Action Of Retinol-binding Protein-4in Development Of Gestational Diabetes Mellitus And Impact Of Its Knockdown Expression On ERK Phosphorylation

Objective：1. To investigate correlation between inflammatory factors IL-6, TNF-α andrelated adipocyte factors(adiponectin, leptin and retinol-binding protein4) ingestational diabetes mellitus patients.2. To prepare retinol binding protein4(RBP4) interfering vector and observe its impacton ERK1/2phosphorylation in human liver LO2cell line.Methods：1. Gestational diabetes mellitus patients(GDM group, n=60) and pregnancies withnormal glucose tolerance(NGT group, n=44) were enrolled. The concentrationsof serum adiponectin and RBP4were determined by ELISA. Serum FIns, leptin,IL-6and TNF-α level were assayed by radioimmunoassay. FBG level wasassayed with glucose oxidase method. ISI and HOMA-IR were calculated,serum TG, TC, ALT, AST and FFA levels were measured with enzymemethods. Late gestational body weight were recorded and BMI was calculated.The Pearson correlation assay was analyzed.2. Two pSilencerTM4.1-RBP4vectors targeting RBP4expressing frame5′or3′terminalregion and the eukaryotic expression vector of pSecTag-RBP4were constructed andidentified by sequencing and enzyme cutting, then they were transfected into LO2cells, the interfering efficiency were evaluated by RT-PCR and Western blot, and pSilencerTM4.1-RBP4-5′was used for follow-up experiment.3. LO2cells were divided into four groups: blank control group, negative controlgroup,pSecTag-RBP4group and pSilencerTM4.1-RBP4-5′group. The proteinexpression and phosphorylation of ERK1/2were evaluated by Western blot, thecontent of glucose remained in LO2cells supernatant was measured by the methodof glucose oxidase peroxides(GOD-POD).Results：1. Compared with those in NGT group, the FPG, TG, TC, FFA, leptin, IL-6andTNF-α were significantly increased in patients of GDM, serum adiponectinlevel was significantly decreased, indicated significant inflammatory activationand insulin resistance; HOMA-IR, RBP4and leptin were positively related withIL-6and TNF-α, ISI and adiponectin were negatively related with IL-6andTNF-α.2. The interfering vector and eukaryotic expression vector were successfully prepared,RBP4expression in LO2was effectively inhibited by both of pSilencerTM4.1-RBP4.pSilencerTM4.1-RBP4-5′was chosen for following experiment.3. After transfection, each group of the protein expression of ERK1/2was nosignificantly decreased compared with the blank control group, but thephosphorylation level of ERK1/2in pSecTag-RBP4group were significantlydecreased, while significantly increased in pSilencerTM4.1-RBP4-5′group.Comparedwith the blank control group, the glucose intake by insulin stimulation wassignificantly decreased in pSecTag-RBP4group, while the glucose uptake wasmarkedly increased in pSilencerTM4.1-RBP4-5′group.Conclusion:1. Highly expressed adipocytokines in GDM patients were positively correlated with inflammatory cytokines, this was possibly one of mechanism for insulinresistance in GDM patients.2. The interfering vector and eukaryotic expression vector are successfully prepared.The phosphorylation levels of ERK1/2and glucose uptake by insulin stimulationwere obviously influenced by the changes of RBP4expression in LO2cells aftertransfection of RBP4interfering vector or expression vector.