HepG2 cells from the European Collection of Authenticated Cell Cultures (ECACC) were cultured according to standard methods and routinely tested for mycoplasma. For experiments, cells were seeded at a density of 5 × 104 cells/cm2, and incubated for 72 h with no change of medium. Cells were transduced with human small hairpin (sh)RNAs (Mission; Sigma, St Louis MO, USA) for MST3 (also known as STK24), MST4 (also known as STK26) or non-targeting (sequences available on request), and were selected with Puromycin (Sigma).
Transient transfections were performed with Lipofectamine 2000 (Life Technologies, Carlsbad, CA, USA). The plasmids used were pGL3-G6P C (− 3.9kb to − 21 kb) , pCMV5-βgal (Clontech, Mountain View CA) and pCMV5HA-MST3.
Treatments were done using insulin (100 nmol/l), LY294,002 (10 μmol/l), H-89 dihydrochloride hydrate (10 μmol/l), N6,2′-O-dibutyryladenosine 3′,5′-cyclic monophosphate (dbcAMP; 1 mmol/l), all from Sigma.
Mst3-deficient mice were generated from embryonic stem cells AM0826 from the International Gene Trap Consortium (IGTC) , and crossed with C57/BL6J mice to obtain a mixed 129sv/C57BL6J background (B6;129). For a pure C57/BL6J genetic background, these mice were backcrossed with C57/BL6J mice five more times. Genotyping was performed as described . Male littermates (6–8 weeks, 20–25 g) were used in all animal experiments. All animals were healthy, negative for pathogens and there were no significant differences in weight at the beginning of the experiments.
Animals were kept in a specific-pathogen-free environment. Where indicated, mice were fed with either chow food and water ad libitum (i.e. normal diet) or with a diet supplying 45% of energy from fat (HFD) (Research Diets, New Brunswick, NJ, USA) for 21 weeks. Unless otherwise indicated, there were nine mice per group fed a normal diet, nine mice per group of wild-type (WT) mice fed an HFD, and ten Mst3-deficient mice per group fed an HFD.
Glucose in animals was measured in whole blood using a Glucocard G+ meter glucometer from Menarini Diagnostics (Firenze, Italy). Liver triacylglycerol was extracted using a protocol adapted from Boyle et al , and measured using a triacylglycerol measurement kit (Spinreact, Girona, Spain). Insulin was measured in blood samples collected from mice after 4 h fasting using an ELISA kit (EZRMI-13K, Millipore, Billerica, MA, USA).
Metabolic phenotyping was performed using individual metabolism cages with ventilation and sensors to register different variables, using an infrared-based activity monitoring system for locomotor activity (PhenoMaster, Sophisticated Life Science Research Instrumentation, TSE Systems, Bad Homburg vor der Höhe, Germany) over 48 h, after an adaptation period of 48 h.
Fat composition was determined in triplicate using NMR (Whole Body Composition Analyzer; EchoMRI, Houston, TX, USA), corrected for body weight.
Mice were fasted for 14 h, glucose was measured, and animals were injected intraperitoneally with pyruvate (1.25 g/kg), glucose (2 g/kg) or insulin (0.75 U/kg). Glucose was then measured at the times indicated after the injection. For the GTT and pyruvate tolerance test (PTT), the AUC from time 0 to 120 min was calculated. To determine the glucose disappearance rate (KITT) in an insulin tolerance test (ITT), glucose concentrations over the first 30 min for each animal were transformed logarithmically, and a linear regression calculated for each mouse, with KITT as the minus slope of the regression. Methods for glucose, triacylglycerol and insulin measurements in mice, metabolic phenotyping and fat composition, are described in electronic supplementary material (ESM) Methods.
