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Sustained expression of the transcription factor GLIS3 is required for normal beta cell function in adults.

Yang Y, Chang BH, Chan L - EMBO Mol Med (2012)

Bottom Line: Genome-wide association studies identified GLIS3 as a susceptibility locus for type 1 and type 2 diabetes.GLIS3 controls beta cell proliferation in response to high-fat feeding at least partly by regulating Ccnd2 transcription.We conclude that normal Glis3 expression is required for pancreatic beta cell function and mass maintenance during adulthood, which impairment leads to diabetes in adults.

View Article: PubMed Central - PubMed

Affiliation: Division of Diabetes, Endocrinology and Metabolism, Department of Medicine, Diabetes and Endocrinology Research Center, Baylor College of Medicine, Houston, TX, USA.

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Glis3 is required for beta cell proliferation and directly regulates Ccnd2 transcriptionA. The mRNA expression of Ccnd1, Ccnd2, Ccnd3, Cdk4, Cdkn1a, Cdkn1b, Cdkn2a and Cdkn2c in the islets of Glis3+/+ and Glis3+/− mice fed with a CD (n = 5) or HFD (n = 6) for 20 weeks. *p < 0.05, versus Glis3+/+ CD. #p < 0.05, ###p < 0.001, versus Glis3+/+ HFD.B. The mRNA expression of Ccnd1, Ccnd2 and Ccnd3 in the islets of Glis3fl/fl/Pdx1CreERT+ mice treated with TAM (newly diabetic, plasma glucose just reaching 250 mg/dl) or vehicle. **p = 0.004 versus vehicle-treated mice. N = 6 for each group.C. The mRNA expression of Glis3 and Ccnd2 in INS-1 derived 832/13 cells transfected with control siRNA or Glis3 siRNA for 48 h. **p = 0.003, ***p = 0.000007, versus control siRNA group. r: rat.D,E. The mRNA expression of Glis3(D) and Ccnd2(E) in INS-1 derived 832/13 cells stably overexpressing c-Myc-YFP or c-Myc-Glis3. ***p = 0.00002 and 0.0007 versus c-Myc-YFP group, respectively. m: mouse, r: rat.F–H. Representative Western blots of CCND2 and densitometri ratios of CCND2/alpha tubulin in the islets of Glis3+/+ and Glis3+/− mice fed with a CD or HFD for 20 weeks (F) (***p = 0.00002 versus Glis3+/+ CD; ###p = 0.00008 versus Glis3+/+ HFD), and in the islets of Glis3fl/fl/Pdx1CreERT+ mice treated with TAM (newly diabetic) or vehicle (G) (***p < 0.0001 versus vehicle-treated group), as well as in INS-1 derived 832/13 cells transfected with control siRNA or Glis3 siRNA for 48 h (H) (n = 4, ***p = 0.0004 versus control siRNA group). Alpha tubulin was used as an internal control.I. Alignment of the 15-bp sequences (Glis3RE) located at −3670, −1095, and −160 in the 10-Kb mouse Ccnd2 gene promoter. The Glis3RE in the mouse Ins2 promoter was used as a comparison.J. EMSA using an in vitro translated GLIS3-ZFD peptide was performed with biotin-labeled probes containing putative Glis3REs sequences at −3670, −1095, and −160 in mouse Ccnd2 gene promoter. Five- or fifty fold corresponding non-biotinylated Glis3REs were added as competitors. The specific band was indicated by an arrow.K. ChIP assays with anti-GLIS3 or IgG control were performed in the islets of wild type C57BL/6 mice. Immunoprecipitated DNA was purified and analyzed by qPCR using primers specifically spanning the putative Glis3RE region at −3690, −1095 and −160 sites and a control fragment located at −9926 of mouse Ccnd2 promoter. Results were analyzed by student's t-test and presented as the mean ± S.E. from three independent experiments. *p = 0.048, **p = 0.007, versus IgG control.
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fig03: Glis3 is required for beta cell proliferation and directly regulates Ccnd2 transcriptionA. The mRNA expression of Ccnd1, Ccnd2, Ccnd3, Cdk4, Cdkn1a, Cdkn1b, Cdkn2a and Cdkn2c in the islets of Glis3+/+ and Glis3+/− mice fed with a CD (n = 5) or HFD (n = 6) for 20 weeks. *p < 0.05, versus Glis3+/+ CD. #p < 0.05, ###p < 0.001, versus Glis3+/+ HFD.B. The mRNA expression of Ccnd1, Ccnd2 and Ccnd3 in the islets of Glis3fl/fl/Pdx1CreERT+ mice treated with TAM (newly diabetic, plasma glucose just reaching 250 mg/dl) or vehicle. **p = 0.004 versus vehicle-treated mice. N = 6 for each group.C. The mRNA expression of Glis3 and Ccnd2 in INS-1 derived 832/13 cells transfected with control siRNA or Glis3 siRNA for 48 h. **p = 0.003, ***p = 0.000007, versus control siRNA group. r: rat.D,E. The mRNA expression of Glis3(D) and Ccnd2(E) in INS-1 derived 832/13 cells stably overexpressing c-Myc-YFP or c-Myc-Glis3. ***p = 0.00002 and 0.0007 versus c-Myc-YFP group, respectively. m: mouse, r: rat.F–H. Representative Western blots of CCND2 and densitometri ratios of CCND2/alpha tubulin in the islets of Glis3+/+ and Glis3+/− mice fed with a CD or HFD for 20 weeks (F) (***p = 0.00002 versus Glis3+/+ CD; ###p = 0.00008 versus Glis3+/+ HFD), and in the islets of Glis3fl/fl/Pdx1CreERT+ mice treated with TAM (newly diabetic) or vehicle (G) (***p < 0.0001 versus vehicle-treated group), as well as in INS-1 derived 832/13 cells transfected with control siRNA or Glis3 siRNA for 48 h (H) (n = 4, ***p = 0.0004 versus control siRNA group). Alpha tubulin was used as an internal control.I. Alignment of the 15-bp sequences (Glis3RE) located at −3670, −1095, and −160 in the 10-Kb mouse Ccnd2 gene promoter. The Glis3RE in the mouse Ins2 promoter was used as a comparison.J. EMSA using an in vitro translated GLIS3-ZFD peptide was performed with biotin-labeled probes containing putative Glis3REs sequences at −3670, −1095, and −160 in mouse Ccnd2 gene promoter. Five- or fifty fold corresponding non-biotinylated Glis3REs were added as competitors. The specific band was indicated by an arrow.K. ChIP assays with anti-GLIS3 or IgG control were performed in the islets of wild type C57BL/6 mice. Immunoprecipitated DNA was purified and analyzed by qPCR using primers specifically spanning the putative Glis3RE region at −3690, −1095 and −160 sites and a control fragment located at −9926 of mouse Ccnd2 promoter. Results were analyzed by student's t-test and presented as the mean ± S.E. from three independent experiments. *p = 0.048, **p = 0.007, versus IgG control.

