Brian T. O'Neill

Contribution of impaired myocardial insulin signaling to mitochondrial dysfunction and oxidative stress in the heart

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.

Diet-induced obesity causes insulin resistance in mouse brown adipose tissue

Diet‐induced obesity (DIO) causes several pathophysiological changes in adipose tissue. Increased inflammation reduces white adipose tissue (WAT) insulin sensitivity and contributes to the development of diabetes. However, little is known about how DIO alters the function of brown adipose tissue (BAT), an organ that consumes calories by β3‐adrenergic receptor (AR)‐mediated thermogenesis and helps regulate energy balance.

Early Mitochondrial Adaptations in Skeletal Muscle to Diet-Induced Obesity Are Strain Dependent and Determine Oxidative Stress and Energy Expenditure But Not Insulin Sensitivity

This study sought to elucidate the relationship between skeletal muscle mitochondrial dysfunction, oxidative stress, and insulin resistance in two mouse models with differential susceptibility to diet-induced obesity. We examined the time course of mitochondrial dysfunction and insulin resistance in obesity-prone C57B and obesity-resistant FVB mouse strains in response to high-fat feeding. After 5 wk, impaired insulin-mediated glucose uptake in skeletal muscle developed in both strains in the absence of any impairment in proximal insulin signaling. Impaired mitochondrial oxidative capacity preceded the development of insulin resistant glucose uptake in C57B mice in concert with increased oxidative stress in skeletal muscle. By contrast, mitochondrial uncoupling in FVB mice, which prevented oxidative stress and increased energy expenditure, did not prevent insulin resistant glucose uptake in skeletal muscle. Preventing oxidative stress in C57B mice treated systemically with an antioxidant normalized skeletal muscle mitochondrial function but failed to normalize glucose tolerance and insulin sensitivity. Furthermore, high fat-fed uncoupling protein 3 knockout mice developed increased oxidative stress that did not worsen glucose tolerance. In the evolution of diet-induced obesity and insulin resistance, initial but divergent strain-dependent mitochondrial adaptations modulate oxidative stress and energy expenditure without influencing the onset of impaired insulin-mediated glucose uptake.

Cardiac PI3K-Akt Impairs Insulin-Stimulated Glucose Uptake Independent of mTORC1 and GLUT4 Translocation

Impaired insulin-mediated glucose uptake characterizes cardiac muscle in humans and animals with insulin resistance and diabetes, despite preserved or enhanced phosphatidylinositol 3-kinase (PI3K) and the serine-threonine kinase, Akt-signaling, via mechanisms that are incompletely understood. One potential mechanism is PI3K- and Akt-mediated activation of mechanistic target of rapamycin (mTOR) and ribosomal protein S6 kinase (S6K), which may impair insulin-mediated activation of insulin receptor substrate (IRS)1/2 via inhibitory serine phosphorylation or proteasomal degradation. To gain mechanistic insights by which constitutive activation of PI3K or Akt may desensitize insulin-mediated glucose uptake in cardiomyocytes, we examined mice with cardiomyocyte-restricted, constitutive or inducible overexpression of a constitutively activated PI3K or a myristoylated Akt1 (myrAkt1) transgene that also expressed a myc-epitope-tagged glucose transporter type 4 protein (GLUT4). Although short-term activation of PI3K and myrAkt1 increased mTOR and S6 signaling, there was no impairment in insulin-mediated activation of IRS1/2. However, insulin-mediated glucose uptake was reduced by 50-80%. Although longer-term activation of Akt reduced IRS2 protein content via an mTORC1-mediated mechanism, treatment of transgenic mice with rapamycin failed to restore insulin-mediated glucose uptake, despite restoring IRS2. Transgenic activation of Akt and insulin-stimulation of myrAkt1 transgenic cardiomyocytes increased sarcolemmal insertion of myc-GLUT4 to levels equivalent to that observed in insulin-stimulated wild-type controls. Despite preserved GLUT4 translocation, glucose uptake was not elevated by the presence of constitutive activation of PI3K and Akt. Hexokinase II activity was preserved in myrAkt1 hearts. Thus, constitutive activation of PI3K and Akt in cardiomyocytes impairs GLUT4-mediated glucose uptake via mechanisms that impair the function of GLUT4 after its plasma-membrane insertion.

