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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 regulation of IRS-2 expression in β-cells is decreased when NFAT is specifically inhibited. Isolated rat pancreatic islets were infected with a recombinant adenovirus expressing a specific peptide inhibitor of NFAT, AdV-VIVIT, or with AdV-FLuc and cultured overnight at normal 5.6 mmol/L glucose. Afterward, the adenovirally infected islets were then incubated at either basal 3 mmol/L or stimulatory 15 mmol/L glucose concentration for 6 h. A: IRS-2 and β-actin (internal reference control) mRNA expression levels were measured by real-time fluorescence-based quantitative RT-PCR. B: IRS-2 and PI3K(p85) (control) protein expression levels were analyzed by immunoblotting, where an example immunoblot (IB) and quantitative measurements are shown. The data are shown as a mean ± SE above basal 3 mmol/L glucose control, where * indicates a statistically significant increase at 15 mmol/L glucose above basal 3 mmol/L glucose and ** indicates statistically significant decrease at 15 mmol/L glucose in AdV-VIVIT–infected versus AdV-FLuc–infected control islets (P ≤ 0.05; n ≥ 4) (A and B).
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Figure 4: Glucose regulation of IRS-2 expression in β-cells is decreased when NFAT is specifically inhibited. Isolated rat pancreatic islets were infected with a recombinant adenovirus expressing a specific peptide inhibitor of NFAT, AdV-VIVIT, or with AdV-FLuc and cultured overnight at normal 5.6 mmol/L glucose. Afterward, the adenovirally infected islets were then incubated at either basal 3 mmol/L or stimulatory 15 mmol/L glucose concentration for 6 h. A: IRS-2 and β-actin (internal reference control) mRNA expression levels were measured by real-time fluorescence-based quantitative RT-PCR. B: IRS-2 and PI3K(p85) (control) protein expression levels were analyzed by immunoblotting, where an example immunoblot (IB) and quantitative measurements are shown. The data are shown as a mean ± SE above basal 3 mmol/L glucose control, where * indicates a statistically significant increase at 15 mmol/L glucose above basal 3 mmol/L glucose and ** indicates statistically significant decrease at 15 mmol/L glucose in AdV-VIVIT–infected versus AdV-FLuc–infected control islets (P ≤ 0.05; n ≥ 4) (A and B).

Mentions: The NFAT family of transcription factors is known to be activated by the Ca2+/calcineurin pathway (28). To assess whether NFAT plays a role in IRS-2 expression, AdV-VIVIT, a synthetic peptide preventing NFAT activation by calcineurin without affecting phosphatase activity of calcineurin, was used (29). In AdV-VIVIT–infected rat islets the specific glucose-induced increase in IRS-2 mRNA levels was significantly decreased by ∼75% compared with control AdV-FLuc–infected islets (P ≤ 0.05) (Fig. 4A). Likewise, the glucose-induced increase in IRS-2 protein levels was effectively blocked in AdV-VIVIT–infected rat islets compared with AdV-FLuc–infected control islets (P ≤ 0.05) (Fig. 4B). VIVIT had no effect on IRS-2 mRNA or protein levels at basal 3 mmol/L glucose (Fig. 4A and B), indicating that this was specific for glucose-induced regulation of IRS-2 mRNA/protein expression in islet β-cells. To further investigate NFAT-mediated control of IRS-2 expression, the effect of increasing ectopic expression of NFATc1 isoform on IRS-2 expression in rat islets using an adenovirus AdV-NFATc1 was examined. In AdV-NFATc1–infected INS-1 cells (Fig. 5A) and rat islets (Fig. 5C), there was an approximate twofold increase in specific IRS-2 mRNA expression at basal 3 mmol/L glucose compared with respective AdV-FLuc–infected control INS-1 or rat islets (P ≤ 0.05), which was further enhanced at 15 mmol/L glucose (Fig. 5A and C). A complementary increase in IRS-2 protein expression was observed at basal 3 mmol/L glucose in AdV-NFATc1–infected INS-1 cells (Fig. 5B) and rat islets (Fig. 5D) compared relative to AdV-FLuc–infected control rat islets. Taken together, these results demonstrate that NFATc1 is implicated in the regulation of glucose-induced IRS-2 expression in β-cells.


