Willis, Wayne T.

Whether obesity influences content and functional capacity of skeletal muscle mitochondria (MITO) remains unresolved. In this study, biopsies of vastus lateralis from lean and obese human subjects were used to isolate subsarcolemmal (SS) and intermyofibrillar (IMF) MITO. IMF MITO were liberated using a protease. The SS and IMF were pelleted separately, using differential centrifugation. Skeletal muscle from obese subjects gave higher MITO protein yield, per g wet muscle vs. skeletal muscle from lean subjects in the SS but not in the IMF. There was a trend for higher isolated yields of citrate synthase (CS) activity, a marker for MITO content, in the muscle of obese subjects vs. lean subjects in SS, but not in IMF MITO. O 2 consumption ( J O ) and ATP production rates measured with NAD‐linked substrates (malate+pyruvate+glutamate), were not different in obese vs. lean in either SS or IMF MITO expressed either per mg MITO protein or per unit CS activity. The similarities in MITO function observed in SS and IMF from both lean and obese human muscle, suggests comparable functional capacity per unit MITO, regardless of the location within the myocyte, or whether the MITO reside in skeletal muscle from lean or obese subjects. However, higher total yield of both protein and CS activities in the SS MITO from the muscle of obese subjects, point to the possibility for greater content of SS MITO in muscle of obese individuals. This conclusion must be qualified; these results may be due to a greater fractional yield of MITO during the isolation procedure of skeletal muscle from obese subjects.

Nagarse (N) is traditionally used to isolate mitochondria (mito) from skeletal muscle (SM). We investigated the effects of N on functional parameters of mechanically liberated mito. Mito from mouse (n = 7) gastrocnemius muscle (119‐175 mg tissue) was isolated using a glass on glass homogenizer followed by differential centrifugation. The 800g supernatant from the first centrifugation, containing mito, was divided into two equal volumes. One was exposed to N and the other was not. In each volume, mito were isolated by standard differential centrifugation. State 3 max ADP‐stimulated O 2 consumption rates (J o ) were measured using polarography with malate + pyruvate + glutamate. N increased state 3 J o , 16.1 ± 1.1 vs. 11.9 ± 1.4 nmol O 2 / ml mito suspension / min; P < 0.004, while it decreased protein concentration in the final mito suspension (typically used to adjust final J o values), 1.9 ± 0.1 vs. 2.5 ± 0.1 mg / ml; P < 0.004. Citrate synthase activity in the final mito suspension, indicative of the available mito, was not different in the preparation treated with N vs. that not treated with N (0.067 ± 0.01 vs. 0.065 ± 0.01μmol / min; P = 0.8). These findings suggest that N treatment: 1) hydrolyzes non‐mito contaminating proteins in the final mito pellet, and 2) induces apparent qualitative changes in mito function. Therefore, calculated functional parameters of mito treated with N are not directly comparable to mito not treated with N. Grant Funding Source : Supported by NIH/NIDDK RO1 DK094062

OBJECTIVE The contribution of mitochondrial dysfunction to skeletal muscle insulin resistance remains elusive. Comparative proteomics are being applied to generate new hypotheses in human biology and were applied here to isolated mitochondria to identify novel changes in mitochondrial protein abundance present in insulin-resistant muscle. RESEARCH DESIGN AND METHODS Mitochondria were isolated from vastus lateralis muscle from lean and insulin-sensitive individuals and from obese and insulin-resistant individuals who were otherwise healthy. Respiration and reactive oxygen species (ROS) production rates were measured in vitro. Relative abundances of proteins detected by mass spectrometry were determined using a normalized spectral abundance factor method. RESULTS NADH- and FADH2-linked maximal respiration rates were similar between lean and obese individuals. Rates of pyruvate and palmitoyl-dl-carnitine (both including malate) ROS production were significantly higher in obesity. Mitochondria from obese individuals maintained higher (more negative) extramitochondrial ATP free energy at low metabolic flux, suggesting that stronger mitochondrial thermodynamic driving forces may underlie the higher ROS production. Tandem mass spectrometry identified protein abundance differences per mitochondrial mass in insulin resistance, including lower abundance of complex I subunits and enzymes involved in the oxidation of branched-chain amino acids (BCAA) and fatty acids (e.g., carnitine palmitoyltransferase 1B). CONCLUSIONS We provide data suggesting normal oxidative capacity of mitochondria in insulin-resistant skeletal muscle in parallel with high rates of ROS production. Furthermore, we show specific abundance differences in proteins involved in fat and BCAA oxidation that might contribute to the accumulation of lipid and BCAA frequently associated with the pathogenesis of insulin resistance.