Dysfunction of endothelium-dependent relaxation to insulin via PKC-mediated GRK2/Akt activation in aortas of ob/ob mice

In diabetic states, hyperinsulinemia may negatively regulate Akt/endothelial nitric oxide synthase (eNOS) activation. Our main aim was to investigate whether and how insulin might negatively regulate Akt/eNOS activities via G protein-coupled receptor kinase 2 (GRK2) in aortas from ob/ob mice. Endothelium-dependent relaxation was measured in aortic rings from ob/ob mice (a type 2 diabetes model). GRK2, β-arrestin2, and Akt/eNOS signaling-pathway protein levels and activities were mainly assayed by Western blotting. Plasma insulin was significantly elevated in ob/ob mice. Insulin-induced relaxation was significantly decreased in the ob/ob aortas [vs. age-matched control (lean) ones]. The response in ob/ob aortas was enhanced by PKC inhibitor or GRK2 inhibitor. Akt (at Thr(308)) phosphorylation and eNOS (at Ser(1177)) phosphorylation, and also the β-arrestin2 protein level, were markedly decreased in the membrane fraction of insulin-stimulated ob/ob aortas (vs. insulin-stimulated lean ones). These membrane-fraction expressions were enhanced by GRK2 inhibitor and by PKC inhibitor in the ob/ob group but not in the lean group. PKC activity was much greater in ob/ob than in lean aortas. GRK2 protein and activity levels were increased in ob/ob and were greatly reduced by GRK2 inhibitor or PKC inhibitor pretreatment. These results suggest that in the aorta in diabetic mice with hyperinsulinemia an upregulation of GRK2 and a decrease in β-arrestin2 inhibit insulin-induced stimulation of the Akt/eNOS pathway and that GRK2 overactivation may result from an increase in PKC activity.     

Nitric oxide-enhanced caspase-3 and acidic sphingomyelinase interaction: a novel mechanism by which airway epithelial cells escape ceramide-induced apoptosis

Reactive nitrogen species (RNS) are implicated in the pathophysiology of inflammatory lung diseases such as asthma and chronic obstructive pulmonary disease. The molecular mechanisms and signaling events involved in lung cell injury by RNS are still poorly understood. In the current study, we observe a novel anti-apoptotic response to nitric oxide (NO) exposure (via the NO donors 3-morpholine-syndnonimine (SIN1) or papa-NONOate) of human airway epithelial (HAE) cells. NO exposure via the NO donors increased cellular ceramide levels via ceramide synthase but did not trigger an apoptotic response. Rather, exposure to the NO donors promoted an increase in the protein-protein interaction between acidic sphingomyelinase (aSMase) and caspase-3, with aSMase sequestering caspase-3 and preventing its cleavage. In contrast, when aSMase was silenced in HAE cells or was knocked out in mice, an increase in cleaved caspase-3 was observed. This elevated caspase-3 cleavage was further augmented upon NO exposure (via SIN1 or papa-NONOate) of HAE cells and could be prevented by an inhibitor to ceramide synthase. These results demonstrate a novel mechanism of NO modulation of apoptosis, in which HAE cells exposed to NO via an NO donor induces ceramide generation via ceramide synthase. However, this ceramide induction does not lead to apoptosis unless aSMase is knocked down, allowing the release of caspase-3, its activation and execution of apoptosis.     

Interleukin-6 impairs the insulin signaling pathway, promoting production of nitric oxide in human umbilical vein endothelial cells