Mentions: Pancreatic beta cell mass expansion is a normal response to an increased demand for insulin, as occurs when mice are fed a HFD, total beta cell mass being modulated by cell proliferation and/or apoptosis (Ackermann & Gannon, 2007; Sachdeva & Stoffers, 2009). As we detected no difference in the number of apoptotic beta cells in Glis3+/+ and Glis3+/− islets (Fig 2I), we examined beta cell proliferation as reflected by proliferating cell nuclear antigen (PCNA) staining and found that HFD feeding led to a significant increase in the number of PCNA+ beta cells in the islets of Glis3+/+ mice but no change in Glis3+/− mice (Fig 2J). D-type cyclins, particularly cyclin D2 (Ccnd2) and D1, are essential for maintaining postnatal pancreatic beta cell mass (Georgia & Bhushan, 2004; Kushner et al, 2005). We therefore examined mRNA expression of D-type cyclins and other cell cycle-related genes in the isolated islets of these mice. qRT-PCR showed that the expression of Ccnd2, a predominant D-type cyclin in pancreatic beta cells (Georgia & Bhushan, 2004; Kushner et al, 2005) which was reported to be crucial for beta cell mass expansion (Georgia et al, 2010), was downregulated in the islets of Glis3+/− mice compared to Glis3+/+ mice fed the same diets (Fig 3A); the expression of cyclin-dependent kinase inhibitor 2a (Cdkn2a) was upregulated in the islets of Glis3+/− mice compared to Glis3+/+ mice fed regular chow; no difference in the expression of Ccnd1, Ccnd3, cyclin-dependent kinase 4 (Cdk4), Cdkn1a, Cdkn1b or Cdkn2c was detected (Fig 3A). We further confirmed by qRT-PCR that the mRNA expression of Ccnd2 was downregulated in the islets of beta cell-specific Glis3-deficient mice (Fig 3B) and in Glis3-knockdown 832/13 cells (Fig 3C), while it was upregulated in Glis3-overexpressing 832/13 cells (Fig 3D and E). To corroborate these findings at the mRNA level, we found that at the protein level CCND2 was lower in the islets of Glis3+/− mice fed either a chow or a HFD (Fig 3F) and in the islets of beta cell-specific Glis3-deficient mice (Fig 3G) as well as in Glis3-knockdown 832/13 cells (Fig 3H).


