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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 dependent on increased cytosolic [Ca2+]i flux. Isolated rat pancreatic islets were cultured overnight at normal 5.6 mmol/L glucose and then incubated at either basal 3 mmol/L (gray bars) or stimulatory 15 mmol/L (black bars) glucose concentration for 6 h in presence or absence of either the voltage-sensitive L-type Ca2+-channel inhibitor nifedipine (50 µmol/L) (A and B) or KCl to depolarize β-cells (30 mmol/L) (C and D). A and C: IRS-2 and β-actin (internal reference control) mRNA expression levels were measured by real-time fluorescence-based quantitative RT-PCR. The data are shown as a mean ± SE above basal 3 mmol/L glucose control, where * indicates statistically significant difference (P ≤ 0.05) at 15 mmol/L glucose in the presence of nifedipine or KCl versus control (n ≥ 4). B and D: IRS-2 and PI3K(p85) (control) protein expression levels were analyzed by immunoblotting. Example immunoblots (IBs) are shown. Quantitative measurements are also shown as a mean ± SE, where * indicates statistically significant increase at 15 mmol/L glucose compared with basal 3 mmol/L glucose control, ** indicates statistically significant inhibition in the presence of nifedipine at 15 mmol/L glucose, and *** indicates statistically significant potentiation by KCl relative to the equivalent glucose concentration in the absence of KCl (P ≤ 0.05; n ≥ 4).
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Figure 2: Glucose regulation of IRS-2 expression in β-cells is dependent on increased cytosolic [Ca2+]i flux. Isolated rat pancreatic islets were cultured overnight at normal 5.6 mmol/L glucose and then incubated at either basal 3 mmol/L (gray bars) or stimulatory 15 mmol/L (black bars) glucose concentration for 6 h in presence or absence of either the voltage-sensitive L-type Ca2+-channel inhibitor nifedipine (50 µmol/L) (A and B) or KCl to depolarize β-cells (30 mmol/L) (C and D). A and C: IRS-2 and β-actin (internal reference control) mRNA expression levels were measured by real-time fluorescence-based quantitative RT-PCR. The data are shown as a mean ± SE above basal 3 mmol/L glucose control, where * indicates statistically significant difference (P ≤ 0.05) at 15 mmol/L glucose in the presence of nifedipine or KCl versus control (n ≥ 4). B and D: IRS-2 and PI3K(p85) (control) protein expression levels were analyzed by immunoblotting. Example immunoblots (IBs) are shown. Quantitative measurements are also shown as a mean ± SE, where * indicates statistically significant increase at 15 mmol/L glucose compared with basal 3 mmol/L glucose control, ** indicates statistically significant inhibition in the presence of nifedipine at 15 mmol/L glucose, and *** indicates statistically significant potentiation by KCl relative to the equivalent glucose concentration in the absence of KCl (P ≤ 0.05; n ≥ 4).

Mentions: It has been indicated previously that control of glucose-induced IRS-2 expression in β-cells is likely Ca2+ dependent (10). It was investigated whether this Ca2+ dependency was based on an influx of extracellular Ca2+. Specifically, blocking L-type voltage-dependent Ca2+ channels (L-type VDCCs) in isolated rat islets with the inhibitor nifedipine had little effect on basal IRS-2 expression at 3 mmol/L glucose, but prevented a 15 mmol/L glucose-induced specific increase in the expression of IRS-2 mRNA (P ≤ 0.05) (Fig. 2A) and protein (P ≤ 0.05) (Fig. 2B). The L-type VDCCs in β-cells can be opened via a depolarization of the plasma membrane (23). Depolarization of primary islet β-cells in vitro, by addition of 30 mmol/L KCl, resulted in a significant increase in IRS-2 mRNA (Fig. 2C) and protein (Fig. 2D) at basal 3 mmol/L glucose and further increased IRS-2 mRNA and protein expression levels at a stimulatory 15 mmol/L glucose relative to control islets. Collectively, these data indicate that a glucose-induced increase in β-cell cytosolic intracellular calcium concentration ([Ca2+]i) flux via depolarization and subsequent opening of L-type VDCCs is required for glucose-induced increases in 