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

<div><p>Aims</p><p>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.</p><p>Methods</p><p>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.</p><p>Results</p><p>IPC exerted significant cardioprotection and markedly improved myocardial glucose uptake 1 h after reperfusion (<i>P</i><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 (<i>P</i><0.05).</p><p>Conclusions</p><p>The present study firstly examined the role of myocardial glucose metabolism during reperfusion in IPC using direct genetic modulation <i>in vivo</i>. 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.</p></div

The effects of magnolol on blood pressure and insulin-induced vasodilatation in rats.

<p>(A) The effect of magnolol of systolic blood pressure (SBP). (B) The effect of magnolol on diastolic pressure (DBP). Impairment of insulin-induced vasodilatation and the effect of magnolol on insulin-induced vasodilatation in aortic segments from SHRs (C). Either ACh (D) or SNAP (E) elicited a significant vasorelaxation effects in aortic vessels from both WKY rats and SHRs, and there was no difference in aortic vasorelaxation between the two strains in response to both vasodilators respectively. (F) Insulin-induced relaxation was abolished either by removal of the endothelium or by pretreatment with L-NAME of the vessels from SHRs by MAG treatment, and also was inhibited by PPARγ antagonist bisphenol A diglycidyl ether (BADGE) pretreatment in vivo. Mag, magnolol; L-NAME, N^ω-nitro-L-argininemethyl ester; E⁻, endothelium denudation. n = 6, **<i>P</i><0.01 vs. WKY; ^##<i>P</i><0.01 vs. SHR; ^$$<i>P</i><0.01 vs. SHR+MAG.</p

Insulin supplementation protects the STZ-treated hearts from myocardial ischemic/reperfusion injury.

<p>(A) Representative photomicrographs of in situ detection of apoptotic myocytes by TUNEL staining; Green fluorescence shows TUNEL-positive nuclei, blue fluorescence shows nuclei of total cardiomyocytes; Original magnification×400, scale bar represents 50 µm; (B) Percentage of TUNEL-positive nuclei in heart tissue sections after 3 h of reperfusion; (C) Representative photographs of heart sections. Blue-stained portion indicates nonischemic, normal region; red-stained portion, ischemic/reperfused but not infarcted region; and negative-stained portion, ischemic/reperfused infarcted region; (D) Myocardial infarct size (INF) expressed as percentage of area at risk (AAR) after 3 h of reperfusion. Values presented are means ± SEM. n = 6/group.<i>**P</i><0.01 vs. MI/R, <i>^##P</i><0.01 vs. STZ+MI/R. Abbreviations as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0069910#pone-0069910-g007" target="_blank">Figure 7</a>.</p

Suppressing GLUT4 with GLUT4 siRNA reduced the cardioprotection of IPC.

<p>(A) GLUT4 mRNA expression (n = 4/group); (B) GLUT4 protein expression, Top images: representative blots (n = 4/group); (C) myocardial infarct size expressed as percentage of area at risk (AAR, n = 6/group). All rats were subjected to IPC and 30 min of ischemia followed by 1 h of reperfusion for qPCR and western analysis or 3h of reperfusion for the determination of myocardial injury. IPC was induced by 2 cycles of 5 min of ischemia/5 min of reperfusion. NC, negative control. Values presented are means ± SEM.<i>**P</i><0.01 vs. NC SiRNA.</p

Preconditioning reduces myocardial injury following myocardial ischemia/reperfusion in normal but not STZ-treated rats.

<p>(A) Myocardial infarct size (INF) expressed as percentage of area at risk (AAR); (B) Plasma creatine kinase (CK) level; (C) Top: Representative photomicrographs of in situ detection of apoptotic myocytes by TUNEL staining; Green fluorescence shows TUNEL-positive nuclei, blue fluorescence shows nuclei of total cardiomyocytes; Original magnification×400, scale bar represents 50 µm; Bottom: Percentage of TUNEL-positive nuclei in heart tissue sections; (D) Myocardial caspase-3 activity. All rats were subjected to 30 min coronary occlusion followed by 3 h of reperfusion (MI/R). IPC was induced by 2 cycles of 5 min of ischemia/5 min of reperfusion. Streptozotocin (STZ, 70 mg/kg) was administrated intraperitoneally 1 week prior to the surgical procedure. Values presented are means ± SEM; n = 6/group.<i>**P</i><0.01 vs. MI/R.</p

Effects of magnolol incubation on PPARγ expression and insulin-stimulated Akt/endothelial NO synthase (eNOS)/NO signaling in human umbilical vein endothelial cells.

