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FoxO feedback control of basal IRS-2 expression in pancreatic β-cells is distinct from that in hepatocytes.

Tsunekawa S, Demozay D, Briaud I, McCuaig J, Accili D, Stein R, Rhodes CJ - Diabetes (2011)

Bottom Line: In contrast, inhibition of phosphatidylinositol 3-kinase (PI3K) or PKB significantly increased IRS-2 levels in β-cells.ChIP assays indicated that transcription factors FoxO1 and FoxO3a associated with the IRE on the IRS-2 promoter in β-cells in a PI3K/PKB-dependent manner, whereas others, such as SREBP-1, the transcription factor binding to immunoglobulin heavy chain enhancer 3', and the aryl hydrocarbon receptor nuclear translocator (ARNT), did not.The molecular mechanism for feedback control of IRS signaling to decrease IRS-2 expression in liver and β-cells is quite distinct, with a predominant role played by FoxO3a in β-cells.

View Article: PubMed Central - PubMed

Affiliation: Kovler Diabetes Center, Department of Medicine, University of Chicago, Chicago, Illinois, USA.

ABSTRACT

Objective: Appropriate regulation of insulin receptor substrate 2 (IRS-2) expression in pancreatic β-cells is essential to adequately compensate for insulin resistance. In liver, basal IRS-2 expression is controlled via a temporal negative feedback of sterol regulatory element-binding protein 1 (SREBP-1) to antagonize transcription factors forkhead box class O (FoxO)1/FoxO3a at an insulin response element (IRE) on the IRS-2 promoter. The purpose of the study was to examine if a similar mechanism controlled IRS-2 expression in β-cells.

Research design and methods: IRS-2 mRNA and protein expression, as well as IRS-2 gene promoter activity, were examined in isolated rat islets. Specific transcription factor association with the IRE on the IRS-2 promoter was examined by chromatin immunoprecipitation (ChIP) assay, and their nuclear translocation was examined by immunofluorescence. A direct in vivo effect of insulin on control of IRS-2 expression in liver and pancreatic islets was also investigated.

Results: In IRS-2 promoter-reporter assays conducted in isolated islets, removal of the IRE decreased basal IRS-2 promoter activity in β-cells up to 80%. Activation of IRS signaling in isolated rat islets by insulin/IGF-I (used as an experimental in vitro tool) or downstream constitutive activation of protein kinase B (PKB) significantly decreased IRS-2 expression. In contrast, inhibition of phosphatidylinositol 3-kinase (PI3K) or PKB significantly increased IRS-2 levels in β-cells. ChIP assays indicated that transcription factors FoxO1 and FoxO3a associated with the IRE on the IRS-2 promoter in β-cells in a PI3K/PKB-dependent manner, whereas others, such as SREBP-1, the transcription factor binding to immunoglobulin heavy chain enhancer 3', and the aryl hydrocarbon receptor nuclear translocator (ARNT), did not. However, only FoxO3a, not FoxO1, was capable of driving IRS-2 promoter activity via the IRE in β-cells. In vivo studies showed insulin was able to suppress IRS-2 expression via activation of SREBP-1 in the liver, but this mechanism was not apparent in pancreatic islets from the same animal.

Conclusions: The molecular mechanism for feedback control of IRS signaling to decrease IRS-2 expression in liver and β-cells is quite distinct, with a predominant role played by FoxO3a in β-cells.

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The contribution of the IRS-2 promoter IRE to basal IRS-2 gene transcription. Isolated islets or INS-1 cells, as indicated, were infected with adenoviral vectors of either WT-IRS-2 promoter reporter (AdV-WT-IRS-2-FLuc) or IRE-mutated IRS-2 promoter reporter (AdV-Mut-IRS-2-FLuc) as indicated, together with the control TK promoter reporter (AdV-TK-RLuc). IRS-2 promoter activity was assessed as described in research design and methods. Results of relative luciferase activities are mean ± SEM (n ≥ 4). A: IRS-2 promoter activity in isolated islet preparations from different species and INS-1 cells incubated for 6 h at basal 3 mmol/L glucose. B: IRS-2 promoter activity in isolated rat islets incubated at basal 3 mmol/L glucose plus or minus insulin (100 nmol/L)/IGF-I (10 nmol/L) or the PI3K inhibitor LY294002 (50 μmol/L). The * indicates statistically significant difference (P ≤ 0.05).
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Figure 2: The contribution of the IRS-2 promoter IRE to basal IRS-2 gene transcription. Isolated islets or INS-1 cells, as indicated, were infected with adenoviral vectors of either WT-IRS-2 promoter reporter (AdV-WT-IRS-2-FLuc) or IRE-mutated IRS-2 promoter reporter (AdV-Mut-IRS-2-FLuc) as indicated, together with the control TK promoter reporter (AdV-TK-RLuc). IRS-2 promoter activity was assessed as described in research design and methods. Results of relative luciferase activities are mean ± SEM (n ≥ 4). A: IRS-2 promoter activity in isolated islet preparations from different species and INS-1 cells incubated for 6 h at basal 3 mmol/L glucose. B: IRS-2 promoter activity in isolated rat islets incubated at basal 3 mmol/L glucose plus or minus insulin (100 nmol/L)/IGF-I (10 nmol/L) or the PI3K inhibitor LY294002 (50 μmol/L). The * indicates statistically significant difference (P ≤ 0.05).

