Test participants (hereafter referred to as post-diabetes mellitus participants) were obese and had a documented history of obesity-associated type 2 diabetes prior to weight loss. Inclusion criteria were: BMI ≥ 30 kg/m2, fasting blood glucose >7.0 mmol/l and HbA1c >7%, or treatment with hypoglycaemic agent(s) prior to weight loss. Age-, sex- and BMI-matched participants who had been similarly obese but normoglycaemic acted as non-diabetic controls. These non-diabetic participants had no family history of type 2 diabetes; however, two of the seven post-diabetes mellitus participants had at least one parent or sibling with type 2 diabetes. Physical activity was measured by questionnaire, which was adapted from a previous publication . There were no differences in physical activity between non-diabetic and post-diabetes mellitus groups (3.3 ± 3.8 h/week and 4.6 ± 3.2 h/week, respectively). Both populations underwent a 26-week standard clinical weight loss programme at the Ottawa Hospital Weight Management Clinic (details, see Electronic supplementary material [ESM] and previous publications [31, 32]). Following weight loss, post-diabetes mellitus participants exhibited consistently normal blood glucose and HbA1c levels (≤6.1 mmol/l and ≤5.7%, respectively). Some participants experienced a modest weight gain after completing the programme; however, all participants had had stable body weight for a minimum of 14 weeks prior to study (weight stability ranged from 14 to 102 weeks). Weight stability was defined as weight maintenance within 5% of body weight. Participant characteristics pre- and post-weight loss as well as at the time of study are described in Table 1.
All participants gave informed consent and these investigations were approved by the Human Research Ethics Committees of the Ottawa Hospital and the University of Ottawa Heart Institute.
Insulin infusate was prepared in isotonic saline, to which 2 ml of participant’s blood was added per 50 ml infusate to prevent absorption of insulin by glass/plastic. Regular insulin was diluted to 300 mU/ml in 0.9% saline (wt/vol.) and infused as 10 min priming infusion, followed by fixed infusion of 40 mU/m2. Glucose was infused as 20% dextrose (wt/vol) and was started 4 min after initiation of insulin infusion at a rate of 2.0 mg kg−1 min−1 for 4 to 10 min and at 2.5 mg kg−1 min−1 for 10 to 15 min. Blood samples were taken every 5 min. Glucose infusion was adjusted to maintain plasma glucose at 5.0 mmol/l .
Whole-body fat mass, fat free mass and regional fat distribution were assessed by dual-energy x-ray absorptiometry (DEXA) (LUNAR Prodigy; GE Medical Systems, Madison, WI, USA). The precision of repeated measurements is expressed as the per cent coefficient of variation (2.2% for fat mass percentage).
Biopsies of vastus lateralis were obtained from seven post-diabetes mellitus and seven non-diabetic participants using a 5 mm Bergstrom needle (Opitek, Glostrup, Denmark) as described previously . Participants had refrained from physical activity for 3 days and had fasted for 12 h prior to biopsy. A sample of muscle (~10 mg) was frozen in optimal cutting temperature compound for determination of fibre type ratio and IMTG. The remainder of muscle biopsy was processed for satellite cell isolation and culture.
Freshly biopsied vastus lateralis was minced, subjected to trypsin digestion and plated in Ham’s F-10 media supplemented with 15% fetal bovine serum (vol./vol.), 0.5 mg/ml BSA, 1 μmol/l dexamethazone, 10 ng/ml EGF, 0.5 mg/ml fetuin and 0.25 pmol/l insulin. Muscle satellite cells were isolated using an immuno-based magnetic sorting technique as previously described . Myoblasts were differentiated for 7 days prior to experimentation in LGI DMEM (5.5 mmol/l glucose, supplemented with 0.25 pmol/l insulin, 2% horse serum (vol./vol.), 1% antibiotic–antimycotic (vol./vol.) and 2.5 mg/ml gentamycin) or in HGI DMEM (25 mmol/l glucose, supplemented with 10 pmol/l insulin, 2% horse serum, 1% antibiotic–antimycotic and 2.5 mg/ml gentamycin).
Fibre type ratio
Frozen tissues were sectioned and mounted on Superfrost slides (Fisher, Ottawa, ON, Canada). Type 1 and type 2 fibres were immunostained sequentially using A4.840 and N2.261 (Developmental Studies Hybridoma Bank, Iowa City, IA, USA) primary antibodies and biotinylated goat anti-mouse (BA-9200; Vector Laboratories, Burlington, ON, Canada) secondary antibody. Type 1 fibres were stained red (SK5100; Vector Laboratories) and type 2 fibres were stained blue (SK5300; Vector Laboratories). The number of type 1 and type 2 fibres was counted in three fields of view in each of three sections per participant. The fibre type ratio was determined from these numbers.
