Berberine Potentiates Insulin Secretion Via CAMP/PKA/calcium Channel Pathway

Objective Berberine(BBR), a protoberberine alkaloid, the main active is isalkaloids, which has been proved to be effective in the treatment of type 2 diabetes mellitus(T2DM) in clinical. Most of realted studies focusd on the effect of BBR improving insulin resistance. But only few controversial reports reveal that BBR also has impacts on insulin secretion. The underlying mechanism is still far to clear. In this study, we conducted an investigation to BBR regulating insulin secrtion via c AMP-PKA-CREB-calcium channel proteins pathways.Methods Rat insulin tumour cell lines INS-1 832/13 cells were cultured. Different concentrations of Berberine(1?M, 5?M, 10?M, 20?M) were added to the cells for 72 hours. After 72 h incubation, MTT was used to determine cytotoxicity of BBR to the cells. Apoptosis of the cells was analyzed by flow cytometry. Quantitative real-time polymerase chain reaction(q PCR) was applied to evaluate expression of genes including bcl-2, L-type calcium channel cav1.2, pdx1, ins1 and ins2, respectively. Western blotting was used to evaluate expression of Cav1.2, AMPK, P-AMPK, CREB and P-CREB, respectively. Insulin content and glucose stimulated insulin secretion(GSIS) of rat islets or the INS-1 832/13 cells treated with different concentration of berberine were measured by ELISA. c AMP content of the cells was measured by ELISA.Results MTT results showed that activity of the INS-1 832/13 cells was not affected significantly by BBR treatment in any concentrationcompared to the control group, which only trated by vehicle(p> 0.05). As showed by flow cytometry assays, the INS-1832/13 cells treated by different concentrations(1?M, 5?M, 10?M, 20?M) of BBR 72 h, the percentage of apoptosis cells(including both early and late apoptotic cells) were 12.9%, 13.2%, 14.3% and 12.9%, respectively. The apoptotic percentage of the cotrol group was 13.9%. This results indicted that BBR treatment did not have impact on the apotosis of the cells(p>0.05). After 20?M BBR treatment for 72 hours, m RNA expression levels of ins1 and ins2 significantly decreased to 47% and 48%, respectively. The difference was statistically significant compared to the control group(p<0.01). However, m RNA expression of pdx1 in the control group and the BBR intervention groups were at similar levels(p>0.05). Insulin secrtion at 2.5 m Mglucose incubation for 2 hours was considered as basal insulin secretion. The islets treated with BBR by selected concentrations were compared to the control group, the basic insulin secretion had no significant different(p>0.05). When the glucose concentration changed to 16.7 m M(GSIS), insulin secretion was significantly increased by the BBR treatment at 1?M, 5?M and10?M(p<0.05). Wherase insulin secretion did not change by the 20?M BBR treatment compared to the control(p>0.05). Quantitative PCR experiment results showed that the cells treated with different concentrations of BBR(1?M, 5?M, 10?M) could significant increased m RNA expression level of cav1.2(p<0.01). Two L-type calcium channel protein Cav1.2 and Cav1.3 were detected by specific monoclonal antibodies(alomone lab). The results showed that the protein expression level of Cav1.2 was higher than that of Cav1.3 in INS-1 832/13 cells. BBR interventions(1?M, 5?M, 10?M) significantly increased the expression of Cav1.2, but the 20?M BBR intervention failed to(p>0.05). This result is consistent with the experiments conducted by q PCR measurement. 5?M and 10?M BBR treatments could significantly increased the c AMP content of the INS-1 832/13 cells(p<0.01). 5?M and 10?M of BBR significantly increased phosphorylation of AMPK(p<0.01). Control group, BBR group(5?M), PKA agonist Forskolin group, BBR + PKA agonist Forskolin group, PKA inhibitor H89 group and the BBR+PKA inhibitor H89 group were designed to evaluate phosphorylation level of CREB and Cav1.2 protein expression. The results showed that BBR, BBR+Forskolin increased P-CREB and Cav1.2 protein, on the other hand, H89, BBR+H89 significantly decreased P-CREB and Cav1.2 protein(p<0.01).Conclusions BBR represents a novel insulinotropic action is not due to its inhibitory effect to apoptosis of pancreatic beta cells and biosynthesis of insulin as reported previously. BBR increases insulin secretion via increasing the expression of calcium channel protein Cav1.2, and this impact might be involved the c AMP-PKA-CREB pathway.