Interleukin 6 (IL-6) is an independent predictor of type 2 diabetes and cardiovascular disease and is correlated with insulin resistance. Insulin stimulates nitric oxide (NO) production through the IRS-1/PI3-kinase/Akt/eNOS pathway (where IRS-1 is insulin receptor substrate 1, PI3-kinase is phosphatidylinositol 3-kinase, and eNOS is endothelial NO synthase). We asked if IL-6 affects insulin vasodilator action both in human umbilical vein endothelial cells (HUVEC) and in the aortas of C57BL/6J mice and whether this inhibitory effect was caused by increased Ser phosphorylation of IRS-1. We observed that IL-6 increased IRS-1 phosphorylation at Ser(312) and Ser(616); these effects were paralleled by increased Jun N-terminal protein kinase (JNK) and extracellular signal-regulated kinase 1/2 (ERK1/2) phosphorylation and reversed by JNK and ERK1/2 inhibition. In addition, IL-6 treatment resulted in impaired IRS-1 phosphorylation at Tyr(612), a site essential for engaging PI3-kinase. Furthermore, IL-6 treatment reduced insulin-stimulated phosphorylation of eNOS at the stimulatory Ser(1177) site and impaired insulin-stimulated eNOS dephosphorylation at the inhibitory Thr(495) site. Insulin-stimulated eNOS activation and NO production were also inhibited by IL-6; these effects were reversed by inhibition of JNK and ERK1/2. Treatment of C57BL/6J mice with IL-6 resulted in impaired insulin-dependent activation of the Akt/eNOS pathway in the aorta as a result of JNK and ERK1/2 activation. Our data suggest that IL-6 impairs the vasodilator effects of insulin that are mediated by the IRS-1/PI3-kinase/Akt/eNOS pathway through activation of JNK and ERK1/2.     

Statins restore ischemic limb blood flow in diabetic microangiopathy via eNOS/NO upregulation but not via PDGF-BB expression

3-Hydroxy-3-methyl-glutaryl CoA reductase inhibitors, or statins, have pleiotropic effects and can protect the vasculature in a manner independent of their lipid-lowering effect. The effectiveness of statins in reducing the risk of coronary events has been shown even in patients with diabetes, and their effects on diabetic complications have been reported. Using a model of severe hindlimb ischemia in streptozotocin-induced diabetic mice (STZ-DM), we investigated the effects and mechanisms of statin therapy in diabetic angiopathy in ischemic hindlimbs. As a result, STZ-DM mice frequently lost their hindlimbs after induced ischemia, whereas non-DM mice did not. Supplementation with statins significantly prevented autoamputation. We previously showed that diabetic vascular complications are caused by impaired expression of PDGF-BB, but statin therapy did not enhance PDGF-BB expression. Statins helped enhance endogenous endothelial nitric oxide (NO) synthase (eNOS) expression. Furthermore, the inhibition of NO synthesis by the administration of N(omega)-nitro-l-arginine methyl ester impaired the ability of statins to prevent STZ-DM mouse limb autoamputation, indicating that the therapeutic effect of statins in hindlimb ischemia in STZ-DM mice occurs via the eNOS/NO pathway. A combination therapy of statins and PDGF-BB gene supplementation was more effective for diabetic angiopathy than either therapy alone. In conclusion, these findings indicate that statin therapy might be useful for preventing intractable diabetic foot disease in patients with diabetic angiopathy.     

Cardiac malformations are associated with altered expression of vascular endothelial growth factor and endothelial nitric oxide synthase genes in embryos of diabetic mice

The aim of this study was to investigate the role of nitric oxide (NO), and the expression of endothelial nitric oxide synthase (eNOS) and vascular endothelial growth factor (VEGF) genes in developing hearts at embryonic day 13.5 of embryos from diabetic mice. The protein and mRNA expression levels of eNOS and VEGF were significantly altered in the developing hearts of embryos from diabetic mice. The NO level was significantly decreased, whereas the VEGF concentration was significantly increased in the developing hearts of the embryos from diabetic mice. In vitro study showed a significant reduction in eNOS expression and cell proliferation in cardiac myoblast cells exposed to high glucose concentrations. Further, high glucose induced apoptosis in myoblast cells. Ultrastructural changes characteristics of apoptosis, including cell blebbing, aggregation of ribosomes and vacuoles in the cytoplasm were also evident in myoblast cells exposed to high glucose. It is suggested that hyperglycemia alters the expression of eNOS and VEGF genes that are involved in the regulation of cell growth and vasculogenesis, thereby contributing to the cardiac malformations seen in embryos from diabetic mice.     

