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A mouse model for monitoring islet cell genesis and developing therapies for diabetes.

Shimajiri Y, Kosaka Y, Scheel DW, Lynn FC, Kishimoto N, Wang J, Zhao S, German MS - Dis Model Mech (2010)

Bottom Line: Treatment with the γ-secretase inhibitor DAPT, which blocks Notch signaling, enhanced SeAP secretion rates and increased the number of EGFP-expressing cells as assayed by fluorescence-activated cell sorting (FACS) and immunohistochemistry in cultured pancreases from embryos at embryonic day 11.5, but not in pancreases harvested 1 day later.By contrast, treatment with growth differentiation factor 11 (GDF11) reduced SeAP secretion rates.This model will be useful for studying signals involved in islet cell genesis in vivo and developing therapies that induce this process.

View Article: PubMed Central - PubMed

Affiliation: Diabetes Center, University of California San Francisco, San Francisco, CA 94143-0534, USA.

ABSTRACT
Transient expression of the transcription factor neurogenin-3 marks progenitor cells in the pancreas as they differentiate into islet cells. We developed a transgenic mouse line in which the surrogate markers secreted alkaline phosphatase (SeAP) and enhanced green florescent protein (EGFP) can be used to monitor neurogenin-3 expression, and thus islet cell genesis. In transgenic embryos, cells expressing EGFP lined the pancreatic ducts. SeAP was readily detectable in embryos, in the media of cultured embryonic pancreases and in the serum of adult animals. Treatment with the γ-secretase inhibitor DAPT, which blocks Notch signaling, enhanced SeAP secretion rates and increased the number of EGFP-expressing cells as assayed by fluorescence-activated cell sorting (FACS) and immunohistochemistry in cultured pancreases from embryos at embryonic day 11.5, but not in pancreases harvested 1 day later. By contrast, treatment with growth differentiation factor 11 (GDF11) reduced SeAP secretion rates. In adult mice, partial pancreatectomy decreased, whereas duct ligation increased, circulating SeAP levels. This model will be useful for studying signals involved in islet cell genesis in vivo and developing therapies that induce this process.

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SeAP activity falls in the serum of adult BAC NEUROG3-SeAP/EGFP transgenic mice after partial pancreatectomy but increases after pancreatic duct ligation. Day 0 is prior to surgery. (A) The white bar at day 0 shows the background signal for SeAP activity in the serum of non-transgenic mice. Gray bars show the activity of SeAP in the serum of transgenic animals that underwent a sham operation. Black bars show the activity of SeAP in the serum of transgenic animals that underwent partial pancreatectomy. Data represent mean + s.e.m. from three mice in each treated group, and four mice in the untreated, non-transgenic group. ***P<0.001 compared with serum from sham-operated mice by Student’s t-test. (B) The white bars show the background signal for SeAP activity in the serum of control transgenic mice that did not have surgery. Gray bars show the activity of SeAP in the serum of animals that underwent a sham operation. Black bars show the activity of SeAP in the serum of animals that underwent duct ligation. Data represent mean + s.e.m. from six mice in the untreated group, and seven mice in each treated group. *P<0.03, **P<0.01, ***P<0.001 by Student’s t-test for the comparisons indicated.
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f5-0040268: SeAP activity falls in the serum of adult BAC NEUROG3-SeAP/EGFP transgenic mice after partial pancreatectomy but increases after pancreatic duct ligation. Day 0 is prior to surgery. (A) The white bar at day 0 shows the background signal for SeAP activity in the serum of non-transgenic mice. Gray bars show the activity of SeAP in the serum of transgenic animals that underwent a sham operation. Black bars show the activity of SeAP in the serum of transgenic animals that underwent partial pancreatectomy. Data represent mean + s.e.m. from three mice in each treated group, and four mice in the untreated, non-transgenic group. ***P<0.001 compared with serum from sham-operated mice by Student’s t-test. (B) The white bars show the background signal for SeAP activity in the serum of control transgenic mice that did not have surgery. Gray bars show the activity of SeAP in the serum of animals that underwent a sham operation. Black bars show the activity of SeAP in the serum of animals that underwent duct ligation. Data represent mean + s.e.m. from six mice in the untreated group, and seven mice in each treated group. *P<0.03, **P<0.01, ***P<0.001 by Student’s t-test for the comparisons indicated.

Mentions: Transgene expression persisted at low levels in the adult, as demonstrated by the presence of detectable levels of SeAP activity in the serum (Fig. 5). It is not clear whether this activity originated from expression in endocrine progenitors in the gut, or persistent expression in the pancreas, because adult islets express low but consistently detectable levels of neurogenin-3 mRNA (Fig. 1B) (Wang et al., 2009). After 50% pancreatectomy, serum SeAP levels initially fell at 24 hours, but recovered within 2 weeks without evidence of a spike in neurogenin-3 gene expression (Fig. 5A). To explore the possibility that the surgery reduced the contribution from the gut to serum SeAP levels, we also measured the tissue SeAP levels in gut. Transgenic mice that underwent a 50% pancreatectomy had no difference in gut SeAP content compared with mice subjected to a sham operation (26672±3207 light units/μg protein in sham-operated animals vs 28674±3055 in pancreatectomized animals). By contrast, when the pancreatic duct was ligated, SeAP levels rose relative to sham-operated animals and peaked at 2 weeks after the placement of the ligation (Fig. 5B).


