Increased reactive oxygen species production and lower abundance of complex I subunits and carnitine palmitoyltransferase 1B protein despite normal mitochondrial respiration in insulin-resistant human skeletal muscle.
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.


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 FADH(2)-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.
Lefort
Natalie
N
Center for Metabolic Biology, Arizona State University, Tempe, Arizona, USA.
Glancy
Brian
B
Bowen
Benjamin
B
Willis
Wayne T
WT
Bailowitz
Zachary
Z
De Filippis
Elena A
EA
Brophy
Colleen
C
Meyer
Christian
C
Højlund
Kurt
K
Yi
Zhengping
Z
Mandarino
Lawrence J
LJ
D001275:ATP Citrate (pro-S)-Lyase / Q000378:metabolism; D000596:Amino Acids / Q000378:metabolism; D002334:Carnitine O-Palmitoyltransferase / Q000096:biosynthesis* / Q000378:metabolism; D003402:Creatine Kinase / Q000378:metabolism; D042967:Electron Transport Complex I / Q000378:metabolism*; D005227:Fatty Acids / Q000378:metabolism; D015309:Glucose Clamp Technique; D006801:Humans; D006946:Hyperinsulinism / Q000473:pathology; D007333:Insulin Resistance / Q000502:physiology*; D013058:Mass Spectrometry; D008931:Mitochondria, Muscle / Q000201:enzymology / Q000378:metabolism*; D018482:Muscle, Skeletal / Q000201:enzymology / Q000378:metabolism* / Q000473:pathology; D009765:Obesity / Q000201:enzymology / Q000378:metabolism; D010084:Oxidation-Reduction; D010101:Oxygen Consumption / Q000502:physiology; D021122:Protein Subunits / Q000378:metabolism; D017382:Reactive Oxygen Species / Q000378:metabolism*; D013816:Thermodynamics; D013851:Thinness / Q000201:enzymology / Q000378:metabolism
D000596:Amino Acids; D005227:Fatty Acids; D021122:Protein Subunits; D017382:Reactive Oxygen Species; D002334:Carnitine O-Palmitoyltransferase; D001275:ATP Citrate (pro-S)-Lyase; D003402:Creatine Kinase; D042967:Electron Transport Complex I