Catheter implantation was performed as previously described by Fisher and Kahn . Only mice that had regained at least 90% of their preoperative body weight after 6 days of recovery were analysed. After fasting for 16 h, awake animals were placed in restrainers for the duration of the experiment. Insulin (human regular insulin [Humulin, Lilly, Indianapolis, IN, USA]) and somatostatin (Sigma) solution containing 0.1% wt/vol. BSA (Sigma) were infused at a fixed rate (4 mU g−1 min−1 for insulin; 3 μg kg−1 min−1 for somatostatin) following a bolus infusion (40 mU/g). Blood glucose levels were determined every 10 min, and physiological blood glucose levels (between 5.6 and 6.8 mmol/l) were maintained by adjusting a 20% wt/vol. glucose infusion (DeltaSelect, Munich, Germany). Steady state was ascertained when glucose measurements were constant for at least 30 min at a fixed glucose infusion rate and was achieved within 150 to 180 min.
No adverse effects were detected during the experiments. All experiments and procedures were reviewed and approved by the Ethics Committee of the Universidade de Santiago de Compostela, in accordance with the EU directive for the use of experimental animals.
Glucose measurement in HepG2 cells
Cell medium from untreated cells was changed to Krebs–Henseleit–HEPES (KHH; composition in ESM Methods ‘Glucose measurement’) in the presence or absence of 100 nmol/l insulin for 8 h. Glucose was determined in the medium using the High Sensitivity Glucose Assay Kit (Sigma).
RNA was extracted in TRIzol Reagent (Fisher Scientific, Waltham, MA, USA). Quantitative PCR was performed using TaqMan Gene Expression Assays from Applied Biosystems (Foster City, CA, USA) for the mouse genes Gapdh, Actb, Mst3, Pck1, G6pc, Slc1a2, Aldh3a2, St3gal5, Acaca and Fasn, and the human genes GAPDH, ACTB, ACACA, FASN, CPT1A, PCK1 and G6PC. Results were normalised to Actb and Gapdh. Details of the probes used are shown in ESM Table 1.
For protein extraction, whole livers and cells were washed twice with ice-cold PBS and collected with a buffer modified from Fuller et al  (‘buffer b’ composition in ESM Methods ‘Protein determination’). For livers, TissueLyser beads (Qiagen, Hilden, Germany) were used. Levels of each protein were determined a minimum of three times from different cells or animals.
Western blots were performed using standard procedures. Immunodetection was either by chemiluminescence with Pierce ECL Western Blotting Substrate (Thermo Fisher, Waltham, MA, USA) or fluorescence (Odyssey; Li-Cor Biosciences, Lincoln, NE, USA). Western blots were performed for the following proteins and post-translational modifications: MST3, MST4, p-MST4 + MST3 + SOK1 (Thr178 + Thr190 + Thr174), insulin receptor (IR)β, p-IRβ (Tyr 1150/1151), IRS1, p-IRS1 (Tyr612), Akt serine/threonine kinase (Akt), p-Akt (Thr308), p-Akt (Ser473), forkhead box (FOX)O1, p-FOXO1 (Thr24), AMP-activated protein kinase (AMPK)α, p-AMPKα (Thr172), phosphorylated extracellular signal-regulated kinase 1/2 (p-ERK1/2) (Thr202/Tyr204), glycogen synthase kinase 3β (GSK-3β), p-GSK-3β (Ser9), cAMP responsive element binding protein 1 (CREB), p-CREB (Ser133), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), tubulin and phosphorylated N-MYC downstream regulated gene 1 (p-NDRG1). A list of the antibodies used can be found in ESM Table 2.
The Human phospho-Kinase Array Kit from R&D Systems (Minneapolis, MN, USA) was used according to the manufacturer instructions. The data were analysed using ImageJ software (https://imagej.nih.gov/ij/).
Immunofluorescence studies were performed using standard procedures in cells grown on poly-l-lysine (Sigma), and fixed with PFA 4% wt/vol. Images were obtained using Confocal Microscopy TCS SP5 (Leica, Wetzlar, Germany). Nuclear-to-cytoplasmic ratio was determined using the ImageJ software.
Statistical analysis was performed using GraphPad software (https://www.graphpad.com/), applying the Mann–Whitney test when two groups were compared and ANOVA analysis with a Bonferroni post hoc correction when comparing more than two groups. A value of p < 0.05 was considered significant. All graphs represent the mean ± SEM for a minimum of three independent experiments.