Inhibition Of PDK4 Enhances Myocardial Glucose Uptake And Protects Against Cardiac Ischemia/Reperfusion Injury

BackgroundThe heart is an energy-consuming organ,requires constant and substantial supply for continuous contraction to maintain its normal contractive and diastolic activity and thus has developed an elaborate metabolic network for utilizing all carbon substrates.For its energy production,the mature heart primarily relies on the oxidation of fatty acids(FA)(60%-90%),and remaining 10%-40% derived from carbohydrates,including glucose,lactic acid and ketone body.However,the myocardial metabolic machinery is highly flexible allowing switch of substrate preference in a variety of physiological and pathological conditions to meet the needs of the body.Research has shown that cardiac metabolic switch is believed to be an adaptive response to cardiac switch of myocardial substrate availability and chronic switch of myocardial substrate preference leads to cardiac disorder and dysfunction in disease.Ischemia/reperfusion may play a causal role in cardiovascular disease.In the condition of myocardial ischemia,the insufficient oxygen supply represses both FA and glucose oxidation,and cardiac metabolism shifts from FA oxidation to glycolysis.For its reperfusion,when oxygen and substrates are restored,FA oxidation is quickly recovered or even exceeded to be the major source of ATP production.The elevated FA oxidation in post-ischemic myocardium results in the subsequent inhibition of glucose oxidation through Randle cycle mechanism,leading to glucose oxidation and glycolysis uncoupling.Pyruvate dehydrogenase kinase(PDK4)is the key enzyme in the process of glucose oxidation,in heart,competition between FA and glucose occurs at the level of pyruvate dehydrogenase(PDH),the enzymatic complex that catalyzes the first irreversible step of glucose oxidation.Glucose oxidation is elevated when PDH is active and glucose oxidation is repressed when PDH is inactive.Inactivation of PDH is mediated by a family of PDKs.Among PDKs,PDK4 responses rapidly to metabolic stimuli,and can be activated by FA,Dichloroacetic acid salt(DCA),the inhibitor of PDK4,by stimulating PDH activity,promotes glucose oxidation.Thus,PDK4 functions as a metabolic switch,altering cardiac substrate preference under stress.In our previous study,we found that insulin can not only participate in adjustment of energy metabolism,but also protect the heart through the PI3K-Akt survival signaling pathway.In fact,studies have reported a tight association between PDK4 and insulin signaling.Insulin can suppress PDK4 expression and its effect is impaired in insulin-resistant state,and its mRNA levels in skeletal muscle negatively correlated with insulin-mediated glucose uptake.Increased PDK activity has been suggested to induce insulin resistance in individuals with obesity and type 2 diabetes.Additionally,stimulation of glucose oxidation in the heart either by directly or indirectly approaches,improves the recovery of post-ischemic cardiac function.Although glycolysis and glucose oxidation during reperfusion is well documented,as the initial step of glucose metabolism,the level of glucose uptake as well as its underlying mechanism during reperfusion remains unclear.AimsThe objective of this study is to explore the level of cardiac glucose uptake and its underlying mechanism and significance in post-ischemic heart.MethodsAcute myocardial ischemia model of rats was developed by ligaturing the left descending anterior coronary artery.PET/CT was used to determine myocardial [18F]-fluorodeoccyglucose(FDG)uptake,which was shown by standardized uptake value(SUVmax).A fluorescent-labeled glucose analog 2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]-2-deoxy-D-glucose(2-NBDG)was also used to determine cardiac glucose uptake in vivo.Myocardial infarct size was determined by a double-staining technique and was analyzed by a digital imaging system.Mitochondria were isolated to measure oxygen consumption with a Clark-type oxygen electrode.Neonatal myocytes were isolated and cultured to establish hypoxia/reoxygenation(H/R)model,and PDK4 adenovirus and siRNA were used to regulate PDK4 