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Pro-oxidant/antioxidant balance controls pancreatic β-cell differentiation through the ERK1/2 pathway.

Hoarau E, Chandra V, Rustin P, Scharfmann R, Duvillie B - Cell Death Dis (2014)

Bottom Line: Surprisingly, decreasing ROS production in vivo reduced β-cell differentiation.Gene transfer and pharmacological manipulations, which diminish cellular ROS levels, also interfered with normal β-cell differentiation.This study highlights the role of the redox balance on β-cell development and provides information that will be useful for improving β-cell production from embryonic stem cells, a step in cell therapy for diabetes.

View Article: PubMed Central - PubMed

Affiliation: 1] INSERM, U1016, Institut Cochin, Paris, France [2] Université Paris Descartes, Sorbonne Paris Cité, Faculté de Médecine, Paris, France.

ABSTRACT
During embryogenesis, the intrauterine milieu affects cell proliferation, differentiation, and function by modifying gene expression in susceptible cells, such as the pancreatic β-cells. In this limited energy environment, mitochondrial dysfunction can lead to overproduction of reactive oxygen species (ROS) and to a decline in β-cell function. In opposition to this toxicity, ROS are also required for insulin secretion. Here we investigated the role of ROS in β-cell development. Surprisingly, decreasing ROS production in vivo reduced β-cell differentiation. Moreover, in cultures of pancreatic explants, progenitors were highly sensitive to ROS stimulation and responded by generating β-cells. ROS enhanced β-cell differentiation through modulation of ERK1/2 signaling. Gene transfer and pharmacological manipulations, which diminish cellular ROS levels, also interfered with normal β-cell differentiation. This study highlights the role of the redox balance on β-cell development and provides information that will be useful for improving β-cell production from embryonic stem cells, a step in cell therapy for diabetes.

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Related in: MedlinePlus

Activation of ERK1/2 phosphorylation by H2O2 is required for the proper development of β-cells. (a) E13.5 rat pancreases were cultured for 0, 5, or 15 min with or without 50 μM H2O2 in association or not with U0126. Glucose 20 mM was used as a positive regulator of ERK1/2. Protein extracts from cultured pancreases were analyzed by western blot to quantify P-ERK1/2. Total ERK is used as loading control. Relative ERK Phosphorylation was also quantified for each condition. (b) E13.5 rat pancreases were cultured with or without H2O2 at 50 μM, in association or not with the ERK1/2 inhibitor U0126. For each condition, NGN3 expression (in brown) was detected by immunohistochemistry and the number of NGN3+ cells was quantified. Each point represents the mean±S.E.M. of three individual data pools. Scale bar: 25 μm. *P<0.05; **P<0.01, ***P<0.001
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fig7: Activation of ERK1/2 phosphorylation by H2O2 is required for the proper development of β-cells. (a) E13.5 rat pancreases were cultured for 0, 5, or 15 min with or without 50 μM H2O2 in association or not with U0126. Glucose 20 mM was used as a positive regulator of ERK1/2. Protein extracts from cultured pancreases were analyzed by western blot to quantify P-ERK1/2. Total ERK is used as loading control. Relative ERK Phosphorylation was also quantified for each condition. (b) E13.5 rat pancreases were cultured with or without H2O2 at 50 μM, in association or not with the ERK1/2 inhibitor U0126. For each condition, NGN3 expression (in brown) was detected by immunohistochemistry and the number of NGN3+ cells was quantified. Each point represents the mean±S.E.M. of three individual data pools. Scale bar: 25 μm. *P<0.05; **P<0.01, ***P<0.001

Mentions: We next characterized the molecular mechanisms by which H2O2 increased β-cell development. We examined the mitogen-activated protein kinase (MAPK) ERK1/2 pathway that was recently shown to be sensitive to ROS production in several cell types.29 Pancreases were cultured with or without H2O2 (50 μM) for 5 or 15 min. In parallel experiments, pancreases were treated with 20 mM glucose, a well-known inducer of ERK1/2 phosphorylation in β-cells and fetal islets.30,31 Western blot analysis showed that H2O2, similar to high glucose, induced the ERK1/2 pathway (Figure 7a). Such an induction of the ERK1/2 pathway by H2O2 was also observed by immunohistochemistry (Supplementary Figure S9).


