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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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Specific glucose-induced regulation of IRS-2 expression in pancreatic islets, but not in hepatocytes in vivo. Normal C57Blk6/6J mice (aged 12 weeks) were fasted overnight and then subjected to an intraperitoneal glucose tolerance test (2 mg/g body wt) or using saline as a control as described (42). Pancreatic islets were isolated, and a liver biopsy was conducted at the 2- and 4-h time point and then subjected to immunoblot (IB) analysis for IRS-2 protein expression relative to PI3K(p85) as a loading control. A: Excursion in circulating glucose in the mice after a glucose (●) or saline (○) injection. A mean ± SE is shown (n ≥ 3). B and C: Example IB analyses of IRS-2 and PI3K(p85) in islets (B) and liver (C) from saline (S)- or glucose (G)-treated mice at 2 or 4 h are shown from two separate experiments. A quantification of a series of experiments is also depicted, where gray bars are S- and black bars are G-treated animals. Data are a mean ± SE (n = 3), where * indicates statistically significant difference (P ≤ 0.05) from the equivalent saline control.
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Figure 1: Specific glucose-induced regulation of IRS-2 expression in pancreatic islets, but not in hepatocytes in vivo. Normal C57Blk6/6J mice (aged 12 weeks) were fasted overnight and then subjected to an intraperitoneal glucose tolerance test (2 mg/g body wt) or using saline as a control as described (42). Pancreatic islets were isolated, and a liver biopsy was conducted at the 2- and 4-h time point and then subjected to immunoblot (IB) analysis for IRS-2 protein expression relative to PI3K(p85) as a loading control. A: Excursion in circulating glucose in the mice after a glucose (●) or saline (○) injection. A mean ± SE is shown (n ≥ 3). B and C: Example IB analyses of IRS-2 and PI3K(p85) in islets (B) and liver (C) from saline (S)- or glucose (G)-treated mice at 2 or 4 h are shown from two separate experiments. A quantification of a series of experiments is also depicted, where gray bars are S- and black bars are G-treated animals. Data are a mean ± SE (n = 3), where * indicates statistically significant difference (P ≤ 0.05) from the equivalent saline control.

Mentions: It has been shown previously that glucose can specifically increase IRS-2 expression in islet β-cells in vitro, predominately at the transcriptional level (10). Here, normal mice were subjected to an intraperitoneal glucose tolerance test using a dose of 2 mg glucose/g body wt or saline as a control where excursions in blood glucose were indicated (Fig. 1A). In islets isolated at 2 h, and more so at 4 h, after the introduction of glucose, IRS-2 protein levels were specifically increased compared with saline-injected controls (Fig. 1B), complementary to previous in vitro observations (10,11). However, in parallel experiments, no increase in IRS-2 expression was found in the liver (Fig. 1C), suggesting that glucose regulation of IRS-2 expression may be unique to pancreatic 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)

Specific glucose-induced regulation of IRS-2 expression in pancreatic islets, but not in hepatocytes in vivo. Normal C57Blk6/6J mice (aged 12 weeks) were fasted overnight and then subjected to an intraperitoneal glucose tolerance test (2 mg/g body wt) or using saline as a control as described (42). Pancreatic islets were isolated, and a liver biopsy was conducted at the 2- and 4-h time point and then subjected to immunoblot (IB) analysis for IRS-2 protein expression relative to PI3K(p85) as a loading control. A: Excursion in circulating glucose in the mice after a glucose (●) or saline (○) injection. A mean ± SE is shown (n ≥ 3). B and C: Example IB analyses of IRS-2 and PI3K(p85) in islets (B) and liver (C) from saline (S)- or glucose (G)-treated mice at 2 or 4 h are shown from two separate experiments. A quantification of a series of experiments is also depicted, where gray bars are S- and black bars are G-treated animals. Data are a mean ± SE (n = 3), where * indicates statistically significant difference (P ≤ 0.05) from the equivalent saline control.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

Figure 1: Specific glucose-induced regulation of IRS-2 expression in pancreatic islets, but not in hepatocytes in vivo. Normal C57Blk6/6J mice (aged 12 weeks) were fasted overnight and then subjected to an intraperitoneal glucose tolerance test (2 mg/g body wt) or using saline as a control as described (42). Pancreatic islets were isolated, and a liver biopsy was conducted at the 2- and 4-h time point and then subjected to immunoblot (IB) analysis for IRS-2 protein expression relative to PI3K(p85) as a loading control. A: Excursion in circulating glucose in the mice after a glucose (●) or saline (○) injection. A mean ± SE is shown (n ≥ 3). B and C: Example IB analyses of IRS-2 and PI3K(p85) in islets (B) and liver (C) from saline (S)- or glucose (G)-treated mice at 2 or 4 h are shown from two separate experiments. A quantification of a series of experiments is also depicted, where gray bars are S- and black bars are G-treated animals. Data are a mean ± SE (n = 3), where * indicates statistically significant difference (P ≤ 0.05) from the equivalent saline control.
Mentions: It has been shown previously that glucose can specifically increase IRS-2 expression in islet β-cells in vitro, predominately at the transcriptional level (10). Here, normal mice were subjected to an intraperitoneal glucose tolerance test using a dose of 2 mg glucose/g body wt or saline as a control where excursions in blood glucose were indicated (Fig. 1A). In islets isolated at 2 h, and more so at 4 h, after the introduction of glucose, IRS-2 protein levels were specifically increased compared with saline-injected controls (Fig. 1B), complementary to previous in vitro observations (10,11). However, in parallel experiments, no increase in IRS-2 expression was found in the liver (Fig. 1C), suggesting that glucose regulation of IRS-2 expression may be unique to pancreatic 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