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Increased systemic glucose tolerance with increased muscle glucose uptake in transgenic mice overexpressing RXRγ in skeletal muscle.

Sugita S, Kamei Y, Akaike F, Suganami T, Kanai S, Hattori M, Manabe Y, Fujii N, Takai-Igarashi T, Tadaishi M, Oka J, Aburatani H, Yamada T, Katagiri H, Kakehi S, Tamura Y, Kubo H, Nishida K, Miura S, Ezaki O, Ogawa Y - PLoS ONE (2011)

Bottom Line: Combination of RXRγ and PPARδ resulted in an increase in Glut1-Luc activity in skeletal muscle in vivo.These results show the importance of skeletal muscle gene regulation in systemic glucose metabolism.Increasing RXRγ expression may be a novel therapeutic strategy against type 2 diabetes.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Medicine and Metabolism, Medical Research Institute, Tokyo Medical and Dental University, Tokyo, Japan.

ABSTRACT

Background: Retinoid X receptor (RXR) γ is a nuclear receptor-type transcription factor expressed mostly in skeletal muscle, and regulated by nutritional conditions. Previously, we established transgenic mice overexpressing RXRγ in skeletal muscle (RXRγ mice), which showed lower blood glucose than the control mice. Here we investigated their glucose metabolism.

Methodology/principal findings: RXRγ mice were subjected to glucose and insulin tolerance tests, and glucose transporter expression levels, hyperinsulinemic-euglycemic clamp and glucose uptake were analyzed. Microarray and bioinformatics analyses were done. The glucose tolerance test revealed higher glucose disposal in RXRγ mice than in control mice, but insulin tolerance test revealed no difference in the insulin-induced hypoglycemic response. In the hyperinsulinemic-euglycemic clamp study, the basal glucose disposal rate was higher in RXRγ mice than in control mice, indicating an insulin-independent increase in glucose uptake. There was no difference in the rate of glucose infusion needed to maintain euglycemia (glucose infusion rate) between the RXRγ and control mice, which is consistent with the result of the insulin tolerance test. Skeletal muscle from RXRγ mice showed increased Glut1 expression, with increased glucose uptake, in an insulin-independent manner. Moreover, we performed in vivo luciferase reporter analysis using Glut1 promoter (Glut1-Luc). Combination of RXRγ and PPARδ resulted in an increase in Glut1-Luc activity in skeletal muscle in vivo. Microarray data showed that RXRγ overexpression increased a diverse set of genes, including glucose metabolism genes, whose promoter contained putative PPAR-binding motifs.

Conclusions/significance: Systemic glucose metabolism was increased in transgenic mice overexpressing RXRγ. The enhanced glucose tolerance in RXRγ mice may be mediated at least in part by increased Glut1 in skeletal muscle. These results show the importance of skeletal muscle gene regulation in systemic glucose metabolism. Increasing RXRγ expression may be a novel therapeutic strategy against type 2 diabetes.

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Transient transfection-reporter assay of the effect of RXRγ on                                Glut1 promoter.(A) Glut1-Luc plasmid, with or without                            RXRγ and/or PPARδ expression vectors, was transfected into the                            quadriceps muscle of C57BL6 mice. Activation of the luciferase reporter                            gene was measured in relative light units and normalized to dual                            luciferase activity. Mean values from experiments                            (n = 5) are shown as fold induction, where the                            activity in the absence of RXRγ is the reference value (set at 100).                                (B) Schematic representations of serial deletion of                                Glut1 promoter constructs are shown in the figure.                            Squares denote the putative PPAR/RXR binding sites. Open bars;                                Glut1-Luc without RXRγ and PPARδ expression                            vectors, and filled bars; Glut1-Luc with RXRγ and                            PPARδ expression vectors. The activity in the absence of RXRγ                            and PPARδ in each experiment for different                            Glut1-Luc construct in the reference value (set at                            100). ** P<0.01, compared with the value of                            wild-type promoter in the absence of RXRγ/PPARδ.
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pone-0020467-g004: Transient transfection-reporter assay of the effect of RXRγ on Glut1 promoter.(A) Glut1-Luc plasmid, with or without RXRγ and/or PPARδ expression vectors, was transfected into the quadriceps muscle of C57BL6 mice. Activation of the luciferase reporter gene was measured in relative light units and normalized to dual luciferase activity. Mean values from experiments (n = 5) are shown as fold induction, where the activity in the absence of RXRγ is the reference value (set at 100). (B) Schematic representations of serial deletion of Glut1 promoter constructs are shown in the figure. Squares denote the putative PPAR/RXR binding sites. Open bars; Glut1-Luc without RXRγ and PPARδ expression vectors, and filled bars; Glut1-Luc with RXRγ and PPARδ expression vectors. The activity in the absence of RXRγ and PPARδ in each experiment for different Glut1-Luc construct in the reference value (set at 100). ** P<0.01, compared with the value of wild-type promoter in the absence of RXRγ/PPARδ.

