Control of NAD+ homeostasis by autophagic flux modulates mitochondrial and cardiac function.

Impaired autophagy is known to cause mitochondrial dysfunction and heart failure, in part due to altered mitophagy and protein quality control. However, whether additional mechanisms are involved in the development of mitochondrial dysfunction and heart failure in the setting of deficient autophagic flux remains poorly explored. Here, we show that impaired autophagic flux reduces nicotinamide adenine dinucleotide (NAD+) availability in cardiomyocytes. NAD+ deficiency upon autophagic impairment is attributable to the induction of nicotinamide N-methyltransferase (NNMT), which methylates the NAD+ precursor nicotinamide (NAM) to generate N-methyl-nicotinamide (MeNAM). The administration of nicotinamide mononucleotide (NMN) or inhibition of NNMT activity in autophagy-deficient hearts and cardiomyocytes restores NAD+ levels and ameliorates cardiac and mitochondrial dysfunction. Mechanistically, autophagic inhibition causes the accumulation of SQSTM1, which activates NF-κB signaling and promotes NNMT transcription. In summary, we describe a novel mechanism illustrating how autophagic flux maintains mitochondrial and cardiac function by mediating SQSTM1-NF-κB-NNMT signaling and controlling the cellular levels of NAD+.

Deficiency of endothelial sirtuin1 in mice stimulates skeletal muscle insulin sensitivity by modifying the secretome.

Downregulation of endothelial Sirtuin1 (Sirt1) in insulin resistant states contributes to vascular dysfunction. Furthermore, Sirt1 deficiency in skeletal myocytes promotes insulin resistance. Here, we show that deletion of endothelial Sirt1, while impairing endothelial function, paradoxically improves skeletal muscle insulin sensitivity. Compared to wild-type mice, male mice lacking endothelial Sirt1 (E-Sirt1-KO) preferentially utilize glucose over fat, and have higher insulin sensitivity, glucose uptake, and Akt signaling in fast-twitch skeletal muscle. Enhanced insulin sensitivity of E-Sirt1-KO mice is transferrable to wild-type mice via the systemic circulation. Endothelial Sirt1 deficiency, by inhibiting autophagy and activating nuclear factor-kappa B signaling, augments expression and secretion of thymosin beta-4 (Tß4) that promotes insulin signaling in skeletal myotubes. Thus, unlike in skeletal myocytes, Sirt1 deficiency in the endothelium promotes glucose homeostasis by stimulating skeletal muscle insulin sensitivity through a blood-borne mechanism, and augmented secretion of Tß4 by Sirt1-deficient endothelial cells boosts insulin signaling in skeletal muscle cells.

MiR-34a and endothelial biology.

MicroRNAs (miRNAs) are endogenous ∼22 nt long RNAs that play important gene-regulatory roles in cells by pairing to the mRNAs of protein-coding genes to direct their posttranscriptional repression. Many miRNAs have been identified in endothelial cells and play important roles in endothelial biology. miR-34a is relatively early identified in endothelial cells and has been involved in regulating endothelial functions, angiogenesis, differentiation, senescence, inflammatory response, responses to shear stress, and mitochondrial function. This review outlines the current understanding of miR-34a in endothelial biology and discusses its potential as a therapeutic target to treat vascular diseases.

Phosphoinositide dependent protein kinase 1 is required for exercise-induced cardiac hypertrophy but not the associated mitochondrial adaptations.

