The Molecular Mechanism Of Clock Gene Regulating The Insulin Secretion In Pancreatic β Cells

Objective: With the gradual clarification of the physiological mechanism of the circadian rhythm disorder,its research on the mechanism of the development of type 2 diabetes has become a hot spot in international research.It has been shown that clock genes play a crucial role in regulating glucose homeostasis.However,the molecular mechanisms by which molecular clocks regulate rhythmic insulin secretion and maintain glucose homeostasis are not fully understood.This study aimed to assess the mechanism of the regulation of insulin secretion by the core member of the clock regulation family.Methods 1.The expression levels of Clock were detected in different organs and tissues of SD rats by q RT-PCR.2.The rhythmic expression levels of Clock in INS-1 cells were detected by q RT-PCR.3.The m RNA expression levels of INS-1 and INS-2 in INS-1 cells were detected by q RT-PCR after inhibition of endogenous Clock expression.4.Insulin content was detected by GSIS assay after inhibition of endogenous Clock expression 5.The rhythmic expression levels of Cacna1 c and Cacna1 d were detected by q RT-PCR.6.The m RNA and protein levels of calcium channel was detected by q RT-PCR and Western blot after inhibition of endogenous Clock expression.7.The intracellular calcium concentration was detected by fluorescent probe assay after inhibition of endogenous Clock expression.8.The binding levels of the Clock protein in the calcium channel Cacna1 c promoter region was detected by the CHIP and Luciferase methods from the DNA-protein binding level.9.The expression levels of c AMP was detected by ELISA.10.The expression levels of c AMP downstream proteins were detected by western blot.11.Construction of chronic sleep disorders in mice.12.Body weight,IPGTT,fasting blood glucose,fasting insulin,GSIS assay were detected.13 the transcriptional expression levels of islet clock gene and calcium channel in mice were detected by q RT-PCR.Results 1.Clock was highly expressed in islet and muscle,and relatively low in spleen.2.The Clock and Bmal1 m RNA expression showed obvious peak and valley values.3.The m RNA expression levels of INS-1 and INS-2 did not show significant changes and the intracellular insulin content did not change significantly.4.There was no significant change in basal insulin secretion under 2.8 m M glucose,while insulin content was significantly reduced under 16.7 m M high glucose.5.The m RNA expression levels of Cacna1 c and Cacna1 d m RNA showed obvious peak-to-valley values,6.The m RNA and protein expression levels of Cacna1 c was significantly decreased after inhibition endogenous expression of Clock,while the expression of Cacna1 d was not significantly changed.7.The intracellular calcium ion content was also significantly reduced.8.CHIP and luciferase reporter gene indicated that the Clock can be combined with the Cacna1 c promoter-444~-454 region.The Clock protein binding of the Cacna1 c promoter region is reduced after inhibition endogenous expression of Clock.9.There was no significant change in intracellular c AMP content and phosphorylated CREB levels in the basal state,but in the case of 16.7 m M high glucose stimulation,intracellular c AMP content and phosphorylation The CREB level was significantly reduced.10.After 4 weeks of chronic sleep disorder,the body weight of C57,C57+CSD and OB mice increased by 22.92%,0.42%,and 26.16%,respectively,and the weight of OB+CSD mice decreased by 0.17%.11.After 4 weeks of chronic sleep disorder,IPGTT results showed that blood glucose levels in C57+CSD were significantly higher than C57 group at 30 min,blood glucose peaks in OB mice were post-shifted at 15 min,30min,and 60 min.The blood glucose at 90 min was significantly higher than that in the C57.The blood glucose of the OB+CSD was significantly increased at 120 min.There was no significant difference in the levels of FBG and FIns between C57 and C57+CSD mice.The levels of FBG and FIns in OB and OB+CSD had no significant changes.(12)GSIS results showed that insulin secretion was decreased in C57 + CSD group than C57 group,insulin secretion was decreased in OB + CSD group than OB group.(13)After 4 weeks of chronic sleep disorder,the m RNA expression levels of CLOCK,BMAL1,PER1 and PER3 in C57 + CSD group were decreased by 45.0%,38.87%,26.81% and 35.57%,respectively.NR1D1 m RNA level was increased by 45.45%.There was no significant difference in PER2 and CRY1 m RNA expression.Compared with OB group,the m RNA expression level of CLOCK m RNA in OB+CSD group was decreased by 28.85%.The m RNA levels of BMAL1,PER1,PER2,PER3 and NR1D1 m RNA were increased by 109.94%,52.42%,26.84%,93.0%,36.72%, respectively.There was no significant difference in Cry1 m RNA expression.Compared with C57 group,the m RNA expression levels of Cav1.2 in C57 + CSD group was decreased by 44.50%;there was no significant difference in Cav1.3 m RNA expression;compared with OB group,Cav1.2 m RNA levels was decreased by 41.50% in OB + CSD group;there was no significant difference in Cav1.3 m RNA levels.Conclusions 1.The expression levels of Clock in different tissues were various and the m RNA expression levels of Clock and Bmal1 has obvious oscillation rhythm in INS-1 cells.2.Clock has no effect on insulin synthesis and basal insulin secretion in INS-1 cells,which may mainly affect the insulin secretion process by damaged the exocytose of insulin particles stimulated by high glucose.3.The m RNA expression levels of Cacna1 c and Cacna1 d has obvious oscillation rhythm,which is similar to that of Clock and Bmal1.Clock regulates the m RNA expression levels of Cacna1 c by binding to the promoter region of it,which affects insulin secretion.4.The mechanism by which Clock regulates insulin secretion under high glucose stimulation may be related to c AMP/PKA signaling pathway.5.Chronic sleep disorder attenuates weight gain of mice and it is more obvious in ob/ob mice.Chronic sleep disorder could damage insulin secretion in normal metabolic mice.It is more obvious in metabolic abnormal ob/ob mice,which may be related to the disorder of the islet clock gene.6.Sleep disorders have become a major health disorder in modern adults and one of the important causes of diabetes development.Clock rhythm regulation is closely related to sleep.By clarifying the relevant mechanisms,we can inspire us to find measures to deal with,especially the promotion of targeted treatment.