Phenylhydrazone

Subjects Informed consent was obtained from all subjects. The study was conducted in accordance with the principles of the Declaration of Helsinki, and was approved by the local ethics committee for Frederiksberg and Copenhagen County. Mitochondrial respiration was measured in permeabilised skeletal muscle fibres obtained from needle biopsies of the vastus lateralis in men with (n = 11) or without (control; n = 8) type 2 diabetes. The characteristics of the subjects are provided in Table 1 and Fig. 1. All subjects were in good health but classified as living a typical Westernised sedentary lifestyle, participating only in routine activities of daily living (walking, gardening, etc.) and not engaged in regular structured or individualised aerobic or strength training programmes or athletics. None of the control subjects had a family history of diabetes and none was receiving treatment for a disease. The diabetic patients were treated for their diabetes with diet or oral glucose-lowering medicine. All medications were withheld 24 h prior to the experiment. The patients with type 2 diabetes had no clinical signs of long-term diabetic complications and were representative of patients treated in the primary care sector.
Table 1

Characteristics of the subjects

	Type 2 diabetic subjects (n = 11)	Control subjects (n = 8)
Age (years)	62 ± 2	58 ± 1
Height (cm)	177 ± 3	179 ± 1
BMI (kg/m2)	32 ± 2*	28 ± 1
Time since diagnosis (years)	5 ± 2	–
Fasting insulin (pmol/l)	61 ± 9*	34 ± 6
Fasting glucose (mmol/l)	9.0 ± 0.5*	5.4 ± 0.1
Complex I activity (nmol min−1 mg protein−1)	50.8 ± 6.0	58.3 ± 4.7
Citrate synthase activity (pmol mg−1 s−1)	1.6 ± 0.1	2.0 ± 0.2
mtDNA (copies/μg tissue) ×103	119 ± 7*	147 ± 12
mtDNA/genomic DNA	2,773 ± 252	3,030 ± 185

Data are means±SEM. *p < 0.05 vs control subjects

Fig. 1

Glucose (a) and insulin (b) concentrations in venous plasma before (t = 0 min) and during an OGTT. The patients with type 2 diabetes had higher fasting glucose levels and were severely insulin resistant compared with healthy control subjects (*p < 0.05). Black and white symbols represent healthy control subjects and patients with type 2 diabetes, respectively