A mouse model for monitoring islet cell genesis and developing therapies for diabetes.

Shimajiri Y, Kosaka Y, Scheel DW, Lynn FC, Kishimoto N, Wang J, Zhao S, German MS - Dis Model Mech (2010)

SeAP activity falls in the serum of adult BAC NEUROG3-SeAP/EGFP transgenic mice after partial pancreatectomy but increases after pancreatic duct ligation. Day 0 is prior to surgery. (A) The white bar at day 0 shows the background signal for SeAP activity in the serum of non-transgenic mice. Gray bars show the activity of SeAP in the serum of transgenic animals that underwent a sham operation. Black bars show the activity of SeAP in the serum of transgenic animals that underwent partial pancreatectomy. Data represent mean + s.e.m. from three mice in each treated group, and four mice in the untreated, non-transgenic group. ***P<0.001 compared with serum from sham-operated mice by Student’s t-test. (B) The white bars show the background signal for SeAP activity in the serum of control transgenic mice that did not have surgery. Gray bars show the activity of SeAP in the serum of animals that underwent a sham operation. Black bars show the activity of SeAP in the serum of animals that underwent duct ligation. Data represent mean + s.e.m. from six mice in the untreated group, and seven mice in each treated group. *P<0.03, **P<0.01, ***P<0.001 by Student’s t-test for the comparisons indicated.
© Copyright Policy - open-access
Related In: Results  -  Collection

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f5-0040268: SeAP activity falls in the serum of adult BAC NEUROG3-SeAP/EGFP transgenic mice after partial pancreatectomy but increases after pancreatic duct ligation. Day 0 is prior to surgery. (A) The white bar at day 0 shows the background signal for SeAP activity in the serum of non-transgenic mice. Gray bars show the activity of SeAP in the serum of transgenic animals that underwent a sham operation. Black bars show the activity of SeAP in the serum of transgenic animals that underwent partial pancreatectomy. Data represent mean + s.e.m. from three mice in each treated group, and four mice in the untreated, non-transgenic group. ***P<0.001 compared with serum from sham-operated mice by Student’s t-test. (B) The white bars show the background signal for SeAP activity in the serum of control transgenic mice that did not have surgery. Gray bars show the activity of SeAP in the serum of animals that underwent a sham operation. Black bars show the activity of SeAP in the serum of animals that underwent duct ligation. Data represent mean + s.e.m. from six mice in the untreated group, and seven mice in each treated group. *P<0.03, **P<0.01, ***P<0.001 by Student’s t-test for the comparisons indicated.
Mentions: Transgene expression persisted at low levels in the adult, as demonstrated by the presence of detectable levels of SeAP activity in the serum (Fig. 5). It is not clear whether this activity originated from expression in endocrine progenitors in the gut, or persistent expression in the pancreas, because adult islets express low but consistently detectable levels of neurogenin-3 mRNA (Fig. 1B) (Wang et al., 2009). After 50% pancreatectomy, serum SeAP levels initially fell at 24 hours, but recovered within 2 weeks without evidence of a spike in neurogenin-3 gene expression (Fig. 5A). To explore the possibility that the surgery reduced the contribution from the gut to serum SeAP levels, we also measured the tissue SeAP levels in gut. Transgenic mice that underwent a 50% pancreatectomy had no difference in gut SeAP content compared with mice subjected to a sham operation (26672±3207 light units/μg protein in sham-operated animals vs 28674±3055 in pancreatectomized animals). By contrast, when the pancreatic duct was ligated, SeAP levels rose relative to sham-operated animals and peaked at 2 weeks after the placement of the ligation (Fig. 5B).

Bottom Line: Treatment with the γ-secretase inhibitor DAPT, which blocks Notch signaling, enhanced SeAP secretion rates and increased the number of EGFP-expressing cells as assayed by fluorescence-activated cell sorting (FACS) and immunohistochemistry in cultured pancreases from embryos at embryonic day 11.5, but not in pancreases harvested 1 day later.By contrast, treatment with growth differentiation factor 11 (GDF11) reduced SeAP secretion rates.This model will be useful for studying signals involved in islet cell genesis in vivo and developing therapies that induce this process.

View Article: PubMed Central - PubMed

Affiliation: Diabetes Center, University of California San Francisco, San Francisco, CA 94143-0534, USA.

ABSTRACT
Transient expression of the transcription factor neurogenin-3 marks progenitor cells in the pancreas as they differentiate into islet cells. We developed a transgenic mouse line in which the surrogate markers secreted alkaline phosphatase (SeAP) and enhanced green florescent protein (EGFP) can be used to monitor neurogenin-3 expression, and thus islet cell genesis. In transgenic embryos, cells expressing EGFP lined the pancreatic ducts. SeAP was readily detectable in embryos, in the media of cultured embryonic pancreases and in the serum of adult animals. Treatment with the γ-secretase inhibitor DAPT, which blocks Notch signaling, enhanced SeAP secretion rates and increased the number of EGFP-expressing cells as assayed by fluorescence-activated cell sorting (FACS) and immunohistochemistry in cultured pancreases from embryos at embryonic day 11.5, but not in pancreases harvested 1 day later. By contrast, treatment with growth differentiation factor 11 (GDF11) reduced SeAP secretion rates. In adult mice, partial pancreatectomy decreased, whereas duct ligation increased, circulating SeAP levels. This model will be useful for studying signals involved in islet cell genesis in vivo and developing therapies that induce this process.

Show MeSH