Effect Of C-peptide On Proliferation And Apoptosis Of Rat Mesangial Cells Cultured In High Glucose

Background and ObjectiveDiabetes mellitus is one of the life-threatening epidemic diseases, which forms the third death rate just descending from the cardiovascular diseases, cerebrovascular diseases and cancer. The high mortality and disability rate characterizes the diabetic complication. The diabetic vascular complication is composed of macrovascular diseases and microvascular diseases and there are some interesting and subtle differences between them. It is generally believed that macrovascular and microvascular complications coexist in type 2 diabetes, while the former is the main fatal reason, however, in typel diabetes the later is more dangerous, for in most cases, the microvascular complications in typel diabetes is obviously faster developed than that in type 2 diabetes. There is now no satisfactory explanation to this phenomenon, yet from these clues it might be reasonable to conclude that the pathogenesis of microvascular complications in type 1 and type 2 diabetes is somewhat different. Recently it is reported that the diabetic patients who preserve some residual pancreas islets were more unlikely to capture the microvascular complications than those whose pancreas are completely destroyed despite almost the same glycemia condition controlled by insulin. It was once believed that this is probably caused by the superiority of endogenesis insulin to exogenesis insulin, but the finding of the biological effect of connecting peptide (C-peptide) obviously is a more reasonable hypothesis. As a product of proinsulin processed to insulin, C-peptideis is secreted by the pancreatic beta cells in equimolar amounts along with insulin. It was thought that C-peptide's main physiological role was in facilitating the folding of the proinsulin molecule in a manner that allowed for the appropriate formation of the disulfide bonds between the cysteine residues of the A- and B-chains of insulin. However recently, it has been demonstrated that C-peptide treatment in type 1 diabetic patients resulted in improved complication conditions.The aim of this study to establish culture technique of rat mesangial cells line HBZY-1 in vitro and to investigate the effects of C-peptide on diabetic nephropathy and the relationship with the proliferation and apoptosis; to testify the biofunction of the C-peptide. The hypothesis of C-peptide will definitely bring us a brand new perspective: the combination use of insulin and C-peptide, a manner which is more physiological, may ultimately replace the conventional use of insulin in the treatment of type 1 diabetes and the type 2 diabetic patients with complete islets destruction.Materials and methodsIn the present study we investigated the biofunction of C-peptide in vitro. rat mesangial cells line HBZY-1 were incubated at different concentration of D-glucose, insulin and C-peptide for 24, 48 and 72 hours, then proliferation was measured by MTT (methyl thiazolyl tetrazolium) method and apoptosis was measured by TUNEL and the cell cycle was detected by flow cytometry. To further investigate the mechanism of apoptosis the level of several protein, Bcl-2 and Bax involved this process were observed by immunocytochemical technique.Results(1) Our findings showed that the high glucose concentration instead of Mannitol significantly enhanced the proliferation of HBZY-1, rat mesangial cell at 48 hours by MTT(methyl thiazolyl tetrazolium) method while the normal glucose concentration did not (P<0.05 respectively). The C-peptide of 10nmol/L concentration suppressed the proliferation and apoptosis caused by high glucose, and the things was the same with higher C-peptide concentration. The case was similar with the combination of the C-peptide with insulin had the same effect (P<0.05). while insulin did not (P>0.05). (2) The apoptosis was measured by TUNEL. The C-peptide suppressed the apoptosis caused by high glucose, and the things was the same with higher C-peptide concentration. The case was similar with insulin, and the combination of the C-peptide with insulin had the sameeffect (P < 0.05 respectively).(3) High glucose concentration also increased the apoptosis and mitosis of the cells simultaneously, that was detected by flow cytometry. The C-peptide suppressed the apoptosis and mitosis caused by high glucose, and the things was the same with the combination of theC-peptide with insulin (P< 0.05 respectively).(4) The level of Bcl-2 and Bax both were increased by high glucose in cells, and when incubated with C-peptide and insulin, the expression of Bax was greatly reduced, however the level of Bcl-2 was also increased (P<0.05).Conclusions(1) The high glucose could induce the proliferation of the cells, and the combination of the C-peptide and insulin may significantly attenuate this trend;(2) The high glucose could induce the apoptosis of the cells at the same time, the mechanism of this phenomenon might be conducted by Bcl-2 and Bax, which are generally believed to play important roles in the apoptosis;(3) The C-peptide and insulin may significantly attenuate this trend. The case was similar with the combination of the C-peptide with insulin had the same effect. namely the C-peptide might possess the biofunction of anti-proliferation and anti-apoptosis simulaneously, and this paradox needs more proof.These results mean that C-peptide may be associated with onset of diabetic microvascular diseases and would be useful in clinical treatment for diabetes.