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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 inactivation in adult beta cell reduces insulin expression independent of glucotoxicityA. The mRNA expression of Glis3, Ins1, Ins2, Glut2 and Gck and islet enriched transcription factors (Pdx1, Nkx6-1, Nkx2-2, Ngn3, Neurod1, Isl1 and MafA) in handpicked islets of Glis3fl/fl/Pdx1CreERT+ mice 10 days after TAM or vehicle administration (n = 6). Results were analyzed by student's t-test and presented as the mean ± S.E. **p < 0.01, ***p < 0.001, versus vehicle-treated mice.B,C. Blood glucose (B) and plasma insulin (C) in Glis3fl/fl/Pdx1CreERT+ mice 10 days after TAM or vehicle administration.D. Immunoreactive Ins and Gcg in Glis3fl/fl/Pdx1CreERT+ mice 10 days after TAM or vehicle administration. Scale bar, 20 µm.E. TUNEL+ beta cells, normalized to total islet cells in Glis3fl/fl/Pdx1CreERT+ mice 10 days after TAM or vehicle administration.F. Representative images and percentage of TUNEL+ cells in INS-1 derived 832/13 cells transfected with control siRNA or Glis3 siRNA for 48 h. Scale bar, 20 µm.G. PCNA+ beta cells, normalized to total islet cells, in the pancreas of 10 days TAM or vehicle-treated Glis3fl/fl/Pdx1CreERT+ mice.H–J. Glucose-induced insulin secretion (GSIS) (H), islet insulin content (I) and the relative GSIS/islet insulin content (J) in Glis3fl/fl/Pdx1CreERT+ mice treated with TAM (newly diabetic) or vehicle (n = 6). **p < 0.01, ***p < 0.001 versus vehicle-treated mice.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 Glis3RE region (−266 MIP) of mouse insulin 2 promoter, the distal region (−6000 MIP) was used as a negative control. Results were analyzed by student's t-test and presented as the mean ± S.E. from three independent experiments. *p = 0.03 versus IgG control.
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fig07: Glis3 inactivation in adult beta cell reduces insulin expression independent of glucotoxicityA. The mRNA expression of Glis3, Ins1, Ins2, Glut2 and Gck and islet enriched transcription factors (Pdx1, Nkx6-1, Nkx2-2, Ngn3, Neurod1, Isl1 and MafA) in handpicked islets of Glis3fl/fl/Pdx1CreERT+ mice 10 days after TAM or vehicle administration (n = 6). Results were analyzed by student's t-test and presented as the mean ± S.E. **p < 0.01, ***p < 0.001, versus vehicle-treated mice.B,C. Blood glucose (B) and plasma insulin (C) in Glis3fl/fl/Pdx1CreERT+ mice 10 days after TAM or vehicle administration.D. Immunoreactive Ins and Gcg in Glis3fl/fl/Pdx1CreERT+ mice 10 days after TAM or vehicle administration. Scale bar, 20 µm.E. TUNEL+ beta cells, normalized to total islet cells in Glis3fl/fl/Pdx1CreERT+ mice 10 days after TAM or vehicle administration.F. Representative images and percentage of TUNEL+ cells in INS-1 derived 832/13 cells transfected with control siRNA or Glis3 siRNA for 48 h. Scale bar, 20 µm.G. PCNA+ beta cells, normalized to total islet cells, in the pancreas of 10 days TAM or vehicle-treated Glis3fl/fl/Pdx1CreERT+ mice.H–J. Glucose-induced insulin secretion (GSIS) (H), islet insulin content (I) and the relative GSIS/islet insulin content (J) in Glis3fl/fl/Pdx1CreERT+ mice treated with TAM (newly diabetic) or vehicle (n = 6). **p < 0.01, ***p < 0.001 versus vehicle-treated mice.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 Glis3RE region (−266 MIP) of mouse insulin 2 promoter, the distal region (−6000 MIP) was used as a negative control. Results were analyzed by student's t-test and presented as the mean ± S.E. from three independent experiments. *p = 0.03 versus IgG control.

