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Specific glucose-induced control of insulin receptor substrate-2 expression is mediated via Ca2+-dependent calcineurin/NFAT signaling in primary pancreatic islet β-cells.

Demozay D, Tsunekawa S, Briaud I, Shah R, Rhodes CJ - Diabetes (2011)

Bottom Line: Specific inhibition of NFAT with the peptide inhibitor VIVIT prevented a glucose-induced IRS-2 transcription.NFATc1 translocation to the nucleus in response to glucose and association of NFATc1 to conserved NFAT binding sites in the IRS-2 promoter were demonstrated.The mechanism behind glucose-induced transcriptional control of IRS-2 gene expression specific to the islet β-cell is mediated by the Ca(2+)/calcineurin/NFAT pathway.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, Section of Endocrinology, Diabetes and Metabolism, Kovler Diabetes Center, University of Chicago, Chicago, Illinois, USA.

ABSTRACT

Objective: Insulin receptor substrate-2 (IRS-2) plays an essential role in pancreatic islet β-cells by promoting growth and survival. IRS-2 turnover is rapid in primary β-cells, but its expression is highly regulated at the transcriptional level, especially by glucose. The aim was to investigate the molecular mechanism on how glucose regulates IRS-2 gene expression in β-cells.

Research design and methods: Rat islets were exposed to inhibitors or subjected to adenoviral vector-mediated gene manipulations and then to glucose-induced IRS-2 expression analyzed by real-time PCR and immunoblotting. Transcription factor nuclear factor of activated T cells (NFAT) interaction with IRS-2 promoter was analyzed by chromatin immunoprecipitation assay and glucose-induced NFAT translocation by immunohistochemistry.

Results: Glucose-induced IRS-2 expression occurred in pancreatic islet β-cells in vivo but not in liver. Modulating rat islet β-cell Ca(2+) influx with nifedipine or depolarization demonstrated that glucose-induced IRS-2 gene expression was dependent on a rise in intracellular calcium concentration derived from extracellular sources. Calcineurin inhibitors (FK506, cyclosporin A, and a peptide calcineurin inhibitor [CAIN]) abolished glucose-induced IRS-2 mRNA and protein levels, whereas expression of a constitutively active calcineurin increased them. Specific inhibition of NFAT with the peptide inhibitor VIVIT prevented a glucose-induced IRS-2 transcription. NFATc1 translocation to the nucleus in response to glucose and association of NFATc1 to conserved NFAT binding sites in the IRS-2 promoter were demonstrated.

Conclusions: The mechanism behind glucose-induced transcriptional control of IRS-2 gene expression specific to the islet β-cell is mediated by the Ca(2+)/calcineurin/NFAT pathway. This insight into the IRS-2 regulation could provide novel therapeutic means in type 2 diabetes to maintain an adequate functional mass.

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NFATc1 binds to rat IRS-2 gene promoter in β-cells. A: Predicted NFAT binding site elements on rat IRS-2 gene promoter. Examination of the rat IRS-2 gene promoter from −3,020 to +1 bp (transcription start point) showed multiple NFATc1 consensus sequences (GGAAA). Black boxes (A–I) represent NFAT consensus binding sites, and arrows indicates the five primer pairs used for the ChIP assay. B and C: Specific binding of NFATc1 to the endogenous rat IRS-2 promoter. INS-1 β-cells were infected with AdV-NFATc1, or AdV-Fluc as control, and the ChIP assay was performed using anti-NFATc1 antibody or Mouse IgG as a negative control. The input (B) corresponds to cross-linked and sheared chromatin. DNA fragments were then submitted to PCR amplification using primers to amplify five regions in the IRS-2 promoter containing the NFAT binding sites A, B and C together, D, E, or F through I as indicated above. PCR products were loaded onto an agarose gel, where an example is depicted (B). A series of ChIP assays were conducted (n = 6), and the combined data are shown as a mean ± SD above AdV-FLuc control for region A, where * indicates a significant increase (P ≤ 0.05) in AdV-NFATc1–infected cells versus control AdV-FLuc–infected cells (C).
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Figure 6: NFATc1 binds to rat IRS-2 gene promoter in β-cells. A: Predicted NFAT binding site elements on rat IRS-2 gene promoter. Examination of the rat IRS-2 gene promoter from −3,020 to +1 bp (transcription start point) showed multiple NFATc1 consensus sequences (GGAAA). Black boxes (A–I) represent NFAT consensus binding sites, and arrows indicates the five primer pairs used for the ChIP assay. B and C: Specific binding of NFATc1 to the endogenous rat IRS-2 promoter. INS-1 β-cells were infected with AdV-NFATc1, or AdV-Fluc as control, and the ChIP assay was performed using anti-NFATc1 antibody or Mouse IgG as a negative control. The input (B) corresponds to cross-linked and sheared chromatin. DNA fragments were then submitted to PCR amplification using primers to amplify five regions in the IRS-2 promoter containing the NFAT binding sites A, B and C together, D, E, or F through I as indicated above. PCR products were loaded onto an agarose gel, where an example is depicted (B). A series of ChIP assays were conducted (n = 6), and the combined data are shown as a mean ± SD above AdV-FLuc control for region A, where * indicates a significant increase (P ≤ 0.05) in AdV-NFATc1–infected cells versus control AdV-FLuc–infected cells (C).