Mentions: In hepatocytes, basal regulation of IRS-2 expression is mediated via an IRE in the IRS-2 gene promoter that is a conserved FoxO1 binding site at −574 to −568 in the human IRS-2 promoter where +1 is the “A” of the ATG start codon (13). We generated recombinant adenoviruses where a FLuc reporter was driven by the WT human (−1051 to −116) IRS-2 promoter (AdV-WT-IRS2-FLuc) or where the IRE had been mutated (from 5′-TGTTTTG-3′ to 5′-AGATCTG-3′; AdV-Mut-IRS2-FLuc). Isolated human, mouse, or rat islets, and INS-1 cells, were infected with either AdV-WT-IRS2-FLuc or AdV-Mut-IRS2-FLuc, together with the control reporter adenovirus (AdV-TK-RLuc). Adenovirally infected islets/INS-1 cells were then incubated for 6 h at basal 3 mmol/L glucose, and the IRS-2 promoter–driven FLuc activity was measured relative to control RLuc reporter activity. Mutation of the IRE specifically decreased IRS-2 gene promoter activity by 60–80% in isolated human, rat, or mouse islets (P ≤ 0.01) and by ∼75% in INS-1 cells (P < 0.01) (Fig. 2A). These data indicate the importance of the IRE in the IRS-2 promoter for driving basal IRS-2 gene transcription.


FoxO feedback control of basal IRS-2 expression in pancreatic β-cells is distinct from that in hepatocytes.

Tsunekawa S, Demozay D, Briaud I, McCuaig J, Accili D, Stein R, Rhodes CJ - Diabetes (2011)

The contribution of the IRS-2 promoter IRE to basal IRS-2 gene transcription. Isolated islets or INS-1 cells, as indicated, were infected with adenoviral vectors of either WT-IRS-2 promoter reporter (AdV-WT-IRS-2-FLuc) or IRE-mutated IRS-2 promoter reporter (AdV-Mut-IRS-2-FLuc) as indicated, together with the control TK promoter reporter (AdV-TK-RLuc). IRS-2 promoter activity was assessed as described in research design and methods. Results of relative luciferase activities are mean ± SEM (n ≥ 4). A: IRS-2 promoter activity in isolated islet preparations from different species and INS-1 cells incubated for 6 h at basal 3 mmol/L glucose. B: IRS-2 promoter activity in isolated rat islets incubated at basal 3 mmol/L glucose plus or minus insulin (100 nmol/L)/IGF-I (10 nmol/L) or the PI3K inhibitor LY294002 (50 μmol/L). The * indicates statistically significant difference (P ≤ 0.05).
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Figure 2: The contribution of the IRS-2 promoter IRE to basal IRS-2 gene transcription. Isolated islets or INS-1 cells, as indicated, were infected with adenoviral vectors of either WT-IRS-2 promoter reporter (AdV-WT-IRS-2-FLuc) or IRE-mutated IRS-2 promoter reporter (AdV-Mut-IRS-2-FLuc) as indicated, together with the control TK promoter reporter (AdV-TK-RLuc). IRS-2 promoter activity was assessed as described in research design and methods. Results of relative luciferase activities are mean ± SEM (n ≥ 4). A: IRS-2 promoter activity in isolated islet preparations from different species and INS-1 cells incubated for 6 h at basal 3 mmol/L glucose. B: IRS-2 promoter activity in isolated rat islets incubated at basal 3 mmol/L glucose plus or minus insulin (100 nmol/L)/IGF-I (10 nmol/L) or the PI3K inhibitor LY294002 (50 μmol/L). The * indicates statistically significant difference (P ≤ 0.05).
Mentions: In hepatocytes, basal regulation of IRS-2 expression is mediated via an IRE in the IRS-2 gene promoter that is a conserved FoxO1 binding site at −574 to −568 in the human IRS-2 promoter where +1 is the “A” of the ATG start codon (13). We generated recombinant adenoviruses where a FLuc reporter was driven by the WT human (−1051 to −116) IRS-2 promoter (AdV-WT-IRS2-FLuc) or where the IRE had been mutated (from 5′-TGTTTTG-3′ to 5′-AGATCTG-3′; AdV-Mut-IRS2-FLuc). Isolated human, mouse, or rat islets, and INS-1 cells, were infected with either AdV-WT-IRS2-FLuc or AdV-Mut-IRS2-FLuc, together with the control reporter adenovirus (AdV-TK-RLuc). Adenovirally infected islets/INS-1 cells were then incubated for 6 h at basal 3 mmol/L glucose, and the IRS-2 promoter–driven FLuc activity was measured relative to control RLuc reporter activity. Mutation of the IRE specifically decreased IRS-2 gene promoter activity by 60–80% in isolated human, rat, or mouse islets (P ≤ 0.01) and by ∼75% in INS-1 cells (P < 0.01) (Fig. 2A). These data indicate the importance of the IRE in the IRS-2 promoter for driving basal IRS-2 gene transcription.