Mentions: As the glycaemic control in the insulin implanted TAM-treated Glis3fl/fl/Pdx1CreERT+ mice was less than perfect, it was not possible to rule out glucotoxicity-related insulin reduction and beta cell apoptosis in these mice. To further explore the role of Glis3 in the adult pancreas, we collected pancreata from TAM-treated Glis3fl/fl/Pdx1CreERT+ mice 10 days after TAM administration. Given that GLIS3 antibodies for are not available for immunocytochemical staining, we have determined the efficiency of Glis3 deletion in isolated islets by q-RT-PCR, which showed that Glis3 mRNA was reduced over 90% in the islets 10 days after TAM administration (Fig 7A). It should be noted that qRT-PCR underestimates Pdx1-Cre mediated Glis3 silencing in beta cells because Glis3 mRNA is also present in the non-beta islet cells (Senee et al, 2006). We further confirmed the efficiency of Glis3 deletion in the pancreatic islets by in situ hybridization 10 days after TAM or vehicle administration (Supporting Information Fig S5). At this early time point, the mice still displayed euglycaemia (Fig 7B) and a tendency towards glucose intolerance (Supporting Information Fig S6), in the presence of normal plasma insulin (Fig 7C). We observed that the mRNA expression of Pdx1, a mature beta cell marker (Jonsson et al, 1994; Offield et al, 1996; Ohlsson et al, 1993), was unchanged in the islets isolated from the two groups (Fig 7A). Importantly, the level of insulin mRNA (Fig 7A) and immunoreactive insulin (Fig 7D) was significantly decreased at this early stage of TAM treatment as compared with vehicle treatment in Glis3fl/fl/Pdx1CreERT+ mice. However, the number of apoptotic islet cells reflected by TUNEL assay was not different between the two groups (Fig 7E). To corroborate the absence of a difference in the very low percentage of TUNEL+ cells in the pancreatic islets of TAM and vehicle-treated Glis3fl/fl/Pdx1CreERT+ mice, we next performed TUNEL assay in Glis3-knockdown INS-1 derived 832/13 cells and again observed no difference in TUNEL-positive cells between control siRNA and Glis3 siRNA transfected cells (Fig 7F). Therefore, impairment in insulin expression at the mRNA and protein levels occurs early in TAM-treated Glis3fl/fl/Pdx1CreERT+ mice, an effect that precedes the onset of hyperglycaemia and the apoptosis that the latter induced.


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 inactivation in adult beta cell reduces insulin expression independent of glucotoxicityA. The mRNA expression of Glis3, Ins1, Ins2, Glut2 and Gck and islet enriched transcription factors (Pdx1, Nkx6-1, Nkx2-2, Ngn3, Neurod1, Isl1 and MafA) in handpicked islets of Glis3fl/fl/Pdx1CreERT+ mice 10 days after TAM or vehicle administration (n = 6). Results were analyzed by student's t-test and presented as the mean ± S.E. **p < 0.01, ***p < 0.001, versus vehicle-treated mice.B,C. Blood glucose (B) and plasma insulin (C) in Glis3fl/fl/Pdx1CreERT+ mice 10 days after TAM or vehicle administration.D. Immunoreactive Ins and Gcg in Glis3fl/fl/Pdx1CreERT+ mice 10 days after TAM or vehicle administration. Scale bar, 20 µm.E. TUNEL+ beta cells, normalized to total islet cells in Glis3fl/fl/Pdx1CreERT+ mice 10 days after TAM or vehicle administration.F. Representative images and percentage of TUNEL+ cells in INS-1 derived 832/13 cells transfected with control siRNA or Glis3 siRNA for 48 h. Scale bar, 20 µm.G. PCNA+ beta cells, normalized to total islet cells, in the pancreas of 10 days TAM or vehicle-treated Glis3fl/fl/Pdx1CreERT+ mice.H–J. Glucose-induced insulin secretion (GSIS) (H), islet insulin content (I) and the relative GSIS/islet insulin content (J) in Glis3fl/fl/Pdx1CreERT+ mice treated with TAM (newly diabetic) or vehicle (n = 6). **p < 0.01, ***p < 0.001 versus vehicle-treated mice.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 Glis3RE region (−266 MIP) of mouse insulin 2 promoter, the distal region (−6000 MIP) was used as a negative control. Results were analyzed by student's t-test and presented as the mean ± S.E. from three independent experiments. *p = 0.03 versus IgG control.
