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Modulation of β-catenin signaling by glucagon receptor activation.

Ke J, Zhang C, Harikumar KG, Zylstra-Diegel CR, Wang L, Mowry LE, Miller LJ, Williams BO, Xu HE - PLoS ONE (2012)

Bottom Line: Thus, glucagon is a key component of glucose homeostasis by counteracting the effect of insulin.Since low-density-lipoprotein receptor-related protein 5 (Lrp5) is an essential co-receptor required for Wnt protein mediated β-catenin signaling, we examined the role of Lrp5 in glucagon-induced β-catenin signaling.Furthermore, we showed that Lrp5 physically interacted with GCGR by immunoprecipitation and bioluminescence resonance energy transfer assays.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Structural Sciences, Van Andel Research Institute, Grand Rapids, Michigan, United States of America. jiyuan.ke@vai.org

ABSTRACT
The glucagon receptor (GCGR) is a member of the class B G protein-coupled receptor family. Activation of GCGR by glucagon leads to increased glucose production by the liver. Thus, glucagon is a key component of glucose homeostasis by counteracting the effect of insulin. In this report, we found that in addition to activation of the classic cAMP/protein kinase A (PKA) pathway, activation of GCGR also induced β-catenin stabilization and activated β-catenin-mediated transcription. Activation of β-catenin signaling was PKA-dependent, consistent with previous reports on the parathyroid hormone receptor type 1 (PTH1R) and glucagon-like peptide 1 (GLP-1R) receptors. Since low-density-lipoprotein receptor-related protein 5 (Lrp5) is an essential co-receptor required for Wnt protein mediated β-catenin signaling, we examined the role of Lrp5 in glucagon-induced β-catenin signaling. Cotransfection with Lrp5 enhanced the glucagon-induced β-catenin stabilization and TCF promoter-mediated transcription. Inhibiting Lrp5/6 function using Dickkopf-1(DKK1) or by expression of the Lrp5 extracellular domain blocked glucagon-induced β-catenin signaling. Furthermore, we showed that Lrp5 physically interacted with GCGR by immunoprecipitation and bioluminescence resonance energy transfer assays. Together, these results reveal an unexpected crosstalk between glucagon and β-catenin signaling, and may help to explain the metabolic phenotypes of Lrp5/6 mutations.

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Lrp5 coexpression enhances glucagon and GLP1-induced β-catenin signaling.A). HEK293 cells cultured in 12-well plate were transfected with a combination of indicated plasmids (GCGR 1000 ng, Lrp5 500 ng) for 24 h and then treated with or without 50 nM GCG1-29 for 1 h. The cells were harvested and lysed, and samples were used for western blot analysis. The blot was first probed with anti-β-catenin antibody and then stripped and reprobed for anti-β-actin antibody as a loading control. B). 293STF cells cultured in 24-well plate were transfected with 100 ng of empty vector (pcDNA3.1) or GCGR plasmid along with the indicated amount of Lrp5 and 5 ng TKRlu plasmids on day 1, and then treated with or without 50 nM GCG1-29 on day 2. Cells were harvested for luciferase activity measurement on day 3. Triplicate samples were used for each treatment. *p<0.005 compared with non-treated group. 4C). 293STF cells cultured in 24-well plate were transfected with 100 ng GLP1R, 100 ng Lrp5 and 5 ng TKRlu plasmids on day 1 and then treated with the GLP1 agonist GLP1(7–36) (50 nM) or the antagonist Exendin(9–39) (50 nM) on day 2. Cells were harvested on day 3 to measure luciferase activity. Duplicate samples were used for each treatment. *p<0.05 compared with the non-treated (NT) group.
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pone-0033676-g004: Lrp5 coexpression enhances glucagon and GLP1-induced β-catenin signaling.A). HEK293 cells cultured in 12-well plate were transfected with a combination of indicated plasmids (GCGR 1000 ng, Lrp5 500 ng) for 24 h and then treated with or without 50 nM GCG1-29 for 1 h. The cells were harvested and lysed, and samples were used for western blot analysis. The blot was first probed with anti-β-catenin antibody and then stripped and reprobed for anti-β-actin antibody as a loading control. B). 293STF cells cultured in 24-well plate were transfected with 100 ng of empty vector (pcDNA3.1) or GCGR plasmid along with the indicated amount of Lrp5 and 5 ng TKRlu plasmids on day 1, and then treated with or without 50 nM GCG1-29 on day 2. Cells were harvested for luciferase activity measurement on day 3. Triplicate samples were used for each treatment. *p<0.005 compared with non-treated group. 4C). 293STF cells cultured in 24-well plate were transfected with 100 ng GLP1R, 100 ng Lrp5 and 5 ng TKRlu plasmids on day 1 and then treated with the GLP1 agonist GLP1(7–36) (50 nM) or the antagonist Exendin(9–39) (50 nM) on day 2. Cells were harvested on day 3 to measure luciferase activity. Duplicate samples were used for each treatment. *p<0.05 compared with the non-treated (NT) group.

