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

Regulation of IRS-2 expression by IRS signaling in pancreatic islet β-cells. Isolated rat islets or INS-1 cells were incubated overnight at normal 5.6 mmol/L glucose and then incubated as indicated. Protein and mRNA expression of IRS-2 and PI3K p85 (control) were examined by immunoblot (IB) and qRT-PCR analyses, respectively, as outlined in research design and methods. A: Isolated rat islets or INS-1 cells, as indicated, were incubated at basal 3 mmol/L glucose for 6 h in the presence or absence of insulin (100 nmol/L) plus IGF-I (10 nmol/L) or the PI3K inhibitor LY294002 (50 μmol/L). An example IB is shown for protein analysis and the qRT-PCR mRNA analysis results are mean ± SEM (n ≥4). B: Isolated rat islets were incubated at basal 3 mmol/L or stimulatory 15 mmol/L glucose concentrations for 6 h in the presence or absence of LY294002 (50 μmol/L). Results of the qRT-PCR analysis are mean ± SEM (n ≥ 4) with an example IB shown. C: Isolated rat islets either uninfected or infected with adenoviruses expressing FLuc, WT, CA, or kinase-dead (KD) PKB-1 as indicated, then incubated for 6 h at either basal 3 mmol/L or stimulatory 15 mmol/L glucose. An example IB of three independent experiments is shown. The * indicates statistically significant difference (P ≤ 0.05).
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

Figure 1: Regulation of IRS-2 expression by IRS signaling in pancreatic islet β-cells. Isolated rat islets or INS-1 cells were incubated overnight at normal 5.6 mmol/L glucose and then incubated as indicated. Protein and mRNA expression of IRS-2 and PI3K p85 (control) were examined by immunoblot (IB) and qRT-PCR analyses, respectively, as outlined in research design and methods. A: Isolated rat islets or INS-1 cells, as indicated, were incubated at basal 3 mmol/L glucose for 6 h in the presence or absence of insulin (100 nmol/L) plus IGF-I (10 nmol/L) or the PI3K inhibitor LY294002 (50 μmol/L). An example IB is shown for protein analysis and the qRT-PCR mRNA analysis results are mean ± SEM (n ≥4). B: Isolated rat islets were incubated at basal 3 mmol/L or stimulatory 15 mmol/L glucose concentrations for 6 h in the presence or absence of LY294002 (50 μmol/L). Results of the qRT-PCR analysis are mean ± SEM (n ≥ 4) with an example IB shown. C: Isolated rat islets either uninfected or infected with adenoviruses expressing FLuc, WT, CA, or kinase-dead (KD) PKB-1 as indicated, then incubated for 6 h at either basal 3 mmol/L or stimulatory 15 mmol/L glucose. An example IB of three independent experiments is shown. The * indicates statistically significant difference (P ≤ 0.05).

Mentions: IRS-2 signaling is critical for β-cell survival and function (2,7), but the biologically relevant ligand/receptor interaction upstream of IRS-2 in β-cells in vivo remains unclear. It is contentious that this is an insulin/insulin receptor engagement on β-cells (21–25) and unlikely to be an autocrine feedback effect of secreted insulin (24,25). An IGF-I/IGF-I receptor interaction is also questionable (22,23,26). As such, the physiological means by which IRS-2 signal transduction is activated in primary β-cells in vivo remains an open question. Nonetheless, one can use the combination of a high concentration of insulin and IGF-I as an experimental tool to activate IRS-2 signaling in β-cells in vitro. Here, incubation of isolated rat islets and INS-1 cells with 100 nmol/L insulin plus 10 nmol/L IGF-I significantly decreased the expression of IRS-2 mRNA and protein, compared with that at basal 3 mmol/L glucose, by ∼40% over a 6-h period (P ≤ 0.05) (Fig. 1A). In contrast, inhibition of the PI3K/PKB branch of IRS signaling using the PI3K inhibitor LY294002 (50 µmol/L) significantly increased IRS-2 mRNA and protein expression from three- to fivefold in rat islets and INS-1 cells (P ≤ 0.01) (Fig. 1A). IRS-2 expression in β-cells is specifically regulated by glucose (10). However, the effect of PI3K inhibition on increasing IRS-2 expression was independent of glucose, with LY294002 significantly increasing IRS-2 mRNA and protein expression in rat islets at both basal 3 mmol/L and stimulatory 15 mmol/L glucose concentrations (Fig. 1B). Inhibition of the extracellular signal–related kinase 1/2 branch of IRS-2 signaling by inhibition of MEK1 with either 50 µmol/L PD98059 or 10 µmol/L U0126 or by inhibition of mammalian target of rapamycin with 10 nmol/L rapamycin had no effect on IRS-2 mRNA (data not shown) or protein levels in isolated rat islets (Supplementary Fig. 1). Downstream of PI3K, the negative regulation of IRS-2 expression in rat islets appeared to be mediated by PKB (Fig. 1C). Adenoviral-mediated expression of wild type (WT) PKB-1 in isolated rat islets specifically decreased IRS-2 protein expression at both basal 3 mmol/L and stimulatory 15 mmol/L glucose levels relative to that in uninfected islets or the FLuc adenoviral-infected control islets (Fig. 1C). Adenoviral-mediated expression of constitutively activated (CA) PKB-1 in islets further depleted IRS-2 protein expression. Conversely, introduction of a kinase-dead PKB-1 variant specifically augmented IRS-2 protein expression independently of the glucose concentration (Fig. 1C).