20-Hydroxyeicosatetraenoic Acid Impairs Endothelial Insulin Signaling by Inducing Phosphorylation of the Insulin Receptor Substrate-1 at Ser616

20-hydroxyeicosatetraenoic acid (20-HETE) induces endothelial dysfunction and is correlated with diabetes. This study was designed to investigate the effects of 20-HETE on endothelial insulin signaling.Human umbilical vein endothelial cells (HUVECs) or C57BL/6J mice were treated with 20-HETE in the presence or absence of insulin, and p-ERK1/2, p-JNK, IRS-1/PI3K/AKT/eNOS pathway, were examined in endothelial cells and aortas by immunoblotting. eNOS activity and nitric oxide production were measured. 20-HETE increased ERK1/2 phosphorylation and IRS-1 phosphorylation at Ser⁶¹⁶; these effects were reversed by ERK1/2 inhibition. We further observed that 20-HETE treatment resulted in impaired insulin-stimulated IRS-1 phosphorylation at Tyr⁶³² and subsequent PI3-kinase/Akt activation. Furthermore, 20-HETE treatment blocked insulin-stimulated phosphorylation of eNOS at the stimulatory Ser¹¹⁷⁷ site, eNOS activation and NO production; these effects were reversed by inhibiting ERK1/2. Treatment of C57BL/6J mice with 20-HETE resulted in ERK1/2 activation and impaired insulin-dependent activation of the IRS-1/PI3K/Akt/eNOS pathway in the aorta. Our data suggest that the 20-HETE activation of IRS-1 phosphorylation at Ser⁶¹⁶ is dependent on ERK1/2 and leads to impaired insulin-stimulated vasodilator effects that are mediated by the IRS-1/PI3K/AKT/eNOS pathway.     

Glucose and insulin improve cardiac efficiency and postischemic functional recovery in perfused hearts from type 2 diabetic (db/db) mice

Hearts from type 2 diabetic (db/db) mice demonstrate altered substrate utilization with high rates of fatty acid oxidation, decreased functional recovery following ischemia, and reduced cardiac efficiency. Although db/db mice show overall insulin resistance in vivo, we recently reported that insulin induces a marked shift toward glucose oxidation in isolated perfused db/db hearts. We hypothesize that such a shift in metabolism should improve cardiac efficiency and consequently increase functional recovery following low-flow ischemia. Hearts from db/db and nondiabetic (db/+) mice were perfused with 0.7 mM palmitate plus either 5 mM glucose (G), 5 mM glucose and 300 microU/ml insulin (GI), or 33 mM glucose and 900 microU/ml insulin (HGHI). Substrate oxidation and postischemic recovery were only moderately affected by GI and HGHI in db/+ hearts. In contrast, GI and particularly HGHI markedly increased glucose oxidation and improved postischemic functional recovery in db/db hearts. Cardiac efficiency was significantly improved in db/db, but not in db/+ hearts, in the presence of HGHI. In conclusion, insulin and glucose normalize cardiac metabolism, restore efficiency, and improve postischemic recovery in type 2 diabetic mouse hearts. These findings may in part explain the beneficial effect of glucose-insulin-potassium therapy in diabetic patients with cardiac complications.     

Role of acidic sphingomyelinase in Fas/CD95-mediated cell death

Engagement of the Fas receptor has been reported to induce ceramide generation via activation of acidic sphingomyelinase (aSMase). However, the role of aSMase in Fas-mediated cell death is controversial. Using genetically engineered mice deficient in the aSMase gene (aSMase(-/-)), we found that thymocytes, concanavalin A-activated T cells, and lipopolysaccharide-activated B cells derived from both aSMase(-/-) and aSMase(+/+) mice were equally sensitive to Fas-mediated cell death, triggered by either anti-Fas antibody or Fas ligand in vitro. Similarly, activation-induced apoptosis of T lymphocytes was unaffected by the status of aSMase, and aSMase(-/-) mice failed to show immunological symptoms seen in animals with defects in Fas function. In vivo, intravenous injection of 3 microg/25 g mouse body weight of anti-Fas Jo2 antibody into aSMase(-/-) mice failed to affect hepatocyte apoptosis or mortality, whereas massive hepatocyte apoptosis and animal death occurred in wild type littermates. Animals heterozygous for aSMase deficiency were also significantly protected. Susceptibility of aSMase(-/-) mice to anti-Fas antibody was demonstrated with higher antibody doses (>/=4 microg/25 g mouse). These data indicate a role for aSMase in Fas-mediated cell death in some but not all tissues.     