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 dependent on increased cytosolic [Ca2+]i flux. Isolated rat pancreatic islets were cultured overnight at normal 5.6 mmol/L glucose and then incubated at either basal 3 mmol/L (gray bars) or stimulatory 15 mmol/L (black bars) glucose concentration for 6 h in presence or absence of either the voltage-sensitive L-type Ca2+-channel inhibitor nifedipine (50 µmol/L) (A and B) or KCl to depolarize β-cells (30 mmol/L) (C and D). A and C: IRS-2 and β-actin (internal reference control) mRNA expression levels were measured by real-time fluorescence-based quantitative RT-PCR. The data are shown as a mean ± SE above basal 3 mmol/L glucose control, where * indicates statistically significant difference (P ≤ 0.05) at 15 mmol/L glucose in the presence of nifedipine or KCl versus control (n ≥ 4). B and D: IRS-2 and PI3K(p85) (control) protein expression levels were analyzed by immunoblotting. Example immunoblots (IBs) are shown. Quantitative measurements are also shown as a mean ± SE, where * indicates statistically significant increase at 15 mmol/L glucose compared with basal 3 mmol/L glucose control, ** indicates statistically significant inhibition in the presence of nifedipine at 15 mmol/L glucose, and *** indicates statistically significant potentiation by KCl relative to the equivalent glucose concentration in the absence of KCl (P ≤ 0.05; n ≥ 4).
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Figure 2: Glucose regulation of IRS-2 expression in β-cells is dependent on increased cytosolic [Ca2+]i flux. Isolated rat pancreatic islets were cultured overnight at normal 5.6 mmol/L glucose and then incubated at either basal 3 mmol/L (gray bars) or stimulatory 15 mmol/L (black bars) glucose concentration for 6 h in presence or absence of either the voltage-sensitive L-type Ca2+-channel inhibitor nifedipine (50 µmol/L) (A and B) or KCl to depolarize β-cells (30 mmol/L) (C and D). A and C: IRS-2 and β-actin (internal reference control) mRNA expression levels were measured by real-time fluorescence-based quantitative RT-PCR. The data are shown as a mean ± SE above basal 3 mmol/L glucose control, where * indicates statistically significant difference (P ≤ 0.05) at 15 mmol/L glucose in the presence of nifedipine or KCl versus control (n ≥ 4). B and D: IRS-2 and PI3K(p85) (control) protein expression levels were analyzed by immunoblotting. Example immunoblots (IBs) are shown. Quantitative measurements are also shown as a mean ± SE, where * indicates statistically significant increase at 15 mmol/L glucose compared with basal 3 mmol/L glucose control, ** indicates statistically significant inhibition in the presence of nifedipine at 15 mmol/L glucose, and *** indicates statistically significant potentiation by KCl relative to the equivalent glucose concentration in the absence of KCl (P ≤ 0.05; n ≥ 4).
Mentions: It has been indicated previously that control of glucose-induced IRS-2 expression in β-cells is likely Ca2+ dependent (10). It was investigated whether this Ca2+ dependency was based on an influx of extracellular Ca2+. Specifically, blocking L-type voltage-dependent Ca2+ channels (L-type VDCCs) in isolated rat islets with the inhibitor nifedipine had little effect on basal IRS-2 expression at 3 mmol/L glucose, but prevented a 15 mmol/L glucose-induced specific increase in the expression of IRS-2 mRNA (P ≤ 0.05) (Fig. 2A) and protein (P ≤ 0.05) (Fig. 2B). The L-type VDCCs in β-cells can be opened via a depolarization of the plasma membrane (23). Depolarization of primary islet β-cells in vitro, by addition of 30 mmol/L KCl, resulted in a significant increase in IRS-2 mRNA (Fig. 2C) and protein (Fig. 2D) at basal 3 mmol/L glucose and further increased IRS-2 mRNA and protein expression levels at a stimulatory 15 mmol/L glucose relative to control islets. Collectively, these data indicate that a glucose-induced increase in β-cell cytosolic intracellular calcium concentration ([Ca2+]i) flux via depolarization and subsequent opening of L-type VDCCs is required for glucose-induced increases in 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