Sustained expression of the transcription factor GLIS3 is required for normal beta cell function in adults.

Yang Y, Chang BH, Chan L - EMBO Mol Med (2012)

Glis3 is required for beta cell proliferation and directly regulates Ccnd2 transcriptionA. The mRNA expression of Ccnd1, Ccnd2, Ccnd3, Cdk4, Cdkn1a, Cdkn1b, Cdkn2a and Cdkn2c in the islets of Glis3+/+ and Glis3+/− mice fed with a CD (n = 5) or HFD (n = 6) for 20 weeks. *p < 0.05, versus Glis3+/+ CD. #p < 0.05, ###p < 0.001, versus Glis3+/+ HFD.B. The mRNA expression of Ccnd1, Ccnd2 and Ccnd3 in the islets of Glis3fl/fl/Pdx1CreERT+ mice treated with TAM (newly diabetic, plasma glucose just reaching 250 mg/dl) or vehicle. **p = 0.004 versus vehicle-treated mice. N = 6 for each group.C. The mRNA expression of Glis3 and Ccnd2 in INS-1 derived 832/13 cells transfected with control siRNA or Glis3 siRNA for 48 h. **p = 0.003, ***p = 0.000007, versus control siRNA group. r: rat.D,E. The mRNA expression of Glis3(D) and Ccnd2(E) in INS-1 derived 832/13 cells stably overexpressing c-Myc-YFP or c-Myc-Glis3. ***p = 0.00002 and 0.0007 versus c-Myc-YFP group, respectively. m: mouse, r: rat.F–H. Representative Western blots of CCND2 and densitometri ratios of CCND2/alpha tubulin in the islets of Glis3+/+ and Glis3+/− mice fed with a CD or HFD for 20 weeks (F) (***p = 0.00002 versus Glis3+/+ CD; ###p = 0.00008 versus Glis3+/+ HFD), and in the islets of Glis3fl/fl/Pdx1CreERT+ mice treated with TAM (newly diabetic) or vehicle (G) (***p < 0.0001 versus vehicle-treated group), as well as in INS-1 derived 832/13 cells transfected with control siRNA or Glis3 siRNA for 48 h (H) (n = 4, ***p = 0.0004 versus control siRNA group). Alpha tubulin was used as an internal control.I. Alignment of the 15-bp sequences (Glis3RE) located at −3670, −1095, and −160 in the 10-Kb mouse Ccnd2 gene promoter. The Glis3RE in the mouse Ins2 promoter was used as a comparison.J. EMSA using an in vitro translated GLIS3-ZFD peptide was performed with biotin-labeled probes containing putative Glis3REs sequences at −3670, −1095, and −160 in mouse Ccnd2 gene promoter. Five- or fifty fold corresponding non-biotinylated Glis3REs were added as competitors. The specific band was indicated by an arrow.K. ChIP assays with anti-GLIS3 or IgG control were performed in the islets of wild type C57BL/6 mice. Immunoprecipitated DNA was purified and analyzed by qPCR using primers specifically spanning the putative Glis3RE region at −3690, −1095 and −160 sites and a control fragment located at −9926 of mouse Ccnd2 promoter. Results were analyzed by student's t-test and presented as the mean ± S.E. from three independent experiments. *p = 0.048, **p = 0.007, versus IgG control.
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fig03: Glis3 is required for beta cell proliferation and directly regulates Ccnd2 transcriptionA. The mRNA expression of Ccnd1, Ccnd2, Ccnd3, Cdk4, Cdkn1a, Cdkn1b, Cdkn2a and Cdkn2c in the islets of Glis3+/+ and Glis3+/− mice fed with a CD (n = 5) or HFD (n = 6) for 20 weeks. *p < 0.05, versus Glis3+/+ CD. #p < 0.05, ###p < 0.001, versus Glis3+/+ HFD.B. The mRNA expression of Ccnd1, Ccnd2 and Ccnd3 in the islets of Glis3fl/fl/Pdx1CreERT+ mice treated with TAM (newly diabetic, plasma glucose just reaching 250 mg/dl) or vehicle. **p = 0.004 versus vehicle-treated mice. N = 6 for each group.C. The mRNA expression of Glis3 and Ccnd2 in INS-1 derived 832/13 cells transfected with control siRNA or Glis3 siRNA for 48 h. **p = 0.003, ***p = 0.000007, versus control siRNA group. r: rat.D,E. The mRNA expression of Glis3(D) and Ccnd2(E) in INS-1 derived 832/13 cells stably overexpressing c-Myc-YFP or c-Myc-Glis3. ***p = 0.00002 and 0.0007 versus c-Myc-YFP group, respectively. m: mouse, r: rat.F–H. Representative Western blots of CCND2 and densitometri ratios of CCND2/alpha tubulin in the islets of Glis3+/+ and Glis3+/− mice fed with a CD or HFD for 20 weeks (F) (***p = 0.00002 versus Glis3+/+ CD; ###p = 0.00008 versus Glis3+/+ HFD), and in the islets of Glis3fl/fl/Pdx1CreERT+ mice treated with TAM (newly