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. 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.

[1]  M. Danson,et al.  Citrate synthase. , 2020, Current topics in cellular regulation.

[2]  Mary-Ellen Harper,et al.  Electron Transport Chain-dependent and -independent Mechanisms of Mitochondrial H2O2 Emission during Long-chain Fatty Acid Oxidation* , 2009, The Journal of Biological Chemistry.

[3]  Benjamin P. Bowen,et al.  Proteomics Analysis of Human Skeletal Muscle Reveals Novel Abnormalities in Obesity and Type 2 Diabetes , 2009, Diabetes.

[4]  K. Nair,et al.  Age, Obesity, and Sex Effects on Insulin Sensitivity and Skeletal Muscle Mitochondrial Function , 2009, Diabetes.

[5]  Atsushi Fukushima,et al.  Reactive oxygen species enhance insulin sensitivity. , 2009, Cell metabolism.

[6]  C. Flynn,et al.  Proteome profile of functional mitochondria from human skeletal muscle using one-dimensional gel electrophoresis and HPLC-ESI-MS/MS. , 2009, Journal of proteomics.

[7]  Michael Stumvoll,et al.  Antioxidants prevent health-promoting effects of physical exercise in humans , 2009, Proceedings of the National Academy of Sciences.

[8]  B. Spiegelman,et al.  PPARδ Agonism Activates Fatty Acid Oxidation via PGC-1α but Does Not Increase Mitochondrial Gene Expression and Function , 2009, The Journal of Biological Chemistry.

[9]  E. B. Tahara,et al.  Tissue-, substrate-, and site-specific characteristics of mitochondrial reactive oxygen species generation. , 2009, Free radical biology & medicine.

[10]  Svati H Shah,et al.  A branched-chain amino acid-related metabolic signature that differentiates obese and lean humans and contributes to insulin resistance. , 2009, Cell metabolism.

[11]  P. Neufer,et al.  Mitochondrial H2O2 emission and cellular redox state link excess fat intake to insulin resistance in both rodents and humans. , 2009, The Journal of clinical investigation.

[12]  M. Febbraio,et al.  Overexpression of Carnitine Palmitoyltransferase-1 in Skeletal Muscle Is Sufficient to Enhance Fatty Acid Oxidation and Improve High-Fat Diet–Induced Insulin Resistance , 2009, Diabetes.

[13]  M. Roden,et al.  Ectopic lipids and organ function , 2009, Current opinion in lipidology.

[14]  R. DeFronzo,et al.  Mitochondrial reactive oxygen species generation in obese non-diabetic and type 2 diabetic participants , 2009, Diabetologia.

[15]  Joris Hoeks,et al.  Lower Intrinsic ADP-Stimulated Mitochondrial Respiration Underlies In Vivo Mitochondrial Dysfunction in Muscle of Male Type 2 Diabetic Patients , 2008, Diabetes.

[16]  Vivian F Su,et al.  A Novel Connexin43-interacting Protein, CIP75, Which Belongs to the UbL-UBA Protein Family, Regulates the Turnover of Connexin43* , 2008, Journal of Biological Chemistry.

[17]  B. Morio,et al.  Mitochondrial dysfunction results from oxidative stress in the skeletal muscle of diet-induced insulin-resistant mice. , 2008, The Journal of clinical investigation.

[18]  E. Ravussin,et al.  Effect of a Polymorphism in the ND1 Mitochondrial Gene on Human Skeletal Muscle Mitochondrial Function , 2008, Obesity.

[19]  K. Sahlin,et al.  Mitochondrial Respiration Is Decreased in Skeletal Muscle of Patients With Type 2 Diabetes , 2007, Diabetes.

[20]  K. Petersen,et al.  Impaired Mitochondrial Substrate Oxidation in Muscle of Insulin-Resistant Offspring of Type 2 Diabetic Patients , 2007, Diabetes.

[21]  R. Boushel,et al.  Patients with type 2 diabetes have normal mitochondrial function in skeletal muscle , 2007, Diabetologia.

[22]  J. Smeitink,et al.  Mitochondrial complex I: Structure, function and pathology , 2006, Journal of Inherited Metabolic Disease.

[23]  M. Sheffield-Moore,et al.  Skeletal muscle protein anabolic response to increased energy and insulin is preserved in poorly controlled type 2 diabetes. , 2006, The Journal of nutrition.

[24]  E. Lander,et al.  Reactive oxygen species have a causal role in multiple forms of insulin resistance , 2006, Nature.

[25]  Giorgio Valle,et al.  Quantitative Proteomic Comparison of Rat Mitochondria from Muscle, Heart, and Liver *S , 2006, Molecular & Cellular Proteomics.

[26]  Alexandros Tzatsos,et al.  Nutrients Suppress Phosphatidylinositol 3-Kinase/Akt Signaling via Raptor-Dependent mTOR-Mediated Insulin Receptor Substrate 1 Phosphorylation , 2006, Molecular and Cellular Biology.

[27]  K. Petersen,et al.  Reduced mitochondrial density and increased IRS-1 serine phosphorylation in muscle of insulin-resistant offspring of type 2 diabetic parents. , 2005, The Journal of clinical investigation.

[28]  K. Petersen,et al.  Decreased Insulin-Stimulated ATP Synthesis and Phosphate Transport in Muscle of Insulin-Resistant Offspring of Type 2 Diabetic Parents , 2005, PLoS medicine.

