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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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Related in: MedlinePlus

FoxO3a predominately drives basal IRS-2 expression in β-cells. Adenovirally infected isolated rat islets were incubated as indicated, where “+” represents an equivalent dose of each adenoviral vector. The rat islet mRNA (A and C) and protein (B and D) levels of IRS-2 and PI3K p85 (control) were examined by qRT-PCR and immunoblot (IB) analyses, respectively, as described in research design and methods. The qRT-PCR data are shown as mean ± SEM (n ≥ 4), and an example immunoblot for protein analysis is shown. A and B: Isolated rat islets were infected with adenoviruses expressing GFP (control), FoxO1-WT, FoxO3a-WT, or in combination as indicated at basal 3 mmol/L glucose for 6 h. C and D: Isolated rat islets were infected with adenoviruses expressing GFP, FoxO1-CA, FoxO3a-CA, or in combination as indicated at basal 3 mmol/L glucose for 6 h plus or minus insulin (100 nmol/L)/IGF-I (10 nmol/L). The * indicates statistically significant difference (P ≤ 0.05).
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Figure 4: FoxO3a predominately drives basal IRS-2 expression in β-cells. Adenovirally infected isolated rat islets were incubated as indicated, where “+” represents an equivalent dose of each adenoviral vector. The rat islet mRNA (A and C) and protein (B and D) levels of IRS-2 and PI3K p85 (control) were examined by qRT-PCR and immunoblot (IB) analyses, respectively, as described in research design and methods. The qRT-PCR data are shown as mean ± SEM (n ≥ 4), and an example immunoblot for protein analysis is shown. A and B: Isolated rat islets were infected with adenoviruses expressing GFP (control), FoxO1-WT, FoxO3a-WT, or in combination as indicated at basal 3 mmol/L glucose for 6 h. C and D: Isolated rat islets were infected with adenoviruses expressing GFP, FoxO1-CA, FoxO3a-CA, or in combination as indicated at basal 3 mmol/L glucose for 6 h plus or minus insulin (100 nmol/L)/IGF-I (10 nmol/L). The * indicates statistically significant difference (P ≤ 0.05).

Mentions: We examined if there was any difference between FoxO1 and FoxO3a in driving IRS-2 gene expression in β-cells. Recombinant adenoviruses were generated to express WT FoxO1 (AdV-FoxO1-WT), WT FoxO3a (AdV-FoxO3a-WT), and green fluorescent protein (GFP) as a control (AdV-GFP). Equivalent levels of FoxO1 and FoxO3a were achieved using these vectors at a similar titer and dosage (Supplementary Fig. 3). In AdV-FoxO1-WT–infected rat islets incubated at basal 3 mmol/L glucose, IRS-2 mRNA levels were only modestly increased above AdV-GFP–infected control islets (Fig. 4A), and IRS-2 protein levels were not appreciably affected (Fig. 4B). In contrast, in AdV-FoxO3a-WT–infected rat islets, there was a threefold increase in basal IRS-2 mRNA levels (P ≤ 0.05) (Fig. 4A) that reflected a similar increase in IRS-2 protein levels (Fig. 4B). In isolated islets infected with both AdV-FoxO1-WT and AdV-FoxO3a-WT, the basal IRS-2 mRNA (Fig. 4A) and protein (Fig. 4B) levels did not appreciably increase above that observed with increased FoxO3a expression alone, suggesting that this effect was mediated only by FoxO3a.


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)

FoxO3a predominately drives basal IRS-2 expression in β-cells. Adenovirally infected isolated rat islets were incubated as indicated, where “+” represents an equivalent dose of each adenoviral vector. The rat islet mRNA (A and C) and protein (B and D) levels of IRS-2 and PI3K p85 (control) were examined by qRT-PCR and immunoblot (IB) analyses, respectively, as described in research design and methods. The qRT-PCR data are shown as mean ± SEM (n ≥ 4), and an example immunoblot for protein analysis is shown. A and B: Isolated rat islets were infected with adenoviruses expressing GFP (control), FoxO1-WT, FoxO3a-WT, or in combination as indicated at basal 3 mmol/L glucose for 6 h. C and D: Isolated rat islets were infected with adenoviruses expressing GFP, FoxO1-CA, FoxO3a-CA, or in combination as indicated at basal 3 mmol/L glucose for 6 h plus or minus insulin (100 nmol/L)/IGF-I (10 nmol/L). The * indicates statistically significant difference (P ≤ 0.05).
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Figure 4: FoxO3a predominately drives basal IRS-2 expression in β-cells. Adenovirally infected isolated rat islets were incubated as indicated, where “+” represents an equivalent dose of each adenoviral vector. The rat islet mRNA (A and C) and protein (B and D) levels of IRS-2 and PI3K p85 (control) were examined by qRT-PCR and immunoblot (IB) analyses, respectively, as described in research design and methods. The qRT-PCR data are shown as mean ± SEM (n ≥ 4), and an example immunoblot for protein analysis is shown. A and B: Isolated rat islets were infected with adenoviruses expressing GFP (control), FoxO1-WT, FoxO3a-WT, or in combination as indicated at basal 3 mmol/L glucose for 6 h. C and D: Isolated rat islets were infected with adenoviruses expressing GFP, FoxO1-CA, FoxO3a-CA, or in combination as indicated at basal 3 mmol/L glucose for 6 h plus or minus insulin (100 nmol/L)/IGF-I (10 nmol/L). The * indicates statistically significant difference (P ≤ 0.05).
Mentions: We examined if there was any difference between FoxO1 and FoxO3a in driving IRS-2 gene expression in β-cells. Recombinant adenoviruses were generated to express WT FoxO1 (AdV-FoxO1-WT), WT FoxO3a (AdV-FoxO3a-WT), and green fluorescent protein (GFP) as a control (AdV-GFP). Equivalent levels of FoxO1 and FoxO3a were achieved using these vectors at a similar titer and dosage (Supplementary Fig. 3). In AdV-FoxO1-WT–infected rat islets incubated at basal 3 mmol/L glucose, IRS-2 mRNA levels were only modestly increased above AdV-GFP–infected control islets (Fig. 4A), and IRS-2 protein levels were not appreciably affected (Fig. 4B). In contrast, in AdV-FoxO3a-WT–infected rat islets, there was a threefold increase in basal IRS-2 mRNA levels (P ≤ 0.05) (Fig. 4A) that reflected a similar increase in IRS-2 protein levels (Fig. 4B). In isolated islets infected with both AdV-FoxO1-WT and AdV-FoxO3a-WT, the basal IRS-2 mRNA (Fig. 4A) and protein (Fig. 4B) levels did not appreciably increase above that observed with increased FoxO3a expression alone, suggesting that this effect was mediated only by FoxO3a.

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