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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

Distinct mechanisms regulate IRS-2 expression in vivo in liver and in islets. Normal rats were either fasted for 12 h (S) or allowed ad libitum feeding and subjected to an intraperitoneal injection of insulin (0.75 mU/g body wt) 2 h prior to tissue harvesting (F). The liver and pancreatic islets from the same rats were then harvested and analyzed in parallel. The protein expression levels of IRS-2, SREBP-1 (precursor and proteolyzed activated forms), FoxO1, FoxO3a, TFE3, ARNT, and PI3K p85 (control) were measured in parallel by immunoblotting. An example immunoblot (IB) analysis of a single animal is shown from three independent experiments.
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Figure 6: Distinct mechanisms regulate IRS-2 expression in vivo in liver and in islets. Normal rats were either fasted for 12 h (S) or allowed ad libitum feeding and subjected to an intraperitoneal injection of insulin (0.75 mU/g body wt) 2 h prior to tissue harvesting (F). The liver and pancreatic islets from the same rats were then harvested and analyzed in parallel. The protein expression levels of IRS-2, SREBP-1 (precursor and proteolyzed activated forms), FoxO1, FoxO3a, TFE3, ARNT, and PI3K p85 (control) were measured in parallel by immunoblotting. An example immunoblot (IB) analysis of a single animal is shown from three independent experiments.

Mentions: In liver, SREBP-1 suppresses IRS-2 gene transcription by preventing FoxO1 from binding to the IRE and its association with TFE3 under fed conditions (15,16). However, in this study, unlike hepatocytes, SREBP-1, TFE3, or ARNT did not associate with the IRE region of the IRS-2 promoter in islet β-cells under conditions where regulated FoxO1 and FoxO3a association were observed (Fig. 3). This discrepancy was further investigated in vivo, in liver and islets isolated from the very same rats that were either starved overnight or allowed to feed ad libitum and subjected to an intraperitoneal insulin injection (0.75 mU/g body wt) 2 h prior. In liver, there was an ∼80% decrease in IRS-2 protein expression in the plus insulin/fed condition, compared with that in the fasted condition (Fig. 6). This was correlated by a massive increase in expression and proteolytic activation of SREBP-1 in liver, a modest increase in TFE3 protein expression, and a reduction in FoxO1 protein levels, but no change in ARNT or FoxO3a protein expression levels relative to the PI3K p85 loading control in the liver plus insulin/fed condition compared with the fasted condition (Fig. 6). In contrast, in isolated islets from the same animal, IRS-2, SREBP-1, FoxO1, FoxO3a, TFE3, and ARNT protein levels did not change relative to the PI3K p85 under the plus insulin/fed condition compared with the fasted condition (Fig. 6). It should also be noted that relative protein expression levels of IRS-2, FoxO1, FoxO3a, and ARNT were higher in islets compared with liver, and SREBP-1, TFE3, and PI3K p85 were lower (Fig. 6). Indeed, to make sure that SREBP-1 was detectably present in islets, the liver X receptor agonist TO-901317 was used as a positive control to increase SREBP-1 expression (31). Although TO-901317 instigated a specific increase in the precursor form of SREBP-1 protein in rat islets, there was no change in proteolytic cleavage activation of SREBP-1 or basal IRS-2 protein expression (Supplementary Fig. 4). These data collectively indicate that there is different regulation of SREBP-1 expression/activation between islets and liver, and SREBP-1 does not participate in the regulation of IRS-2 expression in islet β-cells. As such, the molecular mechanism behind regulation of IRS-2 gene transcription is distinct in these different cell types in vivo.


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)

Distinct mechanisms regulate IRS-2 expression in vivo in liver and in islets. Normal rats were either fasted for 12 h (S) or allowed ad libitum feeding and subjected to an intraperitoneal injection of insulin (0.75 mU/g body wt) 2 h prior to tissue harvesting (F). The liver and pancreatic islets from the same rats were then harvested and analyzed in parallel. The protein expression levels of IRS-2, SREBP-1 (precursor and proteolyzed activated forms), FoxO1, FoxO3a, TFE3, ARNT, and PI3K p85 (control) were measured in parallel by immunoblotting. An example immunoblot (IB) analysis of a single animal is shown from three independent experiments.
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Related In: Results  -  Collection

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

Figure 6: Distinct mechanisms regulate IRS-2 expression in vivo in liver and in islets. Normal rats were either fasted for 12 h (S) or allowed ad libitum feeding and subjected to an intraperitoneal injection of insulin (0.75 mU/g body wt) 2 h prior to tissue harvesting (F). The liver and pancreatic islets from the same rats were then harvested and analyzed in parallel. The protein expression levels of IRS-2, SREBP-1 (precursor and proteolyzed activated forms), FoxO1, FoxO3a, TFE3, ARNT, and PI3K p85 (control) were measured in parallel by immunoblotting. An example immunoblot (IB) analysis of a single animal is shown from three independent experiments.
Mentions: In liver, SREBP-1 suppresses IRS-2 gene transcription by preventing FoxO1 from binding to the IRE and its association with TFE3 under fed conditions (15,16). However, in this study, unlike hepatocytes, SREBP-1, TFE3, or ARNT did not associate with the IRE region of the IRS-2 promoter in islet β-cells under conditions where regulated FoxO1 and FoxO3a association were observed (Fig. 3). This discrepancy was further investigated in vivo, in liver and islets isolated from the very same rats that were either starved overnight or allowed to feed ad libitum and subjected to an intraperitoneal insulin injection (0.75 mU/g body wt) 2 h prior. In liver, there was an ∼80% decrease in IRS-2 protein expression in the plus insulin/fed condition, compared with that in the fasted condition (Fig. 6). This was correlated by a massive increase in expression and proteolytic activation of SREBP-1 in liver, a modest increase in TFE3 protein expression, and a reduction in FoxO1 protein levels, but no change in ARNT or FoxO3a protein expression levels relative to the PI3K p85 loading control in the liver plus insulin/fed condition compared with the fasted condition (Fig. 6). In contrast, in isolated islets from the same animal, IRS-2, SREBP-1, FoxO1, FoxO3a, TFE3, and ARNT protein levels did not change relative to the PI3K p85 under the plus insulin/fed condition compared with the fasted condition (Fig. 6). It should also be noted that relative protein expression levels of IRS-2, FoxO1, FoxO3a, and ARNT were higher in islets compared with liver, and SREBP-1, TFE3, and PI3K p85 were lower (Fig. 6). Indeed, to make sure that SREBP-1 was detectably present in islets, the liver X receptor agonist TO-901317 was used as a positive control to increase SREBP-1 expression (31). Although TO-901317 instigated a specific increase in the precursor form of SREBP-1 protein in rat islets, there was no change in proteolytic cleavage activation of SREBP-1 or basal IRS-2 protein expression (Supplementary Fig. 4). These data collectively indicate that there is different regulation of SREBP-1 expression/activation between islets and liver, and SREBP-1 does not participate in the regulation of IRS-2 expression in islet β-cells. As such, the molecular mechanism behind regulation of IRS-2 gene transcription is distinct in these different cell types in vivo.

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