Bottom Line: In contrast, inhibition of phosphatidylinositol 3-kinase (PI3K) or PKB significantly increased IRS-2 levels in β-cells.ChIP assays indicated that transcription factors FoxO1 and FoxO3a associated with the IRE on the IRS-2 promoter in β-cells in a PI3K/PKB-dependent manner, whereas others, such as SREBP-1, the transcription factor binding to immunoglobulin heavy chain enhancer 3', and the aryl hydrocarbon receptor nuclear translocator (ARNT), did not.The molecular mechanism for feedback control of IRS signaling to decrease IRS-2 expression in liver and β-cells is quite distinct, with a predominant role played by FoxO3a in β-cells.

View Article: PubMed Central - PubMed

Affiliation: Kovler Diabetes Center, Department of Medicine, University of Chicago, Chicago, Illinois, USA.

ABSTRACT

Objective: Appropriate regulation of insulin receptor substrate 2 (IRS-2) expression in pancreatic β-cells is essential to adequately compensate for insulin resistance. In liver, basal IRS-2 expression is controlled via a temporal negative feedback of sterol regulatory element-binding protein 1 (SREBP-1) to antagonize transcription factors forkhead box class O (FoxO)1/FoxO3a at an insulin response element (IRE) on the IRS-2 promoter. The purpose of the study was to examine if a similar mechanism controlled IRS-2 expression in β-cells.

Research design and methods: IRS-2 mRNA and protein expression, as well as IRS-2 gene promoter activity, were examined in isolated rat islets. Specific transcription factor association with the IRE on the IRS-2 promoter was examined by chromatin immunoprecipitation (ChIP) assay, and their nuclear translocation was examined by immunofluorescence. A direct in vivo effect of insulin on control of IRS-2 expression in liver and pancreatic islets was also investigated.

Results: In IRS-2 promoter-reporter assays conducted in isolated islets, removal of the IRE decreased basal IRS-2 promoter activity in β-cells up to 80%. Activation of IRS signaling in isolated rat islets by insulin/IGF-I (used as an experimental in vitro tool) or downstream constitutive activation of protein kinase B (PKB) significantly decreased IRS-2 expression. In contrast, inhibition of phosphatidylinositol 3-kinase (PI3K) or PKB significantly increased IRS-2 levels in β-cells. ChIP assays indicated that transcription factors FoxO1 and FoxO3a associated with the IRE on the IRS-2 promoter in β-cells in a PI3K/PKB-dependent manner, whereas others, such as SREBP-1, the transcription factor binding to immunoglobulin heavy chain enhancer 3', and the aryl hydrocarbon receptor nuclear translocator (ARNT), did not. However, only FoxO3a, not FoxO1, was capable of driving IRS-2 promoter activity via the IRE in β-cells. In vivo studies showed insulin was able to suppress IRS-2 expression via activation of SREBP-1 in the liver, but this mechanism was not apparent in pancreatic islets from the same animal.

Conclusions: The molecular mechanism for feedback control of IRS signaling to decrease IRS-2 expression in liver and β-cells is quite distinct, with a predominant role played by FoxO3a in β-cells.

Show MeSH
Related in: MedlinePlus