Genetic loss of insulin receptors worsens cardiac efficiency in diabetes.

AIMS: To determine the contribution of insulin signaling versus systemic metabolism to metabolic and mitochondrial alterations in type 1 diabetic hearts and test the hypothesis that antecedent mitochondrial dysfunction contributes to impaired cardiac efficiency (CE) in diabetes. METHODS AND RESULTS: Control mice (WT) and mice with cardiomyocyte-restricted deletion of insulin receptors (CIRKO) were rendered diabetic with streptozotocin (WT-STZ and CIRKO-STZ, respectively), non-diabetic controls received vehicle (citrate buffer). Cardiac function was determined by echocardiography; myocardial metabolism, oxygen consumption (MVO(2)) and CE were determined in isolated perfused hearts; mitochondrial function was determined in permeabilized cardiac fibers and mitochondrial proteomics by liquid chromatography mass spectrometry. Pyruvate supported respiration and ATP synthesis were equivalently reduced by diabetes and genotype, with synergistic impairment in ATP synthesis in CIRKO-STZ. In contrast, fatty acid delivery and utilization was increased by diabetes irrespective of genotype, but not in non-diabetic CIRKO. Diabetes and genotype synergistically increased MVO(2) in CIRKO-STZ, leading to reduced CE. Irrespective of diabetes, genotype impaired ATP/O ratios in mitochondria exposed to palmitoyl carnitine, consistent with mitochondrial uncoupling. Proteomics revealed reduced content of fatty acid oxidation proteins in CIRKO mitochondria, which were induced by diabetes, whereas tricarboxylic acid cycle and oxidative phosphorylation proteins were reduced both in CIRKO mitochondria and by diabetes. CONCLUSIONS: Deficient insulin signaling and diabetes mediate distinct effects on cardiac mitochondria. Antecedent loss of insulin signaling markedly impairs CE when diabetes is induced, via mechanisms that may be secondary to mitochondrial uncoupling and increased FA utilization.

Tissue-specific remodeling of the mitochondrial proteome in type 1 diabetic akita mice.

OBJECTIVE: To elucidate the molecular basis for mitochondrial dysfunction, which has been implicated in the pathogenesis of diabetes complications. RESEARCH DESIGN AND METHODS: Mitochondrial 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. RESULTS: In 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)-gamma coactivator (PGC)-1alpha transcripts. CONCLUSIONS: Type 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.

Mechanisms for increased myocardial fatty acid utilization following short-term high-fat feeding.

AIMS: Diet-induced obesity is associated with increased myocardial fatty acid (FA) utilization, insulin resistance, and cardiac dysfunction. The study was designed to test the hypothesis that impaired glucose utilization accounts for initial changes in FA metabolism. METHODS AND RESULTS: Ten-week-old C57BL6J mice were fed a high-fat diet (HFD, 45% calories from fat) or normal chow (4% calories from fat). Cardiac function and substrate metabolism in isolated working hearts, glucose uptake in isolated cardiomyocytes, mitochondrial function, insulin-stimulated protein kinase B (Akt/PKB) and Akt substrate (AS-160) phosphorylation, glucose transporter 4 (GLUT4) translocation, pyruvate dehydrogenase (PDH) activity, and mRNA levels for metabolic genes were determined after 2 or 5 weeks of HFD. Two weeks of HFD reduced basal rates of glycolysis and glucose oxidation and prevented insulin stimulation of glycolysis in hearts and reduced insulin-stimulated glucose uptake in cardiomyocytes. Insulin-stimulated Akt/PKB and AS-160 phosphorylation were preserved, and PDH activity was unchanged. GLUT4 content was reduced by 55% and GLUT4 translocation was significantly attenuated. HFD increased FA oxidation rates and myocardial oxygen consumption (MVO2), which could not be accounted for by mitochondrial uncoupling or by increased expression of peroxisome proliferator activated receptor-alpha (PPAR-alpha) target genes, which increased only after 5 weeks of HFD. CONCLUSION: Rates of myocardial glucose utilization are altered early in the course of HFD because of reduced GLUT4 content and GLUT4 translocation despite normal insulin signalling to Akt/PKB and AS-160. The reciprocal increase in FA utilization is not due to PPAR-alpha-mediated signalling or mitochondrial uncoupling. Thus, the initial increase in myocardial FA utilization in response to HFD likely results from impaired glucose transport that precedes impaired insulin signalling.

Insulin-like growth factor I receptor signaling is required for exercise-induced cardiac hypertrophy.

