BACKGROUND: Type 2 diabetes afflicts 95% of diabetes patients. Recent data suggest that miRNAs play a key role in insulin production, secretion and function. This study aims to explore the specific role of miR-499-5p in hepatic insulin resistance. METHODS: The miRNA expression levels in the livers of db/db mice were analyzed using miRNA chips and were verified by real-time PCR. miR-499-5p mimics and an inhibitor were transfected into NCTC1469 cells. Then, the PI3K/AKT signaling pathway and glycogen level were determined. The target genes of miR-499-5p were predicted by bioinformatics and then confirmed by dual luciferase reporter assay and Western blot. To establish an insulin resistance (IR) animal model, C57BL/6 mice were fed a high-fat diet (HFD). The level of miR-499-5p in the livers of HFD-fed mice was upregulated through tail vein injection of adenovirus vectors (ad) containing miR-499-5p mimics. The glucose tolerance test (GTT) and insulin tolerance test (ITT) were used to determine glucose tolerance and insulin tolerance, respectively. RESULTS: MicroRNA chips and qPCR showed that miR-499-5p was significantly decreased in the livers of db/db mice. Downregulation of miR-499-5p impaired the insulin signaling pathway and glycogen synthesis, whereas upregulation of miR-499-5p promoted the insulin signaling pathway and glycogen synthesis in NCTC1469 cells. The dual luciferase reporter assay and Western blot demonstrated that PTEN was the target gene of miR-499-5p. Compared with the control group, miR-499-5p was increased 2.1-fold in the livers of HFD-fed mice. By tail vein injection of adenovirus vector containing miR-499-5p mimics, GTT and ITT were improved in HFD-fed mice. CONCLUSION: Downregulation of the miR-499-5p level impaired the PI3K/AKT/GSK signaling pathway and glycogen synthesis by targeting PTEN.
BACKGROUND: Type 2 diabetes afflicts 95% of diabetespatients. Recent data suggest that miRNAs play a key role in insulin production, secretion and function. This study aims to explore the specific role of miR-499-5p in hepatic insulin resistance. METHODS: The miRNA expression levels in the livers of db/db mice were analyzed using miRNA chips and were verified by real-time PCR. miR-499-5p mimics and an inhibitor were transfected into NCTC1469 cells. Then, the PI3K/AKT signaling pathway and glycogen level were determined. The target genes of miR-499-5p were predicted by bioinformatics and then confirmed by dual luciferase reporter assay and Western blot. To establish an insulin resistance (IR) animal model, C57BL/6 mice were fed a high-fat diet (HFD). The level of miR-499-5p in the livers of HFD-fed mice was upregulated through tail vein injection of adenovirus vectors (ad) containing miR-499-5p mimics. The glucose tolerance test (GTT) and insulin tolerance test (ITT) were used to determine glucose tolerance and insulin tolerance, respectively. RESULTS: MicroRNA chips and qPCR showed that miR-499-5p was significantly decreased in the livers of db/db mice. Downregulation of miR-499-5p impaired the insulin signaling pathway and glycogen synthesis, whereas upregulation of miR-499-5p promoted the insulin signaling pathway and glycogen synthesis in NCTC1469 cells. The dual luciferase reporter assay and Western blot demonstrated that PTEN was the target gene of miR-499-5p. Compared with the control group, miR-499-5p was increased 2.1-fold in the livers of HFD-fed mice. By tail vein injection of adenovirus vector containing miR-499-5p mimics, GTT and ITT were improved in HFD-fed mice. CONCLUSION: Downregulation of the miR-499-5p level impaired the PI3K/AKT/GSK signaling pathway and glycogen synthesis by targeting PTEN.
Authors: Manal S Fawzy; Essam Al Ageeli; Saeed Awad Al-Qahtani; Baraah T Abu Alsel; Shahad W Kattan; Walla Alelwani; Eman A Toraih Journal: Exp Ther Med Date: 2021-11-22 Impact factor: 2.447