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)

Regulation of IRS-2 expression by IRS signaling in pancreatic islet β-cells. Isolated rat islets or INS-1 cells were incubated overnight at normal 5.6 mmol/L glucose and then incubated as indicated. Protein and mRNA expression of IRS-2 and PI3K p85 (control) were examined by immunoblot (IB) and qRT-PCR analyses, respectively, as outlined in research design and methods. A: Isolated rat islets or INS-1 cells, as indicated, were incubated at basal 3 mmol/L glucose for 6 h in the presence or absence of insulin (100 nmol/L) plus IGF-I (10 nmol/L) or the PI3K inhibitor LY294002 (50 μmol/L). An example IB is shown for protein analysis and the qRT-PCR mRNA analysis results are mean ± SEM (n ≥4). B: Isolated rat islets were incubated at basal 3 mmol/L or stimulatory 15 mmol/L glucose concentrations for 6 h in the presence or absence of LY294002 (50 μmol/L). Results of the qRT-PCR analysis are mean ± SEM (n ≥ 4) with an example IB shown. C: Isolated rat islets either uninfected or infected with adenoviruses expressing FLuc, WT, CA, or kinase-dead (KD) PKB-1 as indicated, then incubated for 6 h at either basal 3 mmol/L or stimulatory 15 mmol/L glucose. An example IB of three independent experiments is shown. The * indicates statistically significant difference (P ≤ 0.05).
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

Figure 1: Regulation of IRS-2 expression by IRS signaling in pancreatic islet β-cells. Isolated rat islets or INS-1 cells were incubated overnight at normal 5.6 mmol/L glucose and then incubated as indicated. Protein and mRNA expression of IRS-2 and PI3K p85 (control) were examined by immunoblot (IB) and qRT-PCR analyses, respectively, as outlined in research design and methods. A: Isolated rat islets or INS-1 cells, as indicated, were incubated at basal 3 mmol/L glucose for 6 h in the presence or absence of insulin (100 nmol/L) plus IGF-I (10 nmol/L) or the PI3K inhibitor LY294002 (50 μmol/L). An example IB is shown for protein analysis and the qRT-PCR mRNA analysis results are mean ± SEM (n ≥4). B: Isolated rat islets were incubated at basal 3 mmol/L or stimulatory 15 mmol/L glucose concentrations for 6 h in the presence or absence of LY294002 (50 μmol/L). Results of the qRT-PCR analysis are mean ± SEM (n ≥ 4) with an example IB shown. C: Isolated rat islets either uninfected or infected with adenoviruses expressing FLuc, WT, CA, or kinase-dead (KD) PKB-1 as indicated, then incubated for 6 h at either basal 3 mmol/L or stimulatory 15 mmol/L glucose. An example IB of three independent experiments is shown. The * indicates statistically significant difference (P ≤ 0.05).
Mentions: IRS-2 signaling is critical for β-cell survival and function (2,7), but the biologically relevant ligand/receptor interaction upstream of IRS-2 in β-cells in vivo remains unclear. It is contentious that this is an insulin/insulin receptor engagement on β-cells (21–25) and unlikely to be an autocrine feedback effect of secreted insulin (24,25). An IGF-I/IGF-I receptor interaction is also questionable (22,23,26). As such, the physiological means by which IRS-2 signal transduction is activated in primary β-cells in vivo remains an open question. Nonetheless, one can use the combination of a high concentration of insulin and IGF-I as an experimental tool to activate IRS-2 signaling in β-cells in vitro. Here, incubation of isolated rat islets and INS-1 cells with 100 nmol/L insulin plus 10 nmol/L IGF-I significantly decreased the expression of IRS-2 mRNA and protein, compared with that at basal 3 mmol/L glucose, by ∼40% over a 6-h period (P ≤ 0.05) (Fig. 1A). In contrast, inhibition of the PI3K/PKB branch of IRS signaling using the PI3K inhibitor LY294002 (50 µmol/L) significantly increased IRS-2 mRNA and protein expression from three- to fivefold in rat islets and INS-1 cells (P ≤ 0.01) (Fig. 1A). IRS-2 expression in β-cells is specifically regulated by glucose (10). However, the effect of PI3K inhibition on increasing IRS-2 expression was independent of glucose, with LY294002 significantly increasing IRS-2 mRNA and protein expression in rat islets at both basal 3 mmol/L and stimulatory 15 mmol/L glucose concentrations (Fig. 1B). Inhibition of the extracellular signal–related kinase 1/2 branch of IRS-2 signaling by inhibition of MEK1 with either 50 µmol/L PD98059 or 10 µmol/L U0126 or by inhibition of mammalian target of rapamycin with 10 nmol/L rapamycin had no effect on IRS-2 mRNA (data not shown) or protein levels in isolated rat islets (Supplementary Fig. 1). Downstream of PI3K, the negative regulation of IRS-2 expression in rat islets appeared to be mediated by PKB (Fig. 1C). Adenoviral-mediated expression of wild type (WT) PKB-1 in isolated rat islets specifically decreased IRS-2 protein expression at both basal 3 mmol/L and stimulatory 15 mmol/L glucose levels relative to that in uninfected islets or the FLuc adenoviral-infected control islets (Fig. 1C). Adenoviral-mediated expression of constitutively activated (CA) PKB-1 in islets further depleted IRS-2 protein expression. Conversely, introduction of a kinase-dead PKB-1 variant specifically augmented IRS-2 protein expression independently of the glucose concentration (Fig. 1C).

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