Experimental animals
All animal procedures were approved by the University of British Columbia Animal Care Committee, following Canadian Council for Animal Care guidelines. The Ins1-null and Ins2-null alleles were generated by Jami and colleagues [26]. Mice had mixed genetic background (predominately C57BL/6 and 129 strains). Ins1
−/−:Ins2
+/+ and Ins1
−/−:Ins2
+/− female mice from each litter were weaned (at 3–4 weeks) and distributed (with matching body weights) between two diet assignments: a moderate-energy chow diet (CD) (25% fat content; LabDiet 5LJ5; PMI Nutrition International, St. Louis, MO, USA) or a high-energy HFD (58% fat content; Research Diets D12330; Research Diets, New Brunswick, NJ, USA) [11] provided ad libitum, except during fasting periods (Fig. 1a). Mice were housed under specific pathogen-free conditions at 21°C, on a 12 h light–dark cycle. We considered in our studies whether the dam or sire of individual mice had the disrupted Ins2 allele, to control for possible parental imprinting [27, 28], but as no obvious parental effects were observed, we combined ‘parental groups’. Experimenters were blind to mouse genotype, diet (where possible), and parental group while performing and analysing each experiment.
Glucose homeostasis and plasma analytes
Mice were fasted for 4 h during the light period to ensure a postprandial state for blood sampling. Fasting and glucose-stimulated (2 g/kg) insulin secretion was assessed, as well as blood glucose response to intraperitoneal injection of glucose (2 g/kg) or insulin analogue (0.75 U/kg of Humalog; Eli Lilly, Indianapolis, IN, USA). Plasma insulin was measured with a mouse insulin ELISA (Alpco Diagnostics, Salem, NH, USA) and C-peptide was measured in a subset of 27-week-old mice with a mouse C-peptide ELISA (Alpco Diagnostics). In plasma from 40-week-old mice, we measured total cholesterol (Cholesterol E kit; Wako Chemicals, Richmond, VA, USA), triacylglycerols (Serum Triacylglycerol kit; Sigma-Aldrich, St Louis, MO, USA) and NEFA levels (NEFA-HR(2) kit; Wako Chemicals), as well as leptin, resistin, interleukin 6, glucose-dependent insulinotropic polypeptide (GIP), peptide YY and glucagon, using a mouse magnetic bead panel assay utilising Luminex technology (Millipore, St Charles, MO, USA).
Body composition and whole body metabolism
A subset of pups was weighed prior to weaning. After weaning, all mice were weighed weekly. Body composition was assessed using dual-energy x-ray absorptiometry (DEXA) (Lunar PIXImus densitometer; GE Medical Systems LUNAR, Madison, WI, USA). A subset of 17-week-old mice on HFD was evaluated in PhenoMaster metabolic cages (TSE Systems, Chesterfield, MO, USA), as described elsewhere [29]. Before data collection, mice were individually housed for at least 1 week and were allowed to become acclimated to the PhenoMaster cage environment for at least 4 days. Data were averaged from 48–84 h of continual acquisition.
Murine INSULIN2 treatment
A cohort of mice were put on HFD at 15 weeks and subcutaneously implanted at 17 weeks with mini osmotic pumps (Alzet 2004; DURECT, Cupertino, CA, USA) designed to release murine INSULIN2 peptide (0.1 U/day; generously provided by Novo Nordisk, Bagsvaerd, Denmark) or vehicle for 28 days.
Tissue analyses
Tissues were dissected from mice 4 h after fasting. Mice were from the 50-week-old HFD-fed mini osmotic pump group and 25-week-old CD or HFD groups. Islets were isolated as described [30], with minor modifications, and cultured at 37°C and 5% CO2 for at least 16 h in RPMI-1640 medium (Invitrogen, Burlington, ON, Canada) supplemented with 11 mmol/l glucose, 100 U/ml penicillin, 100 μg/ml streptomycin and FBS (10% vol./vol.). Islet perifusions were performed as described [31] using 150 size-matched islets per group. Insulin content was measured by ELISA from ten islets per mouse that were lysed at −20°C using 0.02 mol/l HCl in 70% ethanol and sonicated for 30 s.
Other tissues were weighed and flash-frozen in liquid nitrogen before being stored at −80°C, except for half of the gonadal WAT depot, which was fixed in 4% paraformaldehyde, embedded in paraffin and cut into 5 μm-thick sections that were stained for perilipin (antibody 9349S; Cell Signaling Technology, Danvers, MA, USA) using an Alexa fluor 488-conjugated secondary antibody (Life Technologies, Burlington, ON, Canada) and DAPI. Images with identical exposure times were taken with a Zeiss 200 M inverted microscope (Carl Zeiss, Oberkochen, Germany) and lipid droplet areas were determined using CellProfiler 2.1.0 (http://www.cellprofiler.org/) [32].
For real-time PCR, total RNA was extracted from islets using the Qiagen RNeasy Mini kit (Qiagen, Mississauga, ON, Canada), and from adipose tissue using acid guanidinium thiocyanate–phenol–chloroform extraction with TRIzol (Invitrogen) followed by Qiagen RNeasy Mini columns [33]. cDNA was generated using a qScript cDNA synthesis kit (Quanta Biosciences, Gaithersburg, MD, USA). Reactions were performed on default parameters of a StepOnePlus real-time PCR system (Applied Biosystems, Foster City, CA, USA), using TaqMan Fast Universal PCR or Fast SYBR Green Master Mixes (Applied Biosystems). See electronic supplementary material [ESM] Table 1 for primer details. Values were normalised using the \( {2}^{-\Delta {\mathrm{C}}_{\mathrm{t}}} \) method.
Statistical analyses
Statistical analyses were performed with SPSS 15.0 software and a critical α-level of p ≤ 0.05. Two-way ANOVA models were used to assess factors of genotype and diet and a significant interaction led to one-way ANOVAs comparing HFD-fed Ins1
−/−:Ins2
+/+ mice, CD-fed Ins1
−/−:Ins2
+/+ mice, HFD-fed Ins1
−/−:Ins2
+/− mice and CD-fed Ins1
−/−:Ins2
+/− mice, with Bonferroni corrections applied. ANCOVA was used to test energy expenditure with covariates of lean and fat mass. One-way ANOVAs or two-tailed t tests were used to analyse tissue data from the 50-week time point.