Studies On Effects And Roles Of C-peptide In Endothelial Cells Apoptosis Induced By Hyperglycemia And Hyperinsulinemia

Diabetic macrovascular complications are the major cause of mortality and disability among diabetes patients, the incidence of macrovascular disease is higher, the disease severity is greater and the onset is earlier. It was estimated by WHO that the incidence of diabetes mellitus will be double in next 10 years, so it is very necessary to investigate the impairment role of diabetic macrovascular and find out the way for prevention and treatment.Endothelial cells apoptosis is an initiatory and determinant in diabetic macrovascular disease, it can induce a series of changes on structure and function of vessel and it has closely relation with nitric oxide (NO). C-peptide is a kind of active peptide hormone that was realized soon. It had showed exert important physiological effects on regulating glucose utilization and improving renal and nerve function, but the effects and roles of C-peptide in macrovascular disease was unware. Several studies showed endothelial nitric oxide synthase(eNOS) mediated the effect of C-peptide. We would like to know whether C-peptide exerts actions in macrovascular disease, so design the experiment. Objective: To establish culture technique of HUVEC in vitro and to investigate the effects of C-peptide on diabetic macrovascular disease andthe relationship with the expression of eNOS and genes related to apoptosis.Methods: The cultured Human Umbilical Vein Endothelial Cells (HUVEC) were incubated at different concentration of D-glucose, insulin and C-peptide for 72 hours, then EC apoptosis was measured through Cell DNA Fragmentation ELISA and the expression of genes were measured through SQ-RT-PCR.Result: l.ECs present cobblestone-shaped and a positive immunofluorescent reaction.2.The expression of eNOS at low concentration of C-peptide was lower compared with control group, the expressions of eNOS were higher at moderate and high concentration of C-peptide.3. Physiologic concentration of C-peptide may inhibit EC apoptosis induced by hyperglycemia and enhance the expression of eNOS, the effect is similar with hyperglycemia, hyperinsulinemia and high concentration of C-peptide, after C-peptide concentration was reduced, EC apoptosis increased and the expression of eNOS decreased. 4.Low concentration of insulin and C-peptide may inhibit EC apoptosis induced by hyperglycemia and enhance the expression of eNOS, but the effect was alike as insulin alone, the same result had been obtained in High concentration glucose and insulin.S.It was negative correlation between EC apoptosis and the expression of eNOS.6.physiologic concentration of C-peptide may enhance the expression bcl-xL and decrease the expression of bax. High concentration of C-peptide had similar effects, when C-peptide concentration was reduced, theexpression of bax was enhanced and the expression of bcl-xL was decreased. It was positive correlation between bcl-xL and eNOS. Conclusion: l.The culture of HUVEC was successful. 2.HUVEC has response to C-peptide, therefore it can be used in research of C-peptide. 3. The effects of C-peptide on EC apoptosis may be mediated by eNOS and associated with the ratio of bcl-xL/bax.4.C-peptide had no synergistic action with insulin on inhibiting EC apoptosis, that means insulin and C-peptide could exert through same way.5.lower concentration of plasma C-peptide may be contributed to diabetic macrovascular diseases hi Type 2 DM.These results means that C-peptide may be associated with onset of diabetic macrovascular diseases and would be useful in clinical treatment for diabetes.