Subjects were fasted overnight prior to the experiment. A catheter was inserted into an antecubital vein for blood sampling. After local anaesthesia of the skin and the subcutis, a muscle biopsy was taken (Tru-Core; PBN-Medicals, Stenløse, Denmark) and then a 120-min OGTT (75 g glucose dissolved in 300 ml of water) was performed. At t = 30 min, a second muscle biopsy was taken.A portion of the obtained muscle tissue was frozen immediately in liquid nitrogen and stored at −80°C for later analysis (see below), and a smaller piece (2–6 mg) was placed onto a Petri dish on ice with 1 ml of relaxing solution containing Ca²⁺/EGTA buffer (10 mmol/l), free calcium (0.1 μmol/l), imidazole (20 mmol/l), K⁺/4-morpholinoethanesulfonic acid (MES) (50 mmol/l), dithiothreitol (DTT; 0.5 mmol/l), MgCl₂ (6.56 mmol/l), ATP (5.77 mmol/l), phosphocreatine (15 mmol/l), pH 7.1, and individual fibre bundles were separated with two pairs of sharp forceps, achieving a high degree of fibre separation. The fibre bundles were permeabilised for 30 min in 3 ml of ice-cold relaxing solution containing saponin (50 μg/ml) [10 (link)]. After rinsing in respiration medium (MiR05; Oroboros, Innsbruck, Austria) containing sucrose (110 mmol/l), potassium lactobionate (60 mmol/l), EGTA (0.5 mmol/l), MgCl₂.6H₂O (3 mmol/l), taurine (20 mmol/l), KH₂PO₄ (10 mmol/l), HEPES (20 mmol/l), sucrose (110 mmol/l), BSA (1 g/l), pH 7.1, the muscle bundles were blotted and measured for wet weight in a balance controlled for constant relative humidity, so that all biopsy samples were hydrated to the same degree. The muscle bundles were then immediately transferred into a respirometer (Oxygraph-2k; Oroboros) containing air-saturated respiration medium at 37°C.The Oxygraph-2k is a two-chamber titration-injection respirometer with a limit of oxygen flux detection of 1 pmol s⁻¹ ml⁻¹. The instrumentation allows for O₂ flux measurements with only 0.04 mg of mitochondrial protein or 1.5 mg of muscle fibres (wet weight). Standardised instrumental and chemical calibrations were performed to correct for back-diffusion of O₂ into the chamber from the various components, leak from the exterior, O₂ consumption by the chemical medium, and sensor O₂ consumption [11 (link)]. O₂ flux was resolved by software capable of converting nonlinear changes in the negative time derivative of the oxygen concentration signal.
Analysis of muscle tissue Citrate synthase activity and complex I activity were measured spectrophotometrically at 37°C. Citrate synthase activity was determined as described previously [12 (link)], and complex I activity was assessed by measuring the oxidation of NADH (300 μmol/l) using ubiquinone 1 (100 μmol/l) as the acceptor. The complex I rotenone-sensitive activity was measured by the addition of rotenone (1 μmol/l). The protein content, needed to calculate the specific activity, was measured using a commercially available assay (BCA, Sigma Chemicals, St Louis, MO, USA). For measurement of mitochondrial DNA (mtDNA) content, DNA was isolated from muscle biopsy samples (∼10 mg) by proteinase K digestion at 55°C for 3 days. The 100-μl digestion mix contained 50 mU proteinase K (PCR grade, Roche, Basel, Switzerland), 20 mmol/l Tris-HCl (pH 8.4) and 50 mmol/l KCl. After incubation at 80°C for 45 min, the remains were spun down and the supernatant fraction diluted ×200 in triethanolamine titanate (TE) plus 1 ng/μl salmon sperm DNA (Sigma). 5 μl of this dilution was amplified in a 25 μl PCR reaction containing 1×Quantitect SYBR Green Master Mix (Qiagen, Hilden, Germany) and 100 nmol/l of each primer. The amplification was monitored real-time using the MX3000P Real-time PCR machine (Stratagene, La Jolla, CA, USA). The primers were designed to target genomic DNA (Forward: AGG TGC TGT CAG GAA GCA AGG A, Reverse: TAG GGG GAG GAG GGA ACA AGG A) or mtDNA (Forward: CCC CTG CCA TAA CCC AAT ACC A, Reverse: CCA GCA GCT AGG ACT GGG AGA GA). The threshold cycle (C_t) values were related to a standard curve made with the cloned PCR products.
Respirometry protocol All measurements of respiration were made in duplicate, simultaneously. Resting, routine respiration (state 2, absence of adenylates) was assessed by the addition of malate (1.5 mmol/l) and glutamate (19 mmol/l) as the complex I substrate supply, and then state 3 respiration was assessed by the addition of ADP (4.8 mmol/l). The addition of succinate (9.5 mmol/l) provided state 3 respiration with parallel electron input to complexes I and II. The integrity of the outer mitochondrial membrane was established by the addition of cytochrome c (19 μmol/l); no stimulation of respiration was observed. We examined ADP control of coupled respiration and uncoupling control through addition of the protonophore carbonylcyanide-4-(trifluoromethoxy)-phenylhydrazone (FCCP) (0.7 μmol/l). The addition of rotenone (0.1 μmol/l) resulted in inhibition of complex I for examination of O₂ flux with complex II substrate alone, while antimycin A (12 μmol/l) was added to inhibit complex III to observe non-mitochondrial respiration with small contributions from electron leak in the uncoupled state. The concentrations of substrates and inhibitors used were based on prior experiments conducted for optimisation of the titration protocols.
Data analysis All values are given as means±SEM for all experiments, run in duplicate or triplicate. For all statistical evaluations, a p value of less than 0.05 was considered significant. Statistical analysis of differences in oxygen flux between healthy control subjects and patients with type 2 diabetes was carried out with a two-way ANOVA for repeated measures. In the case of a significant main effect and interaction between the variables, the Holm-Sidak method was used for post hoc analysis. All other comparisons between the two groups were performed using the unpaired Student’s t test. SigmaStat version 3.11 (Systat software, Richmond, CA, USA) was used in all analyses.