Animal use and treatment. All animal protocols were approved by the animal care and use committee of the University of Pittsburgh, and experiments were performed in strict adherence to the National Institutes of Health Guidelines for the Use of Laboratory Animals. Male wild-type (WT) C57BL/6 mice were purchased from The Jackson Laboratory (no.000664). HC-HMGB1-/- mice were bred at our facility as described previously (18). Mice were housed four per cage. Starting at 10 weeks of age, mice of similar starting weights were randomized to either get high-fat diet (HFD) (45%kcal%fat; D12451, Research Diets, New Brunswick, NJ, USA) or low-fat diet (LFD) (10%kcal%fat; D12450K, Research Diets, New Brunswick, NJ, USA) for up to 16 weeks. See Supplemental Table 1 for Composition of rodent special diets. Mouse weight and food intake were measured weekly. Housing conditions and access to food and water were the same for all mice. Mice were euthanized under ether anesthesia and blood was obtained by cardiac puncture. Liver tissue was removed after perfusion with cold phosphate-buffered saline, and then either immediately fixed in 2% paraformaldehyde or snap-frozen in liquid nitrogen.
Body fat composition, energy expenditure and glucose tolerance test (GTT).Whole-body fat was measured in conscious mice using magnetic resonance spectroscopy (EchoMRI-100; Echomedical Systems, Houston, TX) (19). To evaluate energy expenditure, O2 consumption was monitored by The Comprehensive Lab Animal Monitoring System (CLAMS) (Columbus Instruments, Columbus, OH). Blood glucose was measured using a TRUEtrack blood glucose meter (TRIVIDIA, Fort Lauderdale, FL, USA) in blood collected from the tails of mice. The TRUEtrack® System exceeds the minimum International Organization for Standardization (ISO) standards for accuracy with 96.5% of results within the ISO defined limits. Mice were fasted for 6h before the test with free access to water. Blood glucose was then measured just before the intraperitoneal glucose injection (1 g/kg body wt in saline) and subsequently at 15, 30, 60, 90, and 120 min post-administration.
Biochemical analyses. Serum alanine amino-transferase (ALT) levels were measured using the DRI-CHEM 4000 Chemistry Analyzer System (Heska, Des Moines, IA). Serum interleukin (IL)-6 levels in mice were detected by an enzyme-linked immunosorbent assay (ELISA) kit (R&D Systems, no.M6000B; sensitivity: 1.8 pg/mL). HMGB1 was quantiﬁed using by ELISA (TECAN, no.ST51011; sensitivity: 2.5 ng/mL).
Histology. Specimens were fixed in 10% neutral buffered formalin, paraffin embedded, and sectioned. Hepatic lipid accumulation was evaluated using hematoxylin and eosin (H&E)-stained histological sections. Steatosis was graded as: 0, 0% to 5% of the hepatocytes in the section are steatotic; 1, greater than 5% to 33% of hepatocytes are steatotic; 2, greater than 33% to 66%; and 3, greater than 66% (20). All histology was assessed by an investigator blinded to treatment group.
Immunofluorescent staining. Liver tissue was removed after perfusion with cold phosphate-buffered saline and 2% paraformaldehyde. Tissue was then placed in 2% paraformaldehyde for an additional 2h of further fixation, followed by 3 changes in 30% sucrose in distilled water over 24h. Tissue sections of 5 μm were incubated with 5% normal goat serum for 45 min. Samples were incubated with 2 μg/ml anti-HMGB1 antibody (rabbit IgG, Abcam, ab18256) for 1 h. Sections were then incubated with Alexa 488-conjugated F-actin phalloidin (1:500, Invitrogen, San Diego, CA, USA); Cy3-conjugated goat anti-rabbit IgG (1:1000, for anti-HMGB1 antibody, Jackson Immunoresearch, no.111165003) for 1 h. A Hoechst nuclear stain was applied for 30 s and slides were prepared for imaging. Imaging conditions were maintained at identical settings within each antibody-labeling experiment with original gating performed using the negative control. Large area images in X and Y using a Nikon A1 confocal microscope (purchased with 1S10OD019973-01 awarded to Dr. Simon C. Watkins).
Mouse hepatocyte isolation and cell culture. Primary mouse hepatocytes from WT and HC-HMGB1-/- mice were isolated and plated as previously described (21). Hepatocytes (150,000 cells/ml) were plated on gelatin-coated culture plates or coverslips precoated with Collagen I (Thermo Fisher Scientific, no.08774383) in Williams E medium with 10% calf serum, 15mM HEPES, 10−6M insulin, 2mM L-glutamine, 100U/mL penicillin, and 100U/mL streptomycin. Palmitic acid (PA; Sigma-Aldrich, no.P0500)-induced fat accumulation in vitro in hepatocytes was established as previously described (13, 16, 22). PA was dissolved in 95% ethanol at 100mM stock solution, which was then mixed with Williams medium E containing 10% calf serum to 8mM stock concentration. PA concentration used ranged from 200 to 800μM, which is similar to fasting plasma total free fatty acid (FFA) concentration in human nonalcoholic steatohepatitis (23). Hepatocytes were allowed to attach to plates for 6h and cultured in serum-free media for 12h prior to the PA treatment. Isolated hepatocytes were exposed to tunicamycin (TM; Sigma-Aldrich, no.654380 ) (2μg/mL for 6h) to induce ER stress, or pretreated with 4-phenylbutyric acid (PBA; Sigma-Aldrich, no.P21005) (200μM for 12h) to inhibit ER stress.
