High Plasma BCAA Level Aggravates Myocardial Is-chemia/Reperfusion Injury And The Underling Molecular Mechanisms Of Disturbed BCAA Catabolism

BackgroundOver the past three decades, the number of people with diabetes mellitus has morethan doubled globally, making it one of the most important public health challenges to allnations. According to the report from International Diabetes Federation (IDA), an esti-mated382million people worldwide had diabetes mellitus in2013. The number of peopleglobally with diabetes mellitus or prediabetes is projected to rise to592or471million by2035. Additionlly, there are510million people worldwide die from diabetes-related dis-eases（8.39%of total deaths）in2013and the worldwide medical expenses in diabetes upto USD548billion, accounting for11%of global health care expenditures. Our countryhas the largest number of patients with diabetes mellitus. The overall prevalence of diabe-tes in the Chinese adult population was estimated to be113.9million in2013and project-ed to rise to143million by2035. Therefore, taking effective measures to prevention andtreatment diabetes and its complications is significant. Diabetes is an important risk factor for coronary heart disease. Cardiovascular com-plications accounts for2/3of the mortality seen in the diabetic population every year anddiabetes equates to an approximately threefold increased risk of myocardial infarctioncompared with the general population. Experimental results have demonstrated that dia-betes patients with increased vulnerability of diabetic hearts[14]. However, that can not befully explained by “toxic” hyperglycemia. Series of clinical trial show that intensive gly-cemic control does not reduce the risk of cardiovascular disease[2,3]. Therefore,“a mul-ti-faceted approach including risk factor control must be employed to prevent the devel-opment of diabetes and its complications, most importantly CVD[15]”.Type2diabetic patients not only reveal disturbed glucose and lipid metabolism, butalso have abnormal protein metabolism. However, protein as one of macronutrients, in-cluding branched-chain amino acids （BCAA）, the role of which in diabetic myocardialinjury is poorly understood. It is reported that plasma BCAA levels were significantly in-creased in diabetic patients[4]. Additonally, BCAA contributes to development of obesi-ty-associated insulin resistance[5]. These results suggest that BCAA may play an causalrole in the development of disease. Moreover, High BCAA concentrations resulting in afivefold increased risk of type2diabetes[6]; plasma BCAA levels are closely related withinsulin resistance[7-9]. It has been known that BCAA and its-ralated metabolites had highlysignificant associations with future diabetes[16]. In addition, independent of insulin re-sistance and diabetic disease, BCAA levels are closely related with the severity of coro-nary heart disease[10]. However, whether BCAA is involved in heart injury, that is unclear.Adiponectin （APN） has effects on the regulation of glucose and lipid metabolism[11]and has vascular/cardioprotective effects[12]. More recently, Liu and colleagues[17]havereported that APN corrects altered muscle BCAA metabolism caused by a HD. In addition,mounting observations have revealed that type2diabetes are associated with decreasedplasma APN levels[13]. However, whether decreased APN is involved in diabetic BCAAaccumulation has never been investigated. More importantly, the underlying molecularmechanisms by which APN affects diabetic BCAA catabolism are completely unknown.Therefore, our scientific questions are （1） independent of other risk factors， whether high BCAA concentrations increased myocardial ischemic/reperfusion injury and themechanisms by which high BCAA levels exert this effect;（2） what are the causes forexcess blood BCAA and the underlying molecular mechanisms.Aims1The aims of section one were to examine（1） whether high BCAA level directlyenhances myocardial ischemic/reperfusion injury;（2） to determine whether oxidativestress mediated this effect.2The purposes of section two were to examine （1） whether decreased APN is in-volved in diabetic BCAA accumulation;（2） the underlying molecular mechanisms bywhich APN affects diabetic BCAA catabolism.Methods1High BCAA concentrations increases myocardial ischemic/reperfusion injury viastimulation of oxidative stress1.1MI/R was produced by placing a silk suture slipknot at the left anterior coronary ar-tery. After40mins of ischemia, the slipknot was released, and the myocardium wasreperfused for3hours or24hours. At10mins before reperfusion, mice were randomizedto receive either vehicle（PBS）or MnTBAP（SOD mimic）by intraperitoneal injection.1.2Cardiac function （left ventricular ejection fraction, LVEF） was determined byechocardiography at the end of24hours reperfusion period.1.3Myocardial infarct size was detected by the Evans blue/TTC double staining methodat the end of24hours reperfusion period.1.4Determination of BCAA concentrations was performed in triplicate using a commer-cially available BCAA detection kit.1.5Mitochondrial morphology was imaged by transmission electron microscopy.1.6After3hours reperfusion, myocardial apoptosis was determined by TUNEL stainingand caspase-3activity assay.1.7Superoxide （ROS） detection was performed with dihydroethidium（DHE）staining. 