Phosphoinositide-dependent protein kinase-1 (PDPK1) is an important mediator of phosphatidylinositol 3-kinase (PI3K) signaling. We previously reported that PI3K but not Akt signaling mediates the increase in mitochondrial oxidative capacity following physiological cardiac hypertrophy. To determine if PDPK1 regulates these metabolic adaptations we examined mice with cardiomyocyte-specific heterozygous knockout of PDPK1 (cPDPK1(+/-)) after 5 wk. exercise swim training. Akt phosphorylation at Thr308 increased by 43% in wildtype (WT) mice but not in cPDPK1(+/-) mice following exercise training. Ventricular contractile function was not different between WT and cPDPK1(+/-) mice at baseline. In addition, exercise did not influence ventricular function in WT or cPDPK1(+/-) mice. Heart weight normalized to tibia length ratios increased by 13.8% in WT mice (6.2±0.2 vs. 7.1±0.2, P=0.001), but not in cPDPK1(+/-) (6.2±0.3 vs. 6.5±0.2, P=0.20) mice after swim training. Diastolic LV dimension increased in WT mice (3.7±0.1 vs. 4.0±0.1 mm, P=0.01) but not in cPDPK1(+/-) (3.8±0.1 vs. 3.7±0.1 mm, P=0.56) following swim training. Maximal mitochondrial oxygen consumption (VADP, nmol/min/mg) using palmitoyl carnitine as a substrate was significantly increased in mice of all genotypes following swim training (WT: 13.6±0.6 vs.16.1±0.9, P=0.04; cPDPK1(+/-): 12.4±0.6 vs.15.9±1.2, P=0.04). These findings suggest that PDPK1 is required for exercise-induced cardiac hypertrophy but does not contribute to exercise-induced increases in mitochondrial function.

Ceramide-Initiated Protein Phosphatase 2A Activation Contributes to Arterial Dysfunction In Vivo.

Prior studies have implicated accumulation of ceramide in blood vessels as a basis for vascular dysfunction in diet-induced obesity via a mechanism involving type 2 protein phosphatase (PP2A) dephosphorylation of endothelial nitric oxide synthase (eNOS). The current study sought to elucidate the mechanisms linking ceramide accumulation with PP2A activation and determine whether pharmacological inhibition of PP2A in vivo normalizes obesity-associated vascular dysfunction and limits the severity of hypertension. We show in endothelial cells that ceramide associates with the inhibitor 2 of PP2A (I2PP2A) in the cytosol, which disrupts the association of I2PP2A with PP2A leading to its translocation to the plasma membrane. The increased association between PP2A and eNOS at the plasma membrane promotes dissociation of an Akt-Hsp90-eNOS complex that is required for eNOS phosphorylation and activation. A novel small-molecule inhibitor of PP2A attenuated PP2A activation, prevented disruption of the Akt-Hsp90-eNOS complex in the vasculature, preserved arterial function, and maintained normal blood pressure in obese mice. These findings reveal a novel mechanism whereby ceramide initiates PP2A colocalization with eNOS and demonstrate that PP2A activation precipitates vascular dysfunction in diet-induced obesity. Therapeutic strategies targeted to reducing PP2A activation might be beneficial in attenuating vascular complications that exist in the context of type 2 diabetes, obesity, and conditions associated with insulin resistance.

Lipid-induced NOX2 activation inhibits autophagic flux by impairing lysosomal enzyme activity.

Autophagy is a catabolic process involved in maintaining energy and organelle homeostasis. The relationship between obesity and the regulation of autophagy is cell type specific. Despite adverse consequences of obesity on cardiac structure and function, the contribution of altered cardiac autophagy in response to fatty acid overload is incompletely understood. Here, we report the suppression of autophagosome clearance and the activation of NADPH oxidase (Nox)2 in both high fat-fed murine hearts and palmitate-treated H9C2 cardiomyocytes (CMs). Defective autophagosome clearance is secondary to superoxide-dependent impairment of lysosomal acidification and enzyme activity in palmitate-treated CMs. Inhibition of Nox2 prevented superoxide overproduction, restored lysosome acidification and enzyme activity, and reduced autophagosome accumulation in palmitate-treated CMs. Palmitate-induced Nox2 activation was dependent on the activation of classical protein kinase Cs (PKCs), specifically PKCßII. These findings reveal a novel mechanism linking lipotoxicity with a PKCß-Nox2-mediated impairment in pH-dependent lysosomal enzyme activity that diminishes autophagic turnover in CMs.

