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Retinoid X receptor α overexpression alleviates mitochondrial dysfunction-induced insulin resistance through transcriptional regulation of insulin receptor substrate 1.

Lee SE, Koo YD, Lee JS, Kwak SH, Jung HS, Cho YM, Park YJ, Chung SS, Park KS - Mol. Cells (2015)

Bottom Line: RXRα overexpression also increased IRS1 transcription and mitochondrial function.Because RXRα overexpression, knock-down, or activation by LG1506 regulated IRS1 transcription mostly independently of mitochondrial function, it is likely that RXRα directly regulates IRS1 transcription.Consistent with the hypothesis, we showed that RXRα bound to the IRS1 promoter as a heterodimer with peroxisome proliferator-activated receptor δ (PPARδ).

View Article: PubMed Central - PubMed

Affiliation: Department of Internal Medicine, Seoul National University College of Medicine, Seoul 110-744, Korea.

ABSTRACT
Mitochondrial dysfunction is associated with insulin resistance and diabetes. We previously showed that retinoid X receptor α (RXRα) played an important role in transcriptional regulation of oxidative phosphorylation (OXPHOS) genes in cells with mitochondrial dysfunction caused by mitochondrial DNA mutation. In this study, we investigated whether mitochondrial dysfunction induced by incubation with OXPHOS inhibitors affects insulin receptor substrate 1 (IRS1) mRNA and protein levels and whether RXRα activation or overexpression can restore IRS1 expression. Both IRS1 and RXRα protein levels were significantly reduced when C2C12 myotubes were treated with the OXPHOS complex inhibitors, rotenone and antimycin A. The addition of RXRα agonists, 9-cis retinoic acid (9cRA) and LG1506, increased IRS1 transcription and protein levels and restored mitochondrial function, which ultimately improved insulin signaling. RXRα overexpression also increased IRS1 transcription and mitochondrial function. Because RXRα overexpression, knock-down, or activation by LG1506 regulated IRS1 transcription mostly independently of mitochondrial function, it is likely that RXRα directly regulates IRS1 transcription. Consistent with the hypothesis, we showed that RXRα bound to the IRS1 promoter as a heterodimer with peroxisome proliferator-activated receptor δ (PPARδ). These results suggest that RXRα overexpression or activation alleviates insulin resistance by increasing IRS1 expression.

No MeSH data available.


Related in: MedlinePlus

RXRα directly binds to the IRS1 promoter. (A) A diagram of three different lengths of IRS1 promoter-luciferase constructs, IRS1 (-1845)-Luc, IRS1 (-1155)-Luc and IRS1 (-998)-Luc. (B) COS7 cells were transfected with one of the IRS1 promoter-Luc constructs and RXRα expression vector (pcDNARXRα). Three hours after the transfection, cells were treated with LG1506 (2 μM) for an additional 18 h. Luciferase activity of the cells transfected with IRS1 (-1845)-Luc was set to 100 and the other values were expressed as the relatives to that (n = 5). *, P < 0.05 vs pcDNA without LG1506 for each construct. #, P < 0.05 vs. pcDNA-RXRα without LG1506. (C) COS7 cells were transfected with IRS1 (-1155) Luc, expression vectors for RXRα, PPARα, PPARδ or PPARγ. Luci ferase activity of the cells transfected with IRS1 (-1155)-Luc without RXRα or PPAR expression vectors was set to 100 and the others were expressed as the relatives to that (n = 4). *, P < 0.05 vs. cells transfected with IRS1 (-1155)-Luc only. #, P < 0.05 vs. cells transfected with IRS1 (-1155)-Luc and pcDNA-RXRα (D) C2C12 myotubes were infected with Ad-RXRα (50 MOI) for 48 h, and then treated with rotenone for an additional 24 h. Cells were subjected to ChIP. Data are the means ± SEM (n = 3). *, P < 0.05 vs. Ad-GFP infected cells not treated with rotenone. #, P < 0.05 vs. Ad-GFP infected cells treated with rotenone.
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f4-molce-38-4-356: RXRα directly binds to the IRS1 promoter. (A) A diagram of three different lengths of IRS1 promoter-luciferase constructs, IRS1 (-1845)-Luc, IRS1 (-1155)-Luc and IRS1 (-998)-Luc. (B) COS7 cells were transfected with one of the IRS1 promoter-Luc constructs and RXRα expression vector (pcDNARXRα). Three hours after the transfection, cells were treated with LG1506 (2 μM) for an additional 18 h. Luciferase activity of the cells transfected with IRS1 (-1845)-Luc was set to 100 and the other values were expressed as the relatives to that (n = 5). *, P < 0.05 vs pcDNA without LG1506 for each construct. #, P < 0.05 vs. pcDNA-RXRα without LG1506. (C) COS7 cells were transfected with IRS1 (-1155) Luc, expression vectors for RXRα, PPARα, PPARδ or PPARγ. Luci ferase activity of the cells transfected with IRS1 (-1155)-Luc without RXRα or PPAR expression vectors was set to 100 and the others were expressed as the relatives to that (n = 4). *, P < 0.05 vs. cells transfected with IRS1 (-1155)-Luc only. #, P < 0.05 vs. cells transfected with IRS1 (-1155)-Luc and pcDNA-RXRα (D) C2C12 myotubes were infected with Ad-RXRα (50 MOI) for 48 h, and then treated with rotenone for an additional 24 h. Cells were subjected to ChIP. Data are the means ± SEM (n = 3). *, P < 0.05 vs. Ad-GFP infected cells not treated with rotenone. #, P < 0.05 vs. Ad-GFP infected cells treated with rotenone.