Altered metabolism causes cardiac dysfunction in perfused hearts from diabetic (db/db) mice

Contractile function and substrate metabolism were characterized in perfused hearts from genetically diabetic C57BL/KsJ-lepr(db)/lepr(db) (db/db) mice and their non-diabetic lean littermates. Contractility was assessed in working hearts by measuring left ventricular pressures and cardiac power. Rates of glycolysis, glucose oxidation, and fatty acid oxidation were measured using radiolabeled substrates ([5-(3)H]glucose, [U-(14)C]glucose, and [9,10-(3)H]palmitate) in the perfusate. Contractile dysfunction in db/db hearts was evident, with increased left ventricular end diastolic pressure and decreased left ventricular developed pressure, cardiac output, and cardiac power. The rate of glycolysis from exogenous glucose in diabetic hearts was 48% of control, whereas glucose oxidation was depressed to only 16% of control. In contrast, palmitate oxidation was increased twofold in db/db hearts. The hypothesis that altered metabolism plays a causative role in diabetes-induced contractile dysfunction was tested using perfused hearts from transgenic db/db mice that overexpress GLUT-4 glucose transporters. Both glucose metabolism and palmitate metabolism were normalized in hearts from db/db-human insulin-regulatable glucose transporter (hGLUT-4) hearts, as was contractile function. These findings strongly support a causative role of impaired metabolism in the cardiomyopathy observed in db/db diabetic hearts.     

Curcumin restores mitochondrial functions and decreases lipid peroxidation in liver and kidneys of diabetic db/db mice

Background

Nitrosative and oxidative stress play a key role in obesity and diabetes-related mitochondrial dysfunction. The objective was to investigate the effect of curcumin treatment on state 3 and 4 oxygen consumption, nitric oxide (NO) synthesis, ATPase activity and lipid oxidation in mitochondria isolated from liver and kidneys of diabetic db/db mice.

Results

Hyperglycaemia increased oxygen consumption and decreased NO synthesis in liver mitochondria isolated from diabetic mice relative to the control mice. In kidney mitochondria, hyperglycaemia increased state 3 oxygen consumption and thiobarbituric acid-reactive substances (TBARS) levels in diabetic mice relative to control mice. Interestingly, treating db/db mice with curcumin improved or restored these parameters to normal levels; also curcumin increased liver mitochondrial ATPase activity in db/db mice relative to untreated db/db mice.

Conclusions

These findings suggest that hyperglycaemia modifies oxygen consumption rate, NO synthesis and increases TBARS levels in mitochondria from the liver and kidneys of diabetic mice, whereas curcumin may have a protective role against these alterations.     

Klotho attenuates diabetic nephropathy in db/db mice and ameliorates high glucose-induced injury of human renal glomerular endothelial cells

Glomerular endothelial cell injury plays an important role in the development and progression of diabetic nephropathy (DN). The expression and function of klotho in glomerular endothelial cells remain unclear. Thus, this study aimed to investigate the expression and the functional role of klotho in DN progression in mice and in high glucose (HG)-induced cell injury of human renal glomerular endothelial cells (HRGECs) and the underlying mechanism. In this study, HRGECs were cultured with media containing HG to induce endothelial cell injury and db/db mice were used as DN model mice. Klotho was overexpressed or knocked down in HRECs to evaluate its role in HG-induced HRGECs injury. klotho-overexpressing adenovirus (rAAV-klotho) was injected into db/db mice via the tail vein to further validate the protective effect of klotho in DN. Decreased klotho expression was observed in DN patients, DN mice, and HG-exposed HRGECs. Furthermore, klotho overexpression significantly abolished the HG-induced HRGECs injury and activation of Wnt/β-catenin pathway and RAAS. In contrast, klotho knockdown exerted the opposite effects. Moreover, klotho attenuated diabetic nephropathy in db/db mice, which was also associated with inhibition of the Wnt/β-catenin pathway and RAAS. In conclusion, klotho attenuates DN in db/db mice and ameliorates HG-induced injury of HRGECs.     

