Animals and Treatments
Mice were housed in a pathogen-free barrier facility accredited by the Association for Assessment and Accreditation of Laboratory Animal Care and procedures were approved by the local Institutional Animal Care and Use Committee. Six-week-old C57BL/6J, TNF knockout (B6.129-Tnfrsf1atm 1 Mak/J; TNFR1−/−), and PAI-1 knockout (B6.129S2-Serpine 1 tm 1 Mlg/J; PAI-1−/−) mice were obtained from Jackson Laboratory (Bar Harbor, ME). All knockout mice used in this study were back-crossed at least 10 times onto C57BL/6, avoiding concerns regarding genetic differences between wild-type strain and the knockouts at nonspecific loci. Food and tap water were allowed ad libitum prior to experimentation.
Acute alcohol exposure model
1,1-Dimethylbiguanide hydrochloride (metformin; Sigma, St. Louis, MO) or vehicle (saline) was given once a day (200 mg/kg IP) for 4 days prior to ethanol administration; this dosage was based on preliminary range-finding studies and previous work published by others.15
One hour after the last metformin dose, mice received ethanol (6 g/kg IG) or isocaloric/isovolumetric maltose-dextrin solution. This model was slightly modified from Enomoto et al16
and has been proposed as a predictive/screening tool for therapies against liver damage due to chronic alcohol intake. With this dose of alcohol, mice were sluggish, but conscious and regained normal behavior within ~6 hours of alcohol feeding. This dose caused no mortality.
Chronic enteral alcohol exposure model
Surgical implantation of the intragastric cannula and enteral feeding was performed as described previously.17
Briefly, a liquid diet described by Thompson and Reitz18
supplemented with lipotropes as detailed by Morimoto et al19
was prepared daily. Either ethanol-containing or isocaloric maltose-dextrin (control) diet was fed for 4 weeks as described elsewhere. The initial rate of ethanol delivery (16 g/kg per day) was increased in a stepwise manner 1 g/kg every 2 days until the end of the first week and then 1 g/kg every 4 days until the end of the experiment. In parallel studies, additional wild-type mice fed enteral diet received metformin (350 mg/kg per day in the diet) during the course of the study. The dose of metformin was based on previous studies investigating the effect of chronic metformin on dietary steatosis in mice,9
and shown to be well-tolerated in preliminary experiments with alcohol.
At sacrifice, animals were anesthetized with sodium pento-barbital (75 mg/kg IP) 0.5, 1, 2, 3, 6, or 12 hours after ethanol administration. Blood was collected from the vena cava just prior to sacrifice by exsanguination and plasma was stored at −80°C for further analysis. Portions of liver tissue were frozen immediately in liquid nitrogen, whereas others were fixed in neutral-buffered formalin, or frozen-fixed in OCT mounting media (Tissue Tek, Hatfield, PA) for subsequent sectioning and mounting on microscope slides.
Cell Culture and Treatment
AML-12 cells (alpha mouse liver 12, American Type Culture Collection, Manassas, VA) were grown in DMEM/HAM's F12 media (Gibco, Carlsbad, CA) supplemented with 10% FBS, 40 ng/mL dexamethasone, 0.005 mg/mL insulin, and 5 ng/mL selenium (Gibco) in a humidified 5% CO2
atmosphere at 37°C until they were 80% confluent. Cells were serum starved for 18 hours and treated with 500 μmol of metformin for 7 hours, analogous to previous work by others.13,20
Medium was subsequently removed and replaced with fresh serum-free medium with or without metformin (500 μmol). Parallel groups were also incubated in the presence and absence of the insulin receptor kinase inhibitor tyrphostin-AG1024 (300 μmol; Sigma). After 20 minutes, 5 ng/mL of TNF-α (mouse TNF-α; Sigma) was added. Cells were incubated for 3 hours, rinsed with phosphate-buffered saline, and lysed with RNA-STAT 60 Tel-Test (Ambion, Austin, TX) for later RNA isolation and analysis by real-time reverse transcriptase polymerase chain reaction (RT-PCR; see below).
All buffers used for protein extraction contained protease, tyrosine phosphatase, and serine/threonine phosphatase inhibitors purchased from Sigma. To prepare total hepatic protein, snap-frozen liver samples were homogenized with lysis buffer (20 mmol MOPS, 150 mmol NaCl, 1 mmol EDTA, 1% Nonidet P-40, 1% sodium deoxycholate, 0.1% SDS) containing protease, tyrosine phosphatase, and serine/threonine phosphatase inhibitors (as described). To prepare cytosolic fractions, livers were homogenized in Dignum A buffer (10 mmol HEPES, 1.5 mmol MgCl2, 5 mmol KCl, 0.5 mmol DTT), and then extracted with Dignam C (10 mmol HEPES, 50% glycerol, 84 mmol NaCl, 1.5 mmol Mg2Cl, 0.2 mmol EDTA, 0.5 mmol DTT) buffer to obtain nuclear proteins. Respective lysates (40−75 μg protein per well) were separated on either 5% or 8% SDS-polyacrylamide gel. Proteins were transferred to Hybond-P polyvinylidene difluoride membranes using a semidry electroblotter. The resulting blots were then probed with antibodies against phospho-c-Met (Cell Signaling Technology, Beverly, MA), apolipoprotein B100 and B48, sterol regulator element binding protein 1 (SREBP-1; both Santa Cruz, Santa Cruz, CA), phospho-AMPK (Cell Signaling Technology), or phospho-acetyl CoA carboxylase (ACC; Upstate, Waltham, MA) and bands were visualized using Amersham Biosciences ECL plus kit (Amersham Biosciences, Piscataway, NJ). To ensure equal loading, all blots were stained with Ponceau red; haptene signals were normalized to β-actin using a commercially available antibody (Sigma).
