Mitochondrial dysfunction results from oxidative stress in skeletal muscle of diet-induced insulin resistant mice

Measurement of enzyme activities

CS activity was measured spectrophotometrically (36) in purified mitochondria from gastrocnemius muscle. For measurement of caspase 3 activity, gastrocnemius muscle was lysed in ice-cold lysis buffer (1% NP40, 20mM Tris-HCl, 138mM NaCl, 2.7mM KCl, 1mM MgCl2, 5% glycerol, pH=8.0, supplemented with 5mM EDTA, 1mM Na₃VO₄, 20mM NaF, 1mM DTT and protease inhibitors). After centrifugation for 10 minutes at 4°C, aliquots of supernatant (100µg) were incubated with 10µl DEVD-pNA for 1 hour at 37°C, according to the instructions of the manufacturer (Clinisciences). The pNA light emission was quantified using a spectrophotometer at 400 nm.

Protein carbonylation

The Oxyblot Oxidized Protein Detection Kit was purchased from Chemicon. The carbonyl groups in the protein side chains are derivatized to DNP-hydrazone by reaction with DNPH, following the manufacturer’s instructions. After the derivatization of the protein sample, one dimensional electrophoresis was carried out on a 10% SDS-PAGE gel. Proteins were transferred to PVDF-membranes. After incubation with anti-DNP antibody, the blot was developed using a chemiluminescence detection system.