Dulbecco’s Modified Eagle Medium (DMEM), penicillin–streptomycin (P/S), fetal bovine serum (FBS), phosphate-buffered saline (PBS) and 0.25% (w/v) trypsin–EDTA were purchased from Gibco (Gaithersburg, MD, USA). ITS-G (5 mg/mL insulin, 5 mg/L transferrin, 5 μg/L selenious acid) was offered by Peiyuan Biotechnology (Shanghai, China). 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), HK, puromycin, oleic acid, palmitic acid, fatty acid free bovine serum albumin, isoproterenol, DAPI, Oil Red-O, and Free Glycerol Reagent were offered by Sigma–Aldrich (St. Louis, MO, USA). Lipofectamine 3000 Reagent, BCA protein assay kit, SuperSignal West Femto Maximum Sensitivity Substrate and Texas Red-conjugated secondary antibodies were bought from Thermo-Fisher (Grand Island, NY, USA). RIPA lysis buffer and ad-mCherry-GFP-LC3 (#C3011) were offered by Beyotime Biotechnology (Shanghai, China). Triton X-100 and PVDF membranes were supplied by Bio-Rad laboratories (Hercules, CA, USA). The shRNA targeting SIRT3 (mouse, sc-61556), scrambled shRNA (mouse, sc-1080600), and shRNA transfection reagent (mouse, sc-108061) were provided by Santa Cruz Biotechnology (Santa Cruz, CA, USA).
Cell culture and treatments
AML12 cells, obtained from ATCC (Rockville, MD, USA), were cultured in DMEM (supplemented with 10% FBS and ITS-G) in a humidified incubator (5% CO2, 37 °C). Palmitic acid and oleic acid were well dissolved with 75% (v/v) ethanol at 55 °C and diluted to 500 μM and 250 μM with DMEM containing 1% fatty acid free bovine serum albumin (w/v), respectively. To make a mixture solution of palmtic acid and oleic acid (P/O), the two solutions were sterilized with 0.2 μm filter membrane after shaking in an incubator for 2 h.
The viability of AML12 cells was determined by MTT as previously described . The working solution of HK was prepared immediately before use through diluting the stock solution (10 mM in DMSO) with fresh complete medium.
Protein concentration was quantified with a BCA Protein Assay Kit after lysing the cells with RIPA lysis buffer (containing 1% protease inhibitor cocktail and 1% phenylmethane sulfonylflfluoride). Equal amount of proteins (20‒30 μg) were separated using 5–12% SDS-PAGE and then transferred to PVDF membranes. The membranes were firstly blocked with 5% defatted milk for 2 h at room temperature, followed by overnight incubation of specific primary antibodies at 4 °C and further incubation of secondary antibodies for 1 h at room temperature. SuperSignal West Femto Maximum Sensitivity Substrate kit was used to develop the signals. Visualization of the specific protein bands were achieved on the ChemiDoc MP Imaging System, and the bands were quantitated with Image Lab 5.1 (Bio-Rad laboratories, Hercules, CA, USA).
RNA transfection and adenovirus infection
Cells were transfected with 2 μg shRNA using Lipofectamine 3000. After 6 h, cells were switched into fresh medium and incubated for 24 h. Then, cells were successively selected with puromycin (2 μg/mL) for 6 days and puromycin (4 μg/mL) for another 6 days. The survived cells were pooled together.
Cells (2 × 105) were seeded in 6-well plates and infected with 10 μL Ad-mCherry-GFP-LC3 (multiplicity of infection = 5) using Lipofectamine 3000. After 24 h, the cells were switched to fresh medium a and incubated for an additional 24 h. Then, cells were pooled together for further investigations.
Confocal immunofluorescence microscopy
Cells were fixed in formalin (10%), blocked with goat serum (2.5%), and incubated with primary antibodies at 4 ºC overnight. Subsequently, cells were incubated with Texas Red-conjugated secondary antibody at room temperature for 2 h. The nuclei were stained with DAPI. Leica TCS SP8 confocal fluorescence microscope (Leica, Buffalo Grove, IL, USA) was used to capture the images.