Pro-oxidant/antioxidant balance controls pancreatic β-cell differentiation through the ERK1/2 pathway.

Hoarau E, Chandra V, Rustin P, Scharfmann R, Duvillie B - Cell Death Dis (2014)

Activation of ERK1/2 phosphorylation by H2O2 is required for the proper development of β-cells. (a) E13.5 rat pancreases were cultured for 0, 5, or 15 min with or without 50 μM H2O2 in association or not with U0126. Glucose 20 mM was used as a positive regulator of ERK1/2. Protein extracts from cultured pancreases were analyzed by western blot to quantify P-ERK1/2. Total ERK is used as loading control. Relative ERK Phosphorylation was also quantified for each condition. (b) E13.5 rat pancreases were cultured with or without H2O2 at 50 μM, in association or not with the ERK1/2 inhibitor U0126. For each condition, NGN3 expression (in brown) was detected by immunohistochemistry and the number of NGN3+ cells was quantified. Each point represents the mean±S.E.M. of three individual data pools. Scale bar: 25 μm. *P<0.05; **P<0.01, ***P<0.001
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4237262&req=5

fig7: Activation of ERK1/2 phosphorylation by H2O2 is required for the proper development of β-cells. (a) E13.5 rat pancreases were cultured for 0, 5, or 15 min with or without 50 μM H2O2 in association or not with U0126. Glucose 20 mM was used as a positive regulator of ERK1/2. Protein extracts from cultured pancreases were analyzed by western blot to quantify P-ERK1/2. Total ERK is used as loading control. Relative ERK Phosphorylation was also quantified for each condition. (b) E13.5 rat pancreases were cultured with or without H2O2 at 50 μM, in association or not with the ERK1/2 inhibitor U0126. For each condition, NGN3 expression (in brown) was detected by immunohistochemistry and the number of NGN3+ cells was quantified. Each point represents the mean±S.E.M. of three individual data pools. Scale bar: 25 μm. *P<0.05; **P<0.01, ***P<0.001
Mentions: We next characterized the molecular mechanisms by which H2O2 increased β-cell development. We examined the mitogen-activated protein kinase (MAPK) ERK1/2 pathway that was recently shown to be sensitive to ROS production in several cell types.29 Pancreases were cultured with or without H2O2 (50 μM) for 5 or 15 min. In parallel experiments, pancreases were treated with 20 mM glucose, a well-known inducer of ERK1/2 phosphorylation in β-cells and fetal islets.30,31 Western blot analysis showed that H2O2, similar to high glucose, induced the ERK1/2 pathway (Figure 7a). Such an induction of the ERK1/2 pathway by H2O2 was also observed by immunohistochemistry (Supplementary Figure S9).

Bottom Line: Surprisingly, decreasing ROS production in vivo reduced β-cell differentiation.Gene transfer and pharmacological manipulations, which diminish cellular ROS levels, also interfered with normal β-cell differentiation.This study highlights the role of the redox balance on β-cell development and provides information that will be useful for improving β-cell production from embryonic stem cells, a step in cell therapy for diabetes.

View Article: PubMed Central - PubMed

Affiliation: 1] INSERM, U1016, Institut Cochin, Paris, France [2] Université Paris Descartes, Sorbonne Paris Cité, Faculté de Médecine, Paris, France.

ABSTRACT
During embryogenesis, the intrauterine milieu affects cell proliferation, differentiation, and function by modifying gene expression in susceptible cells, such as the pancreatic β-cells. In this limited energy environment, mitochondrial dysfunction can lead to overproduction of reactive oxygen species (ROS) and to a decline in β-cell function. In opposition to this toxicity, ROS are also required for insulin secretion. Here we investigated the role of ROS in β-cell development. Surprisingly, decreasing ROS production in vivo reduced β-cell differentiation. Moreover, in cultures of pancreatic explants, progenitors were highly sensitive to ROS stimulation and responded by generating β-cells. ROS enhanced β-cell differentiation through modulation of ERK1/2 signaling. Gene transfer and pharmacological manipulations, which diminish cellular ROS levels, also interfered with normal β-cell differentiation. This study highlights the role of the redox balance on β-cell development and provides information that will be useful for improving β-cell production from embryonic stem cells, a step in cell therapy for diabetes.

Show MeSH
Related in: MedlinePlus