Mouse breeding and genotyping strategies for mice with a floxed allele of Irs2 and for albumin Cre recombinase transgenic mice
Mice with a floxed allele of Irs2 (Irs2lox mice)  were intercrossed with albumin Cre recombinase transgenic mice (AlbCre mice) obtained from The Jackson Laboratory (Bar Harbor, ME, USA)  to generate compound heterozygote mice. Double heterozygote mice were crossed with Irs2lox
+/− mice to obtain wild-type, Irs2lox
+/+, AlbCre and AlbCreIrs2lox
+/+ mice. Mice lacking Irs2 in AlbCre-expressing cells were designated LivIrs2KO mice. Mice were maintained on a 12-h light/dark cycle with free access to water and standard mouse chow (4% fat, RM1; Special Diet Services, Witham, Essex, UK) and housed in specific-pathogen-free barrier facilities. For high-fat diet studies, mice were fed with a diet containing 45% fat, 20% protein and 35% carbohydrate (Special Diet Services) for 3 months. Mice were handled and all in vivo studies performed in accordance with the 1986 Home Office Animal Procedures Act (Home Office, London, UK). LivIrs2KO mice were studied on a mixed 129Sv/C57Bl/6 background with appropriate litter-mate controls. Wild-type, Cre transgenic and Irs2lox
+/+ mice were phenotypically indistinguishable and balanced numbers of mice of these genotypes were used as controls. Genotyping of the mice was performed by PCR amplification of tail DNA as described previously .
Blood samples were collected from mice via tail vein bleeds or from cardiac puncture on terminally anaesthetised mice. Blood glucose, plasma insulin levels and leptin levels were determined as previously described . Adiponectin was measured using a mouse adiponectin ELISA (R & D Systems, Minneapolis, MN, USA). NEFAs were measured using a NEFA kit (Roche Diagnostics, Lewes, UK). Serum and tissue triglyceride levels from fasted and fed animals were assayed using the GPO Trinder kit (Sigma-Aldrich, Dorset, UK). Albumin and liver function tests were measured using a Dimension RXL multi-channel analyzer (Dade-Behring, Milton Keynes, UK). Glucose and insulin tolerance tests were performed as previously described . Body fat mass was determined by dual emission X-ray absorptiometry using a PIXImus densitometer (GE Healthcare, Chalfont St Giles, UK).
RNA isolation and real-time quantitative RT-PCR
RT-PCR was performed as previously described  using FAM/TAMRA-labelled fluorescent probes. RT primers listed in 5’ to 3’ orientation were: for glucokinase (Gck), forward: GGTGCTTTTGAGACCCGTTTT, reverse: GAGTGCTCAGGATGTTAAGGATCTG, probe: TGTCGCAGGTGGAGAGCGACTCTG; and for glucose-6-phosphatase (G6pc) forward: ACTCTTGCTATCTTTCGAGGAAAGA, reverse: CCAACCACAAGATGACGTTCA, probe: AAAGCCAACGTATGGATTCCGGTGT. The relative amount of mRNA was calculated from an internal standard curve following normalisation to 18 S ribosomal RNA levels.
