Association Of Key Promoter Gene In AMPK Signal Pathway And Metal Co-exposure With Type 2 Diabetes Mellitus

Objective:T2DM(type 2 diabetes mellitus)is a complex disease affected by multiple genes and factors.Although each identified gene variation explains only a small part of the T2DM risk in human population,it contributes to us understand the pathogenesis of T2DM.The key promoter gene is the gene that plays a key role in the regulatory network.To explore the key promoter gene of T2DM from many genes is an important direction of the current research on bioinformatics of T2DM.AMPK signaling pathway plays a key role in maintaining metabolic homeostasis.In addition,metals are important elements to maintain the function of the body,and may play an important role in the homeostasis of glucose and fat metabolism.The glucose and fat metabolism disorders are closely related to T2DM.Therefore,the deficiency or excess of a certain metal in the body may play an important role in the occurrence and development of T2DM.Because metals usually exist in the form of co-exposure in the environment,it is more practical to explore the role of metal co-exposure in the occurrence and development of T2DM.To this end,this study explored the association between key promoter genes of AMPK signaling pathway,metal co-exposure,and their interaction with T2DM risk using a case-control study,and provided new clues and scientific basis for further prevention and treatment of T2DM.Methods:The first part:(1)The participants in this study were from a number of communities in Nanning City,Guangxi Zhuang Autonomous Region,which were included in the REACTION study.According to inclusion and exclusion criteria,867 T2DM patients and 977 controls were included.The general demographic data,lifestyle,height,weight and blood samples were collected.(2)The Boolean model algorithm was used to predict the key promoter targets in the T2DM regulatory network,and bioinformatics methods was used to screen out the key promoter genes and SNPs in AMPK signal pathway,including CAMKK2(rs1653590,rs3817190,rs2686367),TSC1(rs10491534)and AKT1S1(rs2353005).Genotyping was performed by the Mass ARRAY molecular weight array platform.(3)Logistic regression was used to analyze the differences between the T2DM group and the control group in allele,genotype and different genetic models.The generalized multifactor dimensionality reduction method was used to analyze the interaction between genes and genes,genes and environmental factors.Logistic regression and generalized linear models were used to analyze the association of multiplicative and additive interactions between the genes and BMI.The regression coefficient of SNPs in the LASSO regression model was used as the weight to construct the weighted GRS(genetic risk score).Logistic regression was used to analyze the association between weighted GRS and T2DM risk with grouping by the quartile of GRS(Q1,Q2,Q3 and Q4).Covariance analysis was used to analyzed the association of gene polymorphism and weighted GRS with T2DM related metabolic traits.The second part:(1)Based on the first part of the study,T2DM patients with serum samples were selected,and the control group was selected according to age(±3 years old)and gender ratio(±3%).Finally,223 T2DM patients and 302controls were included in this part of the study.(2)The serum metal concentrations in participants were determined by inductively coupled plasma mass spectrometry.(3)After using LASSO regression to weigh the correlation between metals,the metals with a greater impact on T2DM risk were screened out,and the regression coefficient of the metals in the LASSO regression model was used as the weight to build the co-exposure model.After grouping by the quartile of metal concentration(Q1,Q2,Q3 and Q4),logistic regression was used to analyze the association between single metal exposure,metal co-exposure and T2DM risk,as well as the association of interaction between metals and metals,metals and BMI.Covariance analysis was used to analyze the association between single metal exposure,metal co-exposure and T2DM related metabolic traits.Meanwhile,the dose-response relationships between single metal exposure,metal co-exposure and T2DM risk and related metabolic traits was analyzed using restricted cubic splines.Logistic regression was used to analyze the interaction between metal co-exposure and weighted GRS.Results:The first part:(1)The results of associated analysis between gene polymorphisms and T2DM risk showed that the A allele of rs1653590 in CAMKK2 gene was associated with reduced T2DM risk in female(OR=0.68,95%CI:0.48-0.96).The T2DM risk in male individuals with TG or GG genotype of rs2686367 in CAMKK2 gene was lower than those with TT genotype(OR=0.72,95%CI:0.52-0.99).In rs10491534 of TSC1 gene,female individuals with TC genotype had a lower T2DM risk than those with the TT genotype(OR=0.72,95%CI:0.52-0.98).