1.8MnSOD expression was detected by Western blot.2Impaired APN-AMPK-PP2Cm signaling contributes to disturbed catabolism ofbranched-chain amino acids in diabetic mice2.1Type2diabetic mode was induced by the following proceduces. WT C57BL/6micewere fed with a HD （60%kcal%fat） for6weeks. After a12-hour fasting period, themice were intraperitoneally injected with a low dose of streptozotocin （25mg/kg STZ in0.05M sodium citrate, pH4.5） at6weeks of age. Blood glucose and body weight weremeasured daily, and a diabetic condition was confrmed at4weeks after STZ injection bya non-fasting blood glucose level of≥200mg/dl.2.2Mouse hepatocytes were transfected with siRNA （AMPKα1and AMPKα2） byusing siRNA-siRNA-Mate according to the manufacturer’s instructions. Effciency ofgene knockdown was confirmed using Western blotting48hours after siRNA transfection.2.3BCKA concentrations were determined by HPLC.2.4Determination of BCAA concentrations was performed using a commercially availa-ble BCAA detection kit.2.5AMPKα、 AMPKα1、 AMPKα2、 P-AMPKα、 PP2Cm、 P-BCKD E1α、 BCKDE1α expression were detected by Western blot.2.6PP2Cm and APN mRNA levels were detected by real time RT-PCR.2.7Glucose tolerance were detected by intraperitoneal glucose tolerance test （IPGTT）.2.8Endogenous plasma APN levels were determined with a mouse APN ELISA kit.2.9Plasma insulin level was performed by the Department of Clinical Laboratory, XijingHospital, The Fourth Military Medical University.2.10Plasma cholesterol concentration was performed by the Department of Clinical La-boratory, Xijing Hospital, The Fourth Military Medical University.Results1High BCAA concentrations increase myocardial ischemic/reperfusion injury viastimulation of oxidative stress1.1Myocardial ischemic/reperfusion injury are markedly increased in mice with highBCAA concentrations Compared with WT mice, MI size was enlarged and cardiac function was further de-pressed in mice with high blood BCAA levels.1.2High BCAA levels markedly increase I/R-induced cardiomyocyte apoptosisCompared with WT mice, I/R-induced cardiomyocyte apoptosis was significantly in-creased in mice with high blood BCAA levels, evidenced by increased TUNEL stainingand caspase-3activity.1.3High BCAA levels significant increase I/R-induced oxidative stressCompared with WT mice, high blood BCAA levels stimulated ROS production andinhibited I/R-induced MnSOD expression; treatment with MnTBAP10mins beforereperfusion reduced ROS production and ameliorated mitochondrial damage.1.4Treatment with MnTBAP attenuates myocardial ischemic/reperfusion injury inmice with high BCAA concentrationsAdministration of MnTBAP10mins before reperfusion markedly reversed cardiacdysfunction, reduced MI size and cardiomyocyte apoptosis.2Impaired AMPK-PP2Cm signaling contributes to disturbed catabolism ofbranched-chain amino acids in diabetic mice2.1PP2Cm is down-regulated in ob/ob and type2diabetic miceCompared with WT mice, plasma BCAA, BCKA and phosphorylation of BCKD lev-els were significantly increased, PP2Cm mRNA and protein expression were markedlydecreased in both ob/ob and type2diabetic mice.2.2Adiponectin deficiency contributes to decreased PP2Cm expression and BCKDactivityAPN-/-mice revealed a significant increase of BCAA, BCKA and hepatic BCKDphosphorylation levels in mice in response to HD. More importantly, PP2Cm was mark-edly decreased in HD-fed APN-/-mice. Additionlly, treatment with APN completely cor-rected BCAA and BCKA levels, hepatic BCKD activity, and PP2Cm protein expression.2.3Adiponectin administration increases BCKD activity and PP2Cm levels in dia-betic mice; PP2Cm deficiency partially inhibits adiponectin-activated BCKDThere were the significant decrease in BCAA and BCKA levels, as well as BCKD phosphorylation in APN-treated mice when compared to those only treated with the vehi-cle. Moreover, APN significantly increased diabetic PP2Cm mRNA levels and protein ex-pression. Additionally, plasma BCAA and BCKA concentrations and BCKD phosphoryla-tion were signifcantly higher in PP2Cm-/-mice treated with APN when compared withthose in APN treated diabetic and WT control groups.2.4AMPK is necessary for adiponectin-mediated BCKA catabolismAICAR treatment resulted in a significant decrease in BCAA and BCKA concentra-tions, while augmenting BCKD activity and PP2Cm expression in diabetic mice andBCKA challenged mice hepatocyte; compound C completely blocked the effect of APN onBCAA and BCKA, and BCKD activity and PP2Cm expression.2.5AMPKα2contributes to total AMPKα activity in adiponectin-stimulated BCKAcatabolismPP2Cm expression and BCKD activity were reduced, and BCKA were accumulatedin the AMPKα2siRNAknockdown group; AMPKα2siRNAcompletely blocked the effectof APN on PP2Cm regulation, BCKD activity, and BCKA levels.Conclusion1We demostrated that high BCAA concentrations increased myocardial ischem-ic/reperfusion injury. The underlying mechanisms were that high BCAA levels inhib-ited I/R-induced MnSOD expression and ROS removal, increased myocardial apopto-sis.2In the present study, we validated for the first time that impaired APN-AMPK-PP2Cmis an important part of the underlying mechanism for excess BCAA in type2diabetesmellitus. Additionlly, we also revealed that APN up-regulates PP2Cm expression viaAMPK pathway. These new insights provide a better understanding of the underlyingregulatory mechanisms involved in BCAA catabolism during diabetes and potentialtherapeutic targets to mitigate BCAA accumulation in metabolic diseases.