Microsomal Triglyceride Transfer Protein Activity
Frozen mouse livers were homogenized in extraction buffer (10 mmol Tris, pH 7.4, 150 mmol NaCl, 2 mmol EDTA) centrifuged at 7000 ×g for 5 minutes. Fluorescent (λex = 465 nm, λem = 535 nm) measurement of microsomal triglyceride protein activity was performed with a commercially available kit according to the instructions of the manufacturer (Roar Biomedical Inc., New York, NY). One hundred micrograms of total protein was utilized in each reaction.
Very Low-Density Lipoprotein Extraction
Plasma samples were mixed with OptiPrep (12% final concentration, AXIS-SHIELD PoC AS, Oslo, Norway), over layered with HEPES-buffered saline (0.85% NaCl; 10 mmol HEPES, pH 7.4), and centrifuged at 350,000 ×g for 3 hours at 16°C. Immediately after centrifugation, samples were collected in 20 μL fractions starting from the bottom of the centrifugation tube. To ascertain appropriate fractionation of lipoproteins, 2 μL from each fraction was separated in an agarose lipoprotein gel (Beckman Coulter, Fullerton, CA), fixed (60% ethanol, 10% glacial acetic acid), and stained overnight (55% ethanol, 0.1 % Paragon Lipo Stain [Beckman Coulter]). Gels were destained (45% ethanol) and dried. The remaining very low-density lipoprotein (VLDL) fraction was stored at 4°C for later detection of triglyceride levels.
Mouse livers were homogenized in 2× phosphate-buffered saline. Tissue lipids were extracted with methanol: chloroform (1:2), dried in an evaporating centrifuge, and re-suspended in 5% fat-free bovine serum albumin. Colorimetric assessment of hepatic and plasma triglyceride levels was carried out using Sigma Diagnostics Triglyceride Reagent (Sigma). Values were normalized to protein in homogenate prior to extraction determined by the Bradford assay (Bio-Rad Laboratories, Hercules, CA).
Oil Red O Staining
Frozen sections of liver (10 μm) were stained with oil Red O (Sigma) for 10 minutes, washed, and counterstained with hematoxylin for 45 seconds (DAKO, Carpinteria, CA).21
A Metamorph image acquisition and analysis system (Chester, PA) incorporating a Nikon microscope (Nikon, Melville, NY) was used to capture and analyze the oil Red O stained tissue sections at 200× magnification.22
The extent of labeling in the liver lobule was defined as the percent of the field area within the default color range determined by the software. Data from each tissue section (5 fields per section) were pooled to determine means.
RNA Isolation and Real-Time RT-PCR
Total RNA was extracted from liver tissue samples by a guanidium thiocyanate-based method (RNA STAT 60 Tel-Test, Ambion). RNA concentrations were determined spectrophotometrically, and 1 μg total RNA was reverse transcribed using an AMV reverse transcriptase kit (Promega, Madison, WI) and random primers. PCR primers for PAI-1 and β-actin were designed using primer 3 (Whitehead Institute for Biomedical Research, Cambridge, MA). Primers were designed to cross introns to ensure that only cDNA and not genomic DNA was amplified (). The fluorogenic MGB probe was labeled with the reporter dye FAM (6-carboxyfluorescein). TaqMan Universal PCR Master Mix (Applied Biosystems, Foster City, CA) was used to prepare the PCR mix. The 2× mixture was optimized for TaqMan reactions and contains AmpliTaq gold DNA polymerase, AmpErase, dNTPs with UTP, and a passive reference. Primers and probe were added to a final concentration of 300 and 100 nmol, respectively. The amplification reactions were carried out in the ABI Prism 7700 sequence detection system (Applied Biosystems) with initial hold steps (50°C for 2 minutes, followed by 95°C for 10 minutes) and 50 cycles of a 2-step PCR (92°C for 15 seconds, 60°C for 1 minute). The fluorescence intensity of each sample was measured at each temperature change to monitor amplification of the target gene. The comparative CT method was used to determine fold differences between samples. The comparative CT method determines the amount of target, normalized to an endogenous reference (β-actin) and relative to a calibrator (2−ΔΔCt). The purity of PCR products were verified by gel electrophoresis.
Primers and Probes Used for Real-Time RT-PCR Detection of PAI-1 and β-Actin
Histologic Analysis and Clinical Chemistry
Formalin-fixed and paraffin-embedded sections (6 μm) were cut and stained with hematoxylin and eosin for pathologic assessment after chronic enteral alcohol feeding. Pathology was scored as described by Nanji et al.23
: steatosis <5%, 1; 25%−50%, 2; 50%−75%, 3; >75%, 4; 1 or 2 for one or more inflammatory or necrotic foci. Plasma alanine aminotransferase (ALT) activity was determined using a commercially available kit (Thermo Electron, Melbourne, Australia). Urine was collected daily and urine alcohol concentrations measured using routine spectrophotometric techniques.24
Results are reported as means ± SEM (n = 4−6). ANOVA with Bonferroni's post hoc test was used for the determination of statistical significance among treatment groups. For comparison of pathologic scores, the Mann–Whitney rank-sum test was used. A P value < .05 was selected before the study as the level of significance.