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

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.

FIG. 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).

FIG. 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).

FIG. 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.)

FIG. 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).

FIG. 5.

FoxO3a predominately drives basal IRS-2 promoter activity β-cells. Relative FLuc activities mediated by either WT (AdV-WT-IRS-2-Luc) or IRE mutated (AdV-Mut-IRS-2-Luc) were measured as outlined in research design and methods in adenovirally infected isolated rat islets expressing GFP (AdV-GFP; control), WT FoxO1 (AdV-FoxO1-WT), or WT FoxO3a (AdV-FoxO3a-WT). Results are mean ± SEM (n ≥ 3). The * indicates statistically significant difference (P ≤ 0.05).

FIG. 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.