A low-carbohydrate/high-fat diet reduces blood pressure in spontaneously hypertensive rats without deleterious changes in insulin resistance.

Previous studies reported that diets high in simple carbohydrates could increase blood pressure in rodents. We hypothesized that the converse, a low-carbohydrate/high-fat diet, might reduce blood pressure. Six-week-old spontaneously hypertensive rats (SHR; n = 54) and Wistar-Kyoto rats (WKY; n = 53, normotensive control) were fed either a control diet (C; 10% fat, 70% carbohydrate, 20% protein) or a low-carbohydrate/high-fat diet (HF; 20% carbohydrate, 60% fat, 20% protein). After 10 wk, SHR-HF had lower (P < 0.05) mean arterial pressure than SHR-C (148 ± 3 vs. 159 ± 3 mmHg) but a similar degree of cardiac hypertrophy (33.4 ± 0.4 vs. 33.1 ± 0.4 heart weight/tibia length, mg/mm). Mesenteric arteries and the entire aorta were used to assess vascular function and endothelial nitric oxide synthase (eNOS) signaling, respectively. Endothelium-dependent (acetylcholine) relaxation of mesenteric arteries was improved (P < 0.05) in SHR-HF vs. SHR-C, whereas contraction (potassium chloride, phenylephrine) was reduced (P < 0.05). Phosphorylation of eNOSSer1177 increased (P < 0.05) in arteries from SHR-HF vs. SHR-C. Plasma glucose, insulin, and homoeostatic model of insulin assessment were lower (P < 0.05) in SHR-HF vs. SHR-C, whereas peripheral insulin sensitivity (insulin tolerance test) was similar. After a 10-h fast, insulin stimulation (2 U/kg ip) increased (P < 0.05) phosphorylation of AktSer473 and S6 in heart and gastrocnemius similarly in SHR-C vs. SHR-HF. In conclusion, a low-carbohydrate/high-fat diet reduced blood pressure and improved arterial function in SHR without producing signs of insulin resistance or altering insulin-mediated signaling in the heart, skeletal muscle, or vasculature.

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.

Heat and α1-adrenergic responsiveness in human skeletal muscle feed arteries: the role of nitric oxide.

Increased local temperature exerts a sympatholytic effect on human skeletal muscle feed arteries. We hypothesized that this attenuated α(1)-adrenergic receptor responsiveness may be due to a temperature-induced increase in nitric oxide (NO) bioavailability, thereby reducing the impact of the α(1)-adrenergic receptor agonist phenylephrine (PE). Thirteen human skeletal muscle feed arteries were harvested, and wire myography was used to generate PE concentration-response curves at 37 °C and 39 °C, with and without the NO synthase (NOS) inhibitor N(G)-monomethyl-L-arginine (L-NMMA). A subset of arteries (n = 4) were exposed to 37 °C or 39 °C, and the protein content of endothelial NOS (eNOS) and α(1)-adrenergic receptors was determined by Western blot analysis. Additionally, cultured bovine endothelial cells were exposed to static or shear stress conditions at 37 °C and 39 °C and assayed for eNOS activation (phosphorylation at Ser(1177)), eNOS expression, and NO metabolites [nitrate + nitrite (NOx)]. Maximal PE-induced vasocontraction (PE(max)) was lower at 39 °C than at 37 °C [39 ± 10 vs. 84 ± 30% maximal response to 100 mM KCl (KCl(max))]. NO blockade restored vasocontraction at 39 °C to that achieved at 37 °C (80 ± 26% KCl(max)). Western blot analysis of the feed arteries revealed that heating increased eNOS protein, but not α(1)-adrenergic receptors. Heating of bovine endothelial cells resulted in greater shear stress-induced eNOS activation and NOx production. Together, these data reveal for the first time that, in human skeletal muscle feed arteries, NO blockade can restore the heat-attenuated α(1)-adrenergic receptor-mediated vasocontraction and implicate endothelium-derived NO bioavailability as a major contributor to heat-induced sympatholysis. Consequently, these findings highlight the important role of vasodilators in modulating the vascular response to vasoconstrictors.