Mentions: NFAT transcription factors bind to a consensus sequence (GGAAA) in target genes promoter to transactivate gene expression. Bioinformatic analysis of the first 3,020 bp of rat IRS-2 promoter (using a basic local alignment search tool [BLAST] search) indicated the presence of nine potential NFAT binding consensus sequences (Fig. 6A). To test whether NFATc1 binds to these consensus sequences, we conducted a ChIP assay using chromatin from INS-1 β-cells expressing NFATc1 or FLuc as a control, using AdV-NFATc1 and AdV-FLuc, respectively. Oligonucleotide DNA primers (Supplementary Fig. 1) were used to amplify specific regions containing NFAT binding sites on the IRS-2 promoter as indicated: sites A, sites B and C together, site D, site E, or sites F through to I together (Fig. 6A). Significant NFATc1 binding to sequences containing NFAT binding sites A, B/C, and D in the IRS-2 promoter was increased in AdV-NFATc1–infected cells compared with AdV-FLuc–infected control cells (P ≤ 0.05) (Fig. 6B and C). This binding was specific for NFATc1, since nothing was detected in rabbit IgG immunoprecipitated controls (Fig. 6B). These results suggest that the four NFAT consensus binding sequences proximal to the transcriptional start site on the endogenous IRS-2 promoter (sites A–D) are indeed binding sites for NFATc1 to control IRS-2 expression. The putative NFAT binding motifs more distal to the transcriptional start site on the IRS-2 promoter (sites E–I) (Fig. 6A) are not specifically recognized by NFATc1. Intriguingly, the NFAT binding sites A and D in the IRS-2 promoter are highly conserved across mammalian species, including human (Supplementary Fig. 1).


Specific glucose-induced control of insulin receptor substrate-2 expression is mediated via Ca2+-dependent calcineurin/NFAT signaling in primary pancreatic islet β-cells.

Demozay D, Tsunekawa S, Briaud I, Shah R, Rhodes CJ - Diabetes (2011)

