MiR-183-5p Induced by Saturated Fatty Acids Hinders Insulin Signaling by Downregulating IRS-1 in Hepatocytes

Abstract

Excessive saturated fatty acids (SFA) uptake is known to be a primary cause of obesity, a widely acknowledged risk factor of insulin resistance and type 2 diabetes. Although specific microRNAs (miRNAs) targeting insulin signaling intermediates are dysregulated by SFA, their effects on insulin signaling and sensitivity are largely unknown. Here, we investigated the role of SFA-induced miR-183-5p in the regulation of proximal insulin signaling molecules and the development of hepatic insulin resistance. HepG2 hepatocytes treated with palmitate and the livers of high-fat diet (HFD)-fed mice exhibited impaired insulin signaling resulting from dramatic reductions in the protein expressions of insulin receptor (INSR) and insulin receptor substrate-1 (IRS-1). Differential expression analysis showed the level of miR-183-5p, which tentatively targets the 3'UTR of IRS-1, was significantly elevated in palmitate-treated HepG2 hepatocytes and the livers of HFD-fed mice. Dual-luciferase analysis showed miR-183-5p bound directly to the 3'UTR of IRS-1 and reduced IRS-1 expression at the post-transcriptional stage. Moreover, transfection of HepG2 hepatocytes with miR-183-5p mimic significantly inhibited IRS-1 expression and hindered insulin signaling, consequently inhibiting insulin-stimulated glycogen synthesis. Collectively, this study reveals a novel mechanism whereby miR-183-5p induction by SFA impairs insulin signaling and suggests miR-183-5p plays a crucial role in the pathogenesis of hepatic insulin resistance in the background of obesity.

Keywords: IRS-1; insulin resistance; miR-183-5p; obesity.

Figure 1

HFD led to impairment of insulin signaling and induced miR-183-5p in the mouse liver. C57BL/6N male mice were fed either NFD or HFD at the age of 6 wk for 14 wks. Mice were injected intraperitoneally with either insulin (1U/kg body wt) or the vehicle for the last 30 min prior to euthanasia. (A) Representative immunoblots (2 from 8 mice/group) of insulin signaling molecules (INSR, IRS-1, Akt2, and GSK3β) and their phosphorylations (pINSR, pIRS-1, pAkt2, and pGSK3β) in the liver are shown. (B–D) The respective densitometry measurements, normalized versus β-Actin. (E) The miR-183-5p levels from the liver of HFD-fed mice (closed column) and NFD-fed mice (open column) were determined using qRT-PCR. The results in immunoblots and qRT-PCR are presented as a relative ratio, normalized to NFD controls set to one. Results are presented as means ± SEMs. **, p < 0.01; ***, p < 0.001 vs. controls (NFD).

Figure 2

Palmitate impaired insulin signaling and elevated miR-183-5p expression in HepG2 cells. HepG2 cells were pretreated with BSA (control) or palmitate (0.5 mM) for 18 h and incubated with or without 100 nM insulin for the last 30 min. (A) Representative immunoblots of insulin signaling molecules (INSR, IRS-1, Akt2, and GSK3β) and their phosphorylations (pINSR, pIRS-1, pAkt2, and pGSK3β) are shown. (B–D) The respective densitometry measurements, normalized versus β-Actin. (E) The miR-183-5p levels were determined using qRT-PCR. The results in immunoblots and qRT-PCR are presented as a relative ratio, normalized to BSA controls set to one. Results are presented as means ± SEMs (n > 3). **, p < 0.01; ***, p < 0.001 vs. BSA controls.

Figure 3

Palmitate regulated IRS-1 and miR-183-5p expressions in a dose- and time-dependent manner. HepG2 cells were preincubated with BSA or palmitate at 0.125–0.5 mM concentrations for 6 to 18 h. The expression levels of INSR and IRS-1 protein (A,B) or miR-183-5p (C) in various doses of palmitate and incubation time are shown. The protein expressions of INSR and IRS-1 were normalized versus β-Actin. The results in immunoblots and qRT-PCR are expressed as a relative ratio, normalized to control to one. Results are presented as means ± SEMs (n > 3). *, p < 0.05; **, p < 0.01; ***, p < 0.001 vs. BSA controls.

Figure 4

MiR-183-5p directly targeted the 3’UTR of IRS-1 and suppressed IRS-1 protein expression. (A) Graphic illustration of the conserved binding site for miR-183-5p within the 3′UTR of IRS-1. (B) The sequence of miR-183-5p binding site with wild-type (wt-IRS1) or mutant (mut-IRS1) 3′UTR of IRS-1. (C) The relative luciferase activity was performed after 24 h of reversed transfection with either the miR-183-5p or scRNA control (200 nM). (D) Representative immunoblots of INSR, IRS-1, and Akt2 after transfection with scRNA, miR-183-5p mimic, or antimiR-183 for 24 h. (E) Transcriptions of IRS-1 were determined by RT-PCR (upper) and qRT-PCR (lower). The results in immunoblots and qRT-PCR are expressed as a relative ratio, normalized to control set to one. Results are presented as means ± SEMs (n > 3). **, p < 0.01; ***, p < 0.001 vs. scRNA controls.

Figure 5

MiR-183-5p inhibited insulin signaling and insulin-stimulated glycogen synthesis. HepG2 cells were reverse transfected with 200 nM of scRNA or miR-183-5p mimic for 24 h and incubated additionally in the presence or absence of 100 nM insulin for the last 30 min before biochemical analysis. (A) Representative immunoblots of insulin signaling molecules (INSR, IRS-1, Akt2, and GSK3β) and their phosphorylations (pINSR, pIRS-1, pAkt2, and pGSK3β) are shown. (B–D) The respective densitometry measurements, normalized versus β-Actin. (E) The results from glycogen assays are displayed as relative ratios against the basal (without insulin stimulation) scRNA controls. Results are presented as means ± SEMs (n > 3). **, p < 0.01; ***, p < 0.001 vs. scRNA controls.