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. 2016 Dec 30;11(12):e0169039.
doi: 10.1371/journal.pone.0169039. eCollection 2016.

Induction of miR-96 by Dietary Saturated Fatty Acids Exacerbates Hepatic Insulin Resistance through the Suppression of INSR and IRS-1

Won-Mo Yang  1 , Kyung-Ho Min  1 , Wan Lee  1   2
Affiliations

Induction of miR-96 by Dietary Saturated Fatty Acids Exacerbates Hepatic Insulin Resistance through the Suppression of INSR and IRS-1

Won-Mo Yang et al. PLoS One. .

Abstract

Obesity is defined as the excessive accumulation of body fat that ultimately leads to chronic metabolic diseases. Diets rich in saturated fatty acids (SFA) exacerbate obesity and hepatic steatosis, which increase the risk of hepatic insulin resistance and type 2 diabetes (T2DM). Although microRNAs (miRNAs) play an important role in a range of biological processes, the implications of SFA-induced miRNAs in metabolic dysregulation, particularly in the pathogenesis of hepatic insulin resistance, are not well understood. This study investigated the implications of miR-96, which is induced strongly by SFA, in the development of hepatic insulin resistance. The liver of HFD mice and the palmitate-treated hepatocytes exhibited an impairment of insulin signaling due to the significant decrease in INSR and IRS-1 expression. According to expression profiling and qRT-PCR analysis of the miRNAs, the expression level of miR-96 was higher in hepatocytes treated with palmitate. Moreover, miR-96 was also upregulated in the liver of HFD mice. Interestingly, miR-96 targeted the 3'UTRs of INSR and IRS-1 directly, and repressed the expression of INSR and IRS-1 at the post-transcriptional level. Accordingly, the overexpression of miR-96 was found to cause a significant decrease in INSR and IRS-1 expression, thereby leading to an impairment of insulin signaling and glycogen synthesis in hepatocytes. These results reveal a novel mechanism whereby miR-96 promotes the pathogenesis of hepatic insulin resistance resulted from SFA or obesity.

