Analyasis of the functional role of microRN as in regulating macrophage mediated inflammatory responses

Abstract

Unrestrained inflammation frequently observed in chronic inflammatory diseases such as insulin resistance, Type 2 Diabetes (T2D) and several types of cancers is prominently due to imbalances in different activation states of macrophages. Delineation of the regulatory mechanisms underlying macrophage polarization may help us to better understand the pathophysiological basis of inflammation linked diseases. MicroRNAs are posttranscriptional regulatory molecules that drive distinct biological processes such as proliferation, cell survival, differentiation and inflammation. However, the functional role of microRNAs in inflammation induced insulin resistance is poorly studied. Thus, there is a necessity to study the involvement of microRNAs in inflammation induced insulin resistance (IR), as IR is the leading cause of diabetes. With this objective we sought to understand the regulatory role of microRNAs in macrophage polarization and insulin resistance. Our microRNA microarray data revealed several microRNAs that were differentially regulated in polarized macrophages. Among them, we investigated the functional roles of two microRNAs: miR-712 and miR-16 whose expression levels were down-regulated in macrophages exposed to pro-inflammatory stimuli such as LPS+IFNγ and palmitate. Additionally, we have observed that miR-16 expression is also down-regulated in palmitate exposed myoblasts, ER stress inducer (tunicamycin and thapsigargin) treated myoblasts and insulin responsive tissues of high sucrose diet (HSD) induced insulin resistant rats. On the other hand unlike in macrophages, miR-712 expression levels were observed to be increased in myoblasts upon exposure to stress inducers such as palmitate, iv tunicamycin and thapsigargin pointing to potential cell specific effects. Investigating the direct role of miR-712 in skeletal muscle may help us in better understanding of insulin mediated effects in physiology and disease. We next noted that ectopic expression of miR-712 and miR-16 in macrophages reduced the production of pro-inflammatory cytokines such as TNF-α, IL-6 and IFN-β which in turn led to improved insulin sensitivity in insulin resistant (IR) skeletal myoblasts suggesting reduced paracrine inhibitory effects of LPS+IFNγ polarized macrophages on skeletal myoblasts insulin sensitivity. In addition we observed that forced expression of miR-16 directly in myoblasts augmented insulin stimulated glucose uptake via upregulation of two key players: GLUT4 and MEF2A that are involved in insulin stimulated glucose uptake. Mechanistic analysis revealed LRRK2 (a serine / threonine protein kinase associated with inflammatory diseases such as Crohn’s and Parkinson’s disease) as the target of miR-712. Further over-expression of miR-712 resulted in reduced phosphorylation of p38 and ERK1/2, key players involved in inflammatory gene expression suggesting that miR-712 is positioned to control macrophage mediated pro-inflammatory responses. Collectively, our data demonstrates the pivotal roles of microRNAs miR-712 and miR-16 in alleviating inflammation induced insulin resistance for the first time. Exploring the pathophysiological roles of these microRNAs may further help us in understanding the progression and treatment of T2D. MicroRNA-712 and miR-16 dampen macrophage mediated pro-inflammatory responses and improve insulin mediated glucose uptake in myoblasts. (A) miR712 expression is down-regulated in LPS +IFNγ polarized macrophages. Ectopic expression of miR-712 attenuated macrophage pro-inflammatory responses and their paracrine inhibitory effects on myoblast insulin sensitivity. Mechanistic analysis showed that miR-712 directly targets LRRK2 resulting in decreased phosphorylation of p38 and ERK1/2 in macrophages. (B) miR-16 expression is down-regulated in LPS+IFNγ and palmitate stimulated macrophages and in palmitate, tunicamycin and thapsigargin treated myoblasts. Importantly, forced expression of miR-16 into macrophages improved myoblast insulin sensitivity by enhancing GLUT4 and MEF2A expression levels.