Chemicals
BHBA was purchased from Sigma-Aldrich (St. Louis, MO). The 10-mM BHBA stock solution was prepared in distilled water and sterilised by filtration. Metformin was purchased from Sigma (D150959, Sigma-Aldrich, St. Louis, US) with a purity of more than 97%. Then, 1 mM of metformin was dissolved in PBS as a stock solution. Compound C (an AMPK inhibitor) was purchased from MedChemExpressTM (BML-275, MCE, NJ), and 10 mM was prepared in DMSO.
Cell culture conditions
The bovine hepatocytes used in this study were obtained and cultured in conditions described in the previous publication [32]. The hepatocytes were briefly isolated from the liver tissue of mid-lactation multiparous Holstein cows (~160 d postpartum). All experiments were performed with cells at the 4 to 6 passage. Cells (2 × 105 ) were seeded in 6-well plates with overnight incubation in complete medium (90% RPMI 1640, 8119417 Gibco, CA, 10% fetal bovine serum) (Gibco, CA) and antibiotics (penicillin 100 IU/ml; streptomycin 100 μg/ml). Finally, the cells were maintained at 37 °C in a humidified 5% CO2 incubator until reaching confluence.
Experimental design
The scale of the BHBA concentration in this study was based on the blood concentration of dairy cows with ketosis. For the pre-experimental treatment, the bovine primary hepatocytes were precultured with serum-free medium for 12 h. For the dose-dependent experiment, cells were treated with metformin for 12 h at concentrations of 0 mM, 0.5 mM, 1.5 mM, 3 mM, 5 mM, and 10 mM. PBS was added to the cells as control group. Cells were treated with BHBA at concentration of 1.2 mM for 12 h as BHBA group. The hepatocytes were pre-treated with metformin at concentrations of 1.5 mM and 3 mM for 12 h before the BHBA treatment. The inhibitor of AMPK, Compound C, at a concentration of 10 μM, was applied to substantiate the AMPK-dependent responses.
Flow cytometry
Bovine hepatocytes were seeded into 6-well plates (2 × 105 cells/well). Cells were maintained in medium with 10% (v/v) fetal bovine serum and the various treatments selected for this study. Cells were treated with metformin at concentrations of 0 mM, 1 mM, 2 mM, 3 mM, 5 mM, and 10 mM. The apoptotic effect of metformin on differentiating hepatocytes was evaluated using an Annexin V staining kit (#11858777001, Sigma-Aldrich). Cells were collected via trypsinisation and stained. The flow cytometry was analysed using the FACSCalibur platform (BD Biosciences, Franklin Lakes, NJ).
5-ethynyl-2’-deoxyuridine (EdU) detection
Following the manufacturer’s protocols, the BeyoClick™ EdU cell proliferation kit with Alexa Fluor 555 (Beyotime, Shanghai, China) was used to measure the ability of bovine hepatocytes to proliferate under different treatments. Cells were incubated with 1X Edu (10 μM) solution for 2 h, subsequently fixed with 4% paraformaldehyde for 20 min at room temperature (RT), and permeabilised with 0.3% Triton X-100 in PBS for 15 min at RT. For nuclear staining, cells were incubated with 1X Hoechst for 10 min at RT protected from light. Finally, cells were imaged with a fluorescence microscope DMi8 Microsystems GmbH (Leica, Wetzlar, Germany) at 200x magnification.
RNA extraction and Real-time quantitative PCR analysis
Total RNA was isolated with the RNA Isolater Total RNA Extraction Reagent (R401-01, Vazyme, Nanjing, China) according to the manufacturer’s instructions. Then, cDNA was synthesised using HiScript III RT SuperMix (R323-01, Vazyme, Nanjing, China) and then purified with a purification kit (Axygen, Tewksbury, MA). In line with a previous study, qRT-PCR was performed using AceQ Universal SYBR qPCR Master Mix (Vazyme, Nanjing, China) on an Applied Biosystems QuantStudio 5 Real-Time PCR System (Applied Biosystems, Foster City, CA) [33]. Furthermore, primers were designed with the Premier 6.0 software (Premier Biosoft International, Palo Alto, CA), as illustrated in earlier publications [34, 31]. The selected genes, specifically GAPDH, RPS9, and UXT, were used as internal control genes. The geometric mean of the internal control genes was utilised to normalise the target gene expression data. Previous reports have validated the use of internal control genes as references for normalising gene expression in hepatocytes samples [43]. The 2−ΔΔCt method was adopted for relative quantification [28].
