Mir-338-3p Mediates Tnf-A-Induced Hepatic Insulin Resistance by Targeting PP4r1 to Regulate PP4 Expression

Abstract

Objective: Insulin resistance is a critical factor contributing to the pathogenesis of type 2 diabetes and other metabolic diseases. Recent studies have indicated that miR-338-3p plays an important role in cancer. Here, we investigated whether miR-338-3p mediates tumour necrosis factor-α (TNF-α)-induced hepatic insulin resistance. Methods: The activation of the insulin signalling pathway and the level of glycogenesis were examined in the livers of the db/db and high fat diet (HFD)-fed mice and in HEP1-6 cells transfected with miR-338-3p mimic or inhibitor. Computational prediction of microRNA target, luciferase assay and Western blot were used to assess the miR-338-3p target. Chromatin immunoprecipitation (ChIP) assay was used to determine the transcriptional regulator of miR-338-3p. Results: miR-338-3p was down-regulated in the livers of the db/db, HFD-fed and TNF-α-treated C57BL/6J mice, as well as in mouse HEP1-6 hepatocytes treated with TNF-α. Importantly the down-regulation of miR-338-3p induced insulin resistance, as indicated by impaired glucose tolerance and insulin tolerance. Further research showed that the down-regulated miR-338-3p resulted in the impaired AKT/ glycogen synthase kinase 3 beta (GSl·Gβ) signalling pathway and glycogen synthesis. In contrast, hepatic over-expression of miR-338-3p rescued the TNF-α-induced insulin resistance. Moreover, protein phosphatase 4 regulator subunit 1 (PP4R1) was identified as a direct target of miR-338-3p that mediated hepatic insulin signalling by regulating protein phosphatase 4 (PP4). Finally we identified hepatic nuclear factor 4 alpha (HNF-4α) as the transcriptional regulator of miRNA-338-3p. Conclusions: Our studies provide novel insight into the critical role and molecular mechanism by which miR-338-3p is involved in TNF-α-induced hepatic insulin resistance. miR-338-3p might mediate TNF-α-induced hepatic insulin resistance by targeting PP4R1 to regulate PP4 expression.

miR‑338‑3p mediates gluconeogenesis via targeting of PP4R1 in hepatocytes

“…In our previous study, it was demonstrated that treatment with TNF-α decreased miR-338-3p expression levels and suppressed the activation of the AKT/GSK pathway in the livers of mice (13). In the present study, treatment with TNF-α significantly decreased the level of p-FOXO1/FOXO1 and significantly increased the protein and mRNA expression levels of genes associated with gluconeogenesis in the livers of mice, including PGC-1α, G6Pase and PEPCK ( Fig.…”

“…targetscan.org/), Pictar (http://www.pictar.org/) and miRanda (http://microrna.org/) databases were used to predict that PP4R1 was a target of miR-338-3p. The miR-338-3p binding site with in the PP4R1 3'-UTR was verified and in previously study (13). The PP4R1 3'-UTR fragment, including the miR-338-3p binding site was cloned from mouse liver cells and inserted into pmirGLO vector (Promega Corporation, Madison, WI, USA).…”

“…In addition, TNF-α promotes inflammation and attenuates insulin resistance in hepatocytes. Furthermore, in vivo and in vitro studies have suggested that treatment with TNF-α blocks the insulin signalling pathway and results in insulin resistance (13).…”

“…In our previous study, PP4R1 was revealed to represent a target gene of miR-338-3p. The 3'-UTR of PP4R1 was cloned and luciferase reporter vectors were subsequently constructed (13). The relative luciferase activity was significantly decreased following transfection with miR-338-3p mimics (Fig.…”

“…Furthermore, in vitro and in vivo studies have suggested that TNF-α treatment leads to increased gluconeogenesis and decreased glycogenesis in hepatocytes (12). Our previous study demonstrated that miR-338-3p overexpression restores impaired glycogenesis induced by TNF-α by directly inhibiting protein phosphatase 4 regulatory subunit 1 (PP4R1) expression (13). However, the association between miR-338-3p and increased levels of gluconeogenesis induced by TNF-α remains unknown.…”