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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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Glucose induces NFATc1 translocation from cytosol to nucleus in β-cells. INS-1 β-cells (A and C) or isolated rat islets (B) were cultured overnight at 5.6 mmol/L glucose and then incubated at either basal 3 mmol/L or stimulatory 15 mmol/L glucose concentration for 6 h ± FK506 (10 µmol/L). A and B: Nuclear proteins were extracted and nuclear NFATc1 protein expression levels were analyzed by immunoblotting relative to a Maf-A (A) or RNA polymerase-II (Pol-II) (B) protein expression as loading controls. Example immunoblots (IBs) of three individual experiments are shown. C: Cellular localization of NFATc1 in INS-1 pancreatic β-cells. NFATc1 (red) localization was analyzed by immunofluorescence and confocal microscopy relative to insulin immunostaining (green) and nuclei staining by DAPI (blue). Example images of three individual experiments are shown. (A high-quality digital representation of this figure is available in the online issue.)
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Figure 7: Glucose induces NFATc1 translocation from cytosol to nucleus in β-cells. INS-1 β-cells (A and C) or isolated rat islets (B) were cultured overnight at 5.6 mmol/L glucose and then incubated at either basal 3 mmol/L or stimulatory 15 mmol/L glucose concentration for 6 h ± FK506 (10 µmol/L). A and B: Nuclear proteins were extracted and nuclear NFATc1 protein expression levels were analyzed by immunoblotting relative to a Maf-A (A) or RNA polymerase-II (Pol-II) (B) protein expression as loading controls. Example immunoblots (IBs) of three individual experiments are shown. C: Cellular localization of NFATc1 in INS-1 pancreatic β-cells. NFATc1 (red) localization was analyzed by immunofluorescence and confocal microscopy relative to insulin immunostaining (green) and nuclei staining by DAPI (blue). Example images of three individual experiments are shown. (A high-quality digital representation of this figure is available in the online issue.)

Mentions: NFAT activity is tightly regulated by calcineurin, which induces its activation by dephosphorylation, leading to NFAT translocation from the cytosol to the nucleus (30). By contrast, phosphorylated NFAT has negligible transcriptional activity and is retained in the cytosol. It was found that a 15 mmol/L stimulatory glucose concentration caused specific accumulation of NFATc1 to the nucleus of isolated rat islets and INS-1 cells, and this glucose-induced translocation of NFATc1 to the nucleus was prevented in the presence of FK506 (Fig. 7A and B). In complementary experiments in INS-1 cells, immunofluorescent staining of NFATc1 revealed that NFATc1 was mainly localized in the cytosolic compartment at basal 3 mmol/L glucose (Fig. 7C). In contrast, NFATc1 was mostly localized in the nucleus at 15 mmol/L stimulatory glucose concentration (Fig. 7C). Moreover, this glucose-induced translocation of NFATc1 from the cytosol to the nucleus of INS-1 cells was completely blocked in the presence of FK506 (Fig. 7C). Collectively, these data indicate that 15 mmol/L stimulatory glucose induces the translocation of NFATc1 from the cytosolic compartment to the nucleus of islet β-cell in a calcineurin-dependent manner.


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)

Glucose induces NFATc1 translocation from cytosol to nucleus in β-cells. INS-1 β-cells (A and C) or isolated rat islets (B) were cultured overnight at 5.6 mmol/L glucose and then incubated at either basal 3 mmol/L or stimulatory 15 mmol/L glucose concentration for 6 h ± FK506 (10 µmol/L). A and B: Nuclear proteins were extracted and nuclear NFATc1 protein expression levels were analyzed by immunoblotting relative to a Maf-A (A) or RNA polymerase-II (Pol-II) (B) protein expression as loading controls. Example immunoblots (IBs) of three individual experiments are shown. C: Cellular localization of NFATc1 in INS-1 pancreatic β-cells. NFATc1 (red) localization was analyzed by immunofluorescence and confocal microscopy relative to insulin immunostaining (green) and nuclei staining by DAPI (blue). Example images of three individual experiments are shown. (A high-quality digital representation of this figure is available in the online issue.)
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3198104&req=5

Figure 7: Glucose induces NFATc1 translocation from cytosol to nucleus in β-cells. INS-1 β-cells (A and C) or isolated rat islets (B) were cultured overnight at 5.6 mmol/L glucose and then incubated at either basal 3 mmol/L or stimulatory 15 mmol/L glucose concentration for 6 h ± FK506 (10 µmol/L). A and B: Nuclear proteins were extracted and nuclear NFATc1 protein expression levels were analyzed by immunoblotting relative to a Maf-A (A) or RNA polymerase-II (Pol-II) (B) protein expression as loading controls. Example immunoblots (IBs) of three individual experiments are shown. C: Cellular localization of NFATc1 in INS-1 pancreatic β-cells. NFATc1 (red) localization was analyzed by immunofluorescence and confocal microscopy relative to insulin immunostaining (green) and nuclei staining by DAPI (blue). Example images of three individual experiments are shown. (A high-quality digital representation of this figure is available in the online issue.)
Mentions: NFAT activity is tightly regulated by calcineurin, which induces its activation by dephosphorylation, leading to NFAT translocation from the cytosol to the nucleus (30). By contrast, phosphorylated NFAT has negligible transcriptional activity and is retained in the cytosol. It was found that a 15 mmol/L stimulatory glucose concentration caused specific accumulation of NFATc1 to the nucleus of isolated rat islets and INS-1 cells, and this glucose-induced translocation of NFATc1 to the nucleus was prevented in the presence of FK506 (Fig. 7A and B). In complementary experiments in INS-1 cells, immunofluorescent staining of NFATc1 revealed that NFATc1 was mainly localized in the cytosolic compartment at basal 3 mmol/L glucose (Fig. 7C). In contrast, NFATc1 was mostly localized in the nucleus at 15 mmol/L stimulatory glucose concentration (Fig. 7C). Moreover, this glucose-induced translocation of NFATc1 from the cytosol to the nucleus of INS-1 cells was completely blocked in the presence of FK506 (Fig. 7C). Collectively, these data indicate that 15 mmol/L stimulatory glucose induces the translocation of NFATc1 from the cytosolic compartment to the nucleus of islet β-cell in a calcineurin-dependent manner.

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