The receptors for IGF-I (IGF-IR) and insulin (IR) have been implicated in physiological cardiac growth, but it is unknown whether IGF-IR or IR signaling are critically required. We generated mice with cardiomyocyte-specific knockout of IGF-IR (CIGF1RKO) and compared them with cardiomyocyte-specific insulin receptor knockout (CIRKO) mice in response to 5 wk exercise swim training. Cardiac development was normal in CIGF1RKO mice, but the hypertrophic response to exercise was prevented. In contrast, despite reduced baseline heart size, the hypertrophic response of CIRKO hearts to exercise was preserved. Exercise increased IGF-IR content in control and CIRKO hearts. Akt phosphorylation increased in exercise-trained control and CIRKO hearts and, surprisingly, in CIGF1RKO hearts as well. In exercise-trained control and CIRKO mice, expression of peroxisome proliferator-activated receptor-gamma coactivator-1alpha (PGC-1alpha) and glycogen content were both increased but were unchanged in trained CIGF1RKO mice. Activation of AMP-activated protein kinase (AMPK) and its downstream target eukaryotic elongation factor-2 was increased in exercise-trained CIGF1RKO but not in CIRKO or control hearts. In cultured neonatal rat cardiomyocytes, activation of AMPK with 5-aminoimidazole-4-carboxamide-1-beta-D-ribofuranoside (AICAR) prevented IGF-I/insulin-induced cardiomyocyte hypertrophy. These studies identify an essential role for IGF-IR in mediating physiological cardiomyocyte hypertrophy. IGF-IR deficiency promotes energetic stress in response to exercise, thereby activating AMPK, which leads to phosphorylation of eukaryotic elongation factor-2. These signaling events antagonize Akt signaling, which although necessary for mediating physiological cardiac hypertrophy, is insufficient to promote cardiac hypertrophy in the absence of myocardial IGF-I signaling.

Type 1 diabetic akita mouse hearts are insulin sensitive but manifest structurally abnormal mitochondria that remain coupled despite increased uncoupling protein 3.

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 and substrate 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. CONCLUSIONS: Insulin-deficient Akita hearts do not exhibit fatty acid-induced mitochondrial uncoupling, indicating important differences in the basis for mitochondrial dysfunction between insulin-responsive type 1 versus insulin-resistant type 2 diabetic hearts. Increased UCP3 levels do not automatically increase mitochondrial uncoupling in the heart, which supports the hypothesis that fatty acid-induced mitochondrial uncoupling as exists in type 2 diabetic hearts requires a concomitant increase in ROS generation.

A conserved role for phosphatidylinositol 3-kinase but not Akt signaling in mitochondrial adaptations that accompany physiological cardiac hypertrophy.

Physiological cardiac hypertrophy is associated with mitochondrial adaptations that are characterized by activation of PGC-1alpha and increased fatty acid oxidative (FAO) capacity. It is widely accepted that phosphatidylinositol 3-kinase (PI3K) signaling to Akt1 is required for physiological cardiac growth. However, the signaling pathways that coordinate physiological hypertrophy and metabolic remodeling are incompletely understood. We show here that activation of PI3K is sufficient to increase myocardial FAO capacity and that inhibition of PI3K signaling prevents mitochondrial adaptations in response to physiological hypertrophic stimuli despite increased expression of PGC-1alpha. We also show that activation of the downstream kinase Akt is not required for the mitochondrial adaptations that are secondary to PI3K activation. Thus, in physiological cardiac growth, PI3K is an integrator of cellular growth and metabolic remodeling. Although PI3K signaling to Akt1 is required for cellular growth, Akt-independent pathways mediate the accompanying mitochondrial adaptations.

Cardiac hypertrophy caused by peroxisome proliferator- activated receptor-gamma agonist treatment occurs independently of changes in myocardial insulin signaling.

Peroxisome proliferator-activated receptor (PPAR)-gamma ligands are insulin sensitizers, widely used in the treatment of type 2 diabetes. A consistent observation in preclinical species is the development of cardiac hypertrophy after short-term treatment with these agents. The mechanisms for this hypertrophy are incompletely understood. Given the important role of insulin signaling in the regulation of myocardial size, we tested the hypothesis that augmentation of myocardial insulin signaling may play a role in PPAR-gamma ligand-induced cardiac hypertrophy. We treated mice with cardiomyocyte-restricted knockout of insulin receptors (CIRKO) and littermate controls (wild type) with 2-(2-(4-phenoxy-2-propylphenoxy) ethyl) indole-5-acetic acid (COOH), which is a non-thiazolidinedione PPAR-gamma agonist for 2 wk. Two weeks of COOH treatment increased heart weights by 22% in CIRKO mice and 16% in wild type, and induced similar fold increase in the expression of hypertrophic markers such as alpha-skeletal actin, brain natriuretic peptide, and atrial natriuretic peptide in CIRKO and wild-type (WT) hearts. COOH treatment increased plasma volume by 10% in COOH-treated WT and CIRKO mice but did not increase systolic or diastolic blood pressure. Echocardiographic analysis was also consistent with volume overload, as evidenced by increased left ventricular diastolic diameters and cardiac output in COOH-treated CIRKO and WT mice. These data indicate that cardiac hypertrophy after PPAR-gamma agonist treatment can occur in the absence of myocardial insulin signaling and is likely secondary to the hemodynamic consequences of plasma volume expansion.