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Glutathione deficiency induces epigenetic alterations of vitamin D metabolism genes in the livers of high-fat diet-fed obese mice

Obesity has been correlating with low levels of glutathione (GSH) and 25-hydroxyvitamin D3 (25(OH)VD3). The liver is the principal site for the 25(OH)VD3 biosynthesis. This study investigated whether GSH deficiency induces epigenetic alterations that impair Vitamin D (VD) metabolism genes in the livers of HFD-fed mice. The expression of the VD metabolism genes CYP2R1 and CYP27A1 (25-hydroxylase), CYP27B1 (1-α-hydroxylase), and vitamin D receptor (VDR) were downregulated in the livers of mice fed an HFD (GSH- deficient) compared with control diet-fed group. The expression of CYP24A1 (24-hydroxylase) was significantly increased, which catabolizes both 25(OH)VD3 and 1α,25-hydroxyvitaminD3. Gene-specific hypermethylation of 25-hydroxylase, 1-α-hydroxylase, and VDR, and hypomethylation of CYP24A1 was observed in HFD-fed mice. GSH deficiency induced in cultured hepatocytes caused an increase in oxidative stress and alterations in VD regulatory genes. Similarly, elevated global DNA methylation, Dnmt activity, and 5-methylcytosine but decreased Tet activity and 5-hydroxymethylcytosine were observed in the GSH-deficient hepatocytes and the liver of HFD-fed mice. Replenishment of GSH by its prodrugs treatment beneficially altered epigenetic enzymes, and VD-metabolism genes in hepatocytes. HFD-induces GSH deficiency and epigenetically alters VD-biosynthesis pathway genes. This provides a biochemical mechanism for the VD-deficiency and potential benefits of GSH treatment in reducing 25(OH)VD3-deficiency.

Cell culture and treatment FL83B mouse hepatocytes (ATCC ® , Manassas, VA) were cultured and maintained in F-12K complete medium. The culture was grown and maintained at 37 °C in a humidified atmosphere containing 5% CO 2 .
Cells were counted using the Trypan Blue method before all treatments. siRNA were purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX): γ-GCSc siRNA, and Control siRNA-A, a scrambled nonspecific RNA duplex that shares no sequence homology with any of the genes, which was used as a negative control. Cells were transiently transfected with 100 nM siRNA complex using Lipofectamine™2000 transfection reagent (Invitrogen, Carlsbad, CA) following the method described earlier 1,2 . In another set of experiments, cells were treated with L-cysteine (300 μM), N-acetyl-L-cysteine (NAC; 300 μM), or GSH ethyl ester (GSHee; 2.5 mM) (Cayman Chemical Company, Ann Arbor, Michigan), respectively, for 24 h. Buthionine sulfoximine (BSO) is an irreversible inhibitor of γglutamylcysteine synthetase (K i <100 μM), the rate-limiting enzyme for L-glutathione (GSH) synthesis, which depletes GSH 3,4 . Cells were exposed to BSO (10 μM) for 12 h in basal medium (without serum or any growth factors). In another set of experiments, after BSO (10 μM) for 12 h in basal medium, cells washed twice with PBS and left either for 6h or 12h in basal media alone without BSO treatment (withdrawal).

Cell viability assay
Cell viability was determined using the Alamar Blue reduction bioassay. This method is based upon Alamar Blue dye reduction by live cells. Briefly, cells were plated into 96 well plates after treatment per the above-described protocols, AlamarBlue® Cell Viability Reagent (ThermoFisher Scientific, Grand Island, NY) was added, and the cells were incubated at 37 °C in the dark for 4 h. Absorbance was read at 590 nm using a plate reader. Data are expressed as a percentage of viable cells.

Analysis of mRNA expression using RT-qPCR
Total RNA was extracted from cells or tissue using either an AllPrep DNA/RNA/Protein Mini Kit  Table 1). The relative amount of mRNA was calculated using the relative quantification (ΔΔCT) method. The relative amount of each mRNA was normalized to housekeeping gene Glyceraldehyde 3-phosphate dehydrogenase (GADPH). Following Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) guidelines, all of our experiments included technical replicates (n=3) and biological replicates (n=4). Data were analyzed using the comparative CT method, and the fold change was calculated using the 2 −ΔΔCT method 5 using a 7900HT Real-Time PCR system and software (Applied Biosystems). The results were expressed as relative quantification (RQ).

Western blot analysis
The tissue homogenates were processed for the immunoblotting studies. ∼100 mg of liver tissue was homogenized in RIPA buffer on ice using a rotor-stator to extract protein from the liver. RIPA buffer (50 mM Tris pH 8, 150 mM NaCl, 1% NP-40, 0.5% deoxycholic acid, and 0.1% SDS) was supplemented with protease and phosphatase inhibitors (1 mM PMSF, 5 μg/mL leupeptin, 2 μg/mL aprotinin, 1 mM EDTA, 10 mM NaF, and 1 mM NaVO4). For whole-cell extraction, after treatment, the hepatocytes (FL83B cells) were washed twice with ice-cold PBS and lysed in RIPA buffer. Lysates were then centrifuged for 10 min at 10,000 x g at 4 °C. Supernatants were collected and the protein concentrations determined using a Pierce BCA assay kit (Thermo Fisher Scientific, Rockford, IL) for Western blot analysis and HPLC assay. Equal amounts of proteins (20 μg) were separated on 10 % SDS-PAGE and transferred to a polyvinyl difluoride (PVDF) membrane. Membranes were blocked at room temperature for 2 h in a blocking buffer containing 1% BSA to prevent non-specific binding and then incubated with an appropriate primary antibody (Supplementary Table 2). The membranes were washed in TBS-T (50 mmol/L Tris-HCl, pH 7.6, 150 mmol/L NaCl, 0.1 % Tween 20) for 30 min and incubated with the appropriate HRP-conjugated secondary antibody (1:5000 dilution) for 2 h at room temperature. The protein bands were detected using ECL detection reagents (Thermo Scientific, Rockford, IL) and exposed to blue X-ray film (Phenix Research Products, Candler, NC). All of our immunoblot experiments included technical replicates (n=2) and biological replicates (n=4). Western blot scans were analyzed using ImageJ software (developed by Wayne Rasband, National Institutes of Health, Bethesda, MD; available at http://rsb.info.nih.gov/ij/index.html). Densitometry analyses of Western blots were normalized to β-actin (ratio).  The relative amounts of methylation were calculated using an Excel-based data analysis template provided by the manufacturer, using delta-Ct values. The mock digest was used for initial DNA input quantification, the Ms digest to quantify methylated DNA and the Md digest for quantifying unmethylated DNA. The Ms+Md digest was used to quantify the undigested amount of DNA as a background control. EpiTect Methyl II PCR methylation-sensitive and methylation-dependent digest 6 control assays were used to test the cutting efficiency of the restriction enzymes and to ensure reliable and reproducible results.  Figure S1. Unpaired Student's t-test was used to compare the control with the HFD group. *p ≤ 0.05 for a statistical test was considered significant. Data are expressed as mean ± SEM (n=6).

Supplementary
8 Figure S2.  was used to compare the control group with the treatment group. *p≤0.05 was considered significant.
Data are expressed as mean ± SEM.