Lele Ji

AMPK-Regulated and Akt-Dependent Enhancement of Glucose Uptake Is Essential in Ischemic Preconditioning-Alleviated Reperfusion Injury

Aims Ischemic preconditioning (IPC) is a potent form of endogenous protection. However, IPC-induced cardioprotective effect is significantly blunted in insulin resistance-related diseases and the underlying mechanism is unclear. This study aimed to determine the role of glucose metabolism in IPC-reduced reperfusion injury. Methods Normal or streptozotocin (STZ)-treated diabetic rats subjected to 2 cycles of 5 min ischemia/5 min reperfusion prior to myocardial ischemia (30 min)/reperfusion (3 h). Myocardial glucose uptake was determined by 18F-fluorodeoxyglucose-positron emission tomography (PET) scan and gamma-counter biodistribution assay. Results IPC exerted significant cardioprotection and markedly improved myocardial glucose uptake 1 h after reperfusion (P<0.01) as evidenced by PET images and gamma-counter biodistribution assay in ischemia/reperfused rats. Meanwhile, myocardial translocation of glucose transporter 4 (GLUT4) to plasma membrane together with myocardial Akt and AMPK phosphorylation were significantly enhanced in preconditioned hearts. Intramyocardial injection of GLUT4 siRNA markedly decreased GLUT4 expression and blocked the cardioprotection of IPC as evidence by increased myocardial infarct size. Moreover, the PI3K inhibitor wortmannin significantly inhibited activation of Akt and AMPK, reduced GLUT4 translocation, glucose uptake and ultimately, depressed IPC-induced cardioprotection. Furthermore, IPC-afforded antiapoptotic effect was markedly blunted in STZ-treated diabetic rats. Exogenous insulin supplementation significantly improved glucose uptake via co-activation of myocardial AMPK and Akt and alleviated ischemia/reperfusion injury as evidenced by reduced myocardial apoptosis and infarction size in STZ-treated rats (P<0.05). Conclusions The present study firstly examined the role of myocardial glucose metabolism during reperfusion in IPC using direct genetic modulation in vivo. Augmented glucose uptake via co-activation of myocardial AMPK and Akt in reperfused myocardium is essential to IPC-alleviated reperfusion injury. This intrinsic metabolic modulation and cardioprotective capacity are present in STZ-treated hearts and can be triggered by insulin.

Acute hyperglycaemia enhances oxidative stress and aggravates myocardial ischaemia/reperfusion injury: role of thioredoxin-interacting protein

Hyperglycaemia during acute myocardial infarction is common and associated with increased mortality. Thioredoxin‐interacting protein (Txnip) is a modulator of cellular redox state and contributes to cell apoptosis. This study aimed to investigate whether or not hyperglycaemia enhances Txnip expression in myocardial ischaemia/reperfusion (MI/R) and consequently exacerbates MI/R injury. Rats were subjected to 30 min. of left coronary artery ligation followed by 4 hrs of reperfusion and treated with saline or high glucose (HG, 500 g/l, 4 ml/kg/h intravenously). In vitro study was performed on cultured rat cardiomyocytes subjected to simulated ischaemia/reperfusion (SI/R) and incubated with HG (25 mM) or normal glucose (5.6 mM) medium. In vivo HG infusion during MI/R significantly impaired cardiac function, aggravated myocardial injury and increased cardiac oxidative stress. Meanwhile, Txnip expression was enhanced whereas thioredoxin activity was inhibited following HG treatment in ischaemia/reperfusion (I/R) hearts. In addition, HG activated p38 MAPK and inhibited Akt in I/R hearts. In cultured cardiomyocytes subjected to SI/R, HG incubation stimulated Txnip expression and reduced thioredoxin activity. Overexpression of Txnip enhanced HG‐induced superoxide generation and aggravated cardiomyocyte apoptosis, whereas Txnip RNAi significantly blunted the deleterious effects of HG. Moreover, inhibition of p38 MAPK or activation of Akt markedly blocked HG‐induced Txnip expression in I/R cardiomyocytes. Most importantly, intramyocardial injection of Txnip siRNA markedly decreased Txnip expression and alleviated MI/R injury in HG‐treated rats. Hyperglycaemia enhances myocardial Txnip expression, possibly through reciprocally modulating p38 MAPK and Akt activation, leading to aggravated oxidative stress and subsequently, amplification of cardiac injury following MI/R.

Insulin inhibits leukocyte-endothelium adherence via an Akt-NO-dependent mechanism in myocardial ischemia/reperfusion.