Quercetin protects against diabetic encephalopathy via SIRT1/NLRP3 pathway in db/db mice

Epidemiological studies have found that diabetes and cognitive dysfunction are closely related. Quercetin has been certified with the effect on improving diabetes mellitus (DM) and cognitive impairment. However, the effect and related mechanism of quercetin on diabetic encephalopathy (DE) are still ambiguous. In this study, we used the db/db mice (diabetic model) to discover whether quercetin could improve DE through the Sirtuin1/NLRP3 (NOD-, LRR- and pyrin domain-containing 3) pathway. Behavioural results (Morris water maze and new object recognition tests) showed that quercetin (70 mg/kg) improved the learning and memory. Furthermore, quercetin alleviated insulin resistance and the level of fasting blood glucose. Besides, Western blot analysis also showed that quercetin increased the protein expressions of nerve- and synapse-related protein, including postsynapticdensity 93 (PSD93), postsynapticdensity 95 (PSD95), brain-derived neurotrophic factor (BDNF) and nerve growth factor (NGF) in the brain of db/db mice. Quercetin also increased the protein expression of SIRT1 and decreased the expression of NLRP3 inflammation-related proteins, including NLRP3, the adaptor protein ASC and cleaved Caspase-1, the pro-inflammatory cytokines IL-1β and IL-18. In conclusion, the present results indicate that the SIRT1/NLRP3 pathway may be a crucial mechanism for the neuroprotective effect of quercetin against DE.     

Losartan improves aortic endothelium-dependent relaxation via proline-rich tyrosine kinase 2/Src/Akt pathway in type 2 diabetic Goto-Kakizaki rats

In diabetic states, endothelial dysfunction is related to vascular complications. We hypothesized that insulin-induced relaxation and the associated proline-rich tyrosine kinase 2 (Pyk2)/Src/Akt pathway would be abnormal in aortas from the Goto-Kakizaki (GK) type 2 diabetic rat, which exhibits hyperglycemia/insulin resistance, and that losartan treatment of such rats (25 mg·kg(-1)·day(-1) for 2 wk) would correct these abnormalities. Endothelium-dependent relaxation was by measuring isometric force in helical strips of aortas from four groups, each of 30 rats: normal Wistar (control), GK (diabetic), losartan-treated normal, and losartan-treated GK. Pyk2, Src, and Akt/endothelial nitric oxide synthase (eNOS) signaling-pathway protein levels and activities were assayed mainly by Western blotting and partly by immunohistochemistry. In GK (vs. age-matched control) aortas, various insulin-stimulated levels [nitric oxide production and the phosphorylations of eNOS at Ser(1177), of Akt at Thr(308), of phosphoinositide-dependent kinase-1 (PDK1) at Ser(241), of Src at Tyr(416), and of Pyk2 at Tyr(579)] were all significantly decreased and unaffected by either Src inhibitor (PP2) or Pyk2 inhibitor (AG17), while the insulin-stimulated levels of insulin receptor substrate (IRS)-1 phosphorylation at Ser(307), total-eNOS, and total-Akt were significantly increased. Losartan treatment normalized these altered levels. The insulin-stimulated phosphorylation levels of Src/PDK1/Akt/eNOS, but not of Pyk2, were decreased by PP2 in control and losartan-treated GK, but not in GK, aortas. These results suggest that in the GK diabetic aorta increased phospho-IRS-1 (at Ser(307)) and decreased Pyk2/Src activity inhibit insulin-induced stimulation of the PDK/Akt/eNOS pathway. The observed increase in phospho-IRS-1 (at Ser(307)) may result from increased angiotensin II activity.     

Role of blood pressure and the renin-angiotensin system in development of diabetic nephropathy (DN) in eNOS-/- db/db mice

Randomized clinical trials have clearly shown that inhibition of the renin-angiotensin system (RAS) will slow the rate of progression of diabetic nephropathy, but controversy remains about whether the observed beneficial effects result from more than control of blood pressure. Deletion of eNOS in a model of type II diabetes, db/db mice (eNOS(-/-) db/db), induces an accelerated nephropathy and provides an excellent model of human diabetic nephropathy. As is frequently seen in type II diabetes, blood pressure is moderately elevated in eNOS(-/-) db/db mice. To determine the role of elevated blood pressure per se vs. additional deleterious effects of the RAS in mediation of disease progression, 8-wk-old eNOS(-/-) db/db mice were randomly divided into three groups: vehicle, treatment with the angiotensin-converting enzyme inhibitor (ACEI) captopril, or treatment with "triple therapy" (hydralazine, resperine, hydrocholorothiazide), and the animals were euthanized after treatment for 12 wk. Blood pressure was reduced to comparable levels with ACE inhibition or triple therapy. Although both treatment regimens decreased development of diabetic nephropathy, ACE inhibition led to more profound reductions in albuminuria, glomerulosclerosis, markers of tubulointerstitial injury, macrophage infiltration, and markers of inflammation. Therefore, this animal model suggests that while there is an important role for blood pressure control, RAS blockade provides additional benefits in slowing the progression of diabetic nephropathy.     

