Study On Diabetic Cardiomyopathy And The Changes Of Insulin Signaling Pathways In The Heart Of Spontaneous Diabetic Rat (OLETF Diabetic Rat)

Part I: The establishment of a model of the spontaneous diabetic rat with diabetic cardiomyopathyDiabetic cardiomyopathy is a cardiac disease that arises as a result of the diabetic state, independent of vascular or valvular pathology. Because of the few measurements in diagnosing the diabetic cardiomyopathy in clinical practice and the difficulty in getting the myocardium tissue for biopsy in type 2 diabetic patients, it is very hard to detect this complication of diabetes. As the dramatically increasing patients of type 2 diabetes in recent years, it is of great significance in studying the mechanism of diabetic cardiomyopathy. The purpose of this part is to focus on establishing an animal model of type 2 diabetes with diabetic cardiomyopathy.The diabetic rat model—OLETF rat, which resembles human type 2 diabetes, was selected. After a long time of raising and observation, we got a lot of knowledge about the characteristics of this rat model, such as the progress of diabetes, the morphological alteration of myocardium andthe deterioration heart function using the histomorphology (ultrathin section transmission electron microscope) and the functional tests (oral glucose tolerance test and electrophysiolograph). Our results demonstrated the characteristics of OLETF rats were as follows: 1) late onset of hyperglycemia; 2) a chronic course of the disease; 3) mild obesity; 4) abnormal lipid metabolism; 5) renal complication (nodular lesions).Our results also showed a series of changes in the heart of OLETF rat model. At 24 weeks of age, the pathological changes in the hearts of OLETF rats included the swelling and foldback of the blood vessel endothelium, the lumina narrowing, the scattering of considerable white lipid droplids in muscle fibers of cadiocyte and the slightly augment of the interstitial collagen. At 70 weeks of age, when accompanied by renal lesions, the basement of the heart muscle capillary became thickening. The prominence of endothelium in the lumen of capillary vessel was observed. The degeneration, confluence and floatation of mitochondria were detected. The deposits of lipofuscin were found in cardiac muscle cells. The collagen deposition in the myocardial interstitium and perivascular area were greater than that of 24 weeks of age. In addition, the contraction of ventricular myocardial cells and the formation of the cellular necrosis were showed. All mentioned above might play a role in the heart diastolic dysfunction independent of the blood pressure andcoronary artery disease.Part II The changes of the insulin signaling components in OLETF rats - differential gene expression analysis of the diabetic heart using gene chipThe pathogenesis of diabetic cardiomyopathy is complicated. With gene chip, we can detect the differential gene expression related to diabetic cardiomyopathy in a short time. In order to approach the pathogenesis of diabetic cardiomyopathy, the messenger RNA sample of three hearts of OLETF and LETO rats were pooled separately and sent to the company of Affymetrix to compare the differential gene expression. The total RNA was extracted and purified from the left ventricular of these rats. The probes of cDNA and cRNA were obtained by reverse transcription test from the two groups. cRNA probe was hybridized with gene expression trip. The results were offered by scan of the gene trip on a scanner, analyzed by GCOS software and then calculated by statistics. To confirm the gene expression patterns of microarray hybridization, reverse-transcription (RT) polymerase chain reaction (PCR) was performed in one important gene, of which the expression level was significantly decreased. The use of gene chip has made it possible to analyze simultaneously gene expression of more than 30000 genes. 