Insulin signaling alters antioxidant capacity in the diabetic heart.

Diabetic cardiomyopathy is associated with an increase in oxidative stress. However, antioxidant therapy has shown a limited capacity to mitigate disease pathology. The molecular mechanisms responsible for the modulation of reactive oxygen species (ROS) production and clearance must be better defined. The objective of this study was to determine how insulin affects superoxide radical (O2•-) levels. O2•- production was evaluated in adult cardiomyocytes isolated from control and Akita (type 1 diabetic) mice by spin-trapping electron paramagnetic resonance spectroscopy. We found that the basal rates of O2•- production were comparable in control and Akita cardiomyocytes. However, culturing cardiomyocytes without insulin resulted in a significant increase in O2•- production only in the Akita group. In contrast, O2•- production was unaffected by high glucose and/or fatty acid supplementation. The increase in O2•- was due in part to a decrease in superoxide dismutase (SOD) activity. The PI3K inhibitor, LY294002, decreased Akita SOD activity when insulin was present, indicating that the modulation of antioxidant activity is through insulin signaling. The effect of insulin on mitochondrial O2•- production was evaluated in Akita mice that underwent a 1-week treatment of insulin. Mitochondria isolated from insulin-treated Akita mice produced less O2•- than vehicle-treated diabetic mice. Quantitative proteomics was performed on whole heart homogenates to determine how insulin affects antioxidant protein expression. Of 29 antioxidant enzymes quantified, thioredoxin 1 was the only one that was significantly enhanced by insulin treatment. In vitro analysis of thioredoxin 1 revealed a previously undescribed capacity of the enzyme to directly scavenge O2•-. These findings demonstrate that insulin has a role in mitigating cardiac oxidative stress in diabetes via regulation of endogenous antioxidant activity.

Diabetes induced decreases in PKA signaling in cardiomyocytes: The role of insulin.

The cAMP-dependent protein kinase (PKA) signaling pathway is the primary means by which the heart regulates moment-to-moment changes in contractility and metabolism. We have previously found that PKA signaling is dysfunctional in the diabetic heart, yet the underlying mechanisms are not fully understood. The objective of this study was to determine if decreased insulin signaling contributes to a dysfunctional PKA response. To do so, we isolated adult cardiomyocytes (ACMs) from wild type and Akita type 1 diabetic mice. ACMs were cultured in the presence or absence of insulin and PKA signaling was visualized by immunofluorescence microscopy using an antibody that recognizes proteins specifically phosphorylated by PKA. We found significant decreases in proteins phosphorylated by PKA in wild type ACMs cultured in the absence of insulin. PKA substrate phosphorylation was decreased in Akita ACMs, as compared to wild type, and unresponsive to the effects of insulin. The decrease in PKA signaling was observed regardless of whether the kinase was stimulated with a beta-agonist, a cell-permeable cAMP analog, or with phosphodiesterase inhibitors. PKA content was unaffected, suggesting that the decrease in PKA signaling may be occurring by the loss of specific PKA substrates. Phospho-specific antibodies were used to discern which potential substrates may be sensitive to the loss of insulin. Contractile proteins were phosphorylated similarly in wild type and Akita ACMs regardless of insulin. However, phosphorylation of the glycolytic regulator, PFK-2, was significantly decreased in an insulin-dependent manner in wild type ACMs and in an insulin-independent manner in Akita ACMs. These results demonstrate a defect in PKA activation in the diabetic heart, mediated in part by deficient insulin signaling, that results in an abnormal activation of a primary metabolic regulator.

Cardiac Insulin Signaling Regulates Glycolysis Through Phosphofructokinase 2 Content and Activity.