Clinical evidence indicates that intensive insulin therapy during critical illness protects the endothelium and contributes to prevention of organ failure and death but the mechanisms involved remain unclear. This study was designed to test the hypothesis that insulin inhibits adherence of polymorphonuclear leukocytes (PMNs) to endothelial cells in myocardial ischemia/reperfusion (MI/R) and to investigate the underlying mechanisms. Anesthetized rabbits were subjected to MI/R (45 min/4 h) and randomly received saline, glucose-insulin-potassium (GIK) or GK respectively (2 mL/kg/h, i.v.). In vitro study was performed on cultured endothelial cells subjected to simulated ischemia/reperfusion. In vivo treatment with GIK but not GK attenuated myocardial injury as evidenced by reduced plasma creatine kinase activity, myocardial apoptosis and infarct size in MI/R rabbits compared with the saline group. Interestingly, GIK but not GK significantly decreased coronary endothelial expression of P-selectin and intercellular adhesion molecule-1 (ICAM-1), inhibited adherence of PMNs to coronary endothelium (107.7+/-7.4 vs. 155.0+/-9.2 PMNs/mm(2) in saline group, n=8, P<0.01), and therefore decreased myocardial PMNs accumulation. In cultured endothelial cells subjected to simulated ischemia/reperfusion, insulin (10(-)(7) M) increased Akt activity and eNOS phosphorylation with subsequent NO production, and concurrently exerted an anti-adhesive effect as manifested by reduced endothelial P-selectin and ICAM-1 surface expression and PMNs adherence (13.7+/-1.3% vs. 22.2+/-1.9% in vehicle, n=9, P<0.01), all of which are abolished by the specific Akt inhibitor. Furthermore, inhibition of insulin-stimulated NO production using either the selective eNOS inhibitor cavtratin or the NOS inhibitor L-NAME blocked the anti-adhesive effect of insulin. These results demonstrate that insulin reduces endothelial P-selectin and ICAM-1 expression, and thus inhibits leukocyte-endothelium adherence in MI/R rabbit hearts. The anti-adhesive property by insulin may be mediated by the Akt-mediated and NO-dependent pathway.

A Novel Mechanism for Vascular Insulin Resistance in Normotensive Young SHRs Hypoadiponectinemia and Resultant APPL1 Downregulation

Vascular insulin resistance contributes to elevated peripheral vascular resistance and subsequent hypertension. Clinical observation showed that lower plasma adiponectin concentration is significantly associated with hypertension. This study was aimed to determine whether hypoadiponectinemia induces vascular insulin resistance before systemic hypertension and the underlying mechanisms. Four-week-old young spontaneously hypertensive rats (ySHRs, normotensive) and adiponectin knockout (KO; APN-/-) mice were used to evaluate the role of hypoadiponectinemia in insulin-induced vasodilation of resistance vessels. ySHRs showed significant vascular insulin resistance as evidenced by the blunted vasorelaxation response to insulin in mesenteric arterioles compared with that of age-matched Wistar-Kyoto controls. Serum adiponectin and mesenteric arteriolar APPL1 (an adaptor protein that mediates adiponectin signaling) expression of ySHRs were significantly reduced. In addition, Akt and endothelial NO synthase phosphorylation and NO production in arterioles were markedly reduced, whereas extracellular signal-regulated protein kinases 1/2 (ERK1/2) phosphorylation and endothelin-1 secretion were augmented in ySHRs. APN-/- mice showed significantly decreased APPL1 expression and vasodilation evoked by insulin. More importantly, treatment of ySHRs in vivo with the globular domain of adiponectin for 1 week increased APPL1 expression and insulin-induced vasodilation, and restored the balance between insulin-stimulated endothelial vasodilator NO and vasoconstrictor endothelin-1. In cultured human umbilical vein endothelial cells, globular domain of adiponectin upregulated APPL1 expression. Suppression of APPL1 expression with small interfering RNA markedly blunted the globular domain of adiponectin-induced insulin sensitization as evidenced by reduced Akt/endothelial NO synthase and potentiated ERK1/2 phosphorylations. In conclusion, hypoadiponectinemia induces APPL1 downregulation in the resistance vessels, contributing to the development of vascular insulin resistance by differentially modulating the Akt/endothelial NO synthase/NO and ERK1/2/endothelin-1 pathways in vascular endothelium in normotensive ySHRs.Vascular insulin resistance contributes to elevated peripheral vascular resistance and subsequent hypertension. Clinical observation showed that lower plasma adiponectin concentration is significantly associated with hypertension. This study was aimed to determine whether hypoadiponectinemia induces vascular insulin resistance before systemic hypertension and the underlying mechanisms. Four-week-old young spontaneously hypertensive rats (ySHRs, normotensive) and adiponectin knockout (KO; APN-/-) mice were used to evaluate the role of hypoadiponectinemia in insulin-induced vasodilation of resistance vessels. ySHRs showed significant vascular insulin resistance as evidenced by the blunted vasorelaxation response to insulin in mesenteric arterioles compared with that of age-matched Wistar-Kyoto controls. Serum adiponectin and mesenteric arteriolar APPL1 (an adaptor protein that mediates adiponectin signaling) expression of ySHRs were significantly reduced. In addition, Akt and endothelial NO synthase phosphorylation and NO production in arterioles were markedly reduced, whereas extracellular signal-regulated protein kinases 1/2 (ERK1/2) phosphorylation and endothelin-1 secretion were augmented in ySHRs. APN-/- mice showed significantly decreased APPL1 expression and vasodilation evoked by insulin. More importantly, treatment of ySHRs in vivo with the globular domain of adiponectin for 1 week increased APPL1 expression and insulin-induced vasodilation, and restored the balance between insulin-stimulated endothelial vasodilator NO and vasoconstrictor endothelin-1. In cultured human umbilical vein endothelial cells, globular domain of adiponectin upregulated APPL1 expression. Suppression of APPL1 expression with small interfering RNA markedly blunted the globular domain of adiponectin-induced insulin sensitization as evidenced by reduced Akt/endothelial NO synthase and potentiated ERK1/2 phosphorylations. In conclusion, hypoadiponectinemia induces APPL1 downregulation in the resistance vessels, contributing to the development of vascular insulin resistance by differentially modulating the Akt/endothelial NO synthase/NO and ERK1/2/endothelin-1 pathways in vascular endothelium in normotensive ySHRs. # Novelty and Significance {#article-title-29}