Nile red staining
Nile red staining was conducted as previously reported . Briefly, AML12 hepatocytes were fixed with formaldehyde (10%) and stained with Nile red (1 μg/mL). After incubating for 30 min at 4 °C and washing with PBS, the stained LDs were observed with fluorescence microscopy, and quantitated with flow cytometer with excitation and emission wavelength at 530 and 590 nm, respectively.
Determination of cellular triglycerides
TG content in cell lysate and the liver tissue was determined by using commercial kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, Jiangsu, China) and normalized by protein concentration.
Molecular docking analysis
Docking was performed on AutoDock 4.2. The crystal structure of the quaternary complex (SIRT3, a substrate, NAD+, and the specific agonist amiodarone hydrochloride; PDB ID: 5H4D)  was employed as the receptor. The protein was firstly prepared at pH 7.4 with all the water molecules removed and corresponding hydrogen atoms added. The 3D structure of HK was downloaded from the PubChem database. Gasteiger charge was calculated and AD4 atom type was assigned, and a 50 Å × 48 Å × 40 Å grid box with 375 Å spacing was placed to include the surface of the catalytic cleft with the assistance of amiodarone hydrochloride. The genetic searching algorithm was chosen for docking calculations, and 50 genetic algorithm runs were performed. Other parameters were set as default. The acquired poses were clustered with a tolerance of 2.0 Å.
Lipid droplets isolation
LDs were isolated from AML12 hepatocytes as described previously . Briefly, AML12 cells were lysed in hypotonic buffer (50 mM HEPES, 1 mM EDTA and 2 mM MgCl2, pH 7.4) supplemented with protease inhibitors after scaping and homogenized with 50 strokes in a Dounce homogenizer. After spinning down at 1,500 g for 5 min, post-nuclear fractions were mixed with equal volume of 1.05 M sucrose in isotonic buffer (50 mM HEPES, 100 mM KCl, 2 mM MgCl2) and centrifuged at 100,000 g for 2 h to remove Golgi, rough endoplasmic reticulum, mitochondria, and peroxisomes. The acquired supernatant was adjusted to 1 M sucrose in hypotonic buffer and layered on a sucrose gradient (1 mL of 0.75, 0.5, 0.25, 0.125, and 0 M sucrose solution, respectively). The sucrose gradient tube was centrifuged at 100,000 g for 4 h at 4 °C afterwards, followed by collection of LD fractions from the top which were delipidated with acetone and washed with acetone/ether (1:1, v:v). The pellet was dried under nitrogen and resuspended in protein lysis buffer. The protein concentration of LD fractions was analyzed by BCA Protein Assay kit, and subsequent western blotting was performed.
Cellular thermal shift assay (CETSA)
Cells were lysed after pretreatment with or without HK (10 μM) for 12 h. The lysates were centrifuged at 12,000 g for 10 min at 4 °C after incubating in ice for 10 min. The protein concentration was determined and adjusted to 3 μg/μL using RIPA lysis buffer. Cell lysates (50 μL) were transferred to new tubes and heated for 3 min at various temperature (50–90 °C) on a thermal cycler. After standing in ice for 10 min, soluble proteins were obtained by centrifugation at 12,000 g for 20 min at 4 °C and analyzed by western blotting .
Mitochondrial membrane potential assay
The fluorescent dye Rhodamine123 was employed to detect the mitochondrial membrane potential. Specifically, AML12 cells were cultured in the presence or absence of HK, and stained with Rhodamine123 (10 μM) for 10 min. Then, cells were washed twice with PBS, trypsinized and collected into a 1.5 mL tube. The change of membrane potential was qualitatively observed on an In Cell Analyzer 2000 (GE Healthcare Life Sciences, Chicago, IL, USA).
Intracellular reactive oxygen species (ROS) detection
Intracellular ROS levels were detected using DCFH-DA as previously described . Briefly, cells (1 × 105) were seeded into 96-well black multitier plates (clear bottom) and then cultured overnight. The cells were treated with or without HK. After 12 h, the cells were incubated with 2',7'-Dichlorodihydrofluorescein diacetate (DCFH-DA, Sigma-Aldrich, 10 μM) at 37 °C in the dark for 15 min. Fluorescence intensity was analyzed through FACS Calibur flow cytometry (BD, Lake Franklin, NJ, USA).