NFATc1 binds to rat IRS-2 gene promoter in β-cells. A: Predicted NFAT binding site elements on rat IRS-2 gene promoter. Examination of the rat IRS-2 gene promoter from −3,020 to +1 bp (transcription start point) showed multiple NFATc1 consensus sequences (GGAAA). Black boxes (A–I) represent NFAT consensus binding sites, and arrows indicates the five primer pairs used for the ChIP assay. B and C: Specific binding of NFATc1 to the endogenous rat IRS-2 promoter. INS-1 β-cells were infected with AdV-NFATc1, or AdV-Fluc as control, and the ChIP assay was performed using anti-NFATc1 antibody or Mouse IgG as a negative control. The input (B) corresponds to cross-linked and sheared chromatin. DNA fragments were then submitted to PCR amplification using primers to amplify five regions in the IRS-2 promoter containing the NFAT binding sites A, B and C together, D, E, or F through I as indicated above. PCR products were loaded onto an agarose gel, where an example is depicted (B). A series of ChIP assays were conducted (n = 6), and the combined data are shown as a mean ± SD above AdV-FLuc control for region A, where * indicates a significant increase (P ≤ 0.05) in AdV-NFATc1–infected cells versus control AdV-FLuc–infected cells (C).
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Figure 6: NFATc1 binds to rat IRS-2 gene promoter in β-cells. A: Predicted NFAT binding site elements on rat IRS-2 gene promoter. Examination of the rat IRS-2 gene promoter from −3,020 to +1 bp (transcription start point) showed multiple NFATc1 consensus sequences (GGAAA). Black boxes (A–I) represent NFAT consensus binding sites, and arrows indicates the five primer pairs used for the ChIP assay. B and C: Specific binding of NFATc1 to the endogenous rat IRS-2 promoter. INS-1 β-cells were infected with AdV-NFATc1, or AdV-Fluc as control, and the ChIP assay was performed using anti-NFATc1 antibody or Mouse IgG as a negative control. The input (B) corresponds to cross-linked and sheared chromatin. DNA fragments were then submitted to PCR amplification using primers to amplify five regions in the IRS-2 promoter containing the NFAT binding sites A, B and C together, D, E, or F through I as indicated above. PCR products were loaded onto an agarose gel, where an example is depicted (B). A series of ChIP assays were conducted (n = 6), and the combined data are shown as a mean ± SD above AdV-FLuc control for region A, where * indicates a significant increase (P ≤ 0.05) in AdV-NFATc1–infected cells versus control AdV-FLuc–infected cells (C).
Mentions: NFAT transcription factors bind to a consensus sequence (GGAAA) in target genes promoter to transactivate gene expression. Bioinformatic analysis of the first 3,020 bp of rat IRS-2 promoter (using a basic local alignment search tool [BLAST] search) indicated the presence of nine potential NFAT binding consensus sequences (Fig. 6A). To test whether NFATc1 binds to these consensus sequences, we conducted a ChIP assay using chromatin from INS-1 β-cells expressing NFATc1 or FLuc as a control, using AdV-NFATc1 and AdV-FLuc, respectively. Oligonucleotide DNA primers (Supplementary Fig. 1) were used to amplify specific regions containing NFAT binding sites on the IRS-2 promoter as indicated: sites A, sites B and C together, site D, site E, or sites F through to I together (Fig. 6A). Significant NFATc1 binding to sequences containing NFAT binding sites A, B/C, and D in the IRS-2 promoter was increased in AdV-NFATc1–infected cells compared with AdV-FLuc–infected control cells (P ≤ 0.05) (Fig. 6B and C). This binding was specific for NFATc1, since nothing was detected in rabbit IgG immunoprecipitated controls (Fig. 6B). These results suggest that the four NFAT consensus binding sequences proximal to the transcriptional start site on the endogenous IRS-2 promoter (sites A–D) are indeed binding sites for NFATc1 to control IRS-2 expression. The putative NFAT binding motifs more distal to the transcriptional start site on the IRS-2 promoter (sites E–I) (Fig. 6A) are not specifically recognized by NFATc1. Intriguingly, the NFAT binding sites A and D in the IRS-2 promoter are highly conserved across mammalian species, including human (Supplementary Fig. 1).

Bottom Line: Specific inhibition of NFAT with the peptide inhibitor VIVIT prevented a glucose-induced IRS-2 transcription.NFATc1 translocation to the nucleus in response to glucose and association of NFATc1 to conserved NFAT binding sites in the IRS-2 promoter were demonstrated.The mechanism behind glucose-induced transcriptional control of IRS-2 gene expression specific to the islet β-cell is mediated by the Ca(2+)/calcineurin/NFAT pathway.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, Section of Endocrinology, Diabetes and Metabolism, Kovler Diabetes Center, University of Chicago, Chicago, Illinois, USA.

ABSTRACT

Objective: Insulin receptor substrate-2 (IRS-2) plays an essential role in pancreatic islet β-cells by promoting growth and survival. IRS-2 turnover is rapid in primary β-cells, but its expression is highly regulated at the transcriptional level, especially by glucose. The aim was to investigate the molecular mechanism on how glucose regulates IRS-2 gene expression in β-cells.

Research design and methods: Rat islets were exposed to inhibitors or subjected to adenoviral vector-mediated gene manipulations and then to glucose-induced IRS-2 expression analyzed by real-time PCR and immunoblotting. Transcription factor nuclear factor of activated T cells (NFAT) interaction with IRS-2 promoter was analyzed by chromatin immunoprecipitation assay and glucose-induced NFAT translocation by immunohistochemistry.

Results: Glucose-induced IRS-2 expression occurred in pancreatic islet β-cells in vivo but not in liver. Modulating rat islet β-cell Ca(2+) influx with nifedipine or depolarization demonstrated that glucose-induced IRS-2 gene expression was dependent on a rise in intracellular calcium concentration derived from extracellular sources. Calcineurin inhibitors (FK506, cyclosporin A, and a peptide calcineurin inhibitor [CAIN]) abolished glucose-induced IRS-2 mRNA and protein levels, whereas expression of a constitutively active calcineurin increased them. Specific inhibition of NFAT with the peptide inhibitor VIVIT prevented a glucose-induced IRS-2 transcription. NFATc1 translocation to the nucleus in response to glucose and association of NFATc1 to conserved NFAT binding sites in the IRS-2 promoter were demonstrated.

Conclusions: The mechanism behind glucose-induced transcriptional control of IRS-2 gene expression specific to the islet β-cell is mediated by the Ca(2+)/calcineurin/NFAT pathway. This insight into the IRS-2 regulation could provide novel therapeutic means in type 2 diabetes to maintain an adequate functional mass.

Show MeSH
Related in: MedlinePlus