Endothelial function and vascular oxidative stress in long-lived GH/IGF-deficient Ames dwarf mice

Hypopituitary Ames dwarf mice have low circulating growth hormone (GH)/IGF-I levels, and they have extended longevity and exhibit many symptoms of delayed aging. To elucidate the vascular consequences of Ames dwarfism we compared endothelial O2(-) and H2O2 production, mitochondrial reactive oxygen species (ROS) generation, expression of antioxidant enzymes, and nitric oxide (NO) production in aortas of Ames dwarf and wild-type control mice. In Ames dwarf aortas endothelial O2(-) and H2O2 production and ROS generation by mitochondria were enhanced compared with those in vessels of wild-type mice. In Ames dwarf aortas there was a less abundant expression of Mn-SOD, Cu,Zn-SOD, glutathione peroxidase (GPx)-1, and endothelial nitric oxide synthase (eNOS). NO production and acetylcholine-induced relaxation were also decreased in aortas of Ames dwarf mice. In cultured wild-type mouse aortas and in human coronary arterial endothelial cells treatment with GH and IGF significantly reduced cellular O2(-) and H2O2 production and ROS generation by mitochondria and upregulated expression of Mn-SOD, Cu,Zn-SOD, GPx-1, and eNOS. Thus GH and IGF-I promote antioxidant phenotypic changes in the endothelial cells, whereas Ames dwarfism leads to vascular oxidative stress.     

Perfused hearts from Type 2 diabetic (db/db) mice show metabolic responsiveness to insulin

Diabetic (db/db) mice provide an animal model of Type 2 diabetes characterized by marked in vivo insulin resistance. The effect of insulin on myocardial metabolism has not been fully elucidated in this diabetic model. In the present study we tested the hypothesis that the metabolic response to insulin in db/db hearts will be diminished due to cardiac insulin resistance. Insulin-induced changes in glucose oxidation (GLUox) and fatty acid (FA) oxidation (FAox) were measured in isolated hearts from control and diabetic mice, perfused with both low as well as high concentration of glucose and FA: 10 mM glucose/0.5 mM palmitate and 28 mM glucose/1.1 mM palmitate. Both in the absence and presence of insulin, diabetic hearts showed decreased rates of GLUox and elevated rates of FAox. However, the insulin-induced increment in GLUox, as well as the insulin-induced decrement in FAox, was similar or even more pronounced in diabetic that in control hearts. During elevated FA and glucose supply, however, the effect of insulin was blunted in db/db hearts with respect to both FAox and GLUox. Finally, insulin-stimulated deoxyglucose uptake was markedly reduced in isolated cardiomyocytes from db/db mice, whereas glucose uptake in isolated perfused db/db hearts was clearly responsive to insulin. These results show that, despite reduced insulin-stimulated glucose uptake in isolated cardiomyocytes, isolated perfused db/db hearts are responsive to metabolic actions of insulin. These results should advocate the use of insulin therapy (glucose-insulin-potassium) in diabetic patients undergoing cardiac surgery or during reperfusion after an ischemic insult.     

Nicorandil alleviates apoptosis in diabetic cardiomyopathy through PI3K/Akt pathway