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fig07: Glis3 inactivation in adult beta cell reduces insulin expression independent of glucotoxicityA. The mRNA expression of Glis3, Ins1, Ins2, Glut2 and Gck and islet enriched transcription factors (Pdx1, Nkx6-1, Nkx2-2, Ngn3, Neurod1, Isl1 and MafA) in handpicked islets of Glis3fl/fl/Pdx1CreERT+ mice 10 days after TAM or vehicle administration (n = 6). Results were analyzed by student's t-test and presented as the mean ± S.E. **p < 0.01, ***p < 0.001, versus vehicle-treated mice.B,C. Blood glucose (B) and plasma insulin (C) in Glis3fl/fl/Pdx1CreERT+ mice 10 days after TAM or vehicle administration.D. Immunoreactive Ins and Gcg in Glis3fl/fl/Pdx1CreERT+ mice 10 days after TAM or vehicle administration. Scale bar, 20 µm.E. TUNEL+ beta cells, normalized to total islet cells in Glis3fl/fl/Pdx1CreERT+ mice 10 days after TAM or vehicle administration.F. Representative images and percentage of TUNEL+ cells in INS-1 derived 832/13 cells transfected with control siRNA or Glis3 siRNA for 48 h. Scale bar, 20 µm.G. PCNA+ beta cells, normalized to total islet cells, in the pancreas of 10 days TAM or vehicle-treated Glis3fl/fl/Pdx1CreERT+ mice.H–J. Glucose-induced insulin secretion (GSIS) (H), islet insulin content (I) and the relative GSIS/islet insulin content (J) in Glis3fl/fl/Pdx1CreERT+ mice treated with TAM (newly diabetic) or vehicle (n = 6). **p < 0.01, ***p < 0.001 versus vehicle-treated mice.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 Glis3RE region (−266 MIP) of mouse insulin 2 promoter, the distal region (−6000 MIP) was used as a negative control. Results were analyzed by student's t-test and presented as the mean ± S.E. from three independent experiments. *p = 0.03 versus IgG control.
Mentions: As the glycaemic control in the insulin implanted TAM-treated Glis3fl/fl/Pdx1CreERT+ mice was less than perfect, it was not possible to rule out glucotoxicity-related insulin reduction and beta cell apoptosis in these mice. To further explore the role of Glis3 in the adult pancreas, we collected pancreata from TAM-treated Glis3fl/fl/Pdx1CreERT+ mice 10 days after TAM administration. Given that GLIS3 antibodies for are not available for immunocytochemical staining, we have determined the efficiency of Glis3 deletion in isolated islets by q-RT-PCR, which showed that Glis3 mRNA was reduced over 90% in the islets 10 days after TAM administration (Fig 7A). It should be noted that qRT-PCR underestimates Pdx1-Cre mediated Glis3 silencing in beta cells because Glis3 mRNA is also present in the non-beta islet cells (Senee et al, 2006). We further confirmed the efficiency of Glis3 deletion in the pancreatic islets by in situ hybridization 10 days after TAM or vehicle administration (Supporting Information Fig S5). At this early time point, the mice still displayed euglycaemia (Fig 7B) and a tendency towards glucose intolerance (Supporting Information Fig S6), in the presence of normal plasma insulin (Fig 7C). We observed that the mRNA expression of Pdx1, a mature beta cell marker (Jonsson et al, 1994; Offield et al, 1996; Ohlsson et al, 1993), was unchanged in the islets isolated from the two groups (Fig 7A). Importantly, the level of insulin mRNA (Fig 7A) and immunoreactive insulin (Fig 7D) was significantly decreased at this early stage of TAM treatment as compared with vehicle treatment in Glis3fl/fl/Pdx1CreERT+ mice. However, the number of apoptotic islet cells reflected by TUNEL assay was not different between the two groups (Fig 7E). To corroborate the absence of a difference in the very low percentage of TUNEL+ cells in the pancreatic islets of TAM and vehicle-treated Glis3fl/fl/Pdx1CreERT+ mice, we next performed TUNEL assay in Glis3-knockdown INS-1 derived 832/13 cells and again observed no difference in TUNEL-positive cells between control siRNA and Glis3 siRNA transfected cells (Fig 7F). Therefore, impairment in insulin expression at the mRNA and protein levels occurs early in TAM-treated Glis3fl/fl/Pdx1CreERT+ mice, an effect that precedes the onset of hyperglycaemia and the apoptosis that the latter induced.

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.

Show MeSH
Related in: MedlinePlus