Effect Of LRP16 On Cell Insulin Resistance And The Molecular Mechanism

Objective:To investigate the effect of LRP16 on insulin resistance in 3T3-L1 adipocyte, C2-C12 sarcoblast and HepG2 hepatoma carcinoma cell and the possible molecular mechanisms.Methods:(1) Lipidosome transfection and lentivirus mediated siRNA technology were used to construct the cell lines with over-expression of LRP16, down-expression of LRP16 and the conrtol cell lines. (2) 3T3-L1 preadipocytes were differentiated using the regular methods, the effect of LRP16 on differentiation of 3T3-L1 preadipocytes were detected using inverted microscope and red oil staining; The effect of LRP16 on mRNA expression of C/EBPα, PPARy, TNFa, IL-6, Resistin and Adiponectin were measured using Quantitative Real-time PCR. (3) 2-deoxy-[3H]-D-glucose was used to detect the effect of LRP16 on insulin stimulated glucose uptake in 3T3-L1 adipocyte, C2-C12 sarcoblast and HepG2 hepatoma carcinoma cell; Effect of LRP16 on the expression of key protein in IRS-1 signaling pathway, such as pIRS-1(Ser307),pIRS-1(Tyr),PI3-K(p85),pAkt(Ser473),pAkt(Thr308), was detected using Western Blot. (4) The strucural expression vectors, including pCMX-PPARy (pCMX-PPARa), pGL3-PPRE, pcDNA-3.1, pcDNA-3.1-16 and the internal conrtol vector pRL-TK were transiently co-transfected into COS-7 cells, then the PPRE relative luciferase activity which reflected the transcriptional activity of PPARy and PPARa was detected using Dual-luciferase Reporter Gene Assay system. Western Blot was used to detect the effect of LRP16 on the expression of PPARy and GLUT-4 in 3T3-L1 adipocyte, C2-C12 sarcoblast, and the expression of PPARa, LPL in HepG2 hepatoma carcinoma cell.Results:(1) We constructed the cell lines with over-expression of LRP16, down-expression of LRP16 and the conrtol cell lines in 3T3-L1 adipocyte, C2-C12 sarcoblast and HepG2 hepatoma carcinoma cell successfully. (2) The cytolipin in 3T3-L1 adipocyte with over-expression of LRP16 was large, few, uneven, the red oil staining showed that the value of OD in 570nm was lower than the control cells; On the contrary, the cytolipin in 3T3-L1 adipocyte with down-expression of LRP16 was small, intensive and even, the whole visual fields was cauliflower-like, the red oil staining showed that the value of OD in 570nm was higher than the control cells. The mRNA expression of C/EBPa, PPARy and Adiponectin in cells with over-expression of LRP16 was lower than the control cells, but the mRNA expression of TNFα, IL-6, Resistin was higher than the control cells. On the contrary, when the LRP16 was down-expressed, the results were just on the opposite. (3) Over-expression of LRP16 decreased the insulin stimulated glucose uptake in 3T3-L1 adipocyte, C2-C12 sarcoblast and HepG2 hepatoma carcinoma cell, but, down-expression of LRP16 increased the insulin stimulated glucose uptake in these cells. Protein expression of pIRS-1(Ser307) was increased when LRP16 was over-expressed, but, the expression of pIRS-1(Tyr). PI3-K(p85),pAkt(Ser473),pAkt(Thr308) were decreased. On the contrary, when the LRP16 was down-expressed, the results were just on the opposite. (4) LRP16 decreased the transcriptional activity of PPARy in a dose dependent manner, the relative luciferase activity of PPRE accounted for 38%(P<0.01) of the control group at pcDNA3.1-16 0.4μg, and 35%(P<0.01) of the control group at pcDNA3.1-16 0.5μg. LRP16 decreased the transcriptional activity of PPARa in a dose dependent manner, the relative luciferase activity of PPRE accounted for 43%(P<0.01) of the control group at pcDNA3.1-16 0.4μg, and 27%(P<0.01) of the control group at pcDNA3.1-16 0.5μg. overexpression of LRP16 decreased the protein expression of PPARy, GLUT-4 in 3T3-L1 adipocyte, C2-C12 sarcoblast, and the protein expression of PPARa,LPL in HepG2 hepatoma carcinoma cell, but, when LRP16 was down-expressed, the results were on the opposite.Conclusions:(1) We successfully constructed the cell lines with over-expression of LRP16, down-expression of LRP16 and control cell lines, which provided cell model for the follow up experiment. (2) LRP16 caused insulin resistance through several pathways, including inhibiting the differentiation of adipocytes, influencing IRS-1 signaling pathway and inhibiting the transcriptional activity of PPARy and PPARa.