(2)The results of interaction between genes and genes,genes and environmental factors showed that the optimal model is the one-factor model of BMI,with the highest test balance accuracy(0.566)and cross-validation consistency rate(10/10).The dominant model of rs2353005 in AKT1S1gene had a multiplicative interaction with BMI in male population(P_interaction=0.026).Compared with the normal BMI male carrying GG genotype,overweight/obese male with GA or AA genotype had a higher T2DM risk(OR=2.16,95%CI:1.27-3.68).(3)The results of associated analysis between weighted GRS and T2DM risk showed that the T2DM risk in Q4 group of weighted GRS was significantly higher than that in Q1 group both in male and female(OR=1.99,95%CI:1.29-3.07;OR=1.50,95%CI:1.08-2.08,respectively).(4)The results of associated analysis between gene polymorphisms and T2DM related metabolic traits showed that individuals carrying GA or AA genotype of rs1653590 in CAMKK gene had lower levels of Hb A1c than those with GG genotype(P<0.05).The male individuals with TG or GG genotype of rs2686367 in CAMKK gene had lower levels of 2h PG than those with TT genotype(P<0.05).In rs10491534 of TSC1gene,female individuals with TC or CC genotypes had higher levels of SBP,DBP,FBG and 2h PG than those carrying TT genotypes(P<0.05).(5)The results of associated analysis between weighted GRS and T2DM related metabolic traits showed that the high scores of weighted GRS in female was associated with higher Hb A1c and HOMA-IR levels(P<0.05).The second part:The factors with a greater impact on the T2DM risk were screened out by LASSO regression,including calcium,selenium and vanadium.(1)The results of associated analysis between single metal exposure and T2DM risk showed that the T2DM risk at Q4 level of serum calcium was significantly higher than that at Q1 level(OR=2.83,95%CI:1.70-4.72).The concentrations of serum calcium had an S-shape relationship with T2DM risk(P_overall<0.001,P_nonliearity<0.001).Compared with Q1 levels of serum selenium,the T2DM risk at Q2,Q3 and Q4 levels were significantly increased(OR=2.60,95%CI:1.54-4.39;OR=1.98,95%CI:1.17-3.37;OR=3.23,95%CI:1.89-5.49,respectively).There was a linear relationship between serum selenium concentration and the T2DM risk(P_overall<0.001,P_nonliearity=0.389).Compared with Q1 levels of serum vanadium,Q2 levels was associated with an increased T2DM risk(OR=3.16,95%CI:1.87-5.34),while Q3 and Q4 levels were associated with the reduced T2DM risk(OR=0.30,95%CI:0.17-0.52;OR=0.52,95%CI:0.31-0.87).The concentrations of serum vanadium had an S-shape relationship with T2DM risk(P_overall<0.001,P_nonliearity<0.001).(2)The results of metal-metal,metal-BMI interactions showed that there was no interaction between calcium,selenium and vanadium,and there was no interaction between calcium and BMI,selenium and BMI,vanadium and BMI(P_interaction>0.05).(3)The results of associated analysis between metal co-exposure and T2DM risk showed that compared with Q1 group of serum calcium,selenium and vanadium co-exposure score,the T2DM risk at Q4 group were significantly increased(OR=4.96,95%CI:2.91-8.44).Moreover,the co-exposure score had a J-shape relationship with T2DM risk(P_overall<0.001,P_nonliearity=0.003).(4)The results of associated analysis between single metal exposure,metal co-exposure and T2DM related metabolic traits showed that compared with Q1 group,the levels of Hb A1c,HOMA-IR,TG and LDL-c in Q4group of serum calcium were significantly increased(P<0.05),the levels of Hb A1c in Q4 group of serum selenium were significantly increased(P<0.05),the levels of Hb A1c,TC,TG and LDL-c of co-exposure score were significantly increased(P<0.05).The levels of HOMA-IR in Q4 group of serum vanadium were significantly lower than those in Q2 group(P<0.05).There were the nonlinear relationship of serum calcium concentration and DBP level,serum selenium concentration and HDL-c level,serum vanadium concentration and the levels of DBP,HOMA-IR and LDL-c(P_overall<0.05,P_nonliearity<0.05).The serum calcium,selenium and vanadium co-exposure score had a linear dose-response relationship with the levels of HOMA-IR(P_overall=0.045,P_nonliearity=0.103).(5)The results of associated analysis between metal co-exposure and weighted GRS showed that there was no interaction between metal co-exposure and weighted GRS both in male and female(P_interaction>0.05).However,T2DM risk in the group of metal co-exposure M_high+weighted GRS M_high was higher than that in the group of metal co-exposure M_low+weighted GRS M_low(OR=2.64,95%CI:1.36-5.14).Conclusions:Our results suggest that(1)The key promoter gene in AMPK signal pathway:the CAMKK2 gene,rs1653590 may be associated with reduced T2DM risk in female,and rs2686367 may be associated with reduced T2DM risk in male.CAMKK2 gene may be considered as a potential key target for the prevention and treatment of T2DM.High scores of weighted GRS may be associated with increased T2DM risk.(2)High concentrations of serum calcium and selenium may be risk factors for T2DM risk,while high concentrations of serum vanadium may be protective factors for T2DM risk.The high co-exposure scores of calcium,selenium and vanadium may be risk factors for T2DM risk,and the co-exposure scores had a J-shape dose-response relationship with T2DM risk.