NAD+ measurement
Using the NAD+/NADH assay kit with WST-8 according to the manufacturer’s instructions (Beyotime, S0175, Beijing, China), after the treatments, hepatocytes (1 × 106 cells/sample) were harvested, and intracellular NAD+ levels were determined. Briefly, cell lysis was collected with 200 μl of pre-cold lysis buffer. In order to measure the total NAD+/NADH, 20 μl of cell lysates was added to a 96-well plate. Then, measuring NADH required the incubation of the lysed cell suspension at 60°C for 30 min, and 20 μl was added to a 96-well plate. Subsequently, 90 μl of alcohol dehydrogenase was added and incubated at 37°C for 10 min. Finally, 10 μl of the chromogenic solution was added to the plate, and the mixture was incubated at 37°C for 30 min. The standard curve was generated and measured with the samples simultaneously. The absorbance was obtained at 450 nm and analysed on a microplate reader (SPARK, TECAN, Switzerland). By subtracting NADH from the total NAD+/NADH, the amount of NAD+ was derived.
Western-blotting analysis
Western blot was performed using the protocols described earlier [3]. Equal amounts of protein isolated from pbMEC by RIPA lysis buffer (Beyotime, Shanghai, China) were briefly separated on 10% SDS polyacrylamide gels (GenScript ExpressPlus™ PAGE Gels, Nanjing, China). Then, proteins were transferred onto nitrocellulose membranes (Millipore, Billerica, MA), which were incubated with primary antibodies overnight at 4 °C. After washing for 6 consecutive times, the blots were incubated with horseradish peroxidase-coupled secondary antibody. Differences in protein transfer efficiency between blots were normalised with GAPDH quantification. The grey values of the bands of each target protein were quantified using the ImageJ system analysis software. Primary antibodies for p-P65, IL1β, TNFα, acetyl-H3K14, acetyl-H3K9, histone H3, p-AMPKα, AMPKα, p-ACCα, ACCα, COX-2 and SIRT1 were purchased from Cell Signaling Technology (Danvers, MA) (#3033, #15101, #3866, #7627, #9649S, #4499, #2535, #5831, #11818, #3676, #12282S, and # 2496S), and p65 was purchased from Abcam (ab16502) and diluted 1:1,000 for incubation. The primary antibody for GAPDH was purchased from Abcam Corporation (ab8245) and diluted 1:5,000 for incubation.
Immunofluorescence
After the treatment, bovine hepatocytes (2 × 104 cells/well) were seeded onto 12-well plates accordingly. Then, the cells were fixed with 4% paraformaldehyde for 15 min, washed with PBS, and incubated with 0.5% Triton X-100 for 15 min at room temperature to increase the permeability. Incubation with 5% BSA at 37 °C for 1 h was needed for blocking. The cells were incubated at 4 °C overnight with the primary antibody (targeting the proteins interested) in PBS containing 1% BSA and 0.3 Triton X-100 (T9284, Sigma-Aldrich). After three PBS washes, the cells were incubated for 1 h with the secondary antibody in a dark 37 °C room and then washed three times with PBS. DAPI (1 μg/mL) (D8417, Sigma-Aldrich) was used for nuclear counterstaining for 5 min, and then the cells were washed three times. Finally, the cells were imaged using the DMi8 Microsystems GmbH (Leica, Wetzlar, Germany).
Chromatin immunoprecipitation assay
The preparation of samples and experiment was performed based on the protocols described previously [31]. The cells were briefly seeded into 6-well plates for treatment and harvested using the PBS containing protease inhibitor cocktail (Cat. #11697498001; Roche, Basel, Switzerland). Formaldehyde at a concentration of 1% was added for the cross-link of protein and DNA. After shaking for 10 min, glycine was used to stop the reaction. The mix was then centrifuged at 4 °C with 4000 × g for 5 min. Chromatin preparations were fragmented at 200 to 500 bp in length with sonication on ice and then incubated with 4 µg of primary antibody (Anti-P65, ab16502, Abcam) at 4°C for 16 h. Rabbit IgG was incubated with the sample as a negative control. Protein A/G agarose beads (40 µL, 50% slurry, sc-2003; Santa Cruz Biotechnology) were utilised to capture immunoprecipitated chromatin complexes, and 200 µL of chromatin preparation served as the input. Promoter fragments harvested during chromatin immunoprecipitation were quantified with qPCR using primers specific to the respective areas of IL1B promoter (forward 5’ GGCT CAGCTTGTAAAGAATC 3’ and reverse 5’ GAATGCACGAAAGTC ATCC 3’).
Statistics
The data were expressed as the means ± standard deviation (mean ± SD) and analysed using one-way ANOVA with Dunnett’s post-test by SAS Statistics (v 9.2, SAS Institute Inc., Cary, NC). Differences with P-values <0.05 were considered statistically significant. All experiments were carried out in triplicate, with three replicates in each experiment.