Apelin Ameliorates Cardiovascular Disorders And Myocardial Glucose Metabolism In High-fat-fed GK Rats

Objective:Metabolic syndrome is characterized by diabetes mellitus,central obesity,hypertension and dyslipidaemia,which has a common pathophysiological basis—insulin resistance.Among those components,diabetes,especially type 2 diabetes has been proved to include most of factors in metabolic syndrome.Its condition is closely related with the occurence and development of arteriosclerosis.With the alteration of life style and dramatic aged tendency of population,morbidity of diabetes is increasing.Risk of cardiovascular disease increased 2-4 fold in diabetic patients,while more than 70% of people with diabetes die from cardiovascular disorders.So diabetes is proved to be an independent risk factor for cardiovascular disorders.Study on mechanism of type 2diabetes at molecular level has been present research tendency.Tatemoto isolated a novel peptide ligand from bovine stomach extracts in 1998,which was proved to be an endogenous ligand for the putative receptor protein related to the angiotensin receptor AT1(APJ).Then the peptide ligand was named Apelin.A research showed Apelin is secreted by adipose tissue in rats and human being,and it is located in endothelial cells of a variety of tissues including heart,lung,kidney,adrenal.Now Apelin has been shown to exhibit many important potential roles in the regulation of cardiovascular system,adipoinsular axis,hypothalamic hormone secretion,fluid homeostasis,stress,feeding behavior and thermoregulation.Not only has Apelin been observed to decrease left ventricular preload and afterload and improve cardiac contractility without myocardial hypertrophy,but the peptide also exhibits regulation of arterial pressure via vasodilator actions in rats.A study showed Apelin plays a role in regulating arterial pressure via endothelium-dependent vasodilator actions,which doesn't depend on angiotensin ?.A growing number of studies have been reported on the relationship between obesity,glucose metabolism and Apelin.A study provided evidence that insulin exerts a direct control on Apelin gene expression in adipocytes.Apelin can also inhibit the secretion of insulin on hyperglycemia.However,the mechanism of the correlation between Apelin and insulin is unclear still.Present studies were involved in temporary Apelin administration both in vitro and in vivo.Study on chronicadministration of Apelin is required for the potential clinical application.There is increasing evidence that insulin resistance and resultant impaired beta-cells function may play important roles in the development of type 2 diabetes.The function of glucose uptake stimulated by insulin is impaired in type 2 diabetic subjects.Cardiac energy uptake depends mainly on glucose oxidation and anaerobic glycolysis under the condition of acute cardiac overload and ischemia.The first rate-limiting step in glucose metabolism in cardiac cells is that glucose on the myocardial cell membrane is inducted into cells by glucose transporter type 4 isoform(GLUT4).The limited GLUT4 expression is proved to be closely associated with the impaired utilization of glucose of heart.A defect in function or number of GLUT4 may mainly result in insulin resistance.Thus,more research on associative influence factors of GLUT4 would be helpful to control insulin resistance.AMP-activated protein kinase(AMPK)not only plays important biological effects on regulating intracellular fatty acid metabolism via monitoring the change of intracellular AMP/ATP ratio,but also has been proved as a positive regulator of insulin sensitivity.Besides regulating insulin sensitivity and fatty acid oxidation,several studies has showed that AMPK may regulate directly the expression of GLUT4.AMPK activator or AMPK overexpression may lead to the increase of insulin sensitivity.The decreased expression of AMPK was also proved to be closely related to the significantly impaired sensitivity of insulin in skeletal muscles,adipose,liver and other tissues induced by hyperlipidemic environment.Thus,as a receptor of energy metabolism,AMPK has been proved to be the link between insulin sensitivity and energy metabolism.This study investigated the effect of Apelin on cardiovascular protection and myocardial glucose metabolism in GK rats by molecular biology from expression level of mRNA and protein in order to elucidate its mechanism in occurance and development of diabetic cardioangiopathy.The result would establish new theoretical basis and target of clinical diagnosis,prevention and treatment for metabolic syndrome.Methods:1.32 male(12 weeks old)GK rats and 8 male(12 weeks old)Wistar rats were randomly divided into 5 groups: Wistar group,GK-control group,GK-metformin group,GK-atorvastatin group and GK-Apelin group.Fasting plasma glucose(FPG),systolic blood pressure(SBP)and body weight were measured every week.2.Fasting for 12 hours after 4 weeks' treatment,body weight(BW)and body length were measured in rats.All rats were anesthetized with pentobarbital sodium(35mg/kg,intraperitoneally).After