Effects And Mechanisms Of Clozapine And Risperidone On Insulin Secretion Of Cells Of Isolated Rat’s Islets

Adverse metabolic effects, such as weight gain, diabetes mellitus and lipid abnormalities, have increasingly been recognized with the use of atypical antipsychotic drugs. However, these agents may differ in their risk to affect weight, glucose-insulin homeostasis and lipid metabolism. Among atypical antipsychotics, clozapine and olanzapine appear to have the highest propensity to induce weight gain, diabetes and lipid abnormalities, quetiapine and risperidone intermediary, aripiprazole and ziprasidone the least propensity. Given that insulin is a hormone that is involved in both the regulation of body weight, as well as in glucose regulation and lipid metabolism, it is worth while studying the potential effects and their possible mechanisms of atypical antipsychotics on pancreatic insulin secretion. In in vitro studies, we have recently demonstrated that clozapine has effect directly on cells of isolated rat’s islets, while risperidone has less effect. However, the mechanism by which clozapine and risperidone influence insulin secretion remain unclear.Objective:The aim of this study was to determine the mechanisms of clozapine’s and risperidone’s effects on insulin secretion of cells of isolated rat’s islets, and to clarify whether insufficient insulin secretion induced by clozapine and its metabolites result from influence the expression of glucose transporter 2 (GLUT2) located in cell membrane of isolated rat’s islets, and to clarify whether clozapine-induced insufficient insulin secretion result from influence the expression of ATP-sensitive potassium channels(KATP channels) located in cell membrane of isolated rat’s islets or influence the Ca2+ influx through the Voltage-dependent calcium channels(VDCC).Methods:The cells of isolated rat’s islets were prepared by a modified collagenase digestion methods. At 5.5 mmol/L glucose, the cells of islets was treated with 1μmol/L clozapine, desmethyl-clozapine(DCLO), clozapine N-oxide(CNO) and risperidone (RIS),respectively, blank control group was also set.①After incubation 1h or 4h or 48h, the insulin in supernatant was assayed by radioimmunoassay. And the level of insulin in the supernants of the experiment groups were compared with the controls.②The cells of islets was treated with 1μmol/L clozapine, DCLO, CNO and RIS, respectively. Following longer exposures to the drugs (48h), the cells of isolated rat’s islets in each group were detected GLUT2 mRNA level with RT—PCR and its protein expression with Western-blot. The Pearson correlation coefficient was used to clarify the correlativity between the mRNA expression of GLUT2 and corresponding insulin secretion of cells of isolated rat’s islets.③The cells of islets were treated with 1μmol/L clozapine, DCLO, CNO and RIS, respectively. Following longer exposures to the drugs (48h), the cells of islets in each group were detected Kir6.2 mRNA level and SUR1 mRNA level with RT—PCR. The Pearson correlation coefficient was used to clarify the correlativity between Kir6.2 mRNA level or SUR1 mRNA level and corresponding insulin secretion of cells of isolated rat’s islets. After cells were loaded with calcium sensitive fluorecent indicator Fluo-4/AM, [Ca2+]i represented by fluorescence intensity was measured by laser scanning confocal microscope(LSCM), and compared the mean fluorescence intensity of each group after 48h of incubation. Following intervening by 5mmol/L acetylcholine (Ach), the mean fluorescence intensity of each group was compared. All the data are statistical analyzed with SPSS 15.0.Results:①At 5.5mmol/L glucose, no difference in insulin secretion was found between clozapine/DCLO/CNO/RIS group and the control group after lh of incubation(P>0.05). Compared to control group, clozapine significantly inhibited insulin secretion at 5.5mmol/L glucose after 4h of incubation (P=0.022<0.05); DCLO has a tendency to inhibit insulin secretion after 4h of incubation, but no significant difference was found (P=0.081>0.05). There was no difference in insulin secretion between CNO/RIS group and the control group after 4h of incubation(P>0.05). Compared to control group, clozapine significantly inhibited insulin secretion at 5.5mmol/L glucose after 48h of incubation(P=0.007<0.01); DCLO has a tendency to inhibit insulin secretion after 48h of incubation, but no significant difference was found (P=0.154>0.05). There was no difference of insulin secretion between CNO/RIS group and the control group after 48h of incubation (P>0.05).②The mRNA expression of GLUT2 located in cell membrane of slets:clozapine group was significantly lower than control group (P=0.017<0.05), DCLO group was also lower than control group, but no significant difference was found (P=0.182>0.05),and no significant difference between CNO/RIS group and control group (P>0.05); The protein expression of GLUT2 located in cell membrane of islets: clozapine group was significantly lower than control group (P=0.035<0.05), DCLO group was also lower than control group, but no significant difference was found (P= 0.294>0.05),and no significant difference between CNO/RIS group and control group (P>0.05); At the same time, we found there was a significant correlation between the mRNA expression of GLUT2 and corresponding insulin secretion of cells of islets (r= 0.337, P=0.024<0.05).③The mRNA expression of Kir6.2 located in cell membrane of islets:there was no significant difference found between clozapine/DCLO/CNO/RIS group and control group (P>0.05); The mRNA expression of SUR1 located in cell membrane of islets:clozapine group was significantly lower than control group (P= 0.029<0.05), DCLO group was also lower than control group, but no significant difference was found (P=0.249>0.05),and no significant difference between CNO/RIS group and control group (P>0.05);Moreover, we found there was a significant correlation between SUR1mRNA expression and corresponding insulin secretion of cells of islets(r=0.524,P<0.01),but no significant correlation was found between Kir6.2 mRNA expression and corresponding insulin secretion of cells of islets(r=-0.015,P=0.921>0.05).④[Ca2+]i fluorescence intensity in cells of islets:clozapine and DCLO group was significantly lower than control group (P<0.01; P= 0.037<0.05),but no significant difference between CNO/RIS group and control group (P>0.05); [Ca+]i fluorescence intensity in cells of islets can be significantly enhanced by 5mmol/L acetylcholine in each group(P<0.01).The curve of [Ca2+]i fluorescence intensity:after intervention of 5mmol/L acetylcholine, there was no change of time to reach peak between clozapine/DCLO group and control group, but a lower peak value and mean fluorescence intensity were found between them (P<0.01; P=0.015<0.05). Furthermore, no significant difference was found between CNO/RIS group and control group.Conclusion:①At 5.5mmol/L glucose, clozapine can inhibit insulin secretion, which concerned with time. DCLO has a tendency to inhibit insulin secretion, while CNO and RIS have no significant effect on insulin secretion of cells of isolated rat’s islets.②Clozapine can inhibit GLUT2 expression of cells of islets, and then hamper glucose transport through cell membrane, which was one of mechanisms to explain the effect of clozapine on insulin secretion.③clozapine can inhibit GLUT2 expression and SUR1 mRNA expression, which play important roles simultaneously with regard to the inhibitive effect of clozapine on insulin secretion, and inhibition of SUR1 mRNA expression was more important (r=0.337<0.524).Through the inhibition of SUR1 mRNA expression, clozapine can down-regulate the sensitivity of Kir6.2 to ATP, then reduce [Ca2+]i influx, finally result in inhibition of insulin secretion. DCLO may be involved in this process. We didn’t find a significant effect of CNO and RIS on GLUT2 expression, KATP channels and [Ca2+]i influx, so they seems didn’t inhibit or stimulate insulin secretion.