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 regulation of IRS-2 expression in β-cells is decreased when NFAT is specifically inhibited. Isolated rat pancreatic islets were infected with a recombinant adenovirus expressing a specific peptide inhibitor of NFAT, AdV-VIVIT, or with AdV-FLuc and cultured overnight at normal 5.6 mmol/L glucose. Afterward, the adenovirally infected islets were then incubated at either basal 3 mmol/L or stimulatory 15 mmol/L glucose concentration for 6 h. A: IRS-2 and β-actin (internal reference control) mRNA expression levels were measured by real-time fluorescence-based quantitative RT-PCR. B: IRS-2 and PI3K(p85) (control) protein expression levels were analyzed by immunoblotting, where an example immunoblot (IB) and quantitative measurements are shown. The data are shown as a mean ± SE above basal 3 mmol/L glucose control, where * indicates a statistically significant increase at 15 mmol/L glucose above basal 3 mmol/L glucose and ** indicates statistically significant decrease at 15 mmol/L glucose in AdV-VIVIT–infected versus AdV-FLuc–infected control islets (P ≤ 0.05; n ≥ 4) (A and B).
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Figure 4: Glucose regulation of IRS-2 expression in β-cells is decreased when NFAT is specifically inhibited. Isolated rat pancreatic islets were infected with a recombinant adenovirus expressing a specific peptide inhibitor of NFAT, AdV-VIVIT, or with AdV-FLuc and cultured overnight at normal 5.6 mmol/L glucose. Afterward, the adenovirally infected islets were then incubated at either basal 3 mmol/L or stimulatory 15 mmol/L glucose concentration for 6 h. A: IRS-2 and β-actin (internal reference control) mRNA expression levels were measured by real-time fluorescence-based quantitative RT-PCR. B: IRS-2 and PI3K(p85) (control) protein expression levels were analyzed by immunoblotting, where an example immunoblot (IB) and quantitative measurements are shown. The data are shown as a mean ± SE above basal 3 mmol/L glucose control, where * indicates a statistically significant increase at 15 mmol/L glucose above basal 3 mmol/L glucose and ** indicates statistically significant decrease at 15 mmol/L glucose in AdV-VIVIT–infected versus AdV-FLuc–infected control islets (P ≤ 0.05; n ≥ 4) (A and B).
Mentions: The NFAT family of transcription factors is known to be activated by the Ca2+/calcineurin pathway (28). To assess whether NFAT plays a role in IRS-2 expression, AdV-VIVIT, a synthetic peptide preventing NFAT activation by calcineurin without affecting phosphatase activity of calcineurin, was used (29). In AdV-VIVIT–infected rat islets the specific glucose-induced increase in IRS-2 mRNA levels was significantly decreased by ∼75% compared with control AdV-FLuc–infected islets (P ≤ 0.05) (Fig. 4A). Likewise, the glucose-induced increase in IRS-2 protein levels was effectively blocked in AdV-VIVIT–infected rat islets compared with AdV-FLuc–infected control islets (P ≤ 0.05) (Fig. 4B). VIVIT had no effect on IRS-2 mRNA or protein levels at basal 3 mmol/L glucose (Fig. 4A and B), indicating that this was specific for glucose-induced regulation of IRS-2 mRNA/protein expression in islet β-cells. To further investigate NFAT-mediated control of IRS-2 expression, the effect of increasing ectopic expression of NFATc1 isoform on IRS-2 expression in rat islets using an adenovirus AdV-NFATc1 was examined. In AdV-NFATc1–infected INS-1 cells (Fig. 5A) and rat islets (Fig. 5C), there was an approximate twofold increase in specific IRS-2 mRNA expression at basal 3 mmol/L glucose compared with respective AdV-FLuc–infected control INS-1 or rat islets (P ≤ 0.05), which was further enhanced at 15 mmol/L glucose (Fig. 5A and C). A complementary increase in IRS-2 protein expression was observed at basal 3 mmol/L glucose in AdV-NFATc1–infected INS-1 cells (Fig. 5B) and rat islets (Fig. 5D) compared relative to AdV-FLuc–infected control rat islets. Taken together, these results demonstrate that NFATc1 is implicated in the regulation of glucose-induced IRS-2 expression in β-cells.

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