Hyperinsulinaemic clamp studies, whole-body glucose turnover rate, and tissue glycogen content
Hyperinsulinaemic clamp studies were performed as previously described . In brief, under general anaesthesia, an indwelling catheter was placed into the left femoral vein, subcutaneously tunnelled and externalised in the interscapular region. The animals were allowed to recover for 5 days and fasted for 6 h on the day of the experiment. For 3 h d-[3-3H]-glucose was infused at a rate of 11 mBq kg−1 min−1 and insulin at a rate of 18 mU kg−1 min−1. Euglycaemia was maintained by a variable infusion of 15% glucose. Whole blood was sampled from the tail every 10 min during the last hour for 3H-Glucose enrichment, which was biochemically determined as follows. Samples were de-proteinised by precipitation with equal volumes (125 μl) of 0.1 mol/l Ba(OH)2 and 0.1 mol/l ZnSO4, followed by centrifugation for 5 min. Total radioactivity in the supernatant (from d-[3-3H]-glucose and glycolysis-derived 3H2O) was measured by scintillation counting. A corresponding aliquot was evaporated to dryness to estimate d-[3-3H]-glucose alone. Total glucose concentration was determined in a third aliquot by the glucose oxidase method (BioMerieux, Marcy l’Etoile, France). Glycogen content was determined in liver and quadriceps muscle using the amyloglucosidase method. In brief, tissues underwent alkaline hydrolysis at 55°C for 1 h (30 mg of liver in 200 μl 1 mol/l NaOH, 20 mg of muscle in 50 μl 1 mol/l NaOH). Equivalent volumes of 1 mol/l HCl were added for neutralisation and the samples spun at 340 g for 2 min. Aliquots of the glycogen-containing supernatant were incubated at 37°C for 1 h with α-amyloglucosidase (Roche Diagnostics) at a concentration of 0.01 mg/ml in 0.2 mol/l Na citrate buffer pH 4.8. The released glucose was determined by the glucose oxidase method (BioMerieux). Baseline hepatic glucose content was measured in an undigested aliquot and subtracted from the value obtained after α-amyloglucosidase incubation. Calculations of glucose turnover were made from parameters obtained during the last 60 min of the infusions under steady-state conditions as described previously .
Preparation of tissue extracts and immunoblot assays
Following an overnight fast, a bolus of insulin (5 IU) or vehicle was injected via the inferior vena cava of terminally anaesthetised mice. Tissues were removed and snap-frozen in liquid nitrogen and stored at −80°C until use. Tissues were homogenised in lysis buffer , solubilised for 30 min on ice and clarified by centrifugation. Supernatants were snap-frozen in aliquots and stored at −80°C. For Western blotting, 50 μg of total protein extract was immunoblotted with the indicated antibodies. For analysis of IRS protein levels and tyrosine phosphorylation or INSR β tyrosine phosphorylation, tissue extracts (2 mg of total protein) were immunoprecipitated for 2 h with the indicated antibodies. Immune complexes were collected with 100 μl of 50% slurry of protein-A or protein-G sepharose, washed with lysis buffer and resolved on 7.5% SDS-PAGE and transferred to nitrocellulose. The blots were probed with polyclonal antibodies against IRS2 and IRS1 or monoclonal antibody to antiphosphotyrosine and enhanced chemiluminescence (Amersham, Little Chalfont, UK) detection performed. Nuclear preparations were performed by homogenisation of liver samples in nuclear buffer  followed by centrifugation over a cushion of nuclear buffer with 1.2 mol/l sucrose and 5% glycerol. Nuclear pellets were re-suspended in nuclear buffer with 0.3 mol/l sucrose and 10% glycerol, purity analysed under a haematocytometer and aliquots frozen. Nuclear preparations were subsequently lysed in lysis buffer, solubilised for 5 min on ice, clarified by centrifugation, after which 50 μg of nuclear extract was immunoblotted with the indicated antibodies. Blots were stripped and re-probed with an antibody against zinc finger and BTB domain containing protein 16 (ZBTB16) as a nuclear loading control.
Sheep polyclonal antibodies to IRS1 and IRS2 were used as previously described . Rabbit anti-IRS1, anti-IRS2, anti-phosphotyrosine (PY) (4G10) and anti-p85 antibodies were from Upstate Biotechnology (Dundee, UK). Anti-AKT, anti-pAKTSer473, anti-pp70S6KThr389, anti-glycogen synthase kinase (GSK)-3β, anti-pGSK3α/βSer9/21, anti-mitogen-activated protein kinase (MAPK), anti-pMAPKThr202/Tyr204, anti-forkhead box O1 (FOXO1) antibodies were from Cell Signalling Technology (Beverly, MA, USA) and anti-forkhead box A2 (FOXA2) antibody was a gift from R. I. Altaba (University of Geneva Medical School, Geneva, Switzerland). Anti-INSR β, and anti-p70S6K antibodies were from Santa Cruz (Insight Biotechnology, Wembley, UK). Mouse anti-ZBTB16 was from Calbiochem-Novachem (San Diego, CA, USA). HRP-conjugated goat anti-rabbit antibody was from DAKO (Ely, UK) and HRP-conjugated sheep anti-mouse antibody was from Amersham Bioscience.