Mentions: We observed an increase in β-catenin protein level and TCF-mediated luciferase activity upon activation of GCGR. Because Lrp5/6 is an essential coreceptor for Wnt/β-catenin signaling, we asked whether cotransfection with Lrp5/6 will potentiate glucagon-induced β-catenin signaling. HEK293 cells transfected with GCGR had a modest increase in β-catenin protein level upon GCG1-29 treatment, whereas cotransfection with GCGR and Lrp5 caused a larger increase (Fig. 4A). Next, we examined the effect of coexpression of GCGR and Lrp5 on glucagon-induced TCF luciferase activity. As expected, we observed a larger increase (2 to 3-fold vs 1.5-fold) in glucagon-induced TCF luciferase activity in HEK293 cells cotransfected with GCGR and Lrp5, relative to cells transfected with GCGR alone (Fig. 4B & 3A). As a control, transfection with high dose of Lrp5 alone also caused a small increase in TCF luciferase activity, which was not responsive to GCG1-29 treatment (Fig. 4B). Similar results were obtained when HEK293 cells were cotransfected with GCGR and Lrp6 plasmids (data not shown). Considering the report that GLP-1 also induced β-catenin signaling in GLP1R expressing cells [13], we examined the role of Lrp5 in GLP-1-induced β-catenin signaling. We found that cotransfection of HEK293 cells with GLP-1R and Lrp5 increased GLP1 agonist induced TCF luciferase activity to a similar level as observed for cotransfection of HEK293 cells with GCGR and Lrp5 (Fig. 4C). This is consistent with the hypothesis that there is a common mechanism for activation of the β-catenin signaling pathway through both GCGR and GLP1R receptors.


Modulation of β-catenin signaling by glucagon receptor activation.

Ke J, Zhang C, Harikumar KG, Zylstra-Diegel CR, Wang L, Mowry LE, Miller LJ, Williams BO, Xu HE - PLoS ONE (2012)

Lrp5 coexpression enhances glucagon and GLP1-induced β-catenin signaling.A). HEK293 cells cultured in 12-well plate were transfected with a combination of indicated plasmids (GCGR 1000 ng, Lrp5 500 ng) for 24 h and then treated with or without 50 nM GCG1-29 for 1 h. The cells were harvested and lysed, and samples were used for western blot analysis. The blot was first probed with anti-β-catenin antibody and then stripped and reprobed for anti-β-actin antibody as a loading control. B). 293STF cells cultured in 24-well plate were transfected with 100 ng of empty vector (pcDNA3.1) or GCGR plasmid along with the indicated amount of Lrp5 and 5 ng TKRlu plasmids on day 1, and then treated with or without 50 nM GCG1-29 on day 2. Cells were harvested for luciferase activity measurement on day 3. Triplicate samples were used for each treatment. *p<0.005 compared with non-treated group. 4C). 293STF cells cultured in 24-well plate were transfected with 100 ng GLP1R, 100 ng Lrp5 and 5 ng TKRlu plasmids on day 1 and then treated with the GLP1 agonist GLP1(7–36) (50 nM) or the antagonist Exendin(9–39) (50 nM) on day 2. Cells were harvested on day 3 to measure luciferase activity. Duplicate samples were used for each treatment. *p<0.05 compared with the non-treated (NT) group.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3306284&req=5

