Protective Effect Of Mibefradil On INS-1 Cells From Glucose Toxicity

Background Type 2 diabetes mellitus is a metabolic disease characterized by the dysfunction of islet β cells and insulin resistance. Long-term high glucose plays negative role on the synthesis/secretion of insulin and the survival of islet cells.The mechanism in above pathological process involves many factors, including the mass of calcium balance. Futhermore, the disorder of calcium balance may inhibit the signal pathway of insulin secretion and promote the apoptosis of islet β cells.Mibefradil is a selective T type calcium channel antagonist, which blocking effect of T-type calcium channel is about 30-100 times stronger than L-type calcium channel. It was used for angina and hypertensive in clinical at early time. And it is widely used in the researches of tumor, pain, epilepsy at present. But it is not generally studied in diabetes nowadays. T-type calcium channel is low-voltage-activated(LVA), which can be activated in a lower voltage(about-70 m V) compared with the HVA. T-type calcium channel is the only one which allows calcium influx into the cells at the membrane resting potential. The previous study of our group showed that the expression of T-type calcium channel in db/db mice(type 2 diabetes model)was markedly higher than wild mice. And Mibefradil significantly reduced the level of blood glucose and lipid in db/db mice. However, the role of T-type calcium channel especially for Mibefradil on the function of β islet cells remains unknown. In order to reveal the effects of Mibefradil on the INS-1 cells and the possible mechanism, we incubated INS-1 cells with high glucose,then they were treated with Mibefradil. NNC 55-0396(T-type calcium channels blocker) and nicardipine(L-type antagonist) were used as control groups. We observed the role of Mibefradil on cell apoptosis and insulin secretion of INS-1 cells at first, then took the following study on the expression of cav3.1 and cav3.2, the main subunits of T-type calcium channel.Methods1. INS-1 cell line was used as the research object. We determined the optimal concentration and time piont of high glucose and drugs(Mibefradil, NNC 55-0396,Nicardipine) affect on the INS-1 cells via CCK-8 experiment. Then the high glucose(33.3mmol/L) was used for inducing high glucose model. Then the INS-1 cells were divided into several groups: control group,high glucose group,drugs groups and high glucose+drugs groups. We detected the cell apoptosis by Annexin V-FITC/PI assay kit and observed the insulin secretion in the culture supernatant by insulin radioimmunoassay kit. In additional,we measured the expression level of cav3.1 and cav3.2 by RT-PCR and western-blot.Results1. Compared with the control group,the cell proliferation was significantly inhibited when the cells incubated with 33.3mmol/L glucose for 72h(P<0.05). When the INS-1 cells treated with 1μmol/LMibefradil,1μmol/LNNC 55-0396,10μmol/L Nicardipine for 24 h, the cell proliferation was not suppressed(P>0.05),and it was inhibited when we increased drug concentration or treatment time based on this outcome(P<0.05).2. Compared with the control group,the cell apoptosis was significantly increased in high glucose group(P<0.05);whereas there was no significant difference between the high glucose group and high glucose+drug groups(P>0.05).3. Compared to the control group, drug groups found no significant difference in insulin secretion(P>0.05). Compared with the high glucose group,the insulin secretion was decreased in high glucose+Mibefradil group separately(P<0.05).4. In order to explore the mechanism of Mibefradil hypoinsulinemia in high glucose circumstance,we detected the expression of cav3.1 and cav3.2 subunits. The gene and protein expression of cav3.1 and cav3.2 subunits increased obviously(P<0.05) when cells had been incubated with 33.3mmol/L glucose for 72 h. Then we treated INS-1 cells with Mibefradil,we found that there was no significant difference between control group and Mibefradil group(P>0.05). But compared with the high glucose group,the expression of cav3.1 and cav3.2 subunits were decreased markedly in high glucose+Mibefradil group(P<0.05).Conclusions1.Mibefradil,NNC 55-0396 and nicardipine could not attenuate the apoptosis of INS-1 cells from high glucose environment in our experiment.2. Compared with the role of NNC 55-0396 and Nicardipine, only Mibefradil could inhibit the insulin secretion of INS-1 cells under high glucose circumstance instead of normal glucose circumstance.3.The gene and protein expression of cav3.1 and cav3.2 subunits were increased when INS-1 cells incubated with high glucose.4.Mibefrail inhibits the expression of cav3.1 and cav3.2 subunits when INS-1 cells incubated with high glucose but not in normal glucose environment.Our data showed that Mibefradil suppressed the insulin secretion of INS-1 cells when it was incubated with high glucose but not in normal glucose,which was consistent with its effect on the expression of cav3.1 and cav3.2 subunits. Thus,it was implied that Mibefradil may inhibits the insulin secretion of INS-1 cells in high glucose environment by down-regulating the expression of T-type calcium channels cav3.1 and cav3.2 subunits. We should provide some evidence for a further research to reveal the role of T-type calcium channel and Mibefradil in the development of type 2 diabetes.