Limits...
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.

Show MeSH

Related in: MedlinePlus

IRS-2 signaling regulates FoxO1 and FoxO3a binding to the IRS-2 promoter IRE in β-cells. Isolated rat islets and INS-1 cells were examined for FoxO transcription factor expression association to the IRS-2 promoter’s IRE and nuclear translocation. A: Schematic representation of candidate transcription factors predicted to associate to the −546 to −593 region of the IRS-2 promoter around the IRE, E-box, and SREBP response element (SRE). B: Relative mRNA expression of the candidate transcription factors in isolated rat islets was assessed by qRT-PCR. A mean ± SEM (n ≥ 4) is shown. C and D: INS-1 cells were incubated at basal 3 mmol/L glucose for 6 h plus or minus insulin (100 nmol/L)/IGF-I (10 nmol/L) or PI3K inhibitor LY294002 (50 μmol/L) as indicated. ChIP assays for binding FoxO1, FoxO3a, SREBP-1, TFE3, or ARNT to the IRS-2 promoter IRE region were conducted (using rabbit IgG as a negative control) as described in research design and methods and analyzed by electrophoresis (C) or qRT-PCR (D). A mean ± SEM (n ≥ 3) is shown (D). The * indicates statistically significant difference (P ≤ 0.05). E: INS-1 cells were incubated at 3 mmol/L glucose for 6 h plus or minus insulin/IGF-I or LY294002 (50 μmol/L) and analyzed by immunofluorescence for FoxO1, FoxO3a, insulin, and DAPI to determine nuclear localization. Example images from three independent experiments are shown. (A high-quality digital representation of this figure is available in the online issue.)
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC3198101&req=5

Figure 3: IRS-2 signaling regulates FoxO1 and FoxO3a binding to the IRS-2 promoter IRE in β-cells. Isolated rat islets and INS-1 cells were examined for FoxO transcription factor expression association to the IRS-2 promoter’s IRE and nuclear translocation. A: Schematic representation of candidate transcription factors predicted to associate to the −546 to −593 region of the IRS-2 promoter around the IRE, E-box, and SREBP response element (SRE). B: Relative mRNA expression of the candidate transcription factors in isolated rat islets was assessed by qRT-PCR. A mean ± SEM (n ≥ 4) is shown. C and D: INS-1 cells were incubated at basal 3 mmol/L glucose for 6 h plus or minus insulin (100 nmol/L)/IGF-I (10 nmol/L) or PI3K inhibitor LY294002 (50 μmol/L) as indicated. ChIP assays for binding FoxO1, FoxO3a, SREBP-1, TFE3, or ARNT to the IRS-2 promoter IRE region were conducted (using rabbit IgG as a negative control) as described in research design and methods and analyzed by electrophoresis (C) or qRT-PCR (D). A mean ± SEM (n ≥ 3) is shown (D). The * indicates statistically significant difference (P ≤ 0.05). E: INS-1 cells were incubated at 3 mmol/L glucose for 6 h plus or minus insulin/IGF-I or LY294002 (50 μmol/L) and analyzed by immunofluorescence for FoxO1, FoxO3a, insulin, and DAPI to determine nuclear localization. Example images from three independent experiments are shown. (A high-quality digital representation of this figure is available in the online issue.)

Mentions: Several transcription factors were predicted to bind in the region of the IRE on the IRS-2 gene promoter in addition to FoxO transcription factors (using rVista, available from http://rvista.dcode.org/), including SREBP-1c and TFE3, as well as the ARNT that has been linked to type 2 diabetes (27) (Fig. 3A). Relative mRNA expression of these transcription factors was examined in rat islets by qRT-PCR that were present at comparable levels, with the exception of FoxO4, which was negligibly expressed (Fig. 3B). ChIP assays were performed in INS-1 cells incubated for 6 h at basal 3 mmol/L glucose alone, with the addition of insulin/IGF-I or LY294002 to examine which of these factors were capable of associating to the IRE of the IRS-2 promoter. Only FoxO1 and FoxO3a were significantly bound to the IRE above that of the control (rabbit IgG) under basal conditions (Fig. 3C). In contrast, ARNT, SREBP-1, and TFE3 were not found to associate with this region of the IRS-2 promoter in β-cells (Fig. 3C), despite doing so in hepatocytes (13–15,27) (Supplementary Fig. 2). The association of FoxO1 and FoxO3a to the IRE of the IRS-2 promoter was decreased by insulin/IGF-I and increased by LY294002 at basal 3 mmol/L glucose (Fig. 3C and D), in correlation with the transcriptional control of basal IRS-2 expression previously observed (Figs. 1 and 2). Quantification of a series of ChIP analyses indicated that insulin/IGF-I decreased FoxO1 and FoxO3a association to the IRS-2 promoter’s IRE by 75–80% (P ≤ 0.05), and LY294002 increased this FoxO1 and FoxO3a association threefold (P ≤ 0.05) (Fig. 3D). These data suggest that the IRS-2 transcriptional control in β-cells via an IRE in the IRS-2 promoter could be mediated by FoxO1 and/or FoxO3a, but unlike hepatocytes (14,15), SREBP-1 and TFE3 do not appear to be involved.


