| BackgroundGlobal type1diabetes mellitus (T1DM) incidence increases2%to5%annually; in the United States, the prevalence of T1DM is approximately1/300by18years of age. Pancreatic inability to produce insulin is the root mechanismfor T1DM, a lifelong disease. Though its onset is possible at any age, T1DM haspropensity for pediatric and young adult populations, and portends poorprognosis concerning cardiovascular disease complications, the most prevalentcause of diabetic-associated morbidity and mortality.T1DM patients and animal models manifest altered adipokine and metabolismprofiles. Of primarily adipocyte origin, the protein adiponectin (APN) normallycirculates at very high plasma concentrations. Attenuating inflammation andregulating glucose/lipid metabolism, APN additionally serves as an antiapoptoticadipokine. Increasing experimental evidence supports APN as a potentialtherapeutic molecule against cardiovascular disease, demonstrating cardioprotective effect against myocardial ischemia-reperfusion (MI/R) injury.APN exerts its effects primarily via two membrane receptors, APN receptors-1and-2(AdipoR1,2), mediating effects through AMP-activated protein kinase(AMPK) and peroxisome proliferator-activated receptor (PPAR).Unlike obesity-linked diseases (such as coronary artery disease and type-2diabetes) which manifest consistently reduced circulating APN levels, T1DMpatients have been reported to harbor increased and decreased plasma APNconcentrations. Comprehensive information regarding dynamic fluctuations inAPN levels during T1DM progression does not exist. A large number of clinicaltrials showed that people with higher adiponectin levels have the lowercardiovascular mortality and the development of diabetes. Plasma adiponectinlevel can also predict the risk of type2diabetes. In addithion, it has been reportedthat plasma adiponectin concentration correlated negatively with insulin levels.Increase adiponectin concentration or administration of exogenous adiponectintreatment can significantly improve insulin resistance or lowering blood glucose.Therefore, adiponection may be one of the key factors associated with insulinresistance and diabetes, and have a good application prospects in the treatment ofdiabetesA clinical study in2004found that the plasma adiponectin concentration andthe incidence of myocardial infarction was a significant negative correlation. Manwith higher adiponectin level has lower incidence of myocardial infarction. Inanimal models, adionectin knockout mice have severe myocardial injurycompared with WT mice after MI/R injury, while exogenous adiponectinadministration could improve heart injury. Meanwhile, it has been confirmed thatpatient with type2diabetes has lower adiponectin levels, its incidence ofischemia heart desease (IHD) and mortality after myocardial infarction is increased. These data suggest that plasma adiponectin levels not only correlatedwith the occurrence of diabetes, but also correlated with the higher incidence ofIHD in type2diabetes. It also has been found that the risk of cardiovascularevents is4.5times higher in type1diabetes than in non-diabetes patients. Atpresent, studies about the trend of plasma adiponectin level in type1diabetes isnot consistent, given that adiponectin plays important role in myocardial andendothelium protection, it is urgent to clarify the relationship of plasmaadiponectin level and myocardial vulnerability in type1diabetes.High-fat diet and obesity have been demonstrated to decrease APNconcentration and AdipoR1/R2expression levels, thereby reducing APNsensitivity. For instance, AdipoR1/2expression was significantly decreased inhigh-fat fed rats, resulting in reduced vascular responsiveness to APN treatment,leading to APN resistance. It is unknown whether APN resistance occurs inT1DM. In the present study, we aimed to test dynamic change of APNconcentration and APN receptor expression levels in the well-establishedstreptozotocin (STZ)-induced type-1diabetic heart model and further to elucidatethe molecular mechanisms responsible for the aggravation myocardial ischemiainjury in T1DM. Our study might provide experimental basis for may potentiallyreduce morbidity and mortality of ischemic heart disease in diabetic patient.Objectives(1) To determine whether APN concentration and APN receptor expressionlevels are altered in the well-established STZ-induced type-1diabetic heart model(and the time-dependency of any observed alteration);(2) To identify consequences of any observed dynamic change in APN/APNreceptor levels in the cardioprotective effects of APN against MI/R injury. (3) To clarify the role of oxidation and nitration stress in type1diabetesafter MI/R.Methods(1) Mice MI/R model: Mice were anesthetized with2%isoflurane, andmyocardial infarction (MI) was produced by temporarily exteriorizing the heartvia a left thoracic incision and placing a6-0silk suture slipknot around the leftanterior descending coronary artery. After30minutes of MI, the slipknot wasreleased, and the myocardium was reperfused for3hours or24hours.Sham-operated control mice (sham MI/R) underwent the same surgicalprocedures, except the suture placed under the left coronary artery was not tied.