<p>Representative western blots showing PPARγ (A), TRB3 (B), phosphorylated Akt / Akt (C), phosphorylated eNOS / eNOS (D), and NO release (E) in the conditioned medium. HG/HF, high glucose/high fat treatment. All values are presented as mean ± SEM. n = 5–6. *<i>P</i><0.05 vs. Control, ^#<i>P</i><0.05 vs. HG/HF.</p

Preconditioning stimulates Akt and AMPK activation and increases GLUT4 translocation in a PI3K-dependent manner.

<p>(A) Representative blots; (B) Myocardial phosphorylation of Akt; (C) Myocardial phosphorylation of GSK3β; (D) Myocardial phosphorylation of AMPK; (E) Myocardial phosphorylation of ACC; (F) GLUT4 on PM fraction; (G) GLUT4 expression. All rats were subjected to a 30-min coronary occlusion followed by 1 h of reperfusion. IPC was induced by 2 cycles of 5 min of ischemia/5 min of reperfusion. Sham-operated control rats (Sham) underwent the same surgical procedures with the exception of left anterior descending coronary artery occlusion. Wortmannin (W: 15 µg/kg) was administered intravenously 15 min before IPC. Values presented are means ± SEM. n = 4/group.<i>*P</i><0.05,<i>**P</i><0.01 vs. Sham, <i>^#P</i><0.05, <i>^##P</i><0.01 vs. MI/R, <i>^τP</i><0.05, <i>^ττP</i><0.01 vs. IPC+MI/R.</p

PPARγ antagonist blocked effects of MAG incubation on PPARγ expression and insulin-stimulated signaling pathway in human umbilical vein endothelial cells.

<p>Representative western blots showing PPARγ (A), TRB3 (B), phosphorylated Akt / Akt (C), and phosphorylated eNOS / eNOS (D), and NO release (E) in the conditioned medium. HG/HF, high glucose/high fat treatment; Mag, magnolol; BADGE, PPARγ antagonist bisphenol A diglycidyl ether (BADGE). All values are presented as mean ± SEM. n = 5–6. *<i>P</i><0.05 vs. Control, ^#<i>P</i><0.05 vs. HG/HF, ^$<i>P</i><0.05 vs. HG/HF+BADGE.</p

Improved insulin signaling of human umbilical vein endothelial cells by magnolol were abolished by upregulating TRB3 expression.

<p>Representative western blots showing TRB3 (A), phosphorylated Akt / Akt (B), and phosphorylated eNOS / eNOS (C). Mag, magnolol, (D) NO release in the conditioned medium. (E) Proposed mechanisms of magnolol attenuates the vascular insulin resistance and reduces blood pressure. All values are presented as mean ± SEM. n = 5–6. *<i>P</i><0.05 vs. Control, ^#<i>P</i><0.05 vs. HG/HF, ^$<i>P</i><0.05 vs. HG/HF+Chariot.</p

Preconditioning enhances glucose uptake during post-ischemic reperfusion in I/R hearts.

<p>(A) Representative positron emission tomography images obtained in rats subjected to MI/R with or without IPC; Top: Sagittal, coronal and transverse images of FDG uptake in MI/R group, respectively; Bottom: Sagittal, coronal and transverse images of FDG uptake in IPC+MI/R group, respectively; At the lower-left corner of the images, A, H, F, R and L indicates anterior, head, foot, right and left, respectively; (B) Bar graphs summarizing the mean data of FDG uptake expressed as standard uptake values (SUVs, y-axis) in vitro gamma-counter tissue biodistribution studies; (C) Blood glucose level; Each rat (∼250g) was injected with 1 mCi FDG in 100 µL 0.9% saline intravenously at the very beginning of reperfusion (n = 12/group). All rats were subjected to 30 min of coronary occlusion followed by 1 h of reperfusion. IPC was induced by 2 cycles of 5 min of ischemia/5 min of reperfusion. Sham-operated control rats (Sham) underwent the same surgical procedures with the exception of left anterior descending coronary artery occlusion. Values presented are means ± SEM.<i>*P</i><0.05 vs. MI/R, <i>^#P</i><0.05 vs. IPC+MI/R.</p