OBJECTIVE-In obesity and diabetes, myocardial fatty acid utilization and myocardial oxygen consumption (MVO 2 ) are increased, and cardiac efficiency is reduced. Mitochondrial uncoupling has been proposed to contribute to these metabolic abnormalities but has not been directly demonstrated. RESEARCH DESIGN AND METHODS-Oxygen consumptionand cardiac function were determined in db/db hearts perfused with glucose or glucose and palmitate. Mitochondrial function was determined in saponin-permeabilized fibers and proton leak kinetics and H 2 O 2 generation determined in isolated mitochondria.RESULTS-db/db hearts exhibited reduced cardiac function and increased MVO 2 . Mitochondrial reactive oxygen species (ROS) generation and lipid and protein peroxidation products were increased. Mitochondrial proliferation was increased in db/db hearts, oxidative phosphorylation capacity was impaired, but H 2 O 2 production was increased. Mitochondria from db/db mice exhibited fatty acid-induced mitochondrial uncoupling that is inhibitable by GDP, suggesting that these changes are mediated by uncoupling proteins (UCPs). Mitochondrial uncoupling was not associated with an increase in UCP content, but fatty acid oxidation genes and expression of electron transfer flavoproteins were increased, whereas the content of the F1 ␣-subunit of ATP synthase was reduced.CONCLUSIONS-These data demonstrate that mitochondrial uncoupling in the heart in obesity and diabetes is mediated by activation of UCPs independently of changes in expression levels. This likely occurs on the basis of increased delivery of reducing equivalents from ␤-oxidation to the electron transport chain, which coupled with decreased oxidative phosphorylation capacity increases ROS production and lipid peroxidation.

Background-Diabetes-associated cardiac dysfunction is associated with mitochondrial dysfunction and oxidative stress, which may contribute to left ventricular dysfunction. The contribution of altered myocardial insulin action, independent of associated changes in systemic metabolism, is incompletely understood. The present study tested the hypothesis that perinatal loss of insulin signaling in the heart impairs mitochondrial function. Methods and Results-In 8-week-old mice with cardiomyocyte deletion of insulin receptors (CIRKO), inotropic reserves were reduced, and mitochondria manifested respiratory defects for pyruvate that was associated with proportionate reductions in catalytic subunits of pyruvate dehydrogenase. Progressive age-dependent defects in oxygen consumption and ATP synthesis with the substrate glutamate and the fatty acid derivative palmitoyl-carnitine were observed. Mitochondria also were uncoupled when exposed to palmitoyl-carnitine, in part as a result of increased reactive oxygen species production and oxidative stress. Although proteomic and genomic approaches revealed a reduction in subsets of genes and proteins related to oxidative phosphorylation, no reductions in maximal activities of mitochondrial electron transport chain complexes were found. However, a disproportionate reduction in tricarboxylic acid cycle and fatty acid oxidation proteins in mitochondria suggests that defects in fatty acid and pyruvate metabolism and tricarboxylic acid flux may explain the mitochondrial dysfunction observed. Conclusions-Impaired myocardial insulin signaling promotes oxidative stress and mitochondrial uncoupling, which, together with reduced tricarboxylic acid and fatty acid oxidative capacity, impairs mitochondrial energetics. This study identifies specific contributions of impaired insulin action to mitochondrial dysfunction in the heart. (Circulation. 2009; 119:1272-1283.)Key Words: insulin Ⅲ metabolism Ⅲ mitochondria Ⅲ oxidative stress R ecent studies have suggested that impaired mitochondrial energetics may contribute to cardiac dysfunction in obesity and diabetes mellitus. [1][2][3][4][5][6][7] The pathogenesis of mitochondrial dysfunction in obesity or diabetes-related heart disease is likely multifactorial but includes fatty acid (FA)-mediated mitochondrial uncoupling and oxidative damage. 3,4,8 -11 A commonly associated finding in the heart in experimental models of obesity and diabetes mellitus is myocardial insulin resistance. [12][13][14][15][16] However, it is not known whether myocardial insulin resistance per se contributes directly to the pathogenesis of myocardial mitochondrial dysfunction. Clinical Perspective p 1283The effects of myocardial insulin signaling on the acute regulation of myocardial metabolism are well known 17,18 and include increasing glucose uptake and glycolysis via regulation of GLUT4 translocation 19,20 and activation of 6-phosphofructo-1-kinase. 21 In perfused hearts, insulin increases glucose oxidation and reduces FA oxidation. 13 In vivo, the antilipolytic ef...

OBJECTIVE-Fatty acid-induced mitochondrial uncoupling and oxidative stress have been proposed to reduce cardiac efficiency and contribute to cardiac dysfunction in type 2 diabetes. We hypothesized that mitochondrial uncoupling may also contribute to reduced cardiac efficiency and contractile dysfunction in the type 1 diabetic Akita mouse model (Akita). RESEARCH DESIGN AND METHODS-Cardiac function andsubstrate utilization were determined in isolated working hearts and in vivo function by echocardiography. Mitochondrial function and coupling were determined in saponin-permeabilized fibers, and proton leak kinetics was determined in isolated mitochondria. Hydrogen peroxide production and aconitase activity were measured in isolated mitochondria, and total reactive oxygen species (ROS) were measured in heart homogenates. RESULTS-Resting cardiac function was normal in Akita mice, and myocardial insulin sensitivity was preserved. Although Akita hearts oxidized more fatty acids, myocardial O 2 consumption was not increased, and cardiac efficiency was not reduced. ADP-stimulated mitochondrial oxygen consumption and ATP synthesis were decreased, and mitochondria showed grossly abnormal morphology in Akita. There was no evidence of oxidative stress, and despite a twofold increase in uncoupling protein 3 (UCP3) content, ATP-to-O ratios and proton leak kinetics were unchanged, even after perfusion of Akita hearts with 1 mmol/l palmitate.

OBJECTIVETo elucidate the molecular basis for mitochondrial dysfunction, which has been implicated in the pathogenesis of diabetes complications.RESEARCH DESIGN AND METHODSMitochondrial matrix and membrane fractions were generated from liver, brain, heart, and kidney of wild-type and type 1 diabetic Akita mice. Comparative proteomics was performed using label-free proteome expression analysis. Mitochondrial state 3 respirations and ATP synthesis were measured, and mitochondrial morphology was evaluated by electron microscopy. Expression of genes that regulate mitochondrial biogenesis, substrate utilization, and oxidative phosphorylation (OXPHOS) were determined.RESULTSIn diabetic mice, fatty acid oxidation (FAO) proteins were less abundant in liver mitochondria, whereas FAO protein content was induced in mitochondria from all other tissues. Kidney mitochondria showed coordinate induction of tricarboxylic acid (TCA) cycle enzymes, whereas TCA cycle proteins were repressed in cardiac mitochondria. Levels of OXPHOS subunits were coordinately increased in liver mitochondria, whereas mitochondria of other tissues were unaffected. Mitochondrial respiration, ATP synthesis, and morphology were unaffected in liver and kidney mitochondria. In contrast, state 3 respirations, ATP synthesis, and mitochondrial cristae density were decreased in cardiac mitochondria and were accompanied by coordinate repression of OXPHOS and peroxisome proliferator–activated receptor (PPAR)-γ coactivator (PGC)-1α transcripts.CONCLUSIONSType 1 diabetes causes tissue-specific remodeling of the mitochondrial proteome. Preservation of mitochondrial function in kidney, brain, and liver, versus mitochondrial dysfunction in the heart, supports a central role for mitochondrial dysfunction in diabetic cardiomyopathy.

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