Effects Of Glucose And Insulin On The H9c2(2-1) Cell Proliferation May Be Mediated Through Regulating GLUT4Expression

BackgroundDiabetes is the most common chronic disease endangering public health; cardiovascular events were the leading causes of death in diabetics. Glucose is an important energy source for myocardial metabolism. Normal glucose transport and metabolism is one of the key factors to maintain the normal physiological function of myocardial cells. And diabetic metabolic disorders caused by high blood glucose, which act as the major cause for diabetic heart disease, could also be the foundation of many kinds of heart vascular lesions.Glucose transporters (GLUTs) family is the rate-limiting factor which mediates the glucose uptake into the cells and intracellular glucose utilization, now there are14kinds of subtype. GLUT1,4,12can be detected in heart cells. And the delivery of glucose from the blood to the myocardial cells is mainly regulated by GLUT4. Studies had shown that chronic hypoglycemia could cause the compensatory up-regulation of GLUTs expressions in cells. Along with GLUTs expression increasing, cells absorb more glucose to adapt the energy deficiency conditions. Our preliminary studies showed that chronic hyperglycemia down regulated GLUTs expression in brain, which could protect the bodies against hyperglycemic toxicity caused by excessive glucose into cells, as a compensatory mechanism of protection which we called hyperglycemia pre-accommodation (HGPA) mechanism. Based on these results, we put forward the hypothesis of hyperglycemia pre-accommodation. Diabetic heart disease is the leading cause of death in diabetics. However, the specific influence of high blood glucose to myocardial cell metabolism is not clear. And whether the HGPA hypothesis exists in myocardial cells is unclear. This study aimed to explore the influence of glucose and insulin at different concentrations on H9c2(2-1) cell proliferation and its glucose transporter4expression in vitro, and evaluate the correlation between myocardial cells proliferation and GLUT4expression. Verify whether there is HGPA in myocardial cells. Materials and methodsAccording to glucose concentrations in culture medium, cultured H9c2rat myocardial cells were divided into five groups:control group (NC), low glucose group (LG), high glucose group1(HG1), high glucose group2(HG2), high glucose group3(HG3). And the glucose concentrations in five groups respectively were5,0.1,10,15,20mmol/L. According to insulin concentrations in culture medium, each group was further divided into two subgroups:Normal insulin subgroup (INSc, insulin concentration3.8mU/L); High insulin subgroup (INSh, insulin concentration7.6mU/L). H9c2(2-1) cells were cultured for1,2,3days, the proliferation of cells were assayed by CCK-8, the expressions of GLUT4mRNA and protein were detected with RT-PCR and Western Blot technique, and the relation between myocardial cells proliferation and GLUT4expression was evaluated.Results1. Compared to NC group, cell proliferation (OD value) was lower in LG, HG2, HG3group but higher in HG1group on the second and the third day (P<0.05). OD value had a negative correlation with the glucose level in HG1, HG2, HG3groups (P<0.05). OD value in INSc subgroup was lower than in INSh subgroup (P<0.05).2. GLUT4mRNA expression was lower in LG, HG2, HG3groups than in NC group (P<0.05). Compared to NC group, GLUT4mRNA expression in HG1group was higher on the first day but lower on the second and third day (P<0.05). In HG1, HG2, HG3groups, GLUT4mRNA expression had a negative correlation with the level of glucose (P<0.05). GLUT4mRNA expression in INSc subgroup was lower than in INSh subgroup (P<0.05).3. The expression trend of GLUT4protein was similar to that of GLUT4mRNA.4. H9c2cell proliferation had a positive correlation with GLUT4expression (P<0.01).Conclusion 1. Glucose levels could influence myocardial cell proliferation in vitro culture model. With the extension of stimulate time, the higher the glucose concentration was, the lower the cell proliferation was. There’s negative correlation between them.2. Glucose levels could influence myocardial cell expressions of GLUT4mRNA and protein. There was a negative correlation between glucose level and GLUT4expression, which meant high glucose level down regulated GLUT4expression.3. High insulin level could improve cell proliferation, also could increase GLUT4expression.4. Effects of glucose on the myocardial cell proliferation might be mediated by GLUT4, and insulin levels might be also one of the influence factors.5. High glucose level down regulated the expression of GLUT4, which might be an adaptive reaction of the myocardial cells to the high blood glucose, indicating that there is a mechanism of hyperglycemia pre-accommodation (HGPA) in myocardial cells.SignificancesOur study used H9c2myocardial cells to explore the influence of glucose and insulin at different concentrations on H9c2(2-1) cell proliferation and its glucose transporter4expression in vitro, found that glucose an insulin effect myocardial cells proliferation through mediate GLUT4expression; There was a negative correlation between glucose level and GLUT4expression, which meant high glucose level down regulated GLUT4expression. And HGPA was initially proved, found out the suitable external environment for the myocardial cell growth. The present and subsequent studies may be helpful for clinical treatment of DM. It may be also worthy to clinical practice and theoretical significance.