(2) Mice T1DM model: Diabetes was induced by intraperitoneal injection of40mg/kg STZ diluted in citrate buffer (pH4.5) for5consecutive days, andage-matched control mice were injected with an equal volume of citrate buffer.Diabetes onset was confirmed by hyperglycemia exceeding10mmol/L5daysafter STZ administration.(3) Measurement of Myocardial Infarct Size: Twenty-four hours afterreperfusion, mice were anesthetized, and the hearts were excised. Myocardialinfarct size was determined by using Evans blue/2,3,5-triphenyltetrazoliumchloride (TTC) staining as previously described(4) Assessment of Myocardial Injury：To quantitatively determinemyocardial injury extent, blood samples were collected. Serum LDH release wasmeasured per manufacturer’s protocol. Values expressed in international units (U)per liter.(5) Determination of Myocardial Apoptosis：Myocardial apoptosis wasdetermined via TUNEL staining and caspase-3activity assay, inclusive of theentire ischemic/reperfused region commonly termed “area-at-risk" as described previously(6) Quantitation of Plasma APN Concentration：Serum APN concentrationswere determined via mouse APN ELISA kit per manufacturer’s instructions(7) Immunoblotting：Cardiac tissue homogenate proteins were separated onSDS-PAGE gels, transferred to nitrocellulose membranes, and Western blottedwith monoclonal antibody against AdipoR1and AdipoR2. Nitrocellulosemembranes were then incubated with HRP-conjugated antirabbitimmunoglobulin G antibody for1hour. The blot was developed with anECL-Plus chemiluminescence reagent kit and visualized with UVP Bio-ImagingSystems. Blot densities were analyzed with Vision Works LS Acquisition andAnalysis Software.(8) Contents of nitric oxide (NO) and its in vivo metabolites NO2and NO3(collectively termed as NOx) were determined using nitrate reductase method.(9) O2￣production was determined by lucigenin-enhanced luminescenceand in situ dihydroethidium (DHE) staining.(10) Myocardial ONOO-formation was determined by competitive ELISA.Results(1) Plasma APN levels (total and HMW form) increased while cardiacAdipoR1expression decreased early after T1DM onset. With T1DM progression,APN levels reduced, and cardiac AdipoR1expression increased.(2) MI/R injury exacerbated with T1DM progression in time-dependentmanner.(3) Administration of globular APN (gAD)10minutes before reperfusionfailed to attenuate myocardial injury in1-week T1DM mice, while anAMP-activated protein kinase (AMPK) activator (AICAR) reduced MI/R injury.(4) Administration of gAD (and AICAR) reduced infarct size and cardiomyocyte apoptosis in7-week T1DM mice.(5) Myocardial NOx production increased in mice with7-week diabeticduration, and MI/R injury further enhanced NOx production.(6) Myocardial O2￣production was increased in7-week diabetic mice, andMI/R injury significantly enchanced O2￣production (P <0.05vs. WT+Sham).(7) ONOO-formation was increased (P <0.05vs. WT+Sham) in7-weekdiabetic mice and MI/R injury further enhanced its production.(8) Intraperitoneal administration of gAd or EUK134（ONOO-scavenger,5mg/kg）decreased ONOO-production and attenuated myocardial injury inducedby MI/R at the7-week diabetic mice.Conclusions(1) Plasma adiponectin level changes during the time course of type1diabetes, it increases in early stage, but decreases in late stage. Its receptor R1expression decreases in early stage, but increases in late stage. These resultssuggest that there may be exist adiponectin resistance in early stage, andhypoadiponectinemia in late stage.(2) Administration of exogenous adiponectin before reperfusion has noeffect on MI/R injury in early stage of type1diabetes, while AICAR exertsmyocardial protection after MI/R. Suggesting that decreased AdipoR1expressionmay account for the uselessness of adniponectin treatment.(3) Administration of exogenous adiponectin attenuates MI/R injury in thelate stage of type1diabetes, suggesting hypoadiponectinemia is the reason forthat the aggregated MI/R injury in late stage of type1diabetes.(4) The oxidation and nitration stress aggravated during the duration of type1diabetes, and MI/R further aggravated it. Exogenous adiponectin and EUK134improve oxidation and nitration stress and MI/R damage， suggesting that oxidation and nitration stress in late stage of type1diabetes is the key factor forAPN’cardioprotection in aggravated MI/R injury. |
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