Ceramide mediates vascular dysfunction in diet-induced obesity by PP2A-mediated dephosphorylation of the eNOS-Akt complex.

Vascular dysfunction that accompanies obesity and insulin resistance may be mediated by lipid metabolites. We sought to determine if vascular ceramide leads to arterial dysfunction and to elucidate the underlying mechanisms. Pharmacological inhibition of de novo ceramide synthesis, using the Ser palmitoyl transferase inhibitor myriocin, and heterozygous deletion of dihydroceramide desaturase prevented vascular dysfunction and hypertension in mice after high-fat feeding. These findings were recapitulated in isolated arteries in vitro, confirming that ceramide impairs endothelium-dependent vasorelaxation in a tissue-autonomous manner. Studies in endothelial cells reveal that de novo ceramide biosynthesis induced protein phosphatase 2A (PP2A) association directly with the endothelial nitric oxide synthase (eNOS)/Akt/Hsp90 complex that was concurrent with decreased basal and agonist-stimulated eNOS phosphorylation. PP2A attenuates eNOS phosphorylation by preventing phosphorylation of the pool of Akt that colocalizes with eNOS and by dephosphorylating eNOS. Ceramide decreased the association between PP2A and the predominantly cytosolic inhibitor 2 of PP2A. We conclude that ceramide mediates obesity-related vascular dysfunction by a mechanism that involves PP2A-mediated disruption of the eNOS/Akt/Hsp90 signaling complex. These results provide important insight into a pathway that represents a novel target for reversing obesity-related vascular dysfunction.

Knockout of insulin receptors in cardiomyocytes attenuates coronary arterial dysfunction induced by pressure overload.

Ablating insulin receptors in cardiomyocytes causes subendocardial fibrosis and left ventricular (LV) dysfunction after 4 wk of transverse aortic constriction (TAC). To determine whether these maladaptive responses are precipitated by coronary vascular dysfunction, we studied mice with cardiomyocyte-restricted knock out of insulin receptors (CIRKO) and wild-type (WT) TAC mice before the onset of overt LV dysfunction. Two weeks of TAC produced comparable increases (P < 0.05 vs. respective sham) in heart weight/body weight (mg/g) in WT-TAC (8.03 ± 1.14, P < 0.05 vs. respective sham) and CIRKO-TAC (7.76 ± 1.25, P < 0.05 vs. respective sham) vs. WT-sham (5.64 ± 0.11) and CIRKO-sham (4.64 ± 0.10) mice. In addition, 2 wk of TAC were associated with similar LV geometry and function (echocardiography) and interstitial fibrosis (picrosirius red staining) in CIRKO and WT mice. Responses to acetylcholine (ACh), N(G)-monomethyl-L-arginine (l-NMMA), and sodium nitroprusside (SNP) were measured in coronary arteries that were precontracted to achieve ∼70% of maximal tension development using the thromboxane A(2) receptor mimetic U-46619 (∼3 × 10(-6) M). ACh-evoked vasorelaxation was absent in WT-TAC but was present in CIRKO-TAC albeit reduced relative to sham-operated animals. l-NMMA-evoked tension development was similar in vessels from CIRKO-TAC mice but was lower (P < 0.05) in WT-TAC animals vs. the respective sham-operated groups, and SNP-evoked vasorelaxation was similar among all mice. Thus estimates of stimulated and basal endothelial nitric oxide release were better preserved in CIRKO vs. WT mice in response to 2 wk of TAC. These findings indicate that maladaptive LV remodeling previously observed in CIRKO-TAC mice is not precipitated by coronary artery dysfunction, because CIRKO mice exhibit compensatory mechanisms (e.g., increased eNOS transcript and protein) to maintain coronary endothelial function in the setting of pressure overload.

