MiR-499-5p Contributes to Hepatic Insulin Resistance by Suppressing PTEN.
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.


CONCLUSIONS
Downregulation of the miR-499-5p level impaired the PI3K/AKT/GSK signaling pathway and glycogen synthesis by targeting PTEN.
Department of Rehabilitation Medicine, Second Medical School of Nanjing University of Chinese Medicine, Nanjing, China.
Lei
L
Wang
Ning
N
Zhang
Hua-Ping
HP
Pan
Zun
Z
Wang
Zhen-Yu
ZY
Cao
D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D003921:Diabetes Mellitus, Experimental / Q000235:genetics; D059305:Diet, High-Fat; D038362:Glycogen Synthase Kinase 3 / Q000378:metabolism; D007333:Insulin Resistance / Q000235:genetics*; D008099:Liver / Q000378:metabolism* / Q000473:pathology; D008297:Male; D008810:Mice, Inbred C57BL; D035683:MicroRNAs / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D020411:Oligonucleotide Array Sequence Analysis; D051059:PTEN Phosphohydrolase / Q000235:genetics / Q000378:metabolism*; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism; D015398:Signal Transduction / Q000235:genetics; D015854:Up-Regulation / Q000235:genetics
C573190:MIRN499 microRNA, mouse; D035683:MicroRNAs; D051057:Proto-Oncogene Proteins c-akt; D038362:Glycogen Synthase Kinase 3; D051059:PTEN Phosphohydrolase