IMTG content was evaluated ex vivo and in vitro. Ex vivo determinations were conducted as described previously . Frozen tissues were sectioned, mounted on Superfrost slides and fixed in 10% neutral buffered formalin (vol./vol.). Sections were stained with 1% osmium tetroxide (wt/vol.) and counter-stained with haematoxylin and eosin. Three fields of view in each of three sections per participant were quantified using Northern Eclipse imaging software (Empix Imaging, Mississauga, ON, Canada) to determine the amount of IMTG relative to the cross-sectional fibre area.
For in vitro analyses, differentiated myotubes were suspended in 25 mmol/l Tris-HCl pH 7.5 with 1 mmol/l EDTA. Lipids were extracted via chloroform–methanol (2:1), evaporated under N2 gas and dissolved in 2-propanol. Triacylglycerol concentration was determined spectrophotometrically (L-Type TG H; Wako, Richmond, VA, USA) and normalised to protein content as determined by bicinchoninic acid assay.
Differentiated myotubes were suspended in PBM buffer (20 mmol/l KH2PO4, 10 μmol/l CaCl2, 1 mmol/l MgCl2, pH 6.1) and lysed by freeze–thaw. Samples were boiled for 20 min in 30% KOH (wt/vol.) saturated with anhydrous Na2SO4. Glycogen was precipitated with 95% ethanol (vol/vol.), dissolved in double-distilled H2O and incubated at 100°C for 20 min with 0.2% anthrone (wt/vol.) in H2SO4. Glycogen concentration was determined spectrophotometrically by measuring absorbance at 620 nm relative to an oyster glycogen standard curve, and normalised to protein content.
Mitochondrial enzyme activities
Intact mitochondria were isolated from differentiated myotubes using a mitochondrial isolation kit (MITOISO2; Sigma, St Louis, MO, USA). Mitochondrial yield was expressed as mitochondrial protein content per cellular protein content. Cytochrome c oxidase activity was assayed in intact mitochondria using a kit (Cytocox1; Sigma). Citrate synthase activity was determined in lysed mitochondria using a kit (CS0720; Sigma). Enzyme activities were normalised to mitochondrial and cellular protein contents.
Mitochondrial membrane potential
Myoblasts were seeded at 20,000 cells/well in 96-well plates and differentiated in LGI/HGI media for 7 days. Cells were incubated with 800 nmol/l tetramethylrhodamine, ethyl ester (TMRE) perchlorate in either LGI-PBS (5.5 mmol/l glucose, 0.25 pmol/l insulin) or HGI-PBS (25 mmol/l glucose, 10 pmol/l insulin). Incubation was for 15 min at 37°C in the dark. Control wells contained no cells. Fluorescence was measured 15 min post-incubation (excitation 544 nm, emission 590 nm). TMRE is a lypophilic cationic dye that accumulates in the mitochondria proportional to membrane potential and through mitochondrial accumulation quenches TMRE fluorescence . Membrane potential was expressed as the absolute value of the decrease in TMRE fluorescence and normalised to mitochondrial protein content.
Electrophoresis of cell lysates or isolated mitochondria was carried out on a 10% polyacrylamide gel (wt/vol.). Proteins were then transferred to a nitrocellulose membrane. Primary antibodies were: uncoupling protein-3 (UCP3) (ab-3477; Abcam, Cambridge, MA, USA), β-actin (4967; Cell Signaling, Danvers, MA, USA), manganese superoxide dismutase (MnSOD) and succinate dehydrogenase (SDH) (sc-30080 and sc-59687, respectively; Santa Cruz Biotechnology, Santa Cruz, CA, USA). Secondary antibodies were: goat anti-rabbit horseradish peroxidase and goat anti-mouse horseradish peroxidase (sc-2030 and sc-2031, respectively; Santa Cruz). Visualisation was achieved using an enhanced chemiluminescence kit (Amersham Pharmacia, Baie d’Urfe, QC, Canada). Spot densitometry was performed using an Alpha multi-image light camera and Alpha imaging software (Alpha Imaging, Willoughby, OH, USA). Values represent the integrated density value (IDV) of UCP3 or MnSOD divided by the IDV of β-actin or SDH.
HNE–histidine adduct content was determined by ELISA (STA-334; Cell Biolabs, San Diego, CA, USA). Protein samples were absorbed on to a 96-well ELISA plate in triplicate for 2 h at 37°C. Wells were probed with an anti-HNE–histidine antibody and horseradish peroxidase-conjugated secondary antibody. Absolute HNE–histidine adduct content was determined spectrophotometrically by comparing samples with a standard curve of HNE–BSA standards at 600 nm.
A Student’s t test was used to assess statistical differences between clinical variables as well as differences in fibre type percentage, MnSOD expression and UCP3 expression. A one-way ANOVA with Tukey’s post-test was used to assess statistical differences in IMTG content, glycogen content, mitochondrial yield, citrate synthase activity, cytochrome c oxidase activity, HNE–histidine adducts and mitochondrial membrane potential (Figs 1, 2, 3 and 4). Confidence intervals were set at 95%.