Exercise improves the dilatation function of mesenteric arteries in postmyocardial infarction rats via a PI3K/Akt/eNOS pathway-mediated mechanism.

Myocardial infarction (MI) has been shown to induce endothelial dysfunction in peripheral resistance arteries and thus increase peripheral resistance. This study was designed to investigate the underlying mechanisms of post-MI-related dysfunctional dilatation of peripheral resistance arteries and, furthermore, to examine whether exercise may restore dysfunctional dilatation of peripheral resistance arteries. Adult male Sprague-Dawley rats were divided into three groups: sham-operated, MI, and MI + exercise. Ultrastructure and relaxation function of the mesenteric arteries, as well as phosphatidylinositol-3 kinase (PI3K), Akt kinases (Akt), endothelial nitric oxide synthase (eNOS) activity, and phosphorylation of PI3K, Akt, and eNOS by ACh were determined. Post-MI rats exhibited pronounced ultrastructural changes in mesenteric artery endothelial cells and endothelial dysfunction. In addition, the activities of PI3K, Akt, and eNOS, and their phosphorylation by ACh were significantly attenuated in mesenteric arteries (P < 0.05-0.01). After 8 wk of exercise, not only did endothelial cells appeared more normal in structure, but also ameliorated post-MI-associated mesenteric arterial dysfunction, which were accompanied by elevated activities of PI3K, Akt, and eNOS, and their phosphorylation by ACh (P < 0.05-0.01). Importantly, inhibition of either PI3K or eNOS attenuated exercise-induced restoration of the dilatation function and blocked PI3K, Akt, and eNOS phosphorylation by ACh in the mesenteric arteries. These data demonstrate that MI induces dysfunctional dilation of peripheral resistance arteries by degradation of endothelial structural integrity and attenuating PI3K-Akt-eNOS signaling. Exercise may restore dilatation function of peripheral resistance arteries by protecting endothelial structural integrity and increasing PI3K-Akt-eNOS signaling cascades.

Exercise benefits cardiovascular health in hyperlipidemia rats correlating with changes of the cardiac vagus nerve.

The role of exercise training on hemodynamic parameters, blood lipid profiles, inflammatory cytokines, cholinesterase-positive nerves and muscarinic cholinergic (M(2)) receptors expression in the heart was investigated in Sprague-Dawley male rats with hyperlipidemia (HL). The rats were subjected to a high-fat diet and exercise training for 8 weeks, and then the hemodynamic parameters, the profiles of blood lipid and inflammatory cytokines, and the expression of cholinesterase-positive nerves and M(2) receptors were measured. HL rats displayed cardiac dysfunction, dysregulation of inflammatory cytokines, and decreased cholinesterase-positive nerves and M(2) receptors expression. The combination of hyperlipidemia with exercise training (AT) restored the profiles of blood lipids and the levels of inflammatory cytokines. In addition, AT and HL + AT improved cardiac function with increasing cholinesterase-positive nerves and M(2) receptors expression. Overall, these data show that the increased expression of cholinesterase-positive nerves and M(2) receptors in the heart is partially responsible for the benefits of exercise training on cardiac function in hyperlipidemia rats.

Endothelial nitric oxide synthase phosphorylation in treadmill-running mice: role of vascular signalling kinases.

