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MiR-3138 deteriorates the insulin resistance of HUVECs via KSR2/AMPK/GLUT4 signaling pathway.

ABSTRACT: Insulin resistance (IR) is a complex pathological condition resulting from the dysregulation of cellular response to insulin hormone in insulin-dependent cells and is recognized as a pathogenic hallmark and strong risk factor for metabolic syndrome. The present study aims to elucidate the molecular mechanism of the pathogenesis of IR. Here, we used human umbilical vein endothelial cells (HUVECs) to establish the IR cell model induced by 1 × 10^-6 mmol/L insulin. After 48 h, reactive oxygen species (ROS) and glucose consumption were measured by DCFH-DA and GOD-POD methods, respectively. The results of Microarray analysis demonstrated that there were 10 differentially expressed miRNAs (DEMs) selected based on Fold change (FC) and P value in the IR cell model compared with HUVECs. The enriched gene ontology (GO) terms analysis showed that the target genes of these 10 DEMs were significantly enriched in biological process, cellular component and molecular function, and the significantly enriched Kyoto Encyclopedia of Genes or Genomes (KEGG) pathways mainly include AMPK signaling pathway and PI3K signaling pathway. Amongst all, the expression level of miR-3138 was highest in the IR cell model evaluated by qRT-PCR. Through Targetscan, KSR2 mRNA was predicted as a target of miR-3138. And mRNA and protein expression levels of miR-3138, KSR2, GLUT4, AMPK, PI3K, Akt were examined using qRT-PCR and Western blotting, respectively. The interaction between miR-3138 and KSR2 was evaluated by dual-luciferase reporter assay. Our results showed that miR-3138 significantly deteriorated the IR of HUVECs via KSR2/AMPK/GLUT4 signaling pathway.

SUBMITTER: Chen Y

PROVIDER: S-EPMC7894458 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:AMP-activated protein kinase (AMPK) enhances glucose transporter GLUT4 regulation. AMPK also suppresses energy-consuming pathways such as cholesterol synthesis. Interestingly, recent in vitro and in vivo data suggest that excess membrane cholesterol impairs GLUT4 regulation. Therefore, this study tested whether a beneficial, GLUT4-regulatory aspect of AMPK stimulation involved cholesterol lowering. Using L6 myotubes stably expressing an exofacial myc-epitope-tagged-GLUT4, AMPK stimulation by 5-aminoimidazole-4-carboxamide-1-?-d-ribonucleoside (AICAR; 45 min, 1 mm) or 2,4-dinitrophenol (DNP; 30 min, 200 ?m) increased cell surface GLUT4myc labeling by approximately ? 25% (P < 0.05). Insulin (20 min, 100 nm) also increased GLUT4myc labeling by about 50% (P < 0.05), which was further enhanced (? 25%, P < 0.05) by AICAR or DNP. Consistent with AMPK-mediated suppression of cholesterol synthesis, AICAR and DNP decreased membrane cholesterol by 20-25% (P < 0.05). Whereas AMPK knockdown prevented the enhanced basal and insulin-stimulated GLUT4myc labeling by AICAR and DNP, cholesterol replenishment only blocked the AMPK-associated enhancement in insulin action. Cells cultured in a hyperinsulinemic milieu, resembling conditions in vivo that promote the progression/worsening of insulin resistance, displayed an increase in membrane cholesterol. This occurred concomitantly with a loss of cortical filamentous actin (F-actin) and defects in GLUT4 regulation by insulin. These derangements were prevented by AMPK stimulation. Examination of skeletal muscle from insulin-resistant Zucker rats revealed a similar elevation in membrane cholesterol and loss of F-actin. Lowering cholesterol to control levels restored F-actin structure and insulin sensitivity. In conclusion, these data suggest a novel aspect of GLUT4 regulation by AMPK involves membrane cholesterol lowering. Moreover, this AMPK-mediated process protected against hyperinsulinemia-induced insulin resistance.

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MiR-3138 deteriorates the insulin resistance of HUVECs via KSR2/AMPK/GLUT4 signaling pathway.

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