[29]  A. Marette,et al.  Modulation of insulin action by dietary proteins and amino acids: role of the mammalian target of rapamycin nutrient sensing pathway , 2005, Current opinion in clinical nutrition and metabolic care.

[30]  Samy I McFarlane,et al.  Abrogation of oxidative stress improves insulin sensitivity in the Ren-2 rat model of tissue angiotensin II overexpression. , 2005, American journal of physiology. Endocrinology and metabolism.

[31]  W. Willis,et al.  Pyruvate and citric acid cycle carbon requirements in isolated skeletal muscle mitochondria. , 2004, American journal of physiology. Cell physiology.

[32]  K. Petersen,et al.  Impaired mitochondrial activity in the insulin-resistant offspring of patients with type 2 diabetes. , 2004, The New England journal of medicine.

[33]  M. Hofker.  Faculty Opinions recommendation of PGC-1alpha-responsive genes involved in oxidative phosphorylation are coordinately downregulated in human diabetes. , 2003 .

[34]  A. Butte,et al.  Coordinated reduction of genes of oxidative metabolism in humans with insulin resistance and diabetes: Potential role of PGC1 and NRF1 , 2003, Proceedings of the National Academy of Sciences of the United States of America.

[35]  Jing He,et al.  Dysfunction of mitochondria in human skeletal muscle in type 2 diabetes. , 2002, Diabetes.

[36]  Gary Fiskum,et al.  Generation of reactive oxygen species by the mitochondrial electron transport chain , 2002, Journal of neurochemistry.

[37]  Simon C Watkins,et al.  Skeletal muscle lipid content and insulin resistance: evidence for a paradox in endurance-trained athletes. , 2001, The Journal of clinical endocrinology and metabolism.

[38]  H V Westerhoff,et al.  A metabolic control analysis of kinetic controls in ATP free energy metabolism in contracting skeletal muscle. , 2000, American journal of physiology. Cell physiology.

[39]  Rena R Wing,et al.  Skeletal muscle fatty acid metabolism in association with insulin resistance, obesity, and weight loss. , 1999, American journal of physiology. Endocrinology and metabolism.

[40]  L. Buja,et al.  Genomic DNA sequence, promoter expression, and chromosomal mapping of rat muscle carnitine palmitoyltransferase I. , 1998, Genomics.

[41]  V. Skulachev,et al.  High protonic potential actuates a mechanism of production of reactive oxygen species in mitochondria , 1997, FEBS letters.

[42]  W. Willis,et al.  Characteristics of mitochondria isolated from type I and type IIb skeletal muscle. , 1996, The American journal of physiology.

[43]  D. Poole,et al.  Determinants of maximal exercise VO2 during single leg knee-extensor exercise in humans. , 1995, The American journal of physiology.

[44]  M. Kushmerick,et al.  Mammalian skeletal muscle fibers distinguished by contents of phosphocreatine, ATP, and Pi. , 1992, Proceedings of the National Academy of Sciences of the United States of America.

[45]  G. Dobson,et al.  Effect of temperature on the creatine kinase equilibrium. , 1992, The Journal of biological chemistry.

[46]  V. Vítek,et al.  Enzyme activities in quadriceps femoris muscle of obese diabetic male patients , 1977, Diabetologia.

[47]  J. Mitchell,et al.  Maximal oxygen uptake. , 1971, The New England journal of medicine.

[48]  C. Lee,et al.  Biochemical studies of skeletal muscle mitochondria. I. Microanalysis of cytochrome content, oxidative and phosphorylative activities of mammalian skeletal muscle mitochondria. , 1968, Archives of biochemistry and biophysics.

[49]  T. Creighton.  Methods in Enzymology , 1968, The Yale Journal of Biology and Medicine.

[50]  P. Björntorp,et al.  Respiration and phosphorylation of mitochondria isolated from the skeletal muscle of diabetic and normal subjects , 1967, Diabetologia.

[51]  Olga Ilkayeva,et al.  Mitochondrial overload and incomplete fatty acid oxidation contribute to skeletal muscle insulin resistance. , 2008, Cell metabolism.

[52]  B. Goodpaster,et al.  Deficiency of subsarcolemmal mitochondria in obesity and type 2 diabetes. , 2005, Diabetes.

[53]  W. Dröge.  Free radicals in the physiological control of cell function. , 2002, Physiological reviews.

[54]  P. Wagner,et al.  Determinants of maximal oxygen transport and utilization. , 1996, Annual review of physiology.

[55]  B. Saltin,et al.  Maximal oxygen uptake: "old" and "new" arguments for a cardiovascular limitation. , 1992, Medicine and science in sports and exercise.

[56]  E. Ravussin,et al.  Muscle mitochondrial morphology, body composition, and energy expenditure in sedentary individuals. , 1991, American Journal of Physiology.

[57]  P. Srere,et al.  [1] Citrate synthase. [EC 4.1.3.7. Citrate oxaloacetate-lyase (CoA-acetylating)] , 1969 .

[58]  R. Steele,et al.  ON THE HORMONAL REGULATION OF CARBOHYDRATE METABOLISM; STUDIES WITH C14 GLUCOSE. , 1963, Recent progress in hormone research.