Enhanced Cardiac Akt/Protein Kinase B Signaling Contributes to Pathological Cardiac Hypertrophy in Part by Impairing Mitochondrial Function via Transcriptional Repression of Mitochondrion-Targeted Nuclear Genes

ABSTRACT Sustained Akt activation induces cardiac hypertrophy (LVH), which may lead to heart failure. This study tested the hypothesis that Akt activation contributes to mitochondrial dysfunction in pathological LVH. Akt activation induced LVH and progressive repression of mitochondrial fatty acid oxidation (FAO) pathways. Preventing LVH by inhibiting mTOR failed to prevent the decline in mitochondrial function, but glucose utilization was maintained. Akt activation represses expression of mitochondrial regulatory, FAO, and oxidative phosphorylation genes in vivo that correlate with the duration of Akt activation in part by reducing FOXO-mediated transcriptional activation of mitochondrion-targeted nuclear genes in concert with reduced signaling via peroxisome proliferator-activated receptor α (PPARα)/PGC-1α and other transcriptional regulators. In cultured myocytes, Akt activation disrupted mitochondrial bioenergetics, which could be partially reversed by maintaining nuclear FOXO but not by increasing PGC-1α. Thus, although short-term Akt activation may be cardioprotective during ischemia by reducing mitochondrial metabolism and increasing glycolysis, long-term Akt activation in the adult heart contributes to pathological LVH in part by reducing mitochondrial oxidative capacity.

Differential Roles of Insulin and IGF-1 Receptors in Adipose Tissue Development and Function.

To determine the roles of insulin and insulin-like growth factor 1 (IGF-1) action in adipose tissue, we created mice lacking the insulin receptor (IR), IGF-1 receptor (IGF1R), or both using Cre-recombinase driven by the adiponectin promoter. Mice lacking IGF1R only (F-IGFRKO) had a ∼25% reduction in white adipose tissue (WAT) and brown adipose tissue (BAT), whereas mice lacking both IR and IGF1R (F-IR/IGFRKO) showed an almost complete absence of WAT and BAT. Interestingly, mice lacking only the IR (F-IRKO) had a 95% reduction in WAT, but a paradoxical 50% increase in BAT with accumulation of large unilocular lipid droplets. Both F-IRKO and F-IR/IGFRKO mice were unable to maintain body temperature in the cold and developed severe diabetes, ectopic lipid accumulation in liver and muscle, and pancreatic islet hyperplasia. Leptin treatment normalized blood glucose levels in both groups. Glucose levels also improved spontaneously by 1 year of age, despite sustained lipodystrophy and insulin resistance. Thus, loss of IR is sufficient to disrupt white fat formation, but not brown fat formation and/or maintenance, although it is required for normal BAT function and temperature homeostasis. IGF1R has only a modest contribution to both WAT and BAT formation and function.

Domain-dependent effects of insulin and IGF-1 receptors on signalling and gene expression

Despite a high degree of homology, insulin receptor (IR) and IGF-1 receptor (IGF1R) mediate distinct cellular and physiological functions. Here, we demonstrate how domain differences between IR and IGF1R contribute to the distinct functions of these receptors using chimeric and site-mutated receptors. Receptors with the intracellular domain of IGF1R show increased activation of Shc and Gab-1 and more potent regulation of genes involved in proliferation, corresponding to their higher mitogenic activity. Conversely, receptors with the intracellular domain of IR display higher IRS-1 phosphorylation, stronger regulation of genes in metabolic pathways and more dramatic glycolytic responses to hormonal stimulation. Strikingly, replacement of leucine973 in the juxtamembrane region of IR to phenylalanine, which is present in IGF1R, mimics many of these signalling and gene expression responses. Overall, we show that the distinct activities of the closely related IR and IGF1R are mediated by their intracellular juxtamembrane region and substrate binding to this region.

Insulin Downregulates the Transcriptional Coregulator CITED2, an Inhibitor of Proangiogenic Function in Endothelial Cells

In patients with atherosclerotic complications of diabetes, impaired neovascularization of ischemic tissue in the myocardium and lower limb limits the ability of these tissues to compensate for poor perfusion. We identified 10 novel insulin-regulated genes, among them Adm, Cited2, and Ctgf, which were downregulated in endothelial cells by insulin through FoxO1. CBP/p300-interacting transactivator with ED-rich tail 2 (CITED2), which was downregulated by insulin by up to 54%, is an important negative regulator of hypoxia-inducible factor (HIF) and impaired HIF signaling is a key mechanism underlying the impairment of angiogenesis in diabetes. Consistent with impairment of vascular insulin action, CITED2 was increased in cardiac endothelial cells from mice with diet-induced obesity and from db/db mice and was 3.8-fold higher in arterial tissue from patients with type 2 diabetes than control subjects without diabetes. CITED2 knockdown promoted endothelial tube formation and endothelial cell proliferation, whereas CITED2 overexpression impaired HIF activity in vitro. After femoral artery ligation, induction of an endothelial-specific HIF target gene in hind limb muscle was markedly upregulated in mice with endothelial cell deletion of CITED2, suggesting that CITED2 can limit HIF activity in vivo. We conclude that vascular insulin resistance in type 2 diabetes contributes to the upregulation of CITED2, which impairs HIF signaling and endothelial proangiogenic function.