Vasonatrin peptide attenuates myocardial ischemia-reperfusion injury in diabetic rats and underlying mechanisms

Diabetes mellitus increases morbidity/mortality of ischemic heart disease. Although atrial natriuretic peptide and C-type natriuretic peptide reduce the myocardial ischemia-reperfusion damage in nondiabetic rats, whether vasonatrin peptide (VNP), the artificial synthetic chimera of atrial natriuretic peptide and C-type natriuretic peptide, confers cardioprotective effects against ischemia-reperfusion injury, especially in diabetic patients, is still unclear. This study was designed to investigate the effects of VNP on ischemia-reperfusion injury in diabetic rats and to further elucidate its mechanisms. The high-fat diet-fed streptozotocin-induced diabetic Sprague-Dawley rats were subjected to ischemia-reperfusion operation. VNP treatment (100 μg/kg iv, 10 min before reperfusion) significantly improved the instantaneous first derivation of left ventricle pressure (±LV dP/dtmax) and LV systolic pressure and reduced LV end-diastolic pressure, apoptosis index, caspase-3 activity, plasma creatine kinase (CK), and lactate dehydrogenase (LDH) activities. Moreover, VNP inhibited endoplasmic reticulum (ER) stress by suppressing glucose-regulated protein 78 (GRP78) and C/EBP homologous protein (CHOP). These effects were mimicked by 8-bromine-cyclic guanosinemonophosphate (8-Br-cGMP), a cGMP analog, whereas they were inhibited by KT-5823, the selective inhibitor of PKG. In addition, pretreatment with tauroursodeoxycholic acid (TUDCA), a specific inhibitor of ER stress, could not further promote the VNPs cardioprotective effect in diabetic rats. In vitro H9c2 cardiomyocytes were subjected to hypoxia/reoxygenation and incubated with or without VNP (10(-8) mol/l). Gene knockdown of PKG1α with siRNA blunted VNP inhibition of ER stress and apoptosis, while overexpression of PKG1α resulted in significant decreased ER stress and apoptosis. VNP protects the diabetic heart against ischemia-reperfusion injury by inhibiting ER stress via the cGMP-PKG signaling pathway. These results suggest that VNP may have potential therapeutic value for the diabetic patients with ischemic heart disease.