expression to observe the relationship between PDK4 and myocardial insulin sensitivity.Results 1.Glucose uptake in post-ischemic myocardium was remarkably decreased.Radioactive signal of FDG was diminished in the ischemia zone of the heart following I/R.The results of the other in vivo approach using fluorescent-labeled analog 2-NBDG showed that glucose uptake in the ischemic zone of the heart was decreased.Then we examined total GLUT1 content and GLUT4 translocation in post-ischemic heart.GLUT1 content at the ischemic zone of the post-ischemic heart was upregulated.GLTU4 translocation as assessed by GLUT4 content on plasma membrane was held constant in both the ischemic and non-ischemic zones.2.Uncoupling of glycolysis and glucose oxidation in post-ischemic myocardium.Although the glucose uptake in post-ischemia was diminished,we found that glycolysis was increased as evidenced by the elevation of lactate.Glycolytic gene such as hexokinase 2,muscle phosphofructokinase,aldolase,GAPDH and PDK4 were upregulated in the ischemic zone of the heart following I/R.Cardiac mitochondria in the ischemic zone displayed depressed state ? respiration and metabolism efficiency as assessed by respiratory control ratio.Thus glucose oxidation was inhibited in post-ischemia myocardium.3.Enhancement of glucose oxidation through inhibition PDK4 increased glucose uptake in post-ischemic myocardium.PDK4,a rate-limiting enzyme for glucose oxidation in the myocardium was increased in the ischemic zone of the post-ischemic heart.Treatment with DCA,a inhibitor of PDK4,restored the lactate level,and increased glucose uptake in post-ischemic myocardium.In addition,treatment with DCA enhanced the mitochondrial state ? respiration and metabolism efficiency in mitochondrial isolated from the ischemic zone of the post-ischemic heart.Moreover we examined the role of glucose oxidation in regulation of glucose uptake in neonatal myoctyes.PDK4 overexpression by adenovirus infection decreased glucose uptake and PDK4 knockdown by siRNA silence increase glucose uptake.To this end,we noticed that PDK4,as a regulator of glucose oxidation,inversely regulates glucose uptake in heart.4.Stimulation of glucose uptake protects heart against I/R injury.We examined the cardiac injury in the heart subjected to I/R after DCA treatment and glucose infusion.Cardiac infarct size was increased in heart subjected to I/R.Treatment with DCA remarkably decreased cardiac infarct size and serum LDH level.In addition,assessment of cardiac function revealed that administration of DCA improved cardiac function as evidenced by the increased LVSP and ±LVdP/dtmax in rats.Glucose was administrated to directly increase glucose uptake.Glucose administration decreased cardiac infarct size and serum LDH level,and increased LVSP and ±LVdP/dtmax following I/R injury.The results showed that inhibiton of PDK4 with DCA exerted similar cardioprotecion as glucose infusion.5.Inhibition of PDK4 increased insulin sensitivity of post-ischemic myocardium.Insulin increases glucose transport by stimulating GLuT4 translocation into plasma membrane which is mediated by Akt signaling.Treatment with DCA showed no significant effect on blood glucose and serum insulin.PDK4 inhibition increased GLUT4 translocation and insulin signaling activation as assessed by phosphorylation of Akt in pots-ischemic myocardium.Next,we examined insulin sensitivity in post-ischemic neonatal myocytes to confirm the effects of PDK4 inhibiton on myocardial insulin sensitivity.Overexpression of PDK4 decreased insulin sensitivity to at baseline in post-anoxic neonatal myocytes and PDK4 knockdown increased insulin sensitivity in post-anoxic neonatal myocytes.These results indicated that inhibition of PDK4 stimulated glucose uptake,through enhancement of myocardial insulin sensitivity following myocardial I/R.Conclusions 1.Glucose uptake was remarkably decreased in post-ischemic myocardium.2.Enhancement of glucose oxidation through inhibiton of PDK4 increased glucose uptake in post-ischemic myocardium.3.The findings suggest that stimulating glucose uptake could be an efficient approach to improve recovery from myocardial ischemia/reperfusion injury.