcloned into pGEM-T Easy vector (Promega, Madison, WI), and then sequenced for verification (Laragen, Culver City, CA).

Cloning of 5′-Regulatory Region for SLC25A32 Gene

Sequence information of the SLC25A32 gene and 5′ flanking sequence (accession no. NM_030780.3) was used to design PCR cloning primers (forward: 5′-GGTACCAGTCGCTTCCGCGTCC-3′, forward: 5′-GGTACCTTAATAACAGAGTTTTTGAGCAGATCC-3′, and reverse: 5′-CTCGAGAGGATGCAGCCCGGTG-3′). To accommodate potential regulatory sequences, the primers were designed to incorporate sequences that are ∼490 and 3,000 bp of SLC25A32 gene. For subsequent cloning, kpnI and XhoI sites were included on the 5′ end of both primers. Using human genomic DNA (Clontech) as a template, specific sequences were amplified by initial denaturation at 94°C for 4 min followed by 30 cycles of denaturation at 95°C for 1 min, annealing at 55°C for 1 min, extension at 72°C for 1 min, and then a final extension at 72°C for 5 min. The amplified PCR products were cloned into pGL3-Basic vector. The identity of the each fragments was verified by sequencing (Laragen).

Transfection and Reporter Gene Assay

HepG2 cells (<80% confluent) were transiently transfected in 12-well plates with 4 μg of the cloned putative 5′-regulatory regions (in pGL3-Basic vector) and 100 ng of the Renilla transfection control plasmid Renilla luciferase-thymidine kinase (pRL-TK) plasmid (Promega, Madison, WI). Transfection was performed with Lipofectamine 2000 reagent (Invitrogen) according to manufacturer's instructions. Forty-eight hours after transfection the activity of the putative promoter was examined. For alcohol study, the transfected cells were treated on the second posttransfection day with 50 mM alcohol in DMEM medium. The cells were incubated in a alcohol-saturated tissue culture incubator containing 5% CO₂ at 37°C for 96 h as described before (37, 38, 41). To minimize the loss of alcohol concentration because of evaporation, the medium was changed at 12 h intervals. At the end of 96 h incubation, firefly luciferase activity was determined and normalized relative to Renilla luciferase activity by using the Dual Luciferase Assay system (Promega) following standard protocol described by us previously (30, 38).

Data Presentation and Statistical Analysis

Uptake data presented in this paper are means ± SE of at least three independent measurements and are expressed as percentage relative to simultaneously performed controls. The Student's t-test was used for statistical analysis; P < 0.05 was set as statistically significant. Carrier-mediated folate uptake was determined by subtracting uptake by passive diffusion; i.e., uptake in the presence of a high folic acid concentration of 1 mM) from total vitamin uptake. Protein, RNA, and hnRNA determinations as well as assays of promoter activity were all run on at least three different occasions using three different preparations; representative images are shown.

Effect of Chronic Alcohol Feeding on Physiological and Molecular Parameters of Folate Uptake by Rat Liver Mitochondria

Effect of Chronic Alcohol Exposure on Physiological and Molecular Parameters of Folate Uptake by Human Liver Mitochondria: Studies with HepG2 Cells

To extend our studies to the human situation and to further confirm the earlier suggestion that the effect of chronic alcohol consumption on mitochondrial folate uptake is (at least in part) being exerted at the transcriptional level, we first examined the effect of chronic alcohol exposure (96 h) of human liver HepG2 cells on mitochondrial carrier-mediated folate uptake. Chronic exposure of confluent monolayers of HepG2 cells was performed as described recently (37, 38, 41). A concentration of alcohol of 50 mM was chosen to mimic the level of alcohol reached in the blood of chronic alcoholic subjects (28). The results showed a significant (P < 0.01) inhibition in carrier-mediated uptake of folic acid (0.25 μM) by mitochondria isolated from HepG2 cells chronically exposed to alcohol compared with uptake by mitochondria of control cells (Fig. 3A). This inhibition was associated with a significant (P < 0.01) reduction in the steady-state level of the hMFT mRNA in the alcohol-exposed HepG2 cells compared with control cells (Fig. 3B).

To examine the effect of chronic alcohol exposure on transcriptional activity of the SLC25A32 gene, we cloned the 5′-regulatory region of this gene. First, we performed a rapid amplification of the cDNA ends (5′-RACE) to identify the potential transcription start site(s) for SLC25A32 gene in HepG2 cells and found only one site located at position −35 (A of the ATG was used as number 1). We then cloned an ∼490 (−303 to +187) and 3,000 bp (−2,813 to +187) promoter fragments into pGL3-Basic vector, transiently transfected the constructs into HepG2 cells, and measured the promoter activity (see materials and methods). The result showed a robust promoter activity for the 490 bp fragment, which was ∼90-fold higher than that of the pGL3-Basic (Fig. 4A); much less promoter activity was observed with the 3,000 bp fragment (data not shown). Subjecting the 490 bp fragment to a computer analysis (Genomatix MatInspector) indeed showed that this region contains CCAAT binding site, GC-box binding site (SP1/GC), Krueppel-like (KLF) transcription factor site, and CpG island. Thus we used this fragment in our investigations of the effect of alcohol on hMFT promoter activity in mitochondria of HepG2 cells. The results showed that chronic alcohol exposure (50 mM, 96 h) of HepG2 cells leads to a significant (P < 0.05) inhibition in SLC25A32 promoter activity (Fig. 4B). These results provide further support for the earlier suggestion that the inhibitory effect of chronic alcohol exposure on mitochondrial folate uptake is at least in part being exerted at the transcriptional level of the SLC25A32 gene.