The lipolysis activity of AML12 cells was measured as described previously . Cells were incubated with 10 μM isoproterenol (stimulated condition) or DMSO (basal condition) at 37 ºC for 2 h. Subsequently, the medium was collected and heated at 85 °C for 10 min. After centrifuged at 2,000 g for 10 min, Clear supernatant (10 μL) was used to determine the free glycerol content using Free Glycerol Reagent. Lipolysis activity was represented by glycerol concentrations and normalized by protein concentration.
Immunoprecipitation was performed as described previously . Briefly, cell lysates (30 µg protein) were mixed with the indicated antibody (2 μg) at 4 °C overnight. Then protein A/G-agarose beads (20 μL) were added to the mixture and incubated on a rotator at 4 °C for 4 h. Immune complexes were washed twice with lysis buffer supplemented with complete mini-protease inhibitor cocktail. Bound proteins were boiled in sample preparation buffer for 5 min and then immunoblotting was conducted.
The procedures and operations involved in the animal experiments were conducted under the Animal Ethical and Welfare Committee of University of Macau (No. ICMS-AEC-2014–06) regulation. The male C57BL/6J mice were maintained in the animal facility of Faculty of Health Science, University of Macau. The mice were fed with normal chow diet (18% protein, 4.5% fat, and 58% carbohydrate, Guangdong Medical Lab Animal Center, Guangzhou, Guangdong, China) and water ad libitum under standard conditions (specific-pathogen-free) with air filtration (22 ± 2 °C, 12-h light/12-h dark).
Animal experimental procedure
According to the body weight, twenty-eight male mice (6‒8 weeks old) were randomly separated into 6 groups (n = 3‒5). The vehicle group of mice (RD) were fed with a regular chow diet and intraperitoneally injected with 10 mL/kg polyethylene glycol 400 (PEG 400, Sigma-Aldrich, St. Louis, MO, USA) solution (PEG 400:0.9% saline, 6:4, v/v). The remaining five groups of mice were fed with a choline-deficient high fat diet (Trophic Animal Feed High‐Tech Co., Nantong, Jiangsu, China) and intraperitoneally injected with PEG 400 solution (CDHFD), 2.5 mg/Kg HK (HKL, 0.25 mg/mL HK in PEG 400 solution), 10 mg/Kg HK (HKH, 1 mg/mL HK in PEG 400 solution), 5 mg/Kg Compound C (CC, 0.5 mg/mL in PEG 400 solution), and the combination of 5 mg/Kg CC and 10 mg/Kg HK (CC + HK, 0.5 mg/mL CC and 1 mg/mL HK in PEG 400 solution), respectively, once a day for consecutive 4 weeks. Blood samples were collected from tail vein under anesthesia (0.5 L/min inhalation of 3% isoflurane). The mice were euthanized by deeply inhaling carbon dioxide, and the livers were dissected.
Determination of aspartate transaminase (AST) and alanine transaminase (ALT) levels
The levels of AST and ALT in mouse serum were determined by using commercial assay kits (Nanjing Jiancheng, Nanjing, Jiangsu, China) in accordance with the manufacturer's protocols.
H&E staining and Oil-red O staining of the liver
After fixation in 4% paraformaldehyde, the liver was embedded in paraffin. 5 μm sections were deparaffinized and rehydrated followed by hematoxylin and eosin (H&E) staining and Oil-red O staining as described previously .
All experimental data were expressed as mean ± S.D., and sample size for each experiment corresponds to three biological replicates. Data analysis was finished on GraphPad Prism-6 (GraphPad Software, San Diego, CA, USA), where significant differences between groups were evaluated by one-way analysis of variance (ANOVA) followed by Dunnet’s multiple comparisons test (p < 0.05 was considered as significant differences). Where statistical significance is evaluated, variance between groups is confirmed to be similar between comparison groups (control vs. experimental) and the statistical analysis is considered appropriate.