Murine breeding and housing conditions
Mice were maintained in a temperature-controlled facility with 12 h light–dark cycle and ad libitum access to water and a standard chow diet (S8022-S005, ssniff diets, Soest, Germany) or a diet with 45% of energy derived from lard (HFD, S9669-E006, ssniff diets, Soest, Germany) with or without 0.1% fenofibrate. All experiments and blood collections were undertaken in the dark phase. As described previously24,25, peripheral tissue inducible IR knockout mice were generated by crossing IRlox/+ (B6.129S4(FVB)-Insrtm1Khn/J) and CreER (B6.129-Gt(ROSA)26Sortm1(cre/ERT2)Tyj/J) mice and backcrossing on C57Bl/6 N background resulting in IRlox/+;CreER/+, which are referred to as PerIRKO+/−, where Per stands for peripheral. IRlox/+;+/+ littermates were used as control (WT) mice. Cre expression was induced at 10 or 18 weeks of age by administration of 2 mg tamoxifen (Cayman Chemicals, Michigan, USA) or carrier via oral gavage for 5 consecutive days. WT and PerIRKO+/− received the same dose of tamoxifen, and this resulted in the partial ablation of the IR in peripheral, but not central tissues of PerIRKO+/− within 10 days (Supplementary Fig. 1 and24). All experiments were approved by the Canton of Zurich Veterinary Office, Switzerland.
Unless stated otherwise, all other reagents were purchased from Sigma-Aldrich Chemicals (St. Louis, MO, USA). Blood glucose was determined using a handheld glucose meter (Bayer Contour XT Meter), plasma insulin by immunoassay (Meso Scale Discovery, Gaithersburg, MD), leptin by ELISA (Crystal Chem Inc., Chicago, Illinois, USA), and plasma FFA, TGs, ALAT, and ASAT by enzymatic reaction (Cobas Mira; Hoffmann-La Roche, Basel, Switzerland). Citrate synthase activity was measured in supernatant by examining the increase of 5,5-dithiobis-2-nitrobenzoate at a wavelength of 412 nm43. Liver TG were extracted from liver samples as previously described7 and quantified using a commercial enzymatic colorimetric assay (Roche Diagnostics), and glycogen was determined by the acid-hydrolysis method44. Hematoxylin–Eosin and Sirius Red staining was performed on paraffin sections from livers fixed in phosphate-buffered 4% formaldehyde. Images were acquired with ×10 objective on Axio Scope.A1 microscope (Oberkochen, Germany) equipped with an AxioCam MRc digital camera (Oberkochen, Germany). Hepatic ATP and AMP levels were determined by reverse phase high performance liquid chromatography (HPLC) as described before45. In brief, tissue samples were removed and snap-frozen with liquid nitrogen. Samples were homogenized in pre-chilled acetonitrile buffer to precipitate protein. Following the removal of protein for later quantification by chloroform-extraction, the metabolite-containing fraction was subjected to HPLC separation and detection. Metabolites were identified by spiking of samples with appropriate standards. Metabolite content was normalized to total protein content.
Immunoblotting was performed as described previously7. In brief, tissues were homogenized using an electrical handheld homogenizer in 10–20 volumes of ice cold RIPA lysis buffer (50 mm HEPES (pH 7.4), 1% (vol/vol) Triton X-100, 1% (vol/vol) sodium deoxycholate, 0.1% (vol/vol) SDS, 150 mm NaCl, 10% (vol/vol) glycerol, 1.5 mm MgCl2, 1 mm EGTA, 50 mm sodium fluoride, protein inhibitor cocktail (Roche, Basel, Switzerland), 1 mm phenylmethysulfonyl fluoride, 1 mm sodium vanadate), incubated for 20 min on ice and centrifuged at 20,000 × g for 20 min at 4 °C. The supernatants were resolved by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and processed for immunoblotting by standard procedures. Antibody details are provided in supplementary table 1, and uncropped western blots can be found in the Source Data File.
Metabolic and body composition measures
Insulin (ITT), glucose (GTT), and pyruvate (PTT) tolerance tests were performed in two (ITT) or 5 h (GTT and PTT) fasted mice by intraperitoneally injecting a bolus of insulin (0.6 mU/g; ITT), d-glucose (2 mg/g; GTT) or sodium pyruvate (1 mg/g; PTT) and tail blood glucose was measured at the time points indicated as described previously7. Meal challenge experiments involved fasting mice overnight (16 h, largely during light cycle) then allowing ad libitum access to food for 4 h before refasting and monitoring blood glucose for the following 6 h. PhenoMaster (TSE systems, Bad Homburg, Germany) open-circuit calorimetry system was used to measure oxygen consumption and ambulatory activity over 48 h (two light–dark cycles) following a 24–48 h acclimation period and body composition by nuclear magnetic resonance (Echo MRI-100 Body Composition Analyzer, Echo Medical Systems, Huston, USA).
Glucose clamp studies
Glucose turnover rate was assessed in freely moving mice after 10 weeks of HFD during an euglycemic–hyperinsulinemic clamp as previously described46. In brief, mice were anesthetized with isoflurane, and a catheter (MRE 025, Braintree Scientific) was inserted into the right jugular vein and exteriorized at the back of the neck. After 7 days of recovery, only mice that had regained >95% of their preoperative weight were studied. After a fasting period of 5 h, 3-[3 H]glucose (0.1 μCi/min; PerkinElmer) was infused for 80 mins, and blood was collected from tail tip for basal turnover calculation. After basal sampling, insulin (18 mU/kg/min) was infused for 2 h. Euglycemia was maintained by periodically adjusting a variable infusion of 20% glucose with a syringe pump (Harvard Apparatus, Holliston, MA, USA). The glucose infusion rate was calculated as the mean of the steady-state infusion (60–90 mins) after 1 h of insulin infusion. A blood sample was collected from tail tip after steady-state infusion. The glucose turnover rate was calculated by dividing the rate of 3-[3 H]glucose infusion by the plasma 3-[3 H]glucose-specific activity. Hepatic glucose production was calculated by subtracting the glucose infusion rate from the glucose turnover rate.
Real-time polymerase chain reaction
RNA was extracted using Trizol reagent (Invitrogen, Carlsbad, CA), and mRNA was reverse transcribed using the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA). Quantitative real-time PCR was performed on a ViiA 7 Real-Time PCR System (Applied Biosystems, Foster City, CA) using the SYBR green select master mix (Applied Biosystems, Foster City, CA) and relative quantification achieved using the ΔΔCt method with 18 S ribosomal RNA as an internal control. Primer sequences used are listed in Supplementary Table 2.
All data were presented as mean ± SEM. Statistical significance was using unpaired two-tailed Student’s t test and two-way ANOVA with fisher’s least significant difference post hoc analysis as indicated. The level of significance was set at p < 0.05 (SPSS version 20, IBM, Armonk, NY, USA).
Further information on research design is available in the Nature Research Reporting Summary linked to this article.