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.

insulin signaling contributes to a dysfunctional PKA response. To do so, we isolated adult 24 cardiomyocytes (ACMs) from wild type and Akita type 1 diabetic mice. ACMs were cultured in 25 the presence or absence of insulin and PKA signaling was visualized by immunofluorescence 26 microscopy using an antibody that recognizes proteins specifically phosphorylated by PKA. We 27 found significant decreases in proteins phosphorylated by PKA in wild type ACMs cultured in the 28 absence of insulin. Akita ACMs also had decreased PKA signaling in the absence of insulin and 29 this was not rescued by insulin. The decrease in PKA signaling was observed regardless of 30 whether the kinase was stimulated with a beta-agonist, a cell-permeable cAMP analog, or with 31 phosphodiesterase inhibitors. PKA content was unaffected, suggesting that the decrease in 32 PKA signaling may be occurring by the loss of specific PKA substrates. Phospho-specific 33 antibodies were therefore used to discern which potential substrates may be sensitive to the 34 loss of insulin. Contractile proteins were phosphorylated similarly in wild type and Akita ACMs 35 regardless of insulin. However, phosphorylation of the glycolytic regulator, PFK-2, was 36 significantly decreased in an insulin-dependent manner in wild type ACMs and in an insulin-37 independent manner in Akita ACMs. These results demonstrate a defect in PKA activation in the

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Heart disease is the leading cause of death for patients with type I or type II diabetes [1].

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This is in part because diabetes directly impacts cardiac function independently of other 45 comorbidities. This is termed diabetic cardiomyopathy and it is a multi-factorial condition 46 resulting from the metabolic stresses of disrupted insulin signaling, hyperglycemia and 47 hyperlipidemia, and mitochondrial dysfunction [2]. In addition, there are also disruptions in 48 protein kinase A (PKA) signaling, the molecular pathway that mediates the metabolic and 49 contractile responses to sympathetic stimulation [3,4]. While the molecular mechanisms 50 contributing to diabetic cardiomyopathy are highly interrelated, the relationship between 51 metabolic perturbances and changes in PKA signaling are not fully understood.

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In the healthy heart the sympathetic nervous system functions through β-adrenergic 53 signaling to increase cardiac contractility. Catecholamines bind to Gα s -coupled β-adrenergic 54 receptors, stimulate adenylate cyclase, and subsequently increase cAMP to activate PKA. PKA 55 then phosphorylates proteins involved in calcium cycling (troponin, SERCA, and 56 phospholamban) and proteins that affect metabolic substrate selection (phosphofructokinase-2 57 (PFK-2) and acetyl-CoA carboxylase-2) [5,6]. Glucose uptake and oxidation are the primary 58 means of meeting the rapid increase in energy demands in response to sympathetic stimulation 59 [6,7]. In this way the increase in contractility is orchestrated with activation of metabolic 60 pathways to ensure energy demands are met.

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As an insulin sensitive tissue, the heart is affected by either decreases in circulating 62 insulin or by the loss of insulin signaling that occur with type 1 or type 2 diabetes [8,9]

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We next examined how the lack of insulin affects PKA signaling. Freshly isolated ACMs 185 were cultured in insulin-free media for 18h and then stimulated with ISO. PKA-substrate 186 phosphorylation was significantly blunted both basally and following ISO treatment ( Fig 1A).

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Insulin signaling is necessary downstream of cAMP production.