BACKGROUND: The healthy heart has a dynamic capacity to respond and adapt to changes in nutrient availability. Diabetes mellitus disrupts this metabolic flexibility and promotes cardiomyopathy through mechanisms that are not completely understood. Phosphofructokinase 2 (PFK-2) is a primary regulator of cardiac glycolysis and substrate selection, yet its regulation under normal and pathological conditions is unknown. This study was undertaken to determine how changes in insulin signaling affect PFK-2 content, activity, and cardiac metabolism. METHODS AND RESULTS: Streptozotocin-induced diabetes mellitus, high-fat diet feeding, and fasted mice were used to identify how decreased insulin signaling affects PFK-2 and cardiac metabolism. Primary adult cardiomyocytes were used to define the mechanisms that regulate PFK-2 degradation. Both type 1 diabetes mellitus and a high-fat diet induced a significant decrease in cardiac PFK-2 protein content without affecting its transcript levels. Overnight fasting also induced a decrease in PFK-2, suggesting it is rapidly degraded in the absence of insulin signaling. An unbiased metabolomic study demonstrated that decreased PFK-2 in fasted animals is accompanied by an increase in glycolytic intermediates upstream of phosphofructokianse-1, whereas those downstream are diminished. Mechanistic studies using cardiomyocytes showed that, in the absence of insulin signaling, PFK-2 is rapidly degraded via both proteasomal- and chaperone-mediated autophagy. CONCLUSIONS: The loss of PFK-2 content as a result of reduced insulin signaling impairs the capacity to dynamically regulate glycolysis and elevates the levels of early glycolytic intermediates. Although this may be beneficial in the fasted state to conserve systemic glucose, it represents a pathological impairment in diabetes mellitus.

Decreased Mitochondrial Pyruvate Transport Activity in the Diabetic Heart: ROLE OF MITOCHONDRIAL PYRUVATE CARRIER 2 (MPC2) ACETYLATION.

Alterations in mitochondrial function contribute to diabetic cardiomyopathy. We have previously shown that heart mitochondrial proteins are hyperacetylated in OVE26 mice, a transgenic model of type 1 diabetes. However, the universality of this modification and its functional consequences are not well established. In this study, we demonstrate that Akita type 1 diabetic mice exhibit hyperacetylation. Functionally, isolated Akita heart mitochondria have significantly impaired maximal (state 3) respiration with physiological pyruvate (0.1 mm) but not with 1.0 mm pyruvate. In contrast, pyruvate dehydrogenase activity is significantly decreased regardless of the pyruvate concentration. We found that there is a 70% decrease in the rate of pyruvate transport in Akita heart mitochondria but no decrease in the mitochondrial pyruvate carriers 1 and 2 (MPC1 and MPC2). The potential role of hyperacetylation in mediating this impaired pyruvate uptake was examined. The treatment of control mitochondria with the acetylating agent acetic anhydride inhibits pyruvate uptake and pyruvate-supported respiration in a similar manner to the pyruvate transport inhibitor α-cyano-4-hydroxycinnamate. A mass spectrometry selective reactive monitoring assay was developed and used to determine that acetylation of lysines 19 and 26 of MPC2 is enhanced in Akita heart mitochondria. Expression of a double acetylation mimic of MPC2 (K19Q/K26Q) in H9c2 cells was sufficient to decrease the maximal cellular oxygen consumption rate. This study supports the conclusion that deficient pyruvate transport activity, mediated in part by acetylation of MPC2, is a contributor to metabolic inflexibility in the diabetic heart.

Increased membrane affinity of the C1 domain of protein kinase Cdelta compensates for the lack of involvement of its C2 domain in membrane recruitment.

Protein kinase C (PKC) family members are allosterically activated following membrane recruitment by specific membrane-targeting modules. Conventional PKC isozymes are recruited to membranes by two such modules: a C1 domain, which binds diacylglycerol (DAG), and a C2 domain, which is a Ca2+-triggered phospholipid-binding module. In contrast, novel PKC isozymes respond only to DAG, despite the presence of a C2 domain. Here, we address the molecular mechanism of membrane recruitment of the novel isozyme PKCdelta. We show that PKCdelta and a conventional isozyme, PKCbetaII, bind membranes with comparable affinities. However, dissection of the contribution of individual domains to this binding revealed that, although the C2 domain is a major determinant in driving the interaction of PKCbetaII with membranes, the C2 domain of PKCdelta does not bind membranes. Instead, the C1B domain is the determinant that drives the interaction of PKCdelta with membranes. The C2 domain also does not play any detectable role in the activity or subcellular location of PKCdelta in cells; in vivo imaging studies revealed that deletion of the C2 domain does not affect the stimulus-dependent translocation or activity of PKCdelta. Thus, the increased affinity of the C1 domain of PKCdelta allows this isozyme to respond to DAG alone, whereas conventional PKC isozymes require the coordinated action of Ca2+ binding to the C2 domain and DAG binding to the C1 domain for activation.