Mentions: To confirm that RXRα directly binds to the IRS1 promoter and activates the transcription of IRS1, DNA fragments containing three different lengths of 5′-flanking region of the mouse IRS1 gene (from -1845, -1155, or -998 bp to -875 bp) were linked to the luciferase reporter gene and then transfected into COS7 cells with an RXRα expression vector (Fig. 4A). RXRα increased and LG1506 further increased the luciferase activities of the (-1845)-Luc and (-1155)-Luc constructs but not the (-998)-Luc construct (Fig. 4B). This finding suggests that a cis-acting element(s) for RXRα is located between -1155 bp and -998 bp in the IRS1 promoter. In contrast, 9cRA did not increase the luciferase activity further (data not shown). Next, we identified the partner of RXRα for binding to the IRS1 promoter region. Based on a report that LG1506 is a specific activator for the RXRα/PPAR heterodimer, we tested whether PPARα, PPARδ, and PPARγ, could increase the luciferase activity of the IRS1 (-1155)-Luc construct. Co-transfection of PPARδ expression vectors further increased luciferase activity, but PPARα and PPARγ vectors did not (Fig. 4C), indicating that the RXRα/PPARδ heterodimer binds to the promoter region of the IRS1 gene. To confirm that RXRα and PPARδ directly bind to the region between -1155 bp and -998 bp, ChIP was performed with RXRα, PPARδ, or RNA Pol II antibodies in C2C12 myotubes, and real-time PCR was performed using primers flanking the region (from -1155 bp to -998 bp). While bindings of RXRα, PPARδ, and RNA Pol II were decreased following rotenone treatment, bindings of these factors to the region were significantly increased when RXRα was overexpressed. These results indicated that IRS1 transcription was augmented by RXRα overexpression (Fig. 4D). Collectively, RXRα forms a heterodimer with PPARδ that directly binds to the IRS1 promoter region between -1155 bp and -998 bp and regulates IRS1 transcription level.


Retinoid X receptor α overexpression alleviates mitochondrial dysfunction-induced insulin resistance through transcriptional regulation of insulin receptor substrate 1.

Lee SE, Koo YD, Lee JS, Kwak SH, Jung HS, Cho YM, Park YJ, Chung SS, Park KS - Mol. Cells (2015)

