Abstract
MicroRNAs (miRNAs) are known to contribute to many metabolic diseases, including diabetes. In this study, we investigated the role of miR199a-5p in the regulation of hepatic insulin sensitivity. Ad-anti-miR199a-5p adenoviruses were injected into male C57BL/6J WT mice fed a high-fat diet to inhibit miR199a-5p expression before the glucose levels and insulin resistance were assessed. Similarly, Ad-miR199a-5p adenoviruses were injected into male C57BL/6J WT mice to cause the overexpression of miR199a-5p. To investigate the roles of autophagy-related protein 14 (ATG14) and miR199a-5p in the regulation of insulin sensitivity, we injected Ad-miR199a-5p with or without Ad-ATG14 viruses into WT C57BL/6J mice before performing functional assays. Moreover, we infected HepG2 cells or primary hepatocytes with Ad-anti-miR199a-5p or Ad-miR199a-5p viruses to determine the effect of miR199a-5p on insulin resistance in vitro. Finally, we explored the clinical relevance of miR199a-5p by examining the expression level of miR199a-5p in liver samples derived from diabetes patients. We first demonstrated that knocking down miR199a-5p led to decreased glucose tolerance and clearance in vivo, whereas the overexpression of miR199a-5p had the opposite effect. We further identified ATG14 as the target of miR199a-5p, and ATG14 partially rescued miR199a-5p-potentiated glucose and insulin tolerance. In addition, transmission electron microscopy data and western blot data regarding ATG14, LC3 and BECLIN1 illustrated that miR199a-5p regulates autophagy via ATG14. Knocking down miR199a-5p in primary hepatocytes and HepG2 cells suppressed the insulin-stimulated phosphorylation of insulin receptor β, glycogen synthase kinase 3β and protein kinase B, whereas the overexpression of miR199a-5p further potentiated their phosphorylation. Finally, we detected upregulated miR199a-5p levels, which were correlated with reduced ATG14 mRNA levels and downregulated autophagy in liver samples obtained from diabetes patients. Our study uncovered a novel biological role of miR199a-5p in the regulation of hepatic insulin sensitivity via ATG14-mediated autophagy.
Highlights
Diabetes, especially type 2 diabetes, has become an alarming global burden
(see figure on previous page) Fig. 4 miR199a-5p leads to hepatic insulin resistance via autophagy-related protein 14 (ATG14) in vivo. a–e Male C57BL/6J WT mice were infected with Ad-miR199a-5p (Ad-miR199a-5p) or the negative control (Ad-miR NC) and with Ad-ATG14 (Ad-ATG14) or the negative control (Ad-NC) via tail vein injection. a Day 9: measurement of hepatic ATG14 expression. b, c Days 6 and 5: examination of fed or fasting blood glucose and serum insulin levels. d HOMA-IR index. e Days 5 and 3: performance of glucose tolerance test (GTT) and insulin tolerance test (ITT)
The two-tailed Student's t-test was applied to determine the difference between the Ad-miR199a-5p group and the corresponding control group (*P < 0.05, **P < 0.01, ***P < 0.001) or between the Ad-miR199a-5p group with Ad-ATG14 and the Ad-miR199a-5p group without Ad-ATG14., n = 6 mice per group. f–j: Male C57BL/6J WT mice were infected with Ad-ATG14 or Ad-NC via tail vein injection. f Day 9: measurement of hepatic ATG14 expression. g, h Days 6 and 5: examination of fed or fasting blood glucose and serum insulin levels. i HOMA-IR index. j Days 5 and 3: performance of GTTs and ITTs
Summary
Burden of Disease report, the prevalence of diabetes rose from approximately 333 million patients in 2005 to approximately 435 million patients in 2015, a dramatic 30.6% increase during the last 10 years[1]. Among several major pathological changes that occur in type 2 diabetes, insulin resistance is the driving factor, which is characterized as a high blood glucose level, hyperinsulinemia and decreased. Li et al Cell Death and Disease (2018)9:405 insulin sensitivity. Because insulin resistance prevents cells from efficiently taking in glucose[3], the regulation of insulin resistance has attracted considerable attention, and it could be a key component of diabetes treatment. Recent studies have identified several miRNAs that are associated with the core components of metabolic diseases[6], such as insulin resistance and lipid metabolism[7,8,9]
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