Mechanism Of Changed Plasma Apolipoprotein M In People Of Type 2 Diabetes Mellitus

Part one:The research about plasma concentration of apo M in T2 DM patients with different clinical types.Objective:To investigate whether low apo M concentration in patients of type 2 diabetes mellitus is associated with hyperlipidemia and whether plasma apo M is altered in patients with diabetic nephropathy.Methods: patients with hyperlipidemia(n=79), T2 DM without hyperlipidemia(n=125), T2 DM with hyperlipidemia(n=98), healthy controls(n=105) were included in first experiment and diabetic nephropathy(DN; n=96), T2 DM without nephropathy(n=100), and healthy controls(n=111) in second experiment. We measured plasma apo M with enzyme linked immunosorbent assay(ELISA).Results:plasma apo M concentration was on average 18% higher in patients with hyperlipidemia(26.63±10.35 ng/mL) compared to healthy controls(22.61±10.81 ng/mL, P=0.025). There was no significant difference between patients with T2 DM with hyperlipidemia(19.83±7.41 ng/mL) and T2 DM without hyperlipidemia(18.54±10.33 ng/mL, P=0.256). It was higher patients with DN(22.23±11.69 ng/mL) compared to T2 DM without nephropathy(18.96±7.85 ng/mL, P=0.023). Apo M was positively correlated with age(r=0.340, P=0.001) and borderline correlated with cystatin C(CYS-C)(r=0.296, P=0.003) in the DN group, but not in the T2 DM without nephropathy group and healthy controls.Conclusion: The concentration of plasma apo M has closed relation with T2 DM and diabetic nephropathy. The effect of hyperglycemia inhibit the apo M expression may be more likely. Part two:A single-nucleotide polymorphism in the promoter region of the apolipoprotein M gene is associated with type 2 diabetes mellitus in eastern Chinese populationsObjective:to establish the association between apo M single-nucleotide polymorphism(SNP)-C724 del and type 2 diabetes mellitus(T2DM) susceptibility in an eastern Han Chinese cohort.Methods:Two hundred and fifty-nine T2 DM patients and seventy-six healthy controls were included in this study.Amplifying DNA of apo M proximal promoter region including SNPs –C724del by Real-Time Polymerase Chain Reaction(RT-PCR) and amplicons sequencing.Plasma apo M concentrations were assayed by enzyme linked immunosorbentassay(ELISA).Results:Four polymorphic sites, rs805297(C-1065A),rs9404941(T-855C) and rs805296(T-778C), C-724 del were confirmed. rs805297(C-1065A) and rs9404941(T-855C) showed no statistical difference in allele frequencies and genotype distributions between T2 DM patients and healthy control as previous studys.It’s worth noting that the difference of rs805296(T-778C) between these two groups was not found in this study.In SNP C-724 del, the frequency of del allele and del/del,C /del genotypes were higher in T2 DM patients than healthy control(p=0.035;p=0.040, respectively),while the plasma apo M of-C724 del mutant allele carriers compared to the wide-type homozygotes carriers in T2 DM patients was not statistically different.Conclusion:the SNP-C724 del in the promoter region of the apo M gene could confer the risk in the development of T2 DM among eastern Han Chinese. Part three:the alteration of apo M expression in Hep G2 cells under different glucose and the inhibition of PI3 K and JAK2 / STAT3 pathwayObjective: to observe the alteration of apo M expression in Hep G2 cells under high glucose with LY 294002(specific blocker of PI3 K signaling pathway) or AG-490(specific blocker of JAK2 / STAT3 signaling pathway)Methods:1.Hep G2 cells after synchronization are divided into: normal glucose concentrations group(N,Glucose 5.5mmol/L); high glucose group(H,Glucose 25 mmol/L); high glucose + LY 294002 group(H + L, LY 294002 concentrations is 10 umol / L). LY 294002 which was joined before one hour in advance and high glucose Hep G2 cells were co-cultured for 24 hours,。Detecting the expression of AKT,P-AKT and apo M in Hep G2 cells by Western Blot.2. Hep G2 cells after synchronization are divided into: normal glucose concentrations group(N,Glucose 5.5mmol/L); high glucose group(H,Glucose 25 mmol/L); high glucose + AG-490 group(H + A, AG-490 concentrations is 10 umol / L). AG-490 which was joined before one hour in advance and high glucose Hep G2 cells were co-cultured for 24 hours. Detecting the expression of JAK2,P-JAK2,STAT3,P-STAT3 and apo M in Hep G2 cells by Western Blot.Results: high glucose increased apo M expression in Hep G2 cells. apo M expression was lower in high glucose + LY 294002 group then pure high glucose group; apo M expression was also lower in high glucose + AG-490 group then pure high glucose group.Conclusion:1.apo M protein expression was reduced in Hep G2 cells by Blocking PI3 K signaling pathway.2.apo M protein expression was also reduced in Hep G2 cells by blocking JAK2/STAT3 signaling pathway.