The intracellular signalling kinases Akt/protein kinase B (Akt), protein kinase A (PKA) and adenosine monophosphate-activated protein kinase (AMPK) are phosphorylated in response to increased mechanical force or perfusion rate in cultured endothelial cells or isolated blood vessels. All three kinases phosphorylate endothelial nitric oxide synthase (eNOS) on serine (S) 1177, while Akt and PKA additionally phosphorylate eNOS on S617 and S635 respectively. Although these kinases might contribute to subsequent activation of eNOS during dynamic exercise, the specific mediators of exercise-induced eNOS phosphorylation and activation in vivo are unknown. We determined the impact of 50 min of treadmill running on the phosphorylation of Akt, AMPK, cyclic adenosine monophosphate response element binding protein (CREB - a target of PKA) and eNOS (S 1177, 635 and 617 and threonine (T) 495) in the presence or absence of pharmacological inhibition of PI3 kinase (PI3K) and Akt signalling using wortmannin. Compared to arteries from sedentary mice, eNOS enzyme activity was greater in vessels from treadmill-running animals and was associated with increased phosphorylation of Akt (S473), CREB (S133), AMPK (T172), and eNOS at S1177 and S617 but not at S635 or T495. These data suggest that Akt signalling is a major mediator of eNOS activation. To confirm this, treadmill-running was performed in the presence of vehicle (DMSO) or PI3K inhibition. Compared to results from sedentary mice, vascular Akt phosphorylation and eNOS phosphorylation at S617 during treadmill-running were prevented by wortmannin but not vehicle treatment, whereas exercise-related increases in AMPK and CREB phosphorylation were similar between groups. Arterial eNOS phosphorylation at S1177 increased during exercise after wortmannin treatment relative to values obtained from sedentary animals, but the elevation was blunted by approximately 50% compared to results from vehicle-treated mice. These findings indicate that Akt and AMPK contribute importantly to vascular eNOS S1177 phosphorylation during treadmill-running, and that AMPK is sufficient to activate p-eNOS S1177 in the presence of PI3K inhibition.

Contribution of insulin and Akt1 signaling to endothelial nitric oxide synthase in the regulation of endothelial function and blood pressure.

Impaired insulin signaling via phosphatidylinositol 3-kinase/Akt to endothelial nitric oxide synthase (eNOS) in the vasculature has been postulated to lead to arterial dysfunction and hypertension in obesity and other insulin resistant states. To investigate this, we compared insulin signaling in the vasculature, endothelial function, and systemic blood pressure in mice fed a high-fat (HF) diet to mice with genetic ablation of insulin receptors in all vascular tissues (TTr-IR(-/-)) or mice with genetic ablation of Akt1 (Akt1-/-). HF mice developed obesity, impaired glucose tolerance, and elevated free fatty acids that was associated with endothelial dysfunction and hypertension. Basal and insulin-mediated phosphorylation of extracellular signal-regulated kinase 1/2 and Akt in the vasculature was preserved, but basal and insulin-stimulated eNOS phosphorylation was abolished in vessels from HF versus lean mice. In contrast, basal vascular eNOS phosphorylation, endothelial function, and blood pressure were normal despite absent insulin-mediated eNOS phosphorylation in TTr-IR(-/-) mice and absent insulin-mediated eNOS phosphorylation via Akt1 in Akt1-/- mice. In cultured endothelial cells, 6 hours of incubation with palmitate attenuated basal and insulin-stimulated eNOS phosphorylation and NO production despite normal activation of extracellular signal-regulated kinase 1/2 and Akt. Moreover, incubation of isolated arteries with palmitate impaired endothelium-dependent but not vascular smooth muscle function. Collectively, these results indicate that lower arterial eNOS phosphorylation, hypertension, and vascular dysfunction following HF feeding do not result from defective upstream signaling via Akt, but from free fatty acid-mediated impairment of eNOS phosphorylation.

Aging-associated insulin resistance predisposes to hypertension and its reversal by exercise: the role of vascular vasorelaxation to insulin.

