Limits...
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.

Show MeSH

Related in: MedlinePlus

Glucose regulation of IRS-2 expression in β-cells is mediated by calcineurin. Isolated rat pancreatic islets were cultured overnight at normal 5.6 mmol/L glucose and then incubated at either basal 3 mmol/L or stimulatory 15 mmol/L glucose concentration for 6 h in presence or absence of calcineurin inhibitors FK506 (10 µmol/L) (A and B) or CsA (10 µmol/L) (C). A: IRS-2 and β-actin (internal reference control) mRNA expression levels were measured by real-time fluorescence-based quantitative RT-PCR. B and C: IRS-2 and PI3K(p85) (control) protein expression levels were analyzed by immunoblotting, where an example immunoblot (IB) is shown. Quantitative measurements are also shown as a mean ± SE above basal 3 mmol/L glucose control, where * indicates a statistically significant increase at 15 mmol/L glucose and ** indicates a statistically significant difference at 15 mmol/L glucose in the presence of FK506 or CsA versus control (P ≤ 0.05; n ≥ 4) (A–C). Isolated rat pancreatic islets were also infected with a recombinant AdV-CAIN (D and E), AdV-CNca (F and G), or 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. D and F: IRS-2 and β-actin (internal reference control) mRNA expression levels were measured by real-time fluorescence-based quantitative RT-PCR. E and G: IRS-2 and PI3K(p85) (control) protein expression levels were analyzed by immunoblotting, where an example immunoblot is 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, ** indicates a statistically significant inhibition with AdV-CAIN versus AdV-FLuc–infected control islets at 15 mmol/L glucose, and *** indicates a statistically significant increase in AdV-CNca–infected versus AdV-FLuc–infected control islets at the respective 3 and 15 mmol/L glucose concentrations (P ≤ 0.05; n ≥ 4) (D–G).
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC3198104&req=5

Figure 3: Glucose regulation of IRS-2 expression in β-cells is mediated by calcineurin. Isolated rat pancreatic islets were cultured overnight at normal 5.6 mmol/L glucose and then incubated at either basal 3 mmol/L or stimulatory 15 mmol/L glucose concentration for 6 h in presence or absence of calcineurin inhibitors FK506 (10 µmol/L) (A and B) or CsA (10 µmol/L) (C). A: IRS-2 and β-actin (internal reference control) mRNA expression levels were measured by real-time fluorescence-based quantitative RT-PCR. B and C: IRS-2 and PI3K(p85) (control) protein expression levels were analyzed by immunoblotting, where an example immunoblot (IB) is shown. Quantitative measurements are also shown as a mean ± SE above basal 3 mmol/L glucose control, where * indicates a statistically significant increase at 15 mmol/L glucose and ** indicates a statistically significant difference at 15 mmol/L glucose in the presence of FK506 or CsA versus control (P ≤ 0.05; n ≥ 4) (A–C). Isolated rat pancreatic islets were also infected with a recombinant AdV-CAIN (D and E), AdV-CNca (F and G), or 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. D and F: IRS-2 and β-actin (internal reference control) mRNA expression levels were measured by real-time fluorescence-based quantitative RT-PCR. E and G: IRS-2 and PI3K(p85) (control) protein expression levels were analyzed by immunoblotting, where an example immunoblot is 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, ** indicates a statistically significant inhibition with AdV-CAIN versus AdV-FLuc–infected control islets at 15 mmol/L glucose, and *** indicates a statistically significant increase in AdV-CNca–infected versus AdV-FLuc–infected control islets at the respective 3 and 15 mmol/L glucose concentrations (P ≤ 0.05; n ≥ 4) (D–G).

Mentions: A potential downstream target that responds to a rise in cytosolic [Ca2+]i in β-cells is the Ca2+/calmodulin-dependent phosphoprotein phosphatase-2B (or calcineurin) (24,25). To assess whether calcineurin was involved in specific glucose regulation of IRS-2 expression, we used two immunosuppressive drugs, FK506 (also named tacrolimus) and CsA that specifically inhibit calcineurin activity (26). In the presence of FK506, glucose-induced IRS-2 mRNA and protein levels were significantly inhibited by ∼90% in rat islets (P ≤ 0.05) (Fig. 3A and B). Likewise, CsA blocked glucose-induced IRS-2 protein levels compared with control rat islets (P ≤ 0.05) (Fig. 2C). It is noteworthy that neither FK506 nor CsA significantly affected islet IRS-2 mRNA or protein expression levels at basal 3 mmol/L glucose (Fig. 3A–C), indicating that calcineurin only mediated glucose-induced regulation of IRS-2 expression in islet β-cells. An alternative, more specific, inhibition of calcineurin was examined using recombinant adenovirus (AdV-CAIN), which expresses an endogenous peptide inhibitor of calcineurin activity (CAIN) (25,27). In AdV-CAIN–infected rat islets, glucose-induced IRS-2 mRNA and protein expression were significantly inhibited ≥90% (P ≤ 0.05) compared with control islets infected with a recombinant adenovirus expressing Firefly luciferase (AdV-FLuc) (Fig. 3D and E). There was no apparent effect on IRS-2 mRNA or protein expression by inhibiting calcineurin with AdV-CAIN at basal 3 mmol/L glucose (Fig. 3D and E). Islets were also infected with a recombinant adenovirus expressing a constitutively activated variant of calcineurin (AdV-CNCa). In AdV-CNca–infected islets there was a significant increase in IRS-2 mRNA and protein expression at basal 3 mmol/L glucose that was further augmented at a stimulatory 15 mmol/L glucose concentration compared with AdV-FLuc–infected control islets (Fig. 3F and G). Collectively, these data show that calcineurin is required to control glucose-induced IRS-2 expression in rat islets.