pone-0033676-g004: Lrp5 coexpression enhances glucagon and GLP1-induced β-catenin signaling.A). HEK293 cells cultured in 12-well plate were transfected with a combination of indicated plasmids (GCGR 1000 ng, Lrp5 500 ng) for 24 h and then treated with or without 50 nM GCG1-29 for 1 h. The cells were harvested and lysed, and samples were used for western blot analysis. The blot was first probed with anti-β-catenin antibody and then stripped and reprobed for anti-β-actin antibody as a loading control. B). 293STF cells cultured in 24-well plate were transfected with 100 ng of empty vector (pcDNA3.1) or GCGR plasmid along with the indicated amount of Lrp5 and 5 ng TKRlu plasmids on day 1, and then treated with or without 50 nM GCG1-29 on day 2. Cells were harvested for luciferase activity measurement on day 3. Triplicate samples were used for each treatment. *p<0.005 compared with non-treated group. 4C). 293STF cells cultured in 24-well plate were transfected with 100 ng GLP1R, 100 ng Lrp5 and 5 ng TKRlu plasmids on day 1 and then treated with the GLP1 agonist GLP1(7–36) (50 nM) or the antagonist Exendin(9–39) (50 nM) on day 2. Cells were harvested on day 3 to measure luciferase activity. Duplicate samples were used for each treatment. *p<0.05 compared with the non-treated (NT) group.
Mentions: We observed an increase in β-catenin protein level and TCF-mediated luciferase activity upon activation of GCGR. Because Lrp5/6 is an essential coreceptor for Wnt/β-catenin signaling, we asked whether cotransfection with Lrp5/6 will potentiate glucagon-induced β-catenin signaling. HEK293 cells transfected with GCGR had a modest increase in β-catenin protein level upon GCG1-29 treatment, whereas cotransfection with GCGR and Lrp5 caused a larger increase (Fig. 4A). Next, we examined the effect of coexpression of GCGR and Lrp5 on glucagon-induced TCF luciferase activity. As expected, we observed a larger increase (2 to 3-fold vs 1.5-fold) in glucagon-induced TCF luciferase activity in HEK293 cells cotransfected with GCGR and Lrp5, relative to cells transfected with GCGR alone (Fig. 4B & 3A). As a control, transfection with high dose of Lrp5 alone also caused a small increase in TCF luciferase activity, which was not responsive to GCG1-29 treatment (Fig. 4B). Similar results were obtained when HEK293 cells were cotransfected with GCGR and Lrp6 plasmids (data not shown). Considering the report that GLP-1 also induced β-catenin signaling in GLP1R expressing cells [13], we examined the role of Lrp5 in GLP-1-induced β-catenin signaling. We found that cotransfection of HEK293 cells with GLP-1R and Lrp5 increased GLP1 agonist induced TCF luciferase activity to a similar level as observed for cotransfection of HEK293 cells with GCGR and Lrp5 (Fig. 4C). This is consistent with the hypothesis that there is a common mechanism for activation of the β-catenin signaling pathway through both GCGR and GLP1R receptors.

Bottom Line: Thus, glucagon is a key component of glucose homeostasis by counteracting the effect of insulin.Since low-density-lipoprotein receptor-related protein 5 (Lrp5) is an essential co-receptor required for Wnt protein mediated β-catenin signaling, we examined the role of Lrp5 in glucagon-induced β-catenin signaling.Furthermore, we showed that Lrp5 physically interacted with GCGR by immunoprecipitation and bioluminescence resonance energy transfer assays.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Structural Sciences, Van Andel Research Institute, Grand Rapids, Michigan, United States of America. jiyuan.ke@vai.org

ABSTRACT
The glucagon receptor (GCGR) is a member of the class B G protein-coupled receptor family. Activation of GCGR by glucagon leads to increased glucose production by the liver. Thus, glucagon is a key component of glucose homeostasis by counteracting the effect of insulin. In this report, we found that in addition to activation of the classic cAMP/protein kinase A (PKA) pathway, activation of GCGR also induced β-catenin stabilization and activated β-catenin-mediated transcription. Activation of β-catenin signaling was PKA-dependent, consistent with previous reports on the parathyroid hormone receptor type 1 (PTH1R) and glucagon-like peptide 1 (GLP-1R) receptors. Since low-density-lipoprotein receptor-related protein 5 (Lrp5) is an essential co-receptor required for Wnt protein mediated β-catenin signaling, we examined the role of Lrp5 in glucagon-induced β-catenin signaling. Cotransfection with Lrp5 enhanced the glucagon-induced β-catenin stabilization and TCF promoter-mediated transcription. Inhibiting Lrp5/6 function using Dickkopf-1(DKK1) or by expression of the Lrp5 extracellular domain blocked glucagon-induced β-catenin signaling. Furthermore, we showed that Lrp5 physically interacted with GCGR by immunoprecipitation and bioluminescence resonance energy transfer assays. Together, these results reveal an unexpected crosstalk between glucagon and β-catenin signaling, and may help to explain the metabolic phenotypes of Lrp5/6 mutations.

Show MeSH
Related in: MedlinePlus