Resveratrol is used in the field of insulin resistance improvement [22, 23]. RSV increases the expression of microencapsulated protein 3 (CAV-3), thereby allowing skeletal muscle cells to carry glucose the protein GLUT4 activates the transfer from the cytoplasm to the cell membrane, which in turn increases the ability of myocytes to transport glucose and improve insulin resistance. In addition, RSV promotes the beta-oxidation process of fatty acids in the cell mitochondria , so that intracellular lipids are better metabolized. RSV also improves IR in skeletal muscle by reducing SNARE proteins in diabetic rats,which is involved in GLUT4 transport. Meanwhile, RSV attenuates insulin-stimulated AKT phosphorylation by eliminating insulin-induced ROS production in skeletal muscle .
A large amount of experiments has verified the therapeutic effect of RSV in IR. In this study, we demonstrated that the influence of RSV in the improvement of insulin resistance in high-fat diet mice as reported previously [24, 25, 26, 27]. Not only did blood glucose, insulin index, and area under the curve (AUC) decreased in high-fat mice after applying RSV, but also improved blood lipid levels. RSV treatment improved high-fat diet-induced mice insulin resistance by restoring insulin signaling pathway gene expression. After the intervention of RSV, the mRNA and protein expression levels of p-AKT, p-GSK3β and GLUT4 increased significantly after RSV application.
The expression of lncRNA has spatiotemporal specificity, and it participates in the process of gene regulation and biological function regulation in epigenetics, transcription, and post-transcription levels . Abnormal expression of lncRNAs could affect the occurrence and development of human diseases, including non-alcoholic fatty liver, various cancers, and T2DM . At present, the function of most lncRNAs is completely unknown. Previous studies[30, 31] by our group have demonstrated that RSV can improve hepatic IR by regulating lncRNA NONMMUT058999.2 and NONMMUT008655.2 in IR models .Whether RSV could improve IR by regulating the expression of lncRNAs in skeletal muscle remains undefined. In this study, we further studied skeletal muscle, a different tissue than previous studies, and found a novel lncRNA NONMMUT044897.2 that may be involved in resveratrol's improvement of skeletal muscle IR in vivo.
High-throughput sequencing showed that there were 3276 differential lncRNAs and 2118 mRNAs in HFD mice compared to the CON group, and 1640 differential lncRNAs and 604 mRNAs compared to the HFD + RSV group. We further provided 338 mRNAs and 629 lncRNAs whose expression was reversed between HFD and HFD + RSV groups, suggesting that RSV is under a strong role in the overall alteration of skeletal muscle gene expression. Moreover, via RT-qPCR uncovered that RSV improved IR by regulating the expression of lncRNAs in skeletal muscle. As mentioned above, the verified lncRNAs were consistent with the sequencing results and NONMMUT044897.2 was highly expressed. GO and KEGG analysis uncovered that the differential genes were part of the insulin signaling pathway. We found that NONMMUT044897.2 was associated with SOCS1, which is critically involved in the insulin signaling pathway, so we selected this lncRNA for further study, which hadn't been reported before.
SOCS1 is a specific negative regulator that regulates the JAK/STAT pathway . The expression of SOCS1 increases under insulin resistance and overexpression of SOCS1 decreases the phosphorylation of IRS-1; overexpression of SOCS-1 can inhibit insulin-induced glycogen synthesis in L6 myotubes . AKT has essential roles in many signaling pathways, such as cell survival and cell metabolism. AKT is the center of the insulin signaling pathway, which regulates glucose and lipid metabolism. Activated AKT can stimulate the translocation of insulin-sensitive GLUT4 to the cell membrane through its downstream substrate ASl60 to increase glucose uptake; it can also phosphorylate GSK3β to inhibit its activity, promote glycogen synthesis, lower blood sugar, and improve IR . A high fat diet can result in the decrease of skeletal muscle IRS-1, P13K, AKT, GLUT4 gene expression, reduce p-AKT (ser473), p- GSK3β protein expression. Studies have reported that overexpression of SOCS1 can inhibit the phosphorylation and activation of IRS-1[32, 33], which in turn inhibits the activation of AKT, indicating that there is an important link between AKT and SOCS1. We found that in IR model mice, mRNA and protein expression levels of SOCS1 was significantly increased, RSV treatment reversed this trend, thereby improving insulin resistance and decreasing blood glucose.
Numerous studies have demonstrated that lncRNAs may involve in human diseases by regulating miRNA expression [35, 36]. The co-expression network diagram uncovered the possible regulatory roles of candidate lncRNA NONMMUT044897.2 could regulate SOCS1 through two different miRNAs. To further clarify the relationship between NONMMUT044897.2 and SOCS1, we constructed NONMMUT044897.2 and miR-7051-5p, miR-7051-5p and SOCS1base pairing maps based on the NonCode and miRBase databases and the Targetscan database. These results indicated that NONMMUT044897.2 might regulate the expression of SOCS1 through miR-7051-5p. Future studies may perform luciferase assays to verify the interactions between NONMMUT044897.2, miR-7051-5p, and SOCS1.
We confirmed that knockdown of NONMMUT044897.2 increased miR-7051-5p levels, promoted gene expression of insulin signaling (p-AKT, p-GSK3β, GLUT4). Meanwhile, the expression of SOCS1 was suppressed by silencing NONMMUT044897.2. Moreover, knockdown of NONMMUT044897.2 led to reduced glucose concentration, similar to the phenotypes induced by RSV treatment, indicating that resveratrol improves skeletal muscle insulin resistance through downregulating the lncRNA NONMMUT044897.2.