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Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. Upregulation of miR-96 by HFD in the liver of DIO mice.
Mice given HFD for 14 wks exhibited hyperglycemia, impaired glucose tolerance, and impaired insulin tolerance, as shown in S1 Fig. (A) The mice were fasted overnight and sacrificed 30 min after an i.p. injection of the vehicle or insulin (2 U/kg of body weight). The representative immunoblots (2 from 7 mice/group) obtained from the liver lysates are shown. (B) The mRNA levels of INSR and IRS-1 were measured by qRT-PCR from the liver of vehicle-injected mice. (C) The protein expression and phosphorylation of INSR was normalized to the amount of Actin. (D) The protein expression and phosphorylation of IRS-1 were normalized to the amount of Actin. (E) The protein expression of Akt2 was normalized to the amount of Actin. The level of Akt2 phosphorylation was normalized to the amount of Akt2. (F) The protein expression of GSK3β was normalized to the amount of Actin. The level of GSK3β phosphorylation was normalized to the amount of GSK3β. (G) The expression of miR-96 was measured by qRT-PCR from the liver and skeletal muscle (SkM). The values are expressed as the relative ratio, where the average intensity of NFD-fed mice was set to one. The values are the means ± SEM. from five individuals. **, P < 0.01; ***, P < 0.001.
Fig 2
Fig 2. Impaired insulin signaling and upregulation of miR-96 by palmitate in HepG2 cells.
HepG2 cells were treated with either the vehicle or palmitate (0.5 mM) for 18 h, and then incubated in the presence or absence of insulin (100 nM) for 30 min. (A) Representative immunoblots obtained from HepG2 cells lysates are shown. (B) The mRNA levels of INSR and IRS-1 from the vehicle or palmitate-treated HepG2 cells were measured by qRT-PCR. (C) The protein expression or phosphorylation of INSR was normalized to the amount of Actin. (D) The protein expression or phosphorylation of IRS-1 was normalized to the amount of Actin. (E) The protein expression of Akt2 was normalized to the amount of Actin. The level of Akt2 phosphorylation was normalized to the amount of Akt2. (F) The protein expression of GSK3β was normalized to the amount of Actin. The level of GSK3β phosphorylation was normalized to the amount of GSK3β. (G) The expression of miR-96 from the vehicle BSA, oleate (0.5 mM for 18 h) or palmitate-treated HepG2 cells was measured by qRT-PCR. The values are expressed as the relative ratio, where the intensity of the vehicle (open column) was set to one. The values are expressed as the means ± SEM. from three independent experiments. **, P < 0.01; ***, P < 0.001.
Fig 3
Fig 3. Targeting sites of miR-96 in the 3’UTRs of INSR and IRS-1, and a target validation by reporter gene assay.
(A) The seed sequence (red box) of miR-96 was predicted to target INSR 3’UTRs. (B) The seed sequence (red box) of miR-96 was predicted to target IRS-1 3’UTRs. (C) The fragment of INSR 3’UTR was inserted downstream of a firefly luciferase open reading frame (wild-type: INSR 3Uwt). The mutated 3’UTR of the INSR gene lacking the miR-96 binding site (INSR 3Umut) was examined (upper panel). Non-containing 3’UTR (Empty), INSR 3Uwt or INSR 3Umut construct was cotransfected with either scRNA control (open column) or designated mature miR-96 mimic (closed column) into the L6 GLUT4myc cells. Luciferase activities was measured at 24 h after transfection (lower panel). (D) The fragment of IRS-1 3’UTR was inserted downstream of a firefly luciferase open reading frame (wild-type: IRS-1 3Uwt). The mutated 3’UTR of the IRS-1 gene lacking the miR-96 binding site (IRS-1 3Umut) was examined (upper panel). Empty, IRS-1 3Uwt or IRS-1 3Umut construct was cotransfected with either scRNA control or designated miR-96 mimic into the L6 GLUT4myc cells. Luciferase activities was measured at 24 h after transfection (lower panel). The relative luciferase activities were plotted against that of each scRNA control, which was set to one. The values are expressed as the means ± SEM. from three independent experiments. **, P < 0.01.
Fig 4
Fig 4. Effect of miR-96 on the INSR and IRS-1 expression.
(A-C) HepG2 cells were reverse-transfected with scRNA (100 nM), AntimiR-96 (100 nM), and/or miR-96 (100 nM) mimic. After 48 h transfection, the expression of insulin signaling intermediates was analyzed by immunoblotting. Representative immunoblots obtained from HepG2 cells lysates were shown in A. (B) The expression of INSR was normalized to the amount of Actin. (C) The expression of IRS-1 was normalized to the amount of Actin. (D) HepG2 cells were reverse-transfected with 200 nM of miR-96 mimic or scRNA control. The mRNA levels were analyzed at 24 h after reverse-transfection by RT-PCR (upper panel) and qRT-PCR (lower panel). Values are means ± SEM. **, P < 0.01; ***, P < 0.001 vs. scRNA control; ###, p < 0.001 scRNA + miR-96 vs. AntimiR-96 + miR-96.
Fig 5
Fig 5. Impairment of insulin signaling and glycogen synthesis by miR-96.
(A-E) HepG2 cells were reverse-transfected with the scRNA (100 nM), AntimiR-96 (100 nM) and/or miR-96 (100 nM) mimic. After 48 h transfection, the cells were incubated in the presence or absence of insulin (100 nM) for 30 min and subjected to immunoblotting. (A) Representative immunoblots obtained from HepG2 cells lysates are shown. (B) The expression and phosphorylation of INSR were normalized to the amount of Actin. (C) The expression and phosphorylation of IRS-1 were normalized to the amount of Actin. (D) The protein expression of Akt2 was normalized to the amount of Actin. The level of Akt2 phosphorylation was normalized to the amount of Akt2. (E) The protein expression of GSK3β was normalized to the amount of Actin. The level of GSK3β phosphorylation was normalized to the amount of GSK3β. (E) The glycogen contents were quantified in the presence or absence of insulin (100 nM) from the scRNA control, AntimiR-96 and/or miR-96 mimic-transfected HepG2 cells. The values are expressed as the relative ratio, where the intensity of the scRNA control was set to one. The values are expressed as the means ± SEM. from three independent experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001 vs. scRNA control; $$, P < 0.01 scRNA + insulin vs. miR-96 + insulin; %%, P < 0.01 AntimiR-96 vs. AntimiR-96 + insulin; &&, P < 0.01 AntimiR-96 + miR-96 vs. AntimiR-96 + miR+96 + insulin; ##, P < 0.01 miR-96 vs. AntimiR-96 + miR-96.
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Grants and funding

This research was supported by National Research Foundation of Korea, South Korea (NRF) grants funded by Ministry of Education (2013R1A1A2057932) and Ministry of Science, ICT and Future Planning, South Korea (2016M2B2A4912473).