horizontal midline incision in the neck was made,dissection was carried down to expose the right common carotid artery,which head end was ligatured.Microcardio catheter(filled with 40U/ml heparin saline)was inserted retrograde through the common carotid artery into the left ventricle.The other end was connected to the multipurpose polygraph through a pressure transducer.Heart rate,left-ventricular end-systolic pressure(LVESP)and left-ventricular end-diastolic pressure(LVEDP),maximum rate of rise in intraventricular pressure(+ dp/dtmax),maximum rate of fall in intraventricular pressure(-dp/dtmax)in left-ventricular were recorded on Powerlab as described.After venous blood was drawn and centrifuged,the supernatant was extracted.The supernatant was preserved at-70?.After all rats were executed,the myocardium and aorta were removed by thoracotomy for immunohistochemistry assay.3.FPG was determined by glucose oxidase method.Fasting serum insulin(FINS),plasma endothelin-1(ET-1),serum leptin,plasma tumor necrosis factor-?(TNF-?)concentrations were determined by commercially available radioimmunoassay kits.Serum nitric oxide(NO)was measured by nitrate reductase method.Nitric oxide synthase(NOS)was determined by chemical colorimetry.Constructive nitric oxide synthase(cNOS)=total nitric oxide synthase(tNOS)-inducible nitric oxide synthase(iNOS).Total cholesterol(TC)and triglyceride(TG)were determined by enzymatic method.High-density lipoprotein cholesterol(HDL-C)and low-density lipoprotein cholesterol(LDL-C)were measured by direct method.Homeostasis model assessment insulin resistance(HOMA-IR)was calculated as FPG multiplied by FINS and divided by 22.5.4.The mRNA expression of Apelin,GLUT4 and AMPK?2 were determined by RT-PCR.The protein level of Apelin,GLUT4 and phospho-AMPK? were measured by Western blot.5.All statistical analyses were made by the SPSS20.0 software package.Univariate variance analysis(ANOVA)and t test were used to compare the results between the groups.A value of P<0.05 was considered statistically significant.Results:1.FPG,SBP,lee's index and HW/BW in GK-control group were significantly higher than in Wistar group.FPG,SBP,lee's index and HW/BW in GK-Apelin group were significantly reduced than in GK-control group.2.Compared with Wistar rats,GK-control rats showed markedly lower LVESP,± dp/dtmax values and higher LVEDP.After administration of Apelin,LVESP and ± dp/dtmax values were increased,while LVEDP values was lower than in GK-control group.3.Compared with Wistar rats,GK-control rats showed significantly higher FINS,HOMA-IR,TNF-?,leptin,TC,TG,LDL-C,ET-1,iNOS and lower HDL-C,NO,cNOS.In GK-Apelin group,FINS,HOMA-IR,TNF-?,leptin,TC,TG,LDL-C,ET-1 and iNOS levels were reduced,while HDL-C,NO and cNOS were elevated.4.HE stain results: compared with Wistar group,myocardial fibers were chaotically lined up;myocardial cells were enlarged;the aortic endothelial cells were swollen;the intima were thickened;the collagen fibers were proliferated;and the internal elastic membrane were broken in GK-control group.In each intervention group,myocardial fibers were orderly arranged;myocardial cells was complete;the aorta intima was smooth;vascular smooth muscle cells were orderly arranged.Immunohistochemistry assay showed that the positive reaction of Apelin was stained brown.Sparse and light brown granules were found in the myocardial cell membrane and intima of the aorta in GK-control.After Apelin/ metformin/ atorvastatin intervention,the positive stained granules of Apelin increased markedly,especially in GK-Apelin group.5.Lower level of Apelin in plasma,ventricular,aortic tissues,lower mRNA level of Apelin in ventricular and aorta,lower level of Apelin protein were showed in GK-control group compared with Wistar group.After Apelin/ metformin/atorvastatin intervention,concentrations of Apelin,Apelin mRNA and Apelin protein were increased than in GK-control group.6.The expression of GLUT4 mRNA,AMPK?2mRNA,protein levels of GLUT4 and P-AMPK? in ventricular in GK-control group were significantly lower than in Wistar group.In GK-Apelin group,the expression of GLUT4 mRNA,AMPK?2 mRNA and protein levels of GLUT4 and P-AMPK? in ventricular were higher than in GK-control group.Conclusion: 1.High fat induced the decline in levels of Apelin,Apelin mRNA,Apelin protein,GLUT4 mRNA,GLUT4 protein,AMPK?2 mRNA and P-AMPK? protein.High-fat-fed GK rats suffered from severe insulin resistance and glucose transport disfunction.2.After intervention of metformin and atorvastatin,levels of Apelin mRNA,Apelin protein were elevated,which indicates that Apelin may be a mediator for metformin and atorvastatin to protect cardic function.3.After administration of Apelin,levels of GLUT4 mRNA,GLUT4 protein,AMPK?2 mRNA and P-AMPK? protein were increased.The result suggests long-term administration of Apelin may have effect on glucose transport so as to alleviate insulin resistance.4.The administration of Apelin improves endothelial dysfunction.5.The administration of Apelin alleviates blood pressure,hemodynamics and myocardial hypertrophy.