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The decrease in PKA substrate phosphorylation in cardiomyocytes cultured without 205 insulin may be due to changes in β-adrenergic receptors or their response to ligand binding, 206 thereby leading to a dampened response to ISO stimulation. We therefore examined direct 207 activation of PKA using the cell permeable cAMP analog, 8Br-cAMP. Like ISO, 8Br-cAMP induced a robust increase in PKA-substrate phosphorylation in cardiomyocytes isolated from 209 control mice cultured with insulin. However, substrate phosphorylation stimulated by 8Br-cAMP 210 was significantly blunted in ACMs cultured overnight without insulin (Fig 2). Likewise, 211 cardiomyocytes isolated from adult Akita mice had significantly blunted response to 8Br-cAMP 212 and this was not rescued by an 18h culture with insulin. This suggests that the defect in PKA 213 signaling induced by the absence of insulin is downstream of the β-adrenergic receptors and 214 cAMP production. ACMs from control and Akita mice to determine whether blocking PDE activity is sufficient to 242 recover PKA signaling. Addition of IBMX, in the absence of other PKA agonists, was sufficient to 243 stimulate PKA signaling by 2.5-fold (Fig 2). However, the effect of IBMX was blunted in wild type

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ACMs cultured without insulin and in Akita ACMs regardless of whether insulin was present.

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This demonstrates PDE inhibition is sufficient to stimulate PKA substrate phosphorylation 246 similarly to a PKA agonist when insulin is present. However, this effect is blunted by acute 247 insulin withdrawal and in Akita cardiomyocytes.

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We next sought to determine the combined effects of ISO and IBMX on PKA signaling.

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ACMs from control or Akita diabetic mice were cultured overnight in the presence or absence of

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Recent work has identified a relationship between cardiac insulin signaling and the 272 content and activity of PDE4 [11]. Specifically, the hyperinsulinemia that occurs with type 2 273 diabetes is associated with increased PDE4B content which thereby decreases cAMP and 274 attenuates β-adrenergic signaling [11]. We therefore tested to see if reciprocally the lack of substrates. However, the most pronounced effect was observed with inhibitors of PDE4 (Fig 4).

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Next, we tested if PDE4 inhibition could restore PKA signaling in the absence of insulin. While

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PKA signaling in ACMs from control and Akita mice treated with RO-20-1724 closely 283 approximated the effects of IBMX, there was no rescue in ACMs cultured in the absence of 284 insulin (S3 Fig). In addition, we also examined PDE4 content by immunofluorescence and 285 determined that the presence or absence of insulin had no effect on its content (S4 Fig). 286 Consistent with previous reports [17,18], this supports that PDE4 is the primary regulator of 287 cAMP degradation in mouse cardiomyocytes, that its content is not affected by acute changes in 288 insulin, and that its inhibition is not sufficient to recover PKA activity when insulin signaling is 289 absent.

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Phosphorylation of PFK2 is decreased in diabetic conditions.

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Our results demonstrate that the absence of insulin affects PKA substrate 302 phosphorylation downstream of β-adrenergic receptors and that this is not mediated by changes 303 in adenylate cyclase or PDE activities. We next evaluated PKA signaling using phospho-specific antibodies to identify substrates that may be differentially phosphorylated in the absence of 305 insulin. The bifunctional enzyme 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFK-2) 306 is a glycolytic regulator and substrate of PKA [19,20]. When the cardiac isoform (gene produce 307 of pfkfb2) is phosphorylated, PFK-2 increases the production of fructose-2,6-bisphosphate, a 308 potent allosteric activator of the glycolytic enzyme phosphofructokinase-1 (PFK-1). Furthermore,

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we have previously reported that the content of PFK-2, is regulated by insulin signaling [4,21].  substrates are also affected by insulin.

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We demonstrate here that the loss of insulin decreases PKA signaling in ACMs.

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Reciprocally, previous studies have shown that PKA signaling can also alter the effects of 441 insulin. In neonatal rat cardiomyocytes and mice with chronic β-adrenergic stimulation there is a 442 PKA-dependent decrease in insulin signaling [27,28]. The mechanism involves insulin receptor there is synergistic enhancement of Akt phosphorylation, GLUT4 translocation, and glucose uptake [28,29]. This mediates the increase in glucose uptake and oxidation in response to 449 acute β-adrenergic stimulation.

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Our results provide new insights into how diabetes may impact the heart. We clearly 451 show a decrease in PKA signaling in adult cardiomyocytes when insulin is absent. A novelty of 452 this study was using immunofluorescence microscopy as a means of monitoring PKA signaling.