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MiR199a-5p inhibits hepatic insulin sensitivity via suppression of ATG14-mediated autophagy.

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.

SUBMITTER: Li B

PROVIDER: S-EPMC5851987 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BackgroundOsteoporosis affects the mandible resulting in bone loss. Though impairments are not life threatening, they affect a person's quality-of-life particularly vulnerable elderly. MicroRNAs (miRNAs) are novel regulatory factors that play an important role in regulating bone metabolism. Autophagy is evolutionarily conserved intracellular self-degradation process and is vital in the maintenance of both miRNA and bone homeostasis. However, the role of autophagy in the pathogenesis of miRNA regulating osteoporosis remains unclear.MethodsIn the study, we established a rat osteoporosis model induced by ovariectomy (OVX) and isolated mesenchymal stem cells from mandible (MMSCs-M). Several miRNAs were identified to regulate osteoporosis in some studies. qRT-PCR was applied to examine the expression of miRNA, autophagy and osteogenic differentiation-related genes. Western blotting assays were performed to detect the expression of autophagy and osteogenic differentiation proteins. Immunofluorescence and transmission electron microscope were used to verify the autophagy activity. Transfecting technology was used to enhance or suppress the expression of miR-152-5p which enable us to observe the relationship between miR-152-5p, autophagy and osteogenic differentiation. Additionally, the measurement of reactive oxygen species was used to investigate the mechanism of autophagy affecting osteogenic differentiation.ResultsWe found an upregulated expression of miR-152-5p in MMSCs-M in OVX group. Downregulated autophagy-related gene, proteins and autophagosome were detected in vitro of OVX group compared with sham group. Moreover, downregulation of miR-152-5p promoted osteogenic differentiation of MMSCs-M as well as enhanced autophagy-related proteins in OVX group. Conversely, overexpression of miR-152-5p showed opposite effect in sham group. Meanwhile, we found Atg14 (autophagy-related protein homolog 14) was identified to be a direct target of miR-152-5p theoretically and functionally. In other words, we confirmed inhibition of miR-152-5p promoted the osteogenic differentiation via promoting ATG14-mediated autophagy. Furthermore, miR-152-5p/ATG14-mediated autophagy regulated osteogenic differentiation by reducing the endogenous ROS accumulation and maintaining cellular redox homeostasis.ConclusionOur data suggest that miR-152-5p is the first identified to regulate osteogenic differentiation by directly targeting autophagy-related protein ATG14 and regulating oxidative stress and therapeutic inhibition of miR-152-5p may be an efficient anabolic strategy for osteoporosis.

Project description:ContextA central/visceral fat distribution and excess free fatty acid (FFA) availability are associated with dyslipidemia and insulin resistance. However, these two characteristics often coexist, making it difficult to detect the independent contributions of each. Whether FFA suppression is more closely linked to metabolic abnormalities is not clear.ObjectiveThe aim of the study was to examine the relationship between FFA suppression, body fat distribution, and fitness as contributors toward insulin resistance and hypertriglyceridemia.DesignWe measured systemic palmitate turnover using an iv infusion of [9,10-(3)H]palmitate; upper body sc adipose tissue (UBSQ) and visceral adipose tissue (VAT) with dual-energy x-ray absorptiometry and a single-slice abdominal computed tomography scan; fitness with a graded exercise treadmill test; and insulin sensitivity with both the iv glucose tolerance test (IVGTT) (SI(IVGTT)) and mixed meal tolerance test (SI(Meal)).SettingThe study was conducted at a General Clinical Research Center.ParticipantsBaseline data were obtained from 140 elderly adults (age, 60-88 yr; 83 males) and 60 young adults (age, 18-31 yr; 31 males) who participated in a previously published trial assessing the effects of 2-yr supplementation of dehydroepiandrosterone or testosterone on body composition, glucose metabolism, and bone density.InterventionsThere were no interventions.Main outcome measuresWe measured fasting plasma triglyceride (TG) concentrations, SI(IVGTT), and SI(Meal).ResultsUsing multivariate regression analysis, the strongest combined predictors of TG concentrations were VAT, postmeal nadir FFA concentrations, sex, and age. The best predictors of SI(IVGTT) were IVGTT nadir palmitate concentration, VAT, UBSQ fat, fitness, and age, whereas the best predictors of SI(Meal) were meal nadir palmitate concentration, UBSQ fat, fitness, and sex.ConclusionsFFA suppression is associated with both fasting TG concentrations and insulin sensitivity, independent of measures of adiposity.

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MiR199a-5p inhibits hepatic insulin sensitivity via suppression of ATG14-mediated autophagy.

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