Sample preparation and enzyme activity assays

The mitochondrial enzyme activities for each individual were measured from the same whole homogenate sample. Eleven milligrams of lyophilized microdissected skeletal muscle were homogenized in 330 μL of buffer (1:30) containing (in mmol/L) 250 sucrose, 10 Tris•HCl pH 7.4, 1 EDTA, and protease inhibitors (Roche 11836170001). Several aliquots of the 1:30 homogenate were frozen in liquid nitrogen and stored at −80°C until assay for NADH dehydrogenase (NADH‐D), succinate dehydrogenase (SDH), coenzyme Q:cytochrome c – oxidoreductase (Complex III), COX, and ATP synthase activities. Thirty microliter of the 1:30 homogenate was diluted to 1:150 in homogenization buffer, frozen in liquid nitrogen and stored at −80°C until assay for citrate synthase, β‐hydroxyacyl CoA (β‐HAD), acyl CoA synthase (ACS), and total protein.

ACS activity was measured as described (Mashek et al. 2006) using 10 μL of the 1:150 homogenate in 190 μL of assay cocktail. The reaction was terminated after 10 min at 37°C by the addition of 1 mL Dole's reagent (isopropanol:heptane:1N H2SO4, 80:20:2. v/v). The labeled substrate for this assay was [1‐¹⁴C]palmitic acid. The labeled product ([¹⁴C]‐acyl‐CoA) was measured by scintillation counting following two 2 mL heptane washes.

Carnitine palmitoyltransferase (CPT‐1) activity was measured as described (Brown 2003) using 10 μL of the 1:30 fresh homogenate in 10 μL 5X substrate cocktail, 25 μL 2X assay cocktail and either 5 μL homogenization buffer or 5 μL 100 μmol/L malonyl‐CoA. The final concentration of palmitoyl‐CoA was 300 μmol/L. The reaction was terminated after 10 min at 30°C by the addition of 0.5 mL 1.2 N HCl. The labeled substrate for this assay was L‐[N‐methyl‐¹⁴C]carnitineHCl. The labeled product (palmitoyl‐[¹⁴C]carnitine) was measured by scintillation counting following the addition of 500 μL 1‐butanol.

β‐HAD and citrate synthase activities were measured by continuous spectrophotometric rate determination as described (Lynen and Wieland 1955; Srere 1969) using 10 μL of the 1:150 homogenate in a final volume of 1 mL.

NADH‐D, SDH, COX, and ATP synthase activity were measured in microplate assay kits (MitoSciences MS141, MS241, MS443, MS543, respectively) using 3–10.5 μL of the 1:150 homogenate per well.

Complex III activity was measured as described (Luo et al. 2008) using 5 μL of the 1:30 homogenate in a final volume of 200 μL that included (final concentration in mmol/L) 0.05 cytochrome c, 50 Tris‐HCl pH 7.4, 4 NaN3, 0.05 decylubiquinol, and 0.05% Tween‐20 in the absence and presence of antimycin A (10 μmol/L).

Catalase, glutathione peroxidase (GPx), and superoxide dismutase (SOD) enzyme activities for each individual were measured from the same supernatant. Five milligrams of lyophilized microdissected skeletal muscle was homogenized in 150 μL of buffer (1:30) containing (in mmol/L) 250 sucrose, 10 Tris•HCl pH 7.4, 1 EDTA, and protease inhibitors (Roche 11836170001). The homogenate was centrifuged at 10,000 × g for 15 min then several aliquots of the 1:30 supernatant were frozen in liquid nitrogen and stored at −80°C until assay. For catalase (abcam ab83464, fluorometric method) and GPx (abcam ab102530, colorimetric method), the 1:30 sample was diluted fourfold with ice‐cold assay buffer. Diluted supernatant of 4 μL was used in the catalase assay (sample and sample high control) and 10 μL in the GPx assay. For SOD (abcam ab65354, colorimetric method), 12 μL of the undiluted supernatant was used.

ATP and intramuscular triglyceride (IMTG): Five milligrams of lyophilized microdissected skeletal muscle was homogenized in 375 μL of cell lysis reagent (Roche). Whole homogenate of 25 μL was removed to determine ATP content as described (Wang et al. 2011). The sample for ATP was diluted 10‐fold in cell lysis reagent before undergoing six freeze–thaw cycles (liquid nitrogen to room temperature) followed by boiling for 15 min to inactivate ATPase activity. The sample was centrifuged at 17,900 × g for 5 min and the supernatant further diluted 10‐fold in cell lysis reagent. The ATP Bioluminescence Assay Kit HS II (Roche) and a SpectraMax L luminescence microplate reader (Molecular Devices) were used to determine ATP levels. Lipids were extracted (Bligh and Dyer 1959) from the remaining homogenate (350 μL) and solubilized in PBS containing 5% Triton X‐100. IMTG was determined using the EnzyChrom Triglyceride Assay Kit (BioAssay Systems).

Hydrogen peroxide (H₂O₂), glycogen synthase (GS), glucose 6‐phosphate (G6P), and glycogen: Two milligrams of lyophilized microdissected skeletal muscle was homogenized in 300 μL of buffer containing (in mmol/L) 50 tricine pH 7.4, 100 NaF, 10 EDTA, and protease inhibitors (Roche 11836170001). The homogenate was centrifuged at 13,000 × g for 5 min at 4°C. An aliquot of supernatant was removed for GS activity and total protein determination and the remaining supernatant was frozen and boiled for 5 min, and centrifugation repeated. This last supernatant was used for fluorometric determination of H₂O₂ using the Hydrogen Peroxide Assay Kit (abcam), G6P using the High Sensitivity Glucose‐6‐Phosphate Assay Kit (Sigma), and glycogen using the Glycogen Assay Kit (Sigma). GS activity was measured as previously described (Ryan et al. 2012).