MicroRNA-494, Upregulated by Tumor Necrosis Factor-alpha, Desensitizes Insulin Effect in C2C12 Muscle Cells

Abstract

Chronic inflammation is fundamental for the induction of insulin resistance in the muscle tissue of vertebrates. Although several miRNAs are thought to be involved in the development of insulin resistance, the role of miRNAs in the association between inflammation and insulin resistance in muscle tissue is poorly understood. Herein, we investigated the aberrant expression of miRNAs by conducting miRNA microarray analysis of TNF-alpha-treated mouse C2C12 myotubes. We identified two miRNAs that were upregulated and six that were downregulated by a > 1.5-fold change compared to normal cells. Among the findings, qRT-PCR analysis confirmed that miR-494 is consistently upregulated by TNF-alpha-induced inflammation. Overexpression of miR-494 in CHOIR/IRS1 and C2C12 myoblasts suppressed insulin action by down-regulating phosphorylations of GSK-3 alpha/beta, AS160 and p70S6K, downstream of Akt. Moreover, overexpression of miR-494 did not regulate TNF-alpha-mediated inflammation. Among genes bearing the seed site for miR-494, RT-PCR analysis showed that the expression of Stxbp5, an inhibitor of glucose transport, was downregulated following miR-494 inhibition. In contrast, the expression of PTEN decreased in the cells analyzed, thus showing that both positive and negative regulators of insulin action may be simultaneously controlled by miR-494. To investigate the overall effect of miR-494 on insulin signaling, we performed a PCR array analysis containing 84 genes related to the insulin signaling pathway, and we observed that 25% of genes were downregulated ( P<0.05) and 11% were upregulated ( P<0.05). These results confirm that miR-494 might contribute to insulin sensitivity by positive and negative regulation of the expression of diverse genes. Of note, PCR array data showed downregulation of Slc2A4, a coding gene for Glut4. Altogether, the present study concludes that the upregulation of miR-494 expression by TNF-alpha-mediated inflammation exacerbates insulin resistance. Therefore, we suggest that miR-494 could prove an important target for the diagnosis and therapy of inflammation-mediated insulin resistance in muscle.