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

Measurement of metabolites and hormones

Blood glucose levels were measured using a glucometer (Roche Diagnostics). Serum levels of insulin (Linco Research) and leptin (BioVendor) were determined using the murine ELISA kits. Total serum triglycerides (Biomérieux) and FFA (Roche Diagnostics) were assayed by using enzymatic methods. Plasma H₂O₂ levels were measured using an Amplex Red hydrogen peroxide assay kit (Invitrogen).

Transmission electronic microscopy

Gastrocnemius muscle was cut into small pieces and fixed in 2% glutaraldehyde for 2h at 4°C, postfixed in 1% Osmium tetroxide for 1h at 4°C, dehydrated and embedded in Epon at eitheir a longitudinal or transverse orientation. The tissue was then cut using a RMC/MTX ultramicrotome (Elexience) and ultrathin sections (60-80nm) were mounted on copper grids, contrasted with 8% uranyl acetate and lead citrate, and observed with a Jeol 1200 EX transmission electron microscope (Jeol LTD) equipped with MegaView II high resolution TEM camera. Analysis was performed with Soft Imaging System (Eloïse SARL). Selection of oxidative fibers was based on the size of fibers and the amount of mitochondria.