Achyranthes bidentata Polypeptides Reduces Oxidative Stress and Exerts Protective Effects against Myocardial Ischemic/Reperfusion Injury in Rats

Achyranthes bidentata, a Chinese medicinal herb, is reported to be neuroprotective. However, its role in cardioprotection remains largely unknown. Our present study aimed to investigate the effects of Achyranthes bidentata polypeptides (ABPP) preconditioning on myocardial ischemia/reperfusion (MI/R) injury and to test the possible mechanisms. Rats were treated with ABPP (10 mg/kg/d, i.p.) or saline once daily for one week. Afterward, all the animals were subjected to 30 min of myocardial ischemia followed by 4 h of reperfusion. ABPP preconditioning for one week significantly improved cardiac function following MI/R. Meanwhile, ABPP reduced infarct size, plasma creatine kinase (CK)/lactate dehydrogenase (LDH) activities and myocardial apoptosis at the end of reperfusion in rat hearts. Moreover, ABPP preconditioning significantly inhibited superoxide generation, gp91phox expression, malonaldialdehyde formation and enhanced superoxide dismutase activity in I/R hearts. Furthermore, ABPP treatment inhibited PTEN expression and increased Akt phosphorylation in I/R rat heart. PI3K inhibitor wortmannin blocked Akt activation, and abolished ABPP-stimulated anti-oxidant effect and cardioprotection. Our study demonstrated for the first time that ABPP reduces oxidative stress and exerts cardioprotection against MI/R injury in rats. Inhibition of PTEN and activation of Akt may contribute to the anti-oxidant capacity and cardioprotection of ABPP.

Alpha-linolenic acid intake prevents endothelial dysfunction in high-fat diet-fed streptozotocin rats and underlying mechanisms.

BACKGROUND Endothelial dysfunction is an important factor in the pathogenesis of diabetes related vascular complications, and acute alpha-linolenic acid (ALA) intake can increase flow-mediated dilation of the diabetic artery at 4 h postprandially. However, whether chronic ALA supplementation may prevent endothelial dysfunction in the process of diabetes and underlying mechanisms remains largely unknown. MATERIALS AND METHODS The high-fat diet-fed streptozotocin (HFD-STZ) rats provided an animal model for T2DM. Age-matched normal and HFD-STZ rats randomly received normal diet or ALA (500 mg/kg per day). After 5 weeks of feeding, endothelial function was determined. RESULTS Diabetes caused significant endothelial dysfunction (maximal vasorelaxation responses to ACh) in aortic segments, and ALA intake alleviated endothelial dysfunction. Superoxide production and peroxynitrite (ONOO-) formation were reduced with ALA supplement in diabetic vascular segments. Interestingly, ALA intake enhanced eNOS but inhibited iNOS activity in diabetic vessels. Moreover, ALA intake significantly increased eNOS phosphorylation. On the other hand, gp91phox and iNOS overexpression were reduced moderately with ALA intake in diabetic vessels. CONCLUSIONS We concluded that ALA prevents diabetes-induced endothelial dysfunction by enhancing eNOS activity and attenuates oxidative/nitrative stress by inhibiting iNOS and NADPH oxidase expression and ONOO- production.

Rotenone partially reverses decreased BKCa currents in cerebral artery smooth muscle cells from streptozotocin‐induced diabetic mice

1 Reactive oxygen species (ROS) cause vascular complications and impair vasodilation in diabetes mellitus. Large‐conductance Ca2+‐activated potassium channels (BKCa) modulate vascular tone and play an important negative feedback role in vasoconstriction. In the present study, we tested the hypothesis that ROS regulate the function of BKCa in diabetic cerebral artery smooth muscle cells. 2 Diabetes was induced in male BALB/c mice by injection of streptozotocin (STZ; 180 mg/kg, i.p., dissolved in sterile saline). Control and diabetic mice were treated with 12.7 µmol/L rotenone, an inhibitor of the mitochondrial electron transport chain complex I, or placebo every other day for 5 weeks. The whole‐cell patch clamp‐technique and functional vasomotor methods were used to record BKCa currents and myogenic tone of cerebral artery smooth muscle cells. 3 In the diabetic group, there was a significant decrease in spontaneous transient outward currents in cerebral artery smooth muscle cells compared with control. Although the currents were only moderately increased in rotenone‐treated diabetic mice, they remained significantly lower than in the control group. Furthermore, the macroscopic BKCa currents that were decreased in diabetic mice were partially recovered in rotenone‐treated diabetic mice (P < 0.05 vs untreated diabetic group). 4 The posterior cerebral artery from diabetic mice had a significantly higher myogenic tone than the control group, but this impaired contraction was partially reversed in the rotenone‐treated diabetic group (P < 0.05 vs untreated diabetic group). 5 The H2O2 concentration was significantly increased in cerebral arteries from diabetic mice compared with control. This increase in H2O2 was significantly blunted by rotenone treatment. 6 In conclusion, rotenone partially reverses the decreased macroscopic BKCa currents in STZ‐induced Type 1 diabetic mice and this reversal of BKCa currents may be related to the inhibitory effects of rotenone on H2O2 production. Reactive oxygen species, particularly H2O2, are important regulators of BKCa channels and myogenic tone in diabetic cerebral artery.

