Cell culture
HepG2 cells (ECACC, UK) were maintained under a mixture of 95% air and 5% CO2 at 37℃ in six-well plates containing high-glucose (25 mmol/l) DMEM supplemented with 10% heat-inactivated FBS. They were exposed to metformin (Merck, Germany), imeglimin (provided by Sumitomo Pharma Co. Ltd., Japan), or vehicle before analysis.
siRNA transfection
Cells were transfected for 72 h in six-well plates with 25 nmol/l small interfering RNAs (siRNAs) targeting LKB1 (STK11 siRNA-SMART pool, Horizon, UK) or control siRNAs (nontargeting siRNA pool #2, Horizon) and with the use of the Lipofectamine RNAiMAX reagent (Invitrogen, USA). They were then stimulated with metformin or imeglimin for 3 h before immunoblot analysis.
Mice
This study was performed in accordance with relevant guidelines and regulations. Animal experiments were carried out in accordance with the ARRIVE guidelines and approved by the animal experimentation committee of Kobe University Graduate School of Medicine. C57BL/6J mice were obtained from CLEA Japan and maintained on a normal diet and on a 12-h-light/12-h-dark cycle. At 8 weeks of age, male mice were deprived of food overnight, allowed to refeed for 5 h, and then injected i.p. with vehicle or with metformin or imeglimin at 250 mg/kg. A blood sample was collected from the tail vein at 1 h after the injection for determination of the blood glucose level with a glucose meter (Sanwa Chemical, Japan) and of the plasma insulin concentration with an ELISA kit for mouse insulin (Morinaga, Japan).
Immunoblot analysis
Cells were solubilized with a lysis buffer consisting of 0.2% Triton X-100, a protease inhibitor cocktail (Merck, Germany), and a phosphatase inhibitor cocktail (Nacalai Tesque, Japan) in PBS. Mouse liver was homogenized in a lysis buffer comprising 20 mmol/l Tris-HCl (pH 7.5), 150 mmol/l NaCl, 2 mmol/l EDTA, 1% Nonidet P-40, 10% glycerol, and protease inhibitor and phosphatase inhibitor cocktails. The protein concentration of each sample was measured with a BCA protein assay kit (Thermo Fisher Scientific, USA), and equal amounts of protein were fractionated by SDS-PAGE. The separated proteins were transferred to a nitrocellulose membrane (Cytiva, Japan), which was then exposed to 3% BSA (Fuji Film Wako, Japan) before incubation with primary antibodies including those to α subunits of AMPK (AMPKα), to phosphorylated AMPKα (Thr172), to acetyl-CoA carboxylase (ACC), and to phosphorylated ACC (Ser79), all of which were obtained from Cell Signaling Technology (USA). Immune complexes were detected with Anti-Rabbit IgG (H+L) HRP Conjugate (Promega, USA) and chemiluminescence reagents (ECL prime, Cytiva) and were quantified with the use of ImageJ software. Original blots are shown in Fig. S1-7.
Mitochondrial respiration
HepG2 cells in culture medium were seeded at a density of 1.2 × 105 per well in a Seahorse XFe24-well plate (Agilent Technologies, USA). After 24 h, the medium was changed to Seahorse XF DMEM containing 10 mmol/l glucose, 1 mmol/l pyruvate, and 2 mmol/l L-glutamine, and the cells were stimulated with metformin or imeglimin for 3 h. Mitochondrial respiratory chain activity was measured with a Seahorse XFe24 extracellular flux analyzer. Oxygen consumption was determined under the basal condition and after treatment with oligomycin (2 μmol/l), carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP, 1 μmol/l), and both rotenone (0.5 μmol/l) and antimycin A (0.5 μmol/l), and was normalized by protein concentration measured with a BCA protein assay kit (Thermo Fisher Scientific, USA).
RNA-seq and RT-qPCR analysis
Cells were incubated in 12-well plates and stimulated with 0.25 or 3 mmol/l metformin or imeglimin, with 1 mmol/l 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR), or with vehicle for 12 h. Total RNA was extracted from the cells with the use of an RNeasy Mini Kit (Qiagen, Germany) and was subjected to RNA-sequencing (RNA-seq) analysis by Macrogen Japan. An mRNA paired-end library was prepared from each RNA sample with the use of a TruSeq Stranded mRNA Library Preparation Kit (Illumina, USA), and each library was sequenced with an Illumina NovaSeq 6000 system. The number of reads was 4 × 107 per sample, and GRCh38 was used as the human reference sequence genome. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis of genes (https://www.kegg.jp/kegg/kegg1.html) was performed to identify relevant biological pathways with the use of the Database for Annotation, Visualization, and Integrated Discovery (DAVID) v6.8 online tool (https://david.ncifcrf.gov). Genes with expression values of Transcripts Per kilobase Million (TPM) > 10 and with a fold-change > |2| were used for the pathway analysis. P-value less than 0.05 are considered to be statistically significant. For RT and quantitative PCR (qPCR) analysis, isolated RNA was subjected to RT with the use of a high-capacity RT kit (Applied Biosystems, USA) and the resulting cDNA was subjected to qPCR with specific primers (Table S2) in a real-time PCR system (Applied Biosystems, USA). The abundance of target mRNAs was normalized by that of 36B4 mRNA.
Statistical analysis
Quantitative data are presented as means ± SEM and were analyzed with Student’s t test or by one-way ANOVA followed by Tukey’s test. A p value of <0.05 was considered statistically significant.