br Material and methods br

Material and methods
Results
Discussion T2D is a substantial threat to global human health [31]. Obesity-associated chronic low-grade inflammation promotes insulin resistance and T2D [32]. In obese individuals, the production of numerous inflammatory molecules including IL-6 and TNF-a is upregulated, which not only localy induces adipocyte dysfunction, but also affects other organs [33], and then causes insulin resistance. miRNAs regulate a wide range of biological processes including insulin resistance and inflammatory responses. Here, we investigated the role of miR-17 in obesity-associated inflammatory responses and insulin resistance. In this study, we found that expression of miR-17 was lower in macrophages of diabetic mice than in that of normal animals. Moreover, HG or LPS treatment also decreased the expression level of miR-17 in macrophages. These data suggested that miR-17 may play a negative role in diabetes-associated inflammatory responses. Chronic inflammation contributes to insulin resistance and T2D [28,34]. At the beginning of obesity-associated chronic low-grade inflammation, macrophages infiltrated and accumulated in the adipose tissues, which lead to an increased production of a BIIE 0246 synthesis of inflammatory molecules including IL-6 and TNF-a and inflammatory response [35]. Our results showed that miR-17 overexpression inhibited LPS-induced macrophages migration. It has been reported that miR-17 suppresses TNF-α-induced inflammatory cytokines production in rheumatoid arthritis synovial fibroblasts [36]. Accordingly, our results showed that miR-17 overexpression reduced inflammatory cytokines (IL-6, IL-1β and TNF-α) secretion in LPS-stimulated macrophages. Taken together, our observation suggested that miR-17 has anti-inflammatory properties in diabetic macrophages. In adipose tissue, macrophages secreted proinflammatory cytokines can impair insulin sensitivity by directly inhibiting insulin signaling pathway [37,38]. For instance, TNF-α induced insulin resistance via directly preventing insulin signaling in muscle [39]. Recently, it was reported that CM from LPS-treated macrophage impaired insulin-stimulated glucose uptake [26], which is consistent with our observation. Meanwhile, Our results showed that miR-17 abrogated the inhibitory effect of CM from LPS-treated macrophage (CM-LPS) on insulin-stimulated glucose uptake. These data suggested that miR-17 improved macrophages-induced insulin resistance via preventing macrophages inflammatory cytokines secretion. Generally, miRNAs play a key role in various biological functions through directly binding targeting mRNAs and inhibiting gene expression [40]. Using Targetscan Database, it was predicted that miR-17 potentially targeted 3′UTR of ASK1, which are confirmed by the luciferase reporter assay. In line with our observation, it is documented that miR-17 suppresses TNF-α-induced inflammatory cytokines secretion by downregulation of ASK1 in rheumatoid arthritis synovial fibroblasts [36]. As a component of the TLR4 pathway, ASK1-mediated p38 and JNK pathways play a critical role in inflammatory response [36,41]. ASK1 can be activated by TNF-α and LPS [[42], [43], [44], [45]]. Ablation of ASK1 expression in splenocytes and dendritic cells inhibits LPS-induced inflammatory cytokines secretion including IL-6, IL-1β and TNF-α [41]. Accordingly, our results showed that knocking down of ASK1 impaired migration and inflammatory cytokines release of macrophages as well as improved inflammation-related insulin resistance. Moreover, miR-17-induced inhibitory effect on migration and inflammatory cytokines release of macrophages as well as improvement of inflammation-related insulin resistance was reversed by ASK1 overexpression. Taken previous studies with our findings together, miR-17 offsets infiltration and inflammatory cytokines production of macrophages by directly targeting ASK1. In addition, miR-17 indirectly increased insulin-stimulated glucose uptake by reducing the production of inflammatory cytokine levels. These findings have suggested that miR-17 prevents macrophage-mediated adipose tissue inflammation and improves insulin resistance, which provides a therapeutic target for T2D treatment.