RXRα directly binds to the IRS1 promoter. (A) A diagram of three different lengths of IRS1 promoter-luciferase constructs, IRS1 (-1845)-Luc, IRS1 (-1155)-Luc and IRS1 (-998)-Luc. (B) COS7 cells were transfected with one of the IRS1 promoter-Luc constructs and RXRα expression vector (pcDNARXRα). Three hours after the transfection, cells were treated with LG1506 (2 μM) for an additional 18 h. Luciferase activity of the cells transfected with IRS1 (-1845)-Luc was set to 100 and the other values were expressed as the relatives to that (n = 5). *, P < 0.05 vs pcDNA without LG1506 for each construct. #, P < 0.05 vs. pcDNA-RXRα without LG1506. (C) COS7 cells were transfected with IRS1 (-1155) Luc, expression vectors for RXRα, PPARα, PPARδ or PPARγ. Luci ferase activity of the cells transfected with IRS1 (-1155)-Luc without RXRα or PPAR expression vectors was set to 100 and the others were expressed as the relatives to that (n = 4). *, P < 0.05 vs. cells transfected with IRS1 (-1155)-Luc only. #, P < 0.05 vs. cells transfected with IRS1 (-1155)-Luc and pcDNA-RXRα (D) C2C12 myotubes were infected with Ad-RXRα (50 MOI) for 48 h, and then treated with rotenone for an additional 24 h. Cells were subjected to ChIP. Data are the means ± SEM (n = 3). *, P < 0.05 vs. Ad-GFP infected cells not treated with rotenone. #, P < 0.05 vs. Ad-GFP infected cells treated with rotenone.
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f4-molce-38-4-356: RXRα directly binds to the IRS1 promoter. (A) A diagram of three different lengths of IRS1 promoter-luciferase constructs, IRS1 (-1845)-Luc, IRS1 (-1155)-Luc and IRS1 (-998)-Luc. (B) COS7 cells were transfected with one of the IRS1 promoter-Luc constructs and RXRα expression vector (pcDNARXRα). Three hours after the transfection, cells were treated with LG1506 (2 μM) for an additional 18 h. Luciferase activity of the cells transfected with IRS1 (-1845)-Luc was set to 100 and the other values were expressed as the relatives to that (n = 5). *, P < 0.05 vs pcDNA without LG1506 for each construct. #, P < 0.05 vs. pcDNA-RXRα without LG1506. (C) COS7 cells were transfected with IRS1 (-1155) Luc, expression vectors for RXRα, PPARα, PPARδ or PPARγ. Luci ferase activity of the cells transfected with IRS1 (-1155)-Luc without RXRα or PPAR expression vectors was set to 100 and the others were expressed as the relatives to that (n = 4). *, P < 0.05 vs. cells transfected with IRS1 (-1155)-Luc only. #, P < 0.05 vs. cells transfected with IRS1 (-1155)-Luc and pcDNA-RXRα (D) C2C12 myotubes were infected with Ad-RXRα (50 MOI) for 48 h, and then treated with rotenone for an additional 24 h. Cells were subjected to ChIP. Data are the means ± SEM (n = 3). *, P < 0.05 vs. Ad-GFP infected cells not treated with rotenone. #, P < 0.05 vs. Ad-GFP infected cells treated with rotenone.
Mentions: To confirm that RXRα directly binds to the IRS1 promoter and activates the transcription of IRS1, DNA fragments containing three different lengths of 5′-flanking region of the mouse IRS1 gene (from -1845, -1155, or -998 bp to -875 bp) were linked to the luciferase reporter gene and then transfected into COS7 cells with an RXRα expression vector (Fig. 4A). RXRα increased and LG1506 further increased the luciferase activities of the (-1845)-Luc and (-1155)-Luc constructs but not the (-998)-Luc construct (Fig. 4B). This finding suggests that a cis-acting element(s) for RXRα is located between -1155 bp and -998 bp in the IRS1 promoter. In contrast, 9cRA did not increase the luciferase activity further (data not shown). Next, we identified the partner of RXRα for binding to the IRS1 promoter region. Based on a report that LG1506 is a specific activator for the RXRα/PPAR heterodimer, we tested whether PPARα, PPARδ, and PPARγ, could increase the luciferase activity of the IRS1 (-1155)-Luc construct. Co-transfection of PPARδ expression vectors further increased luciferase activity, but PPARα and PPARγ vectors did not (Fig. 4C), indicating that the RXRα/PPARδ heterodimer binds to the promoter region of the IRS1 gene. To confirm that RXRα and PPARδ directly bind to the region between -1155 bp and -998 bp, ChIP was performed with RXRα, PPARδ, or RNA Pol II antibodies in C2C12 myotubes, and real-time PCR was performed using primers flanking the region (from -1155 bp to -998 bp). While bindings of RXRα, PPARδ, and RNA Pol II were decreased following rotenone treatment, bindings of these factors to the region were significantly increased when RXRα was overexpressed. These results indicated that IRS1 transcription was augmented by RXRα overexpression (Fig. 4D). Collectively, RXRα forms a heterodimer with PPARδ that directly binds to the IRS1 promoter region between -1155 bp and -998 bp and regulates IRS1 transcription level.

Bottom Line: RXRα overexpression also increased IRS1 transcription and mitochondrial function.Because RXRα overexpression, knock-down, or activation by LG1506 regulated IRS1 transcription mostly independently of mitochondrial function, it is likely that RXRα directly regulates IRS1 transcription.Consistent with the hypothesis, we showed that RXRα bound to the IRS1 promoter as a heterodimer with peroxisome proliferator-activated receptor δ (PPARδ).

View Article: PubMed Central - PubMed

Affiliation: Department of Internal Medicine, Seoul National University College of Medicine, Seoul 110-744, Korea.

ABSTRACT
Mitochondrial dysfunction is associated with insulin resistance and diabetes. We previously showed that retinoid X receptor α (RXRα) played an important role in transcriptional regulation of oxidative phosphorylation (OXPHOS) genes in cells with mitochondrial dysfunction caused by mitochondrial DNA mutation. In this study, we investigated whether mitochondrial dysfunction induced by incubation with OXPHOS inhibitors affects insulin receptor substrate 1 (IRS1) mRNA and protein levels and whether RXRα activation or overexpression can restore IRS1 expression. Both IRS1 and RXRα protein levels were significantly reduced when C2C12 myotubes were treated with the OXPHOS complex inhibitors, rotenone and antimycin A. The addition of RXRα agonists, 9-cis retinoic acid (9cRA) and LG1506, increased IRS1 transcription and protein levels and restored mitochondrial function, which ultimately improved insulin signaling. RXRα overexpression also increased IRS1 transcription and mitochondrial function. Because RXRα overexpression, knock-down, or activation by LG1506 regulated IRS1 transcription mostly independently of mitochondrial function, it is likely that RXRα directly regulates IRS1 transcription. Consistent with the hypothesis, we showed that RXRα bound to the IRS1 promoter as a heterodimer with peroxisome proliferator-activated receptor δ (PPARδ). These results suggest that RXRα overexpression or activation alleviates insulin resistance by increasing IRS1 expression.

No MeSH data available.


Related in: MedlinePlus