Catalpol decreases peroxynitrite formation and consequently exerts cardioprotective effects against ischemia/reperfusion insult.

Context: Peroxynitrite (ONOO−) formation triggers oxidative/nitrative stress and contributes to exacerbated myocardial ischemia/reperfusion (MI/R) injury. Catalpol, an iridoid glycoside, abundantly found in the roots of Rehmannia glutinosa L. that is included in the family Phrymaceae in the order Lamiales, endemic to China, was found to have neuroprotective effects. However, the effect of catalpol on MI/R injury has not been identified. Objective: This study investigated whether catalpol attenuates oxidative/nitrative stress in acute MI/R. Materials and methods: Adult male rats were subjected to 30 min of myocardial ischemia and 3 h of reperfusion and were treated with saline, catalpol (5 mg/kg, i.p., 5 min before reperfusion) or catalpol plus wortmannin (15 µg/kg intraperitoneally injected 15 min before reperfusion). Results: Pretreatment with catalpol significantly improved cardiac functions, reduced myocardial infarction, apoptosis and necrosis of cardiomyocytes after MI/R (all p < 0.05). Meanwhile, ONOO− formation was markedly reduced after catalpol treatment (3.01 ± 0.22 vs. 4.66 ± 0.53 pmol/mg protein in vehicle, p < 0.05). In addition, catalpol increased Akt and endothelial nitric oxide synthase phosphorylation, nitric oxide (NO) production, anti-oxidant capacity and reduced MI/R-induced inducible nitric oxide synthase expression and superoxide anion (·O2−) production in I/R hearts. PI3K inhibitor wortmannin not only blocked catalpol-induced Akt activation, but also attenuated all the beneficial effects of catalpol. Suppression of ONOO− formation by either catalpol or an ONOO− scavenger uric acid (5 mg/kg) reduced myocardial infarct size in MI/R rats. Discussion and conclusion: In conclusion, catalpol affords cardioprotection against MI/R insult by attenuating ONOO− formation, which is attributable to increased physiological NO and decreased ·O2− production.

Semen Cassiae Attenuates Myocardial Ischemia and Reperfusion Injury in High-Fat Diet Streptozotocin-Induced Type 2 Diabetic Rats

Obese patients with type 2 diabetes mellitus (T2DM), which is characterized by hyperglycemia, are liable to more severe myocardial infarction. Semen Cassiae is proven to reduce serum lipid levels. This study investigated whether the Semen Cassiae extract (SCE) reduces myocardial ischemia and reperfusion (MI/R) injury with or without diabetes and the underlying mechanisms. The high-fat diet-fed streptozotocin (HFD-STZ) rat model was created as a T2DM model. Normal and DM rats received SCE treatment orally (10 mg/kg/day) for one week. Subsequently these animals were subjected to MI/R. Compared with the normal animals, DM rats showed increased plasma total cholesterol (TC) and triacylglycerol (TG), and more severe MI/R injury and cardiac functional impairment. SCE treatment significantly reduced the plasma TC and TG, improved the instantaneous first derivation of left ventricle pressure and reduced infarct size, decreased plasma creatine kinase and lactate dehydrogenase levels, and apoptosis index at the end of reperfusion in diabetic rats. Moreover, SCE treatment increased the antiapoptotic protein Akt and ERK1/2 phosphorylation levels. Pretreatment with a PI3K inhibitor wortmannin or an ERK1/2 inhibitor PD98059 not only blocked Akt and ERK1/2 phosphorylation respectively, but also inhibited the cardioprotective effects of SCE. However, SCE treatment did not show any effects on the MI/R injury in the normal rats. Our data suggest that SCE effectively improves myocardial function and reduces MI/R-induced injury in diabetic but not normal animals, which is possibly attributed to the reduced TC/TG levels and the triggered cell survival signaling Akt and ERK1/2.