Effects Of Circadian Rhythm Disruption And RBP4 In Shifts Increasing The Risk Of Type 2 Diabetes

Objective: To understand the prevalence and related risk factors of type 2 diabetes in shift population.To explore the influence of shift/day night changes on the circadian rhythm and RBP4 expression.To preliminarily reveal the mechanism of regulation of RBP4-MAPK signal axis by circadian rhythm disorders,and provide theory and basis for the prevention and treatment of type 2 diabetes in shift or light pollution exposed population.Methods:(1)In this study,a multistage stratified sampling method was used to selected the study population.The general situation,shift situation,sleep quality,the prevalence of type 2 diabetes,and physical examination were carried out.(2)Selected 30 cases of control group,30 cases of shift group and 30 cases of diabetes randomly as the research object.Used qRT-PCR to determine the expression of clock genes including BMAL1,CLOCK,CRY1,PER2 and REVERB-α.Measured the expression of RBP4 gene and RBP4 protein respectively by qRT-PCR and ELISA.Established shift models of C57BL/6J mice by four weeks light shift.Normal control and mutual control(diabetes mouse model induced by high-fat diet+STZ)used normal LD cycle.Detected the changes of body weight and fasting blood glucose among the groups before and after four weeks.Used HE staining to observe the morphological changes of liver in different groups after 4weeks.Used qRT-PCR to detect the expression of Clock,Bmal1,Cry1,and Per2 mRNA in the liver tissues of different groups of mice at ZT0,ZT6,ZT12,and ZT18 points.The expressions of Rbp4 mRNA and RBP4 protein were detected by qRT-PCR and Western blot respectively.(3)RNA-seq was used to detect the impact of overexpression of RBP4 in Huh-7 cell lines.Then analyzed the possible regulatory pathways of RBP4 genes.Used qRT-PCR to detect the expression of Irs1,Ras,and Erk1 genes in liver tissues of different mice groups.Western blot was used to detect the expression of IRS1,RAS,ERK1/2proteins and the phosphorylation levels of IRS1,ERK1/2 proteins in the liver tissues.Results:(1)50.8% of the surveyed petroleum workers were engaged in shift work.And compared with day workers,the age of shift workers was lower and BMI was higher(P<0.05).The distributions of gender,monthly income,smoking history,and drinking history between shift and day workers were different(P<0.05).The total PSQI score of oil workers in this survey was 6.45 ± 3.15.However,the sleep quality was worse in shift workers,regular smokers and people with family history of diabetes/ hypertension/coronary heart disease(P<0.05).The sleep quality varied in people with different ages,BMI,and educational levels(P<0.05).The prevalence of type 2 diabetes was 5.4%.And the average age and BMI of type 2 diabetes ones were higher than those of non type 2diabetes patients(P<0.05).Univariate analysis showed that aged≥40,male,overweight and obesity,bachelor’s degree,monthly income>4000 yuan,occasional smoking and frequent smoking,frequent drinking,family history of diabetes/hypertension/coronary heart disease,and shift work were all risk factors of type 2 diabetes(P<0.05).And multivariate logistic regression analysis showed that the risk factors of type 2 diabetes mellitus were associated with people aged≥40,overweight and obesity,occasional and frequent smoking,family history of diabetes/hypertension/coronary heart disease,and shift work(P<0.05).It was found that shift workers had a higher risk of type 2 diabetes than day workers,and the OR of model 4 was 1.91(95% CI: 1.17-3.14)in the model adjusted for different variables.(2),The mRNA expression of circadian clock genes CRY1 and PER2 were decreased in the shift group compared with the control group(P<0.05).However there was no statistical difference between the shift group and the diabetes group(P>0.05).The RBP4 mRNA expression were upregulated in the shift population(P<0.05).Multiple linear regression analysis showed that the factors affecting RBP4 protein level were shift work and diabetes.The RBP4 protein level of the shift population was10.88μg/m L higher than the day population(P<0.05).And the levels of RBP4 protein in diabetes population were 5.11 μg /m L higher than the non diabetes population(P<0.05).The shift mice group with 4-weeks diurnal changes led to less weight gain(P<0.05)and higher fasting blood glucose(P<0.05)compared with the control group.The liver morphology of the shift mice changed and was different from control and diabetes mice group.Compared with the control group,the phase and amplitude of Bmal1 and Per2 of shift mice were changed(P<0.05),which was no statistical difference with the diabetes group(P>0.05).The Rbp4 mRNA expression of liver tissue among control group,shift group and diabetes group was significantly different,and there was grouping and time interaction(P<0.05).Compared with the control group,the expression of Rbp4 mRNA of the shift group and diabetes group mice increased at ZT0 point(P<0.05).The expression of Rbp4 mRNA in shift group was higher at ZT6(P<0.05),and lower at ZT18 than that in the diabetes group(P<0.05).The level of RBP4 protein was increased in the shift group and the diabetes group compared with the control group at ZT0(P<0.05).Then compared with the diabetes group,the expression of RBP4 protein was decreased in the shift group(P<0.05).(3)Overexpression of RBP4 in the Huh-7 cell line resulted in 7210 genes changed.Among them,3931 genes were upregulated and 3927 genes were downregulated.GO analysis and KEGG analysis showed that the upregulated genes enriched in multiple biological functions and pathways,included the MAPK signaling pathway.The day and night changes caused the increased expression of Irs1 mRNA in the liver of mice,which was different from the control and diabetes mice group(P<0.05).The expression of Ras and Erk1 mRNA increased in the liver of shift mice group,which was different to the control group(P<0.05).And the expression of IRS1 protein increased in the liver of shift group which was distincted from the control mice group(P<0.05).The phosphorylation levels of IRS1 and ERK1/2 protein increased in the liver of shift group,which was different from the control and diabetes group(P<0.05).Conclusions: Shift work increased the risk of type 2 diabetes by 91.3%,which might be an independent risk factor for type 2 diabetes.Shift/day and night changes could lead to circadian rhythm disorders and upregulated of RBP4 expression.Overexpression of RBP4 could upregulate the MAPK signaling pathway through activated IRS1 and ERK1/2 phosphorylation.It was suggested that the RBP4-MAPK signal axis might play a role in the upregulation of RBP4 expression caused by circadian rhythm disorder andultimately lead to the occurrence of type 2 diabetes.It might be a potential target for the prevention and treatment of type 2diabetes in people with circadian rhythm disorder.