Wenchong Liu

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.

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.

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.

Insulin alleviates posttrauma cardiac dysfunction by inhibiting tumor necrosis factor-α-mediated reactive oxygen species production.

Objective:Clinical evidence indicates that intensive insulin treatment prevents the incidence of multiple organ failures in surgical operation and severe trauma, but the mechanisms involved remain elusive. This study was designed to test the hypothesis that insulin may exert anti-inflammatory and antioxidative effects and thus alleviate cardiac dysfunction after trauma. Design:Prospective, randomized experimental study. Setting:Animal research laboratory. Subjects:Sprague Dawley rats. Interventions:Anesthetized rats were subjected to 200 revolutions at a rate of 35 rpm in Noble-Collip drum to induce a nonlethal mechanical trauma and were randomized to receive vehicle, insulin, and insulin + wortmannin treatments. An in vitro study was performed on cultured cardiomyocytes subjected to sham-traumatic serum (SS), traumatic serum (TS), SS + tumor necrosis factor (TNF)-&agr;, SS + H2O2, TS + neutralizing anti-TNF-&agr; antibody, or TS + tempol treatments. Measurements and Main Results:Immediate cardiac dysfunction occurred 0.5 hr after trauma without significant cardiomyocyte necrosis and apoptosis, while serum TNF-&agr; and cardiac reactive oxygen species (ROS) production was increased. Importantly, incubation of cardiomyocytes with TS or SS + TNF-&agr; significantly increased ROS generation together with dampened cardiomyocyte contractility and Ca2+ transient, all of which were rescued by TNF-&agr; antibody. Administration of insulin inhibited TNF-&agr; and ROS overproduction and alleviated cardiac dysfunction 2 hours after trauma. Scavenging ROS with tempol also attenuated cardiac dysfunction after trauma, whereas insulin combined with tempol failed to further improve cardiac functional recovery compared with insulin treatment alone. Moreover, the aforementioned anti-TNF-&agr;, antioxidative, and cardioprotective effects afforded by insulin were almost abolished by the phosphatidylinositol 3-kinase inhibitor wortmannin. Conclusions:These results demonstrate for the first time that mechanical trauma induces a significant increase in TNF-&agr; and ROS production, resulting in immediate cardiac dysfunction. Early posttrauma insulin treatment alleviates cardiac dysfunction by inhibiting TNF-&agr;-mediated ROS production via a phosphatidylinositol 3-kinase/Akt-dependent mechanism.