Oxygen consumption rate (OCR) measurement. WT and HC-HMGB1-/- mouse hepatocytes were plated in XF-96 cell culture plates (104 cells/well; Agilent, no.101085) overnight. Hepatocytes were then washed and incubated for 1 h in XF assay medium (unbuffered DMEM pH 7.4) in a non-CO2 incubator at 37 °C as per manufacturer’s instructions (Agilent). Real time measurements of hepatocyte OCR were performed using an XF-96 Extracellular Flux Analyzer (Agilent). Three consecutive measurements were obtained under basal conditions and after the sequential addition of 4 μM oligomycin, to inhibit mitochondrial ATP synthase (complex V); 1 μM FCCP (fluoro-carbonyl cyanide phenylhydrazone), a protonophore that uncouples ATP synthesis from oxygen consumption by the electron-transport chain providing a maximal OCR value; and 1 µM rotenone which inhibits the electron transport chain providing a baseline/minimum value.
Human hepatocytes isolation and culture. Human hepatocytes were isolated from histologically normal liver and were kindly provided by Dr. David Geller (University of Pittsburgh Department of Surgery, Pittsburgh, PA) according to a protocol approved by the Institutional Review Board (24). Human hepatocytes were prepared by a three-step collagenase perfusion technique. Isolated human hepatocytes were cultured in William's medium E (Invitrogen, no.12551032) supplemented with 5% calf serum (GE Healthcare Life Sciences), penicillin (100 U/ml), streptomycin (100 U/ml), 2 mM L-glutamine, and 15 mM HEPES.
LDH cytotoxicity Assay. Culture medium from treated hepatocytes (50 μl) was transferred to a 96-well plate and the LDH reaction was performed using Pierce LDH Cytotoxicity Assay Kit (Thermo Fisher Scientific, Waltham, MA, USA) following manufacturer's instructions. Absorbance at 680 nm (background signal) was subtracted from the absorbance at 490 nm. LDH activity was normalized to protein concentration and results are shown as fold of controls. At least three independent experiments with three replicates each were performed.
Oil red O staining. Hepatocytes from each experimental group plated in six-well plates were rinsed three times with PBS, fixed in 4% paraformaldehyde for 30min, stained for 60 min at RT in freshly diluted Oil Red O solution (0.5% Oil Red O in isopropanol: H2O = 3:2), rinsed three times with PBS, redyed for 30sec in hematoxylin staining solution and rinsed with PBS twice. Intracellular lipid droplets were imaged with Nikon TS100 inverted microscope connected to a digital camera.
Real-time polymerase chain reaction (RT-PCR) analyses. Total RNA was extracted from liver tissue or hepatocytes using RNeasy Mini Kit (Qiagen) according to the manufacturer's instructions. Total RNA (1 μg) was reverse transcribed using an iScript cDNA Synthesis Kit (Bio-Rad Laboratories, no. 1708891), according to the manufacturer's instructions. The samples were then diluted, and the same amount of cDNA was added to each reaction on each plate. The iTaq universal SYBR Green supermix (Bio-Rad Laboratories, no. 1725121) and a different primer were used for gene expression analyses. All samples were run in triplicate, and the experiment was repeated three times. Primers used for carnitine-palmitoyltransferase 1α (CPT-1α), medium-chain acyl-CoA dehydrogenase (MCAD), long-chain acyl-CoA dehydrogenase (LCAD), very-long chain acyl-CoA dehydrogenase (VLCAD), fatty acid synthase (FAS), lipoprotein lipase (LPL), sterol regulatory element-binding protein 1 (SREBP-1), peroxisome proliferator-activated receptor gamma (PPAR-γ), adipophilin, collagen type 1A1 (COL1A1), collagen type 1A2 (COL1A2), tissue inhibitors of metalloproteinases (TIMP), α-smooth muscle actin (α-SMA), stearoyl-CoA desaturase-1 (SCD-1), and β-actin were ordered from Qiagen (Hilden, Germany). See Supplemental Table 2 for PCR primer sequences. Thermal cycling conditions were 10 min at 95 ° C, followed by 40 cycles of 95 ° C for 15 s and 60 ° C for 1 min on a sequence detection system (ABI PRISM 7000; Applied Biosystems). Each gene expression was normalized with β-actin mRNA content.
Immunoblotting. Western blot was performed using whole-cell lysates from either liver tissue or hepatocytes, as previously described (21). Total protein concentration of 1 µg /µl was loaded into the 12% gel for each specific western blot analysis. Membranes were incubated overnight with antibodies against Akt (Cell Signaling, no. 9272), phospho-Akt (p-Akt) (Ser473) (Cell Signaling, no. 9271), HMGB1 (Abcam, ab18256), β-actin (Abcam, ab8226), GAPDH (Abcam, ab8245), ATF6 (Abcam, ab37149) and CHOP (Abcam, ab11419). Secondary antibodies (no. 31460 and no. 31430) were from Thermo Fisher Scientific. For Western blot analyses, cell lysis buffer (1:10, Cell Signaling, no. 9803) was used for whole cell lysis and tissue lysis together with protease inhibitors. Western gel images were quantified by densitometry analyses using ImageJ software and presented as a ratio of loading controls.
Statistical analyses. Data are presented as mean ± standard error of mean (SEM). Experimental results are analyzed for their significance by Student’s t-test. Significance was established at the 95% confidence level (P <0.05).