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 (Bar Harbor, ME, USA). HC-HMGB1-/- mice were bred at our facility as described previously (18). Starting at 10 weeks of age, mice of similar starting weights were randomized to either get high-fat diet (HFD) (45%kcal%fat; Research diet, New Brunswick, NJ, USA) or low-fat diet (LFD) (10%kcal%fat; Research diet, New Brunswick, NJ, USA) for up to 16 weeks. 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 and blood was obtained by cardiac puncture. Liver was removed, rinsed with ice-cold saline and either immediately fixed in formalin 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. Mice were fasted for 6h before the test with free access to water. Blood glucose was then measured just before the intraperitoneal glucose injection (1g/kg body wt in saline) and subsequently at 15, 30, 60, 90, and 120min post-administration.
Biochemical analysis. 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, Minneapolis, MN). HMGB1 was quantiﬁed using by ELISA (IBL International Corp., Toronto, Ontario, Canada).
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. All histology was assessed by an investigator blinded to treatment group.
Immunofluorescent staining. The whole animal was perfused with PBS. Followed by 2% paraformaldehyde. Tissue was then placed in 2% paraformaldehyde for an additional 2hrs of further fixation, followed by 3 changes in 30% sucrose in distilled water over 24 hrs. Tissue sections of 5 μm were incubated with 5% normal goat serum (NGS) for 45 min. Samples were incubated with 2 μg/ml HMGB1 (rabbit IgG, Abcam, ab18256) for 1 hr. 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 111-165-003) 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 (20). Hepatocytes (150,000 cells/mL) were plated on gelatin-coated culture plates or coverslips precoated with Collagen I (BD Pharmingen, San Diego, CA) in Williams medium E with 10% calf serum, 15mM HEPES, 10−6M insulin, 2mM L-glutamine, 100U/mL penicillin, and 100U/mL streptomycin. Palmitic acid (PA; Sigma-Aldrich, St Louis, MO, USA)-induced fat accumulation in vitro in hepatocytes was established as previously described (13, 16, 21). 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 FFA concentration in human nonalcoholic steatohepatitis(22). Hepatocytes were allowed to attach to plates for 6h and prior to PA treatments cell culture media was changed to serum-free media for 12h. Isolated hepatocytes were exposed to tunicamycin (TM; Sigma-Aldrich) (2μg/mL for 6h) to induce ER stress, or pretreated with 4-phenylbutyric acid (PBA; Sigma-Aldrich) (200 M for 12h) to inhibit ER stress. olation of human hepatocytes
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. Human hepatocytes were prepared by a three-step collagenase perfusion technique. Isolated human hepatocytes were cultured in William's medium E (Invitrogen Life Technologies) supplemented with 5 % calf serum (GE Healthcare Life Sciences), penicillin (100 U/ml), streptomycin (100 U/m l), 2 mM L-glutamine, and 15 mM HEPES.
LDH Cytotoxicity Assay. Culture medium from treated hepatocytes (50μl) 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 680nm (background signal) was subtracted from the absorbance at 490nm. LDH activity was normalized to protein concentration and results are shown as fold of controls.
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 60min at RT in freshly diluted oil red O, 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 RT-PCR analysis. Total RNA was extracted from liver tissue or hepatocytes using RNeasy Mini Kit (Qiagen). mRNA for carnitine-palmitoyl transferase 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 b-actin were quantiﬁed in triplicate by SYBR Green quantitative reverse-transcriptase polymerase chain reaction (RT-PCR). PCR reaction mixture was prepared using SYBR Green PCR Master Mix (Applied Biosystems, Foster City, CA), using above-described primers. See Supplemental Table for PCR primer sequences.
Immunoblotting. Western blot was performed using whole-cell lysates from either liver tissue or hepatocytes, as previously described (20). Membranes were incubated overnight with antibodies against Akt, phospho-Akt (p-Akt) (Ser473)( Cell Signaling), β-actin, GAPDH, ATF6 and CHOP (Abcam). Blot density was quantified using Image J.
Statistical analysis. 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).