Aging is an independent risk factor for hypertension, and hypertension and insulin resistance commonly coexist in the elderly. This study was designed to examine the effects of aging-related insulin resistance on blood pressure (BP) and its underlying mechanisms, with specific focus on the role of exercise in reversing hypertensive response. Adult (6-month-old) and aging (24-month-old) male Sprague-Dawley rats were subjected to a 10 weeks free-of-loading swim training (60 min/day, 5 days/week). Arterial vasorelaxation, cardiac contraction, eNOS activation, and iNOS and gp91(phox) expression were determined. Under aging-related insulin resistance conditions, insulin infusion significantly elevated BP (P < 0.05). Aging caused significant endothelial dysfunction (P < 0.05 - 0.01), which was responsible for decreased arterial vasorelaxation to insulin. Aging attenuated myocardial contractile response to insulin, decreased eNOS expression and its phosphorylation by insulin, and increased iNOS and gp91(phox) expression in aging arteries (P < 0.01). Exercise improved insulin sensitivity, potentiated insulin's positive inotropic effects, facilitated arterial vasorelaxation to insulin, increased arterial eNOS activation in adult and aging rats, and thus attenuated insulin resistance-related hypertensive response to insulin. Moreover, exercise markedly reversed increased iNOS and gp91(phox) expression in aging arteries. Inhibition of eNOS with Cavtratin or L-NAME significantly blocked exercise-facilitated arterial vasorelaxation to insulin and exercise-lowered BP response to insulin. In conclusion, these results demonstrate that endothelial dysfunction in response to insulin, but not insulin's positive inotropic effects, plays an important role in the development of aging-related hypertension. The reversal of hypertensive response to insulin by exercise is most likely associated with improved insulin sensitivity in an eNOS-dependent manner and reduced oxidative and nitrative stresses.

Reduced cardiotropic response to insulin in spontaneously hypertensive rats: role of peroxisome proliferator-activated receptor-gamma-initiated signaling.

OBJECTIVES: Vascular insulin resistance plays a crucial pathogenic role in the development of genetic hypertension. However, it is not known whether hypertension-associated myocardial insulin resistance also exists, and whether this is involved in the development of related diseases, such as hypertensive heart failure. The present study aimed to determine whether hypertension-associated myocardial insulin resistance exists, in addition to any underlying mechanism. METHODS: Ventricular myocytes were enzymatically isolated from male Wistar-Kyoto (WKY) rats or spontaneously hypertensive rats (SHR) and myocyte shortening and intracellular Ca2+ transient were assessed, as was any signaling mechanism. RESULTS: Compared with WKY rats, insulin-stimulated myocardial glucose uptake was blunted in SHR. More importantly, the positive inotropic effect of insulin was significantly reduced in SHR myocytes. Moreover, peroxisome proliferator-activated receptor (PPAR)gamma and phosphatidylinositol 3 (PI-3) kinase p85 expression and insulin-induced Akt phosphorylation were reduced in SHR cardiomyocytes, but were markedly restored when animals were treated with rosiglitazone for 14 days. Pretreatment with an Akt inhibitor abolished the inotropic effect induced by insulin in rosiglitazone-treated SHR cardiomyocytes. CONCLUSIONS: The data obtained demonstrate that insulin resistance exists in SHR cardiomyocytes as manifested by both reduced insulin-stimulated glucose uptake and an impaired contractile response to insulin, which is attributable to decreased PPARgamma expression and subsequent impairment in PI-3 kinase/Akt signaling.

Insulin protects isolated hearts from ischemia/reperfusion injury: cross-talk between PI3-K/Akt and JNKs.