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)

IRS-2 signaling regulates FoxO1 and FoxO3a binding to the IRS-2 promoter IRE in β-cells. Isolated rat islets and INS-1 cells were examined for FoxO transcription factor expression association to the IRS-2 promoter’s IRE and nuclear translocation. A: Schematic representation of candidate transcription factors predicted to associate to the −546 to −593 region of the IRS-2 promoter around the IRE, E-box, and SREBP response element (SRE). B: Relative mRNA expression of the candidate transcription factors in isolated rat islets was assessed by qRT-PCR. A mean ± SEM (n ≥ 4) is shown. C and D: INS-1 cells were incubated at basal 3 mmol/L glucose for 6 h plus or minus insulin (100 nmol/L)/IGF-I (10 nmol/L) or PI3K inhibitor LY294002 (50 μmol/L) as indicated. ChIP assays for binding FoxO1, FoxO3a, SREBP-1, TFE3, or ARNT to the IRS-2 promoter IRE region were conducted (using rabbit IgG as a negative control) as described in research design and methods and analyzed by electrophoresis (C) or qRT-PCR (D). A mean ± SEM (n ≥ 3) is shown (D). The * indicates statistically significant difference (P ≤ 0.05). E: INS-1 cells were incubated at 3 mmol/L glucose for 6 h plus or minus insulin/IGF-I or LY294002 (50 μmol/L) and analyzed by immunofluorescence for FoxO1, FoxO3a, insulin, and DAPI to determine nuclear localization. Example images from three independent experiments are shown. (A high-quality digital representation of this figure is available in the online issue.)
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3198101&req=5

Figure 3: IRS-2 signaling regulates FoxO1 and FoxO3a binding to the IRS-2 promoter IRE in β-cells. Isolated rat islets and INS-1 cells were examined for FoxO transcription factor expression association to the IRS-2 promoter’s IRE and nuclear translocation. A: Schematic representation of candidate transcription factors predicted to associate to the −546 to −593 region of the IRS-2 promoter around the IRE, E-box, and SREBP response element (SRE). B: Relative mRNA expression of the candidate transcription factors in isolated rat islets was assessed by qRT-PCR. A mean ± SEM (n ≥ 4) is shown. C and D: INS-1 cells were incubated at basal 3 mmol/L glucose for 6 h plus or minus insulin (100 nmol/L)/IGF-I (10 nmol/L) or PI3K inhibitor LY294002 (50 μmol/L) as indicated. ChIP assays for binding FoxO1, FoxO3a, SREBP-1, TFE3, or ARNT to the IRS-2 promoter IRE region were conducted (using rabbit IgG as a negative control) as described in research design and methods and analyzed by electrophoresis (C) or qRT-PCR (D). A mean ± SEM (n ≥ 3) is shown (D). The * indicates statistically significant difference (P ≤ 0.05). E: INS-1 cells were incubated at 3 mmol/L glucose for 6 h plus or minus insulin/IGF-I or LY294002 (50 μmol/L) and analyzed by immunofluorescence for FoxO1, FoxO3a, insulin, and DAPI to determine nuclear localization. Example images from three independent experiments are shown. (A high-quality digital representation of this figure is available in the online issue.)
Mentions: Several transcription factors were predicted to bind in the region of the IRE on the IRS-2 gene promoter in addition to FoxO transcription factors (using rVista, available from http://rvista.dcode.org/), including SREBP-1c and TFE3, as well as the ARNT that has been linked to type 2 diabetes (27) (Fig. 3A). Relative mRNA expression of these transcription factors was examined in rat islets by qRT-PCR that were present at comparable levels, with the exception of FoxO4, which was negligibly expressed (Fig. 3B). ChIP assays were performed in INS-1 cells incubated for 6 h at basal 3 mmol/L glucose alone, with the addition of insulin/IGF-I or LY294002 to examine which of these factors were capable of associating to the IRE of the IRS-2 promoter. Only FoxO1 and FoxO3a were significantly bound to the IRE above that of the control (rabbit IgG) under basal conditions (Fig. 3C). In contrast, ARNT, SREBP-1, and TFE3 were not found to associate with this region of the IRS-2 promoter in β-cells (Fig. 3C), despite doing so in hepatocytes (13–15,27) (Supplementary Fig. 2). The association of FoxO1 and FoxO3a to the IRE of the IRS-2 promoter was decreased by insulin/IGF-I and increased by LY294002 at basal 3 mmol/L glucose (Fig. 3C and D), in correlation with the transcriptional control of basal IRS-2 expression previously observed (Figs. 1 and 2). Quantification of a series of ChIP analyses indicated that insulin/IGF-I decreased FoxO1 and FoxO3a association to the IRS-2 promoter’s IRE by 75–80% (P ≤ 0.05), and LY294002 increased this FoxO1 and FoxO3a association threefold (P ≤ 0.05) (Fig. 3D). These data suggest that the IRS-2 transcriptional control in β-cells via an IRE in the IRS-2 promoter could be mediated by FoxO1 and/or FoxO3a, but unlike hepatocytes (14,15), SREBP-1 and TFE3 do not appear to be involved.

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