diabetic) or vehicle (G) (***p < 0.0001 versus vehicle-treated group), as well as in INS-1 derived 832/13 cells transfected with control siRNA or Glis3 siRNA for 48 h (H) (n = 4, ***p = 0.0004 versus control siRNA group). Alpha tubulin was used as an internal control.I. Alignment of the 15-bp sequences (Glis3RE) located at −3670, −1095, and −160 in the 10-Kb mouse Ccnd2 gene promoter. The Glis3RE in the mouse Ins2 promoter was used as a comparison.J. EMSA using an in vitro translated GLIS3-ZFD peptide was performed with biotin-labeled probes containing putative Glis3REs sequences at −3670, −1095, and −160 in mouse Ccnd2 gene promoter. Five- or fifty fold corresponding non-biotinylated Glis3REs were added as competitors. The specific band was indicated by an arrow.K. ChIP assays with anti-GLIS3 or IgG control were performed in the islets of wild type C57BL/6 mice. Immunoprecipitated DNA was purified and analyzed by qPCR using primers specifically spanning the putative Glis3RE region at −3690, −1095 and −160 sites and a control fragment located at −9926 of mouse Ccnd2 promoter. Results were analyzed by student's t-test and presented as the mean ± S.E. from three independent experiments. *p = 0.048, **p = 0.007, versus IgG control.
Mentions: Pancreatic beta cell mass expansion is a normal response to an increased demand for insulin, as occurs when mice are fed a HFD, total beta cell mass being modulated by cell proliferation and/or apoptosis (Ackermann & Gannon, 2007; Sachdeva & Stoffers, 2009). As we detected no difference in the number of apoptotic beta cells in Glis3+/+ and Glis3+/− islets (Fig 2I), we examined beta cell proliferation as reflected by proliferating cell nuclear antigen (PCNA) staining and found that HFD feeding led to a significant increase in the number of PCNA+ beta cells in the islets of Glis3+/+ mice but no change in Glis3+/− mice (Fig 2J). D-type cyclins, particularly cyclin D2 (Ccnd2) and D1, are essential for maintaining postnatal pancreatic beta cell mass (Georgia & Bhushan, 2004; Kushner et al, 2005). We therefore examined mRNA expression of D-type cyclins and other cell cycle-related genes in the isolated islets of these mice. qRT-PCR showed that the expression of Ccnd2, a predominant D-type cyclin in pancreatic beta cells (Georgia & Bhushan, 2004; Kushner et al, 2005) which was reported to be crucial for beta cell mass expansion (Georgia et al, 2010), was downregulated in the islets of Glis3+/− mice compared to Glis3+/+ mice fed the same diets (Fig 3A); the expression of cyclin-dependent kinase inhibitor 2a (Cdkn2a) was upregulated in the islets of Glis3+/− mice compared to Glis3+/+ mice fed regular chow; no difference in the expression of Ccnd1, Ccnd3, cyclin-dependent kinase 4 (Cdk4), Cdkn1a, Cdkn1b or Cdkn2c was detected (Fig 3A). We further confirmed by qRT-PCR that the mRNA expression of Ccnd2 was downregulated in the islets of beta cell-specific Glis3-deficient mice (Fig 3B) and in Glis3-knockdown 832/13 cells (Fig 3C), while it was upregulated in Glis3-overexpressing 832/13 cells (Fig 3D and E). To corroborate these findings at the mRNA level, we found that at the protein level CCND2 was lower in the islets of Glis3+/− mice fed either a chow or a HFD (Fig 3F) and in the islets of beta cell-specific Glis3-deficient mice (Fig 3G) as well as in Glis3-knockdown 832/13 cells (Fig 3H).

Bottom Line: Genome-wide association studies identified GLIS3 as a susceptibility locus for type 1 and type 2 diabetes.GLIS3 controls beta cell proliferation in response to high-fat feeding at least partly by regulating Ccnd2 transcription.We conclude that normal Glis3 expression is required for pancreatic beta cell function and mass maintenance during adulthood, which impairment leads to diabetes in adults.

View Article: PubMed Central - PubMed

Affiliation: Division of Diabetes, Endocrinology and Metabolism, Department of Medicine, Diabetes and Endocrinology Research Center, Baylor College of Medicine, Houston, TX, USA.

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Related in: MedlinePlus