关键词: 褪黑素；INS-1细胞；葡萄糖；胰岛素；G&alpha, i/o

Abstract: 【Objective】To demonstrate the role of melatonin in the circadian secretion of insulin, the effect of melatonin on insulin and Gαi/o expression in rat insulinoma cell line (INS-1) and molecular mechanism were studied. As melatonin can regulate insulin secretion in a circadian manner, maintaining a homeostasis of blood glucose by regulating diurnal glucose metabolism, thus study on the effect of melatonin on expression of insulin may provide a basis for regulation of melatonin in circadian secretion of insulin.【Method】The cryopreserved INS-1 cells were passaged for at least three generations. After the passage of cells, INS-1 cells were cultured in RPMI1640 medium for about 24-48 h. When the cell confluency reached 60%-70%, washed with RPMI1640 without serum and glucose for two times. INS-1 cells were cultured in media supplemented with different concentrations of glucose(0, 10, 20, 30, and 50 mmol·L^-1) and 100 nmol·L^-1 melatonin for 12 h treatment, then observation of morphological changes of INS-1 cells and statistical analysis were performed. The total RNA extracted from INS-1 cells by Easypure RNA kit, measuring the cells total RNA concentration and purity, then the quality of total RNA was measured by formaldehyde denaturing agarose gel electrophoresis. TranScript One-step Removal and Synthesis Supermix were used to synthesize cDNA by reverse transcription. Primer Premier 5.0 primer design software, reference gene sequence of GenBank were used to design primers. qRT-PCR was used to detect the changes of Insulin1, Gαi1, Gαi2, Gαo, PKA and PKCα inmRNA level.【Result】After incubation of INS-1 cells with different concentrations of glucose, it was observed that the growth of cell synapse first increased and then decreased along with the increase of the concentration of glucose in the culture medium. In the 20 mmol·L^-1 glucose treatment group, the synaptic growth of INS-1 cells was increased significantly; in the 20 mmol·L^-1glucose and melatonin co-treatment group, the area of INS-1 cells was increased, but the synaptic growth was not obvious. In the glucose treatment group, the mRNA level of Insulin1 showed a trend of increase and then reduction, especially was increased at 20 mmol·L^-1 (P＜0.05). The mRNA level of Gαi1 showed a trend of reduction and then increase, especially was reduced at 20 mmol·L^-1 (P＜0.05). The mRNA level of Gαi2 and Gαo was not significantly reduced (P＞0.05). Thus, Gαi1 but not Gαi2 and Gαo showed a negative correlation with Insulin1 mRNA level. Compared with 20 mmol·L^-1 glucose treatment group, the mRNA level of Gαi1 was significantly increased (P＜0.05) at 20 mmol·L^-1 glucose and melatonin co-treatment group, the mRNA level of Insulin1 and PKCα was significantly reduced (P＜0.05), whereas the mRNA level of PKA was not significantly reduced (P＞0.05).【Conclusion】Melatonin inhibits the expression of Insulin1 in INS-1 cells and reduces insulin secretion resulting in the adaptive hyperplasia of INS-1 cells. The binding of melatonin to its receptor can promote the increase of Gαi1 mRNA levels and inhibit the expression of PKCα, which leads to the inhibited expression of Insulin1 and decreased insulin secretion, so that blood glucose remains homeostatic during the night.

Key words: melatonin, INS-1 cells, glucose, insulin, Gαi/o