Our previous results have demonstrated that insulin reduces myocardial ischemia/reperfusion (MI/R) injury and increases the postischemic myocardial functions via activating the cellular survival signaling, i.e., phosphatidylinositol 3-kinase (PI3-K)-Akt-endothelial nitric oxide synthase (eNOS)-nitric oxide (NO) cascade. However, it remains largely controversial whether c-Jun NH2-terminal kinase (JNK) is involved in the effects of insulin on MI/R injury. Therefore, the aims of the present study were to investigate the role of JNK, especially the cross-talk between JNK and previously expatiated Akt signaling, in the protective effect of insulin on I/R myocardium. Isolated hearts from adult Sprague-Dawley rats were subjected to 30 min of regional ischemia and followed by 2 or 4 h of reperfusion (n=6). The hearts were pretreated with PI3-K inhibitor LY294002, or phosphorylated-JNK inhibitor SP600125, respectively, then perfused retrogradely with insulin, and the mechanical functions of hearts, including the heart rate (HR), left ventricular developed pressure (LVDP) and instantaneous first derivation of left ventricular pressure (+/-LVdp/dt(max)) were measured. At the end of reperfusion, the infarct size (IS) and apoptotic index (AI) were examined. MI/R caused significant cardiac dysfunction and myocardial apoptosis (strong TUNEL-positive staining). Compared with the control group, insulin treatment in MI/R rats exerted protective effects as evidenced by reduced myocardial IS [(28.9 +/- 2.0)% vs (45.0 +/- 4.0) %, n=6, P<0.01], inhibited cardiomyocyte apoptosis [decreased AI: (16.0 +/- 0.7) % vs (27.6 +/- 1.3) %, n=6, P<0.01] and improved recovery of cardiac systolic/diastolic function (including LVDP and +/-LVdp/dt(max)) at the end of reperfusion. Moreover, insulin resulted in 1.7-fold and 1.5-fold increases in Akt and JNK phosphorylation in I/R myocardium, respectively (n=6, P<0.05). Inhibition of Akt activation with LY294002 abolished, and inhibition of JNK activation with SP600125 enhanced the cardioprotection by insulin, respectively. And the abolishment by LY294002 could be partly converted by SP600125 pretreatment. In addition, SP600125 also decreased the Akt phosphorylation (n=6, P<0.05). These results demonstrate that insulin simultaneously activates both Akt and JNK, and the latter further increases the phosphorylation of Akt which attenuates MI/R injury and improves heart function; this cross-talk between Akt and JNK in the insulin signaling is involved in insulin-induced cardioprotective effect.

Swim training sensitizes myocardial response to insulin: role of Akt-dependent eNOS activation.

OBJECTIVES: Physical activity has been well known to benefit heart function. The improved autonomic nervous activity is considered to be mainly responsible for this beneficial effect. However, the precise mechanism behind the intrinsic myocardial responsiveness to exercise is still unclear. This study was designed to examine the effect of swim training on myocardial response to insulin with a special focus on the endogenous endothelial nitric oxide synthase (eNOS)-nitric oxide (NO) cascade. METHODS: Adult male Sprague-Dawley (SD) rats were subjected to a 10-week free-loading swim training (3 h/day, 5 days/week). Contractile response to insulin at the levels of cardiomyocytes and isolated perfused heart, myocardial glucose uptake and post-insulin receptor signaling cascades were evaluated. RESULTS: Swim training enhanced cardiac contractile response to insulin in cardiomyocytes and isolated perfused heart, respectively. The improved cardiac response was accompanied by facilitated insulin-stimulated glucose uptake, GLUT4 translocation and upregulation of Akt and eNOS expression (p<0.01). Treatment with insulin resulted in a 3.6- and 2.2-fold increase of eNOS phosphorylation (p<0.01), as well as a 3.0- and 1.9-fold increase of Akt phosphorylation in exercise and sedentary groups, respectively (p<0.01). In addition, exercise significantly facilitated insulin-induced myocardial NO production (p<0.01 vs. sedentary). Moreover, pretreatment with either LY294002, a phosphatidylinositol-3 kinase (PI-3K) inhibitor or L-NAME, a NOS inhibitor, abolished the exercise-induced sensitization of myocardial contractile response to insulin, insulin-induced NO production and phosphorylation of Akt and eNOS. CONCLUSION: These results demonstrate that swim training is capable of sensitizing myocardial contractile response to insulin via upregulation of Akt- and eNOS signaling cascades.