The Effects Of LRP16 Inhibition In Vitro On Adiponectin Functions And Insulin Resistance

Objective:This work aims to study the role of LRP16 in adiponectin and insulin signaling pathways, and investigate the mechanism underlying the effect of LRP16 on insulin resistance.Methods:HepG2 stable cell lines with altered LRP16 expression were generated by slow-virus infection:LRP16 stable overexpression HepG2 cell line and its control cell line, LRP16 stable knockdown HepG2 cell line and its control cell line. Each cell line was treated with PBS, insulin (100 nM,30min), adiponectin (2g/mL,15min), or pretreatment with adiponectin (2 g/mL,15 min) followed by insulin (100 nM,30min). Cellular glucose uptake rate was determined with colorimetric assay. The intracellular triglyceride and cholesterol contents were measured using enzymatic assay. Q-PCR was performed to test the expression of glucose and lipid metabolism enzyme Fatp2, ACC, and Acs15. Western blot was performed to examine the phosphorylation and protein expression of signaling molecules in PI3K and AMPK pathways. Finally, transcriptome analysis was performed in LRP16 stable overexpression HepG2 cell line and its control cell line.Results:1. Overexpression of LRP16 inhibited insulin-and adiponectin-stimulated glucose uptake by HepG2 cells. In contrast, suppressing LRP16 expression enhanced insulin-and adiponectin-induced glucose uptake by HepG2 cells. Overexpression of LRP16 up-regulated the phosphorylation of IRS-1 (Ser307), and down-regulated the expression of PI3K (p85) and the phosphorylation of AKT (Ser473). On the other hand, suppressing LRP16 expression down-regulated the phosphorylation of IRS-1 (Ser307) and up-regulated the expression of PI3K (p85) and the phosphorylation of AKT (Ser473).2. Overexpression of LRP16 reduced the intracellular triglyceride and cholesterol content stimulated by adiponectin. In contrast, suppressing the expression of LRP16 increased the intracellular triglyceride and cholesterol content stimulated by adiponectin. Overexpression of LRP16 attenuated adiponectin's down-regulation of Fatp2, Fatp5, and Acs15 at mRNA level. On the other hand, suppressing the expression of LRP16 enhanced adiponectin's down-regulation of Fatp2, Fatp5, and Acs15 at mRNA level. Overexpression of LRP16 down-regulated the phosphorylation of AMPK (Thr172), and suppressing LRP16 expression up-regulated the phosphorylation of AMPK (Thr172).3. Transcriptome analyses. Gene differential expression analysis demonstrated that the overexpression of LRP16 not only down-regulated the expression of INSR, IRS-1, PI3K, and GLUT4 in the insulin signaling pathway, but also down-regulated the expression of Adipo-R, AMPK, and PPAR in the adiponectin signaling pathway. Differential enrichment analysis demonstrated that the overexpression of LRP16 inhibited the sterol sulfatase enzyme, alcohol sulfotransferase enzyme and cholesterol monooxygenase enzyme of steroid hormone biosynthesis.Conclusion:LRP16 contributed to insulin resistance in HepG2 cells by inhibiting the PI3K signaling pathway, and suppressed the insulin-sensitizing effect of adiponectin by inhibiting the AMPK signaling pathway. It impacted adiponectin's effects on glucose and lipid metabolism by inhibiting the steroid hormone biosynthesis. Together, our data suggest that LRP16 expression contributes insulin resistance and adiponectin signaling pathway plays an important role in mediating the effects of LRP16 on insulin resistance.