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.

研究目的
缺血预处理（Ischemic Preconditioning, IPC）是一种强效的内源性保护手段，但胰岛素抵抗相关疾病中IPC的心肌保护效应会显著受损，其潜在机制尚不明确。本研究旨在明确葡萄糖代谢在IPC减轻再灌注损伤中的作用。

研究方法
将正常大鼠及链脲佐菌素（Streptozotocin, STZ）诱导的糖尿病大鼠，在心肌缺血（30分钟）/再灌注（3小时）造模前，先进行2轮5分钟缺血/5分钟再灌注的预处理。通过18F-氟代脱氧葡萄糖正电子发射断层扫描（18F-fluorodeoxyglucose-positron emission tomography, PET）及γ计数器生物分布实验，检测心肌葡萄糖摄取水平。

研究结果
在缺血/再灌注大鼠中，PET成像与γ计数器生物分布实验结果证实，IPC可发挥显著的心肌保护作用，并在再灌注1小时后显著提升心肌葡萄糖摄取水平（P<0.01）。同时，预处理后的心脏中，葡萄糖转运蛋白4（Glucose Transporter 4, GLUT4）向细胞膜的转位，以及心肌Akt与AMPK的磷酸化水平均显著升高。心肌内注射GLUT4小干扰RNA（siRNA）可显著降低GLUT4的表达，并阻断IPC的心肌保护作用，表现为心肌梗死面积增大。此外，磷脂酰肌醇3-激酶（PI3K）抑制剂渥曼青霉素（wortmannin）可显著抑制Akt与AMPK的激活，减少GLUT4转位与葡萄糖摄取，最终削弱IPC介导的心肌保护作用。进一步研究发现，在STZ诱导的糖尿病大鼠中，IPC所具备的抗凋亡效应显著受损。外源性胰岛素补充可通过协同激活心肌AMPK与Akt通路提升葡萄糖摄取，并减轻缺血/再灌注损伤，表现为STZ大鼠的心肌细胞凋亡与梗死面积均显著降低（P<0.05）。

研究结论
本研究首次在体通过直接基因调控手段，探讨了再灌注阶段心肌葡萄糖代谢在IPC中的作用。再灌注心肌中，通过协同激活AMPK与Akt通路以增强葡萄糖摄取，是IPC减轻再灌注损伤的核心机制。这一内在的代谢调控与心肌保护能力在STZ诱导的糖尿病大鼠心脏中依然存在，且可通过外源性胰岛素触发激活。