Nicorandil exerts myocardial protection through its antihypoxia and antioxidant effects. Here, we investigated whether it plays an anti‐apoptotic role in diabetic cardiomyopathy. Sprague‐Dawley rats were fed with high‐fat diet; then single intraperitoneal injection of streptozotocin was performed. Rats with fasting blood glucose (FBG) higher than 11.1 mmol/L were selected as models. Eight weeks after the models were built, rats were treated with nicorandil (7.5 mg/kg day and 15 mg/kg day respectively) for 4 weeks. H9c2 cardiomyocytes were treated with nicorandil and then stimulated with high glucose (33.3 mmol/L). TUNEL assay and level of bcl‐2, bax and caspase‐3 were measured. 5‐HD was used to inhibit nicorandil. Also, PI3K inhibitor (Miltefosine) and mTOR inhibitor (rapamycin) were used to inhibit PI3K/Akt pathway. The results revealed that nicorandil (both 7.5 mg/kg day and 15mg/kg day) treatment can increase the level of NO in the serum and eNOS in the heart of diabetic rats compared with the untreated diabetic group. Nicorandil can also improve relieve cardiac dysfunction and reduce the level of apoptosis. In vitro experiments, nicorandil (100 µmol) can attenuate the level of apoptosis stimulated by high glucose significantly in H9C2 cardiomyocyte compared with the untreated group. The effect of nicorandil on apoptosis was blocked by 5‐HD, and it was accompanied with inhibition of the phosphorylation of PI3K, Akt, eNOS, and mTOR. After inhibition of PI3K/Akt pathway, the protective effect of nicorandil is restrained. These results verified that as a NO donor, nicorandil can also inhibit apoptosis in diabetic cardiomyopathy which is mediated by PI3K/Akt pathway.     

Sildenafil-mediated acute cardioprotection is independent of the NO/cGMP pathway

Sildenafil, a potent inhibitor of phosphodiesterase type 5, has recently been investigated in animal models of myocardial ischemia-reperfusion (MI/R) injury. Previous studies have suggested that the protective effects of sildenafil are mediated via activation of endothelial nitric oxide (NO) synthesis (eNOS) and inducible NOS (iNOS). To further investigate the protective mechanism of sildenafil, we subjected wild-type, eNOS, and iNOS null animals to 30 min of myocardial ischemia and 24 h of reperfusion. Treatment with 0.06 mg/kg sildenafil 5 min before reperfusion significantly reduced myocardial infarct size in wild-type, eNOS null mice (eNOS(-/-)), and iNOS(-/-) animals. Additionally, the low dose utilized in this study did not alter myocardial cGMP. These results suggest that acute low-dose sildenafil-mediated cardioprotection is independent of eNOS, iNOS, and cGMP. In a second series of experiments, we investigated sildenafil in db/db diabetic mice subjected to MI/R. We found that sildenafil failed to protect diabetic mice against MI/R. However, NO(.) donor therapy was found to significantly protect against MI/R injury in both nondiabetic and diabetic mice, suggesting that protection could be conferred in diabetic mice and that the upstream modulator of soluble guanylyl cyclase, NO(.), may mediate protection independent of cGMP signaling. The present study suggests that further research is needed to delineate the precise mechanisms by which sildenafil exerts cardioprotection.     

Alterations of the Ca2+ signaling pathway in pancreatic beta-cells isolated from db/db mice

Upon glucose elevation, pancreatic beta-cells secrete insulin in a Ca²⁺-dependent manner. In diabetic animal models, different aspects of the calcium signaling pathway in beta-cells are altered, but there is no consensus regarding their relative contributions to the development of beta-cell dysfunction. In this study, we compared the increase in cytosolic Ca²⁺ ([Ca²⁺]_i) via Ca²⁺ influx, Ca²⁺ mobilization from endoplasmic reticulum (ER) calcium stores, and the removal of Ca²⁺ via multiple mechanisms in beta-cells from both diabetic db/db mice and nondiabetic C57BL/6J mice. We refined our previous quantitative model to describe the slow [Ca²⁺]_i recovery after depolarization in beta-cells from db/db mice. According to the model, the activity levels of the two subtypes of the sarco-endoplasmic reticulum Ca²⁺-ATPase (SERCA) pump, SERCA2 and SERCA3, were severely down-regulated in diabetic cells to 65% and 0% of the levels in normal cells. This down-regulation may lead to a reduction in the Ca²⁺ concentration in the ER, a compensatory up-regulation of the plasma membrane Na⁺/Ca²⁺ exchanger (NCX) and a reduction in depolarizationevoked Ca²⁺ influx. As a result, the patterns of glucosestimulated calcium oscillations were significantly different in db/db diabetic beta-cells compared with normal cells. Overall, quantifying the changes in the calcium signaling pathway in db/db diabetic beta-cells will aid in the development of a disease model that could provide insight into the adaptive transformations of beta-cell function during diabetes development.