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 mediated by calcineurin. Isolated rat pancreatic islets were cultured overnight at normal 5.6 mmol/L glucose and then incubated at either basal 3 mmol/L or stimulatory 15 mmol/L glucose concentration for 6 h in presence or absence of calcineurin inhibitors FK506 (10 µmol/L) (A and B) or CsA (10 µmol/L) (C). A: IRS-2 and β-actin (internal reference control) mRNA expression levels were measured by real-time fluorescence-based quantitative RT-PCR. B and C: IRS-2 and PI3K(p85) (control) protein expression levels were analyzed by immunoblotting, where an example immunoblot (IB) is shown. Quantitative measurements are also shown as a mean ± SE above basal 3 mmol/L glucose control, where * indicates a statistically significant increase at 15 mmol/L glucose and ** indicates a statistically significant difference at 15 mmol/L glucose in the presence of FK506 or CsA versus control (P ≤ 0.05; n ≥ 4) (A–C). Isolated rat pancreatic islets were also infected with a recombinant AdV-CAIN (D and E), AdV-CNca (F and G), or 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. D and F: IRS-2 and β-actin (internal reference control) mRNA expression levels were measured by real-time fluorescence-based quantitative RT-PCR. E and G: IRS-2 and PI3K(p85) (control) protein expression levels were analyzed by immunoblotting, where an example immunoblot is 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, ** indicates a statistically significant inhibition with AdV-CAIN versus AdV-FLuc–infected control islets at 15 mmol/L glucose, and *** indicates a statistically significant increase in AdV-CNca–infected versus AdV-FLuc–infected control islets at the respective 3 and 15 mmol/L glucose concentrations (P ≤ 0.05; n ≥ 4) (D–G).
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

Figure 3: Glucose regulation of IRS-2 expression in β-cells is mediated by calcineurin. Isolated rat pancreatic islets were cultured overnight at normal 5.6 mmol/L glucose and then incubated at either basal 3 mmol/L or stimulatory 15 mmol/L glucose concentration for 6 h in presence or absence of calcineurin inhibitors FK506 (10 µmol/L) (A and B) or CsA (10 µmol/L) (C). A: IRS-2 and β-actin (internal reference control) mRNA expression levels were measured by real-time fluorescence-based quantitative RT-PCR. B and C: IRS-2 and PI3K(p85) (control) protein expression levels were analyzed by immunoblotting, where an example immunoblot (IB) is shown. Quantitative measurements are also shown as a mean ± SE above basal 3 mmol/L glucose control, where * indicates a statistically significant increase at 15 mmol/L glucose and ** indicates a statistically significant difference at 15 mmol/L glucose in the presence of FK506 or CsA versus control (P ≤ 0.05; n ≥ 4) (A–C). Isolated rat pancreatic islets were also infected with a recombinant AdV-CAIN (D and E), AdV-CNca (F and G), or 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. D and F: IRS-2 and β-actin (internal reference control) mRNA expression levels were measured by real-time fluorescence-based quantitative RT-PCR. E and G: IRS-2 and PI3K(p85) (control) protein expression levels were analyzed by immunoblotting, where an example immunoblot is 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, ** indicates a statistically significant inhibition with AdV-CAIN versus AdV-FLuc–infected control islets at 15 mmol/L glucose, and *** indicates a statistically significant increase in AdV-CNca–infected versus AdV-FLuc–infected control islets at the respective 3 and 15 mmol/L glucose concentrations (P ≤ 0.05; n ≥ 4) (D–G).
Mentions: A potential downstream target that responds to a rise in cytosolic [Ca2+]i in β-cells is the Ca2+/calmodulin-dependent phosphoprotein phosphatase-2B (or calcineurin) (24,25). To assess whether calcineurin was involved in specific glucose regulation of IRS-2 expression, we used two immunosuppressive drugs, FK506 (also named tacrolimus) and CsA that specifically inhibit calcineurin activity (26). In the presence of FK506, glucose-induced IRS-2 mRNA and protein levels were significantly inhibited by ∼90% in rat islets (P ≤ 0.05) (Fig. 3A and B). Likewise, CsA blocked glucose-induced IRS-2 protein levels compared with control rat islets (P ≤ 0.05) (Fig. 2C). It is noteworthy that neither FK506 nor CsA significantly affected islet IRS-2 mRNA or protein expression levels at basal 3 mmol/L glucose (Fig. 3A–C), indicating that calcineurin only mediated glucose-induced regulation of IRS-2 expression in islet β-cells. An alternative, more specific, inhibition of calcineurin was examined using recombinant adenovirus (AdV-CAIN), which expresses an endogenous peptide inhibitor of calcineurin activity (CAIN) (25,27). In AdV-CAIN–infected rat islets, glucose-induced IRS-2 mRNA and protein expression were significantly inhibited ≥90% (P ≤ 0.05) compared with control islets infected with a recombinant adenovirus expressing Firefly luciferase (AdV-FLuc) (Fig. 3D and E). There was no apparent effect on IRS-2 mRNA or protein expression by inhibiting calcineurin with AdV-CAIN at basal 3 mmol/L glucose (Fig. 3D and E). Islets were also infected with a recombinant adenovirus expressing a constitutively activated variant of calcineurin (AdV-CNCa). In AdV-CNca–infected islets there was a significant increase in IRS-2 mRNA and protein expression at basal 3 mmol/L glucose that was further augmented at a stimulatory 15 mmol/L glucose concentration compared with AdV-FLuc–infected control islets (Fig. 3F and G). Collectively, these data show that calcineurin is required to control glucose-induced IRS-2 expression in rat islets.

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