Investigation Of The Effects Of Pyruvate Carboxylase On Islet β Cells Proliferation

Background:With the development of society and economy and the change of people’s lifestyle,type 2 diabetes(T2DM)and its complications have brought an increasingly serious burden on human health.β cells proliferation and compensatory proliferation are important for maintaining blood glucose homeostasis under pathological conditions.Promoting the proliferation of residual β cells,increasing endogenous insulin level and preventing hyperglycemia is a significant underlying therapeutic target for diabetes.Pyruvate carboxylase is one of the key enzymes in the mitochondrial tricarboxylic acid cycle and involved in regulation insulin secretion in beta cells.Recently,we have found that PC is upregulated in the β cells of female pregnant mice and obesity mice induced by high-fat diet.Overexpression of PC may induce β cell proliferation,which may be blocked by knocking down the PC.This phenomenon may be caused by the inhibitory effect of PC on p53.Based on these findings,we propose that PC are vital for maintaining β cell proliferation.In the early stages of insulin resistance,short-term metabolic stress(such as obesity or over nutrients)can induce PC expression and inhibit the expression of p53,thereby promoting the compensatory proliferation of β cells.The purpose of this study was to investigate the effect of pyruvate carboxylase on the proliferation of islet β cells and the possible regulatory mechanism.Methods:Immunofluorescence staining of paraffin section was used to detect the expression of PC in islets of diabetic mice,pregnant female mice and the positive rate of ki67 in islets of pregnant female mice.Islet β cell line MIN6 cells were cultured in vitro and treated with different glucose concentrations(5mM,15mM,25mM)for 6h,12h,18h and 24h to establish an high glucose stimulated model.Western blot and real-time quantitive PCR were used to detect the effects of different glucose concentrations on PC expression from protein and mRNA level,respectively.Establishing the over-expression and knock-down model of PC in vitro by having MIN6 transfected with PC/GFP adenovirus and PC/GFP lentivirus.CCK-8 was used to detect cell proliferation.Immunofluorescence staining was used to detect the positive rate of BrdU.Western blotting was used to detect the changes of p53 in the cases of PC overexpression and knockdown on the protein level.Results:Immunofluorescence staining of paraffin sections from mouse pancreas showed that the expression of PC in the islets of diabetic mice was lower than that of the normal control mice,and the expression level dropped gradually along with the progress of diabetes.The positive rate of ki67 in the islets of pregnant mice was higher than that in the female mice,especially in the second trimester(P<0.05),and the expression of PC in the islets of pregnant mice was higher than that of the female mice.After high-glucose treatment of MIN6,PC expression began to increase at 6h and reached the peak at 24h treated with 15mM glucose(P<0.0001).After MIN6 cells transfected with PC/Flag adenovirus,the CCK-8 assay showed that the cell proliferation in the overexpression group was significantly higher than that in the control group(P<0.0001).The immunofluorescence showed that the BrdU positive rate in overexpression PC group was higher than the control group(P<0.05),western blotting showed that the p53 expression was downregulated after overexpressing PC.After MIN6 cells transfected with PC/GFP lentivirus,the CCK-8 assay showed that the cell proliferation in the knockdown group was significantly lower than that in the control group(P<0.0001).The immunofluorescence showed that the BrdU positive rate in knockdown PC group was lower than the control group(P<0.0001),western blotting showed that MDM2 expression was downregulated and the p53 expression was upregulated after knocking down PC.Conclusion:Increasing PC expression can promote islet β cells proliferation in mice and this effect can be blocked by knocking down PC.Overexpression of PC can facilitate the proliferation of islet β cell by down-regulating the expression of p53 protein.