102 genes were 2-fold greater downregulated and 103 genes were 2-foldgreater upregulated in the hearts of OLETF rats than that of LETO rats. Of the known genes, the genes of insulin signaling, the regulatory subunit of phosphatidylinositol 3-kinase, p85 and the IRS—1 was significantly downregulated. Furthermore, the expression of enolase 1, alpha, Acyl Coenzyme A dehydrogenase, 3-hydroxy-3-methylglutaryl-Coenzyme A lyase, esterase 2, ubiquitin-activating enzyme El-domain containing 1 and solute carrier family 3, member 1 gene were decreased. Myosin, a protein with the ATP enzymatic activity, which can bind to calcium and take part in the movement and contraction of myofilament, was underexpressed. Other kinds of genes related to the induction of smooth muscle cell proliferation, angiogenesis, cell differentiation, cell adhesion and migration were overexpressed, such as Kruppel-like factor 5, epidermal growth factor nuclear factor, interleukin 3 regulated, integrin alpha 1, angiopoietin-like protein 4, Glutamyl aminopeptidase, cysteine rich protein 61 gene and so on. Kruppel-like factor 5 is a positive regulator transcription factor. When simulated by angiotensin II, it can bind to PDGF-A resulting in myocardial hypertrophy. Also, it can activate MEK pathway like epidermal growth factor.Part III— The study of cardiac leptin JAK2/IRS-2/PI-3K and insulin IRS-2/PI-3K signaling pathways in the hearts of OLETF ratsThe hyperinsulinemia and hyperleptinemia always exist in type 2 diabetes with obesity. Some studies showed that insulin receptor and some kinds of leptin receptors located simultaneously in the myocardial cells. So, we suppose there are complex interaction between these two hormonal signaling systems in the heart as in liver and pancreas. In present study, we are aim to exam the insulin-stimulated IRS-1/PI-3K mediated signaling for translocation of GLUT4 and the cross talk between leptin and insulin signaling pathways in myocardial cells. By using the methods of aorta retrograde catheterization and heart perfusing, cardiomyocytes isolated from the rats were cultured in DMEM medium. The existence of leptin OB-Ra and OB-Rb receptors in the myocardium of OLETF, LETO, SD and STZ rats was validated. Western-blot was used to detect different GLUT4 expression in the cytomembrane of cardiomyocyte. Result showed: 1) After the stimulation of insulin or leptin alone or together, the levels of GLUT4 protein in cytomembrane of myocardial cells of OLETF rats were less increased than that of LETO rats. Our data supported the view that insulin can improve the gene expression or translocation of GLUT4. Leptin might act through the cross talk with insulin signaling pathway to effect the gene expression or translocation of GLUT4; 2) The pretreatment of inhibitor of PI-3K, ly294002, for 30 minutes, decreased GLUT4 content in cytomembrane of myocardial cells with the stimulation of insulin or leptin or thecombination of them in LETO rats. But in OLETF rats, this effect was only observed in the group stimulated by insulin. Meanwhile, the pretreatment of inhibitor of JAK2, AG490, for 30 minutes, resulted in the decrease of GLUT4 content after the stimulation of leptin in the cytomembrane of myocardial cells in LETO rats. However, it had no effect on that of the OLETF rats. These results suggested these signaling pathways might be primary impaired in OLETF rats.Conclusions1) The diabetic cardiomyopathy in type 2 diabetic model was identified with characteristics of ① having the traits of type 2 diabetes; ② without hypertension; ③slight damage of heart diastolic function; ④ predominance cardiac pathological changes including interstitial fibrosis and microangiopathy; ⑤without coronary artery disease.2) The methods of myocardial cell culture of adult OLETF and LETO rats were successfully established. The viability of these cells reached the requirement for experiments.3) The existence of leptin OB-Ra and OB-Rb receptors in the myocardium of SD, STZ, OLETF or LETO rats was validated.4) In OLETF rats at 70 weeks of age, the regulatory subunit of phosphatidylinositol 3-kinase, p85, was 8-fold greater downregulated and the expression of IRS—1 mRNA also decreased, strongly suggesting the impairment of insulin signaling pathway in diabetic heart. Second, the gene expression levels of some important enzymes related for glycolysis, lipid metabolism, proteometabolism were significantly decreased expression, which suggested the metabolic disturbance in diabetic heart. Third, some well-known genes for smooth muscle cell proliferation, angiogenesis, cell differentiation, cell adhesion and migration are higher expressed. These changes in gene expression may be the foundation of the pathological impairments in diabetic cardiomyopathy.5) The Insulin signaling pathway of IRS-1/PI-3K and the leptin signaling pathway of JAK2/IRS-2/PI-3K may exist in the hearts of OLETF and LETO rats, which may have cross talk with each other. Leptin can mimic insulin action through the cross talk with insulin signaling. But in OLETF rats, these pathways might be impaired. LY294002 and AG490, which block the insulin and leptin signaling pathways separately, may be harmful to cardiac glucose metabolism.