Mentions: To examine whether RXRγ directly activates mRNA expression of Glut1, we performed the in vivo luciferase reporter analysis using Glut1 promoter (Glut1-Luc). In this study, the activity of Glut1-Luc was marginally enhanced by RXRγ alone (Fig. 4A). Moreover, we found no significant activation of the Glut1-Luc by PPARδ, an important regulator of glucose as well as lipid metabolism in the skeletal muscle [7]. Interestingly, combination of RXRγ and PPARδ resulted in significant increase in Glut1-Luc activity in the skeletal muscle in vivo (P<0.01) (Fig. 4A). Motif search analysis revealed two putative PPAR-responsive elements (PPRE1 and PPRE2) (−657/−645 and −520/−508, respectively) in the mouse Glut1 promoter. A series of deletion mutant analysis showed that combination of RXRγ and PPARδ activates the regions of −1500/+75, and −647/+75, but not the region of −152/+75 in the Glut1 promoter (Fig. 4B), suggesting that the second putative PPRE (−520/−508) is involved in the RXRγ/PPARδ-induced Glut1 promoter activation.


Increased systemic glucose tolerance with increased muscle glucose uptake in transgenic mice overexpressing RXRγ in skeletal muscle.

Sugita S, Kamei Y, Akaike F, Suganami T, Kanai S, Hattori M, Manabe Y, Fujii N, Takai-Igarashi T, Tadaishi M, Oka J, Aburatani H, Yamada T, Katagiri H, Kakehi S, Tamura Y, Kubo H, Nishida K, Miura S, Ezaki O, Ogawa Y - PLoS ONE (2011)

Transient transfection-reporter assay of the effect of RXRγ on                                Glut1 promoter.(A) Glut1-Luc plasmid, with or without                            RXRγ and/or PPARδ expression vectors, was transfected into the                            quadriceps muscle of C57BL6 mice. Activation of the luciferase reporter                            gene was measured in relative light units and normalized to dual                            luciferase activity. Mean values from experiments                            (n = 5) are shown as fold induction, where the                            activity in the absence of RXRγ is the reference value (set at 100).                                (B) Schematic representations of serial deletion of                                Glut1 promoter constructs are shown in the figure.                            Squares denote the putative PPAR/RXR binding sites. Open bars;                                Glut1-Luc without RXRγ and PPARδ expression                            vectors, and filled bars; Glut1-Luc with RXRγ and                            PPARδ expression vectors. The activity in the absence of RXRγ                            and PPARδ in each experiment for different                            Glut1-Luc construct in the reference value (set at                            100). ** P<0.01, compared with the value of                            wild-type promoter in the absence of RXRγ/PPARδ.
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Related In: Results  -  Collection

