All animals received care according to the Canadian Council on Animal Care and all procedures were approved by the University of Alberta Health Sciences Animal Welfare Committee. All animal husbandry was performed by University of Alberta Health Sciences Laboratory Animal Services staff. Six-week-old HSA-Cre male mice [Jackson Laboratory, Bar Harbor, ME, USA; stock no. 025750; Tg(ACTA1-cre/Esr1*)2Kesr] and their wild-type (WT) littermates (Jackson Laboratory, stock no. 000664, C57BL/6J) received daily i.p. injections with either vehicle (corn oil) or tamoxifen (50 mg/kg) for 5 days. All mice were allowed a 1 week washout post-tamoxifen, following which 8-week-old mice were placed on either a low-fat diet (LFD) (10% kJ from lard; Research Diets, New Brunswick, NJ, USA, D12450J) or a high-fat diet (HFD) (60% kJ from lard; Research Diets D12492) for 12 weeks. Animals were euthanised at 20 weeks of age via i.p. injection of sodium pentobarbital (12 mg) following a 2 h fast. No randomisation was carried out and experimenters were not blinded to group assignment and outcome assessment.
C2C12 skeletal muscle myoblasts (ATCC, Rockville, MD, USA) were cultured and differentiated into myotubes as previously described . Following 4 days of differentiation, C2C12 myotubes underwent Lipofectamine-mediated transfection with either a control or Cre recombinase-expressing plasmid (Addgene, Cambridge, MA, USA, plasmid no. 13775) for 24 h. Media was replaced with serum-free media for 24 h and collected. INS-1 832/3 cells (referred to, herein, as INS-1) were cultured and used for the assessment of glucose-stimulated insulin secretion (GSIS) as previously described . As a positive control for stimulating GSIS, separate groups of INS-1 cells were treated with D-Ala2 glucose-dependent insulinotropic polypeptide (D-Ala2 GIP).
Assessment of glucose homeostasis
I.p. glucose (2 g/kg body weight) and insulin (0.7 U/kg body weight) tolerance tests were performed in 16- and 17-week-old overnight-fasted WT and HSA-Cre mice, respectively. Blood glucose measurements were performed via tail bleed with the Accu-Chek Advantage system (Roche, Indianapolis, IN, USA) in blood samples obtained at the end of the fast (0 min), followed by blood samples at 15, 30, 60, 90 and 120 min following glucose/insulin administration. Plasma was extracted from whole blood collected from mouse tails for measuring insulin levels as previously described .
Whole-body in vivo metabolic assessment
Indirect calorimetry was performed in 18-week-old WT or HSA-Cre mice using Oxymax metabolic cages (Columbus Instruments, Columbus, OH, USA). Animals were initially acclimatised in the system for 24 h, following which the subsequent 24 h was used for data collection (e.g. oxygen consumption) as previously described .
Determination of muscle triacylglycerol (TAG) levels
Frozen powdered gastrocnemius muscle tissue was extracted in a 2:1 chloroform:methanol solution, following which the supernatant phase was retained for the assessment of TAG content with an enzymatic assay kit (Wako Pure Chemical Industries, Osaka, Japan) as previously described .
Protein samples were prepared from powdered frozen gastrocnemius tissue (20 mg) homogenised in protein lysis buffer, following which they were subjected to western blotting protocols as previously described . Akt and phospho (p)-Akt (9272S and 4060L); glycogen synthase kinase (GSK)3β and p-GSK3β (5676S and 9331L); mammalian target of rapamycin (mTOR) and p-mTOR (2983S and 5536); and Cre recombinase (15036) antibodies were purchased from Cell Signaling (Danvers, MA, USA) and prepared in a 1:500 dilution in 5% bovine serum albumin (BSA).
Real-time PCR analysis
First-strand cDNA was synthesised from total RNA using the SuperScript III synthesis system (Invitrogen, Carlsbad, CA, USA). Real-time PCR was subsequently carried out using TaqMan gene expression assays (Applied Biosystems, Foster City, CA, USA) for quantification of relative mRNA transcript levels (see electronic supplementary material [ESM] Table 1 for primer list) as previously described .
Pancreases were fixed in 4% formalin and embedded in paraffin, sliced (5 μm) and mounted on slides. Citrate buffer was used for antigen retrieval, and immunohistochemistry was performed using either guinea pig anti-insulin antibody (Abcam, Cambridge, MA, USA) at a 1:250 dilution or rabbit anti-glucagon antibody (Millipore, Billerica, MA, USA) at a 1:5000 dilution in 5% BSA. Antibodies were visualised using the 3,3′-diaminobenzidine colorimetric system and images were captured using a Leica DMRE microscope (Leica, Wetzlar, Germany) as previously described .
Nuclear and cytoplasmic protein fractions from gastrocnemius tissue were extracted using the Nuclear Extract Kit (Active Motif, Carlsbad, CA) according to the manufacturer’s protocol as previously described . For western blotting analyses, voltage-dependent anion-selective channel (VDAC)1 and histone H3 were used as cytoplasmic and nuclear markers, respectively.
Equal amounts of protein lysates were incubated with anti-Cre antibody and a non-specific IgG antibody as a negative control overnight at 4°C. To pull down the antigen–antibody complex, 40 μl protein G Dynabeads (Thermo Fisher Scientific, Waltham, MA, USA) were added to all samples and incubated at 4°C for 2 h. The beads were washed three times with ice-cold PBS, and bound proteins were eluted by boiling at 95°C for 5 min with 20 μl of SDS protein loading buffer, following which they were subsequently processed for western blotting as previously described .
No samples were excluded from our data analyses. All values are presented as means ± SEM. Significant differences were determined using an unpaired, two-tailed Student’s t test or one- or two-way ANOVA followed by Bonferroni post hoc analysis.