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pone-0020467-g004: Transient transfection-reporter assay of the effect of RXRγ on Glut1 promoter.(A) Glut1-Luc plasmid, with or without RXRγ and/or PPARδ expression vectors, was transfected into the quadriceps muscle of C57BL6 mice. Activation of the luciferase reporter gene was measured in relative light units and normalized to dual luciferase activity. Mean values from experiments (n = 5) are shown as fold induction, where the activity in the absence of RXRγ is the reference value (set at 100). (B) Schematic representations of serial deletion of Glut1 promoter constructs are shown in the figure. Squares denote the putative PPAR/RXR binding sites. Open bars; Glut1-Luc without RXRγ and PPARδ expression vectors, and filled bars; Glut1-Luc with RXRγ and PPARδ expression vectors. The activity in the absence of RXRγ and PPARδ in each experiment for different Glut1-Luc construct in the reference value (set at 100). ** P<0.01, compared with the value of wild-type promoter in the absence of RXRγ/PPARδ.
Mentions: To examine whether RXRγ directly activates mRNA expression of Glut1, we performed the in vivo luciferase reporter analysis using Glut1 promoter (Glut1-Luc). In this study, the activity of Glut1-Luc was marginally enhanced by RXRγ alone (Fig. 4A). Moreover, we found no significant activation of the Glut1-Luc by PPARδ, an important regulator of glucose as well as lipid metabolism in the skeletal muscle [7]. Interestingly, combination of RXRγ and PPARδ resulted in significant increase in Glut1-Luc activity in the skeletal muscle in vivo (P<0.01) (Fig. 4A). Motif search analysis revealed two putative PPAR-responsive elements (PPRE1 and PPRE2) (−657/−645 and −520/−508, respectively) in the mouse Glut1 promoter. A series of deletion mutant analysis showed that combination of RXRγ and PPARδ activates the regions of −1500/+75, and −647/+75, but not the region of −152/+75 in the Glut1 promoter (Fig. 4B), suggesting that the second putative PPRE (−520/−508) is involved in the RXRγ/PPARδ-induced Glut1 promoter activation.

Bottom Line: Combination of RXRγ and PPARδ resulted in an increase in Glut1-Luc activity in skeletal muscle in vivo.These results show the importance of skeletal muscle gene regulation in systemic glucose metabolism.Increasing RXRγ expression may be a novel therapeutic strategy against type 2 diabetes.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Medicine and Metabolism, Medical Research Institute, Tokyo Medical and Dental University, Tokyo, Japan.

ABSTRACT

Background: Retinoid X receptor (RXR) γ is a nuclear receptor-type transcription factor expressed mostly in skeletal muscle, and regulated by nutritional conditions. Previously, we established transgenic mice overexpressing RXRγ in skeletal muscle (RXRγ mice), which showed lower blood glucose than the control mice. Here we investigated their glucose metabolism.

Methodology/principal findings: RXRγ mice were subjected to glucose and insulin tolerance tests, and glucose transporter expression levels, hyperinsulinemic-euglycemic clamp and glucose uptake were analyzed. Microarray and bioinformatics analyses were done. The glucose tolerance test revealed higher glucose disposal in RXRγ mice than in control mice, but insulin tolerance test revealed no difference in the insulin-induced hypoglycemic response. In the hyperinsulinemic-euglycemic clamp study, the basal glucose disposal rate was higher in RXRγ mice than in control mice, indicating an insulin-independent increase in glucose uptake. There was no difference in the rate of glucose infusion needed to maintain euglycemia (glucose infusion rate) between the RXRγ and control mice, which is consistent with the result of the insulin tolerance test. Skeletal muscle from RXRγ mice showed increased Glut1 expression, with increased glucose uptake, in an insulin-independent manner. Moreover, we performed in vivo luciferase reporter analysis using Glut1 promoter (Glut1-Luc). Combination of RXRγ and PPARδ resulted in an increase in Glut1-Luc activity in skeletal muscle in vivo. Microarray data showed that RXRγ overexpression increased a diverse set of genes, including glucose metabolism genes, whose promoter contained putative PPAR-binding motifs.

Conclusions/significance: Systemic glucose metabolism was increased in transgenic mice overexpressing RXRγ. The enhanced glucose tolerance in RXRγ mice may be mediated at least in part by increased Glut1 in skeletal muscle. These results show the importance of skeletal muscle gene regulation in systemic glucose metabolism. Increasing RXRγ expression may be a novel therapeutic strategy against type 2 diabetes.

Show MeSH
Related in: MedlinePlus