Oxygen tension regulates pancreatic beta-cell differentiation through hypoxia-inducible factor 1alpha

Abstract

Objective: Recent evidence indicates that low oxygen tension (pO2) or hypoxia controls the differentiation of several cell types during development. Variations of pO2 are mediated through the hypoxia-inducible factor (HIF), a crucial mediator of the adaptative response of cells to hypoxia. The aim of this study was to investigate the role of pO2 in beta-cell differentiation.

Research design and methods: We analyzed the capacity of beta-cell differentiation in the rat embryonic pancreas using two in vitro assays. Pancreata were cultured either in collagen or on a filter at the air/liquid interface with various pO2. An inhibitor of the prolyl hydroxylases, dimethyloxaloylglycine (DMOG), was used to stabilize HIF1alpha protein in normoxia.

Results: When cultured in collagen, embryonic pancreatic cells were hypoxic and expressed HIF1alpha and rare beta-cells differentiated. In pancreata cultured on filter (normoxia), HIF1alpha expression decreased and numerous beta-cells developed. During pancreas development, HIF1alpha levels were elevated at early stages and decreased with time. To determine the effect of pO2 on beta-cell differentiation, pancreata were cultured in collagen at increasing concentrations of O2. Such conditions repressed HIF1alpha expression, fostered development of Ngn3-positive endocrine progenitors, and induced beta-cell differentiation by O2 in a dose-dependent manner. By contrast, forced expression of HIF1alpha in normoxia using DMOG repressed Ngn3 expression and blocked beta-cell development. Finally, hypoxia requires hairy and enhancer of split (HES)1 expression to repress beta-cell differentiation.

Conclusions: These data demonstrate that beta-cell differentiation is controlled by pO2 through HIF1alpha. Modifying pO2 should now be tested in protocols aiming to differentiate beta-cells from embryonic stem cells.

FIG. 1.

Differential development of β-cells between pancreata cultured in collagen and at the air-medium interface. A: E13.5 pancreata were cultured in collagen or on a filter at the air-medium interface for 3 or 7 days. At day 3, NGN3 was detected by immunohistochemistry (in brown). At day 7, anti-insulin (INS) (in red) and anti-carboxypeptidase A (CPA) (in green) antibodies were used to detect β- and acinar cell development, respectively. B: Hypoxia was analyzed in pancreata cultured in collagen and at the air-medium interface. After 1 or 7 days, pimonidazole was added to the medium for the last 2 h of the culture period and hypoxia was analyzed using anti-Mab1 antibodies (in green). Bar: 50 μm. (A high-quality digital representation of this figure is available in the online issue.)

FIG. 2.

Induction of β-cell differentiation by increasing pO₂ in collagen. A: Pancreata were cultured for 7 days at 21, 60, and 80% O₂. β- and acinar cells were detected by immunohistochemistry with antibodies directed against insulin (INS) (in red) and carboxypeptidase A (CPA) (in green). Next, the absolute surface area occupied by insulin-positive cells was quantified. B: Apoptotic cells (in green) were detected using a TUNEL reaction assay in pancreata cultured for 1 day at 21 or 80% O₂. The epithelial cells were detected using an anti–E-cadherin antibody (in red). Note the absence of apoptotic cells within the epithelium. C: Real-time PCR quantification of Ngn3 mRNA in pancreata cultured at 21% () or 80% (■) O₂ at different time points. Gene expression is presented as a percentage of the highest sample. Each point represents the mean ± SEM of three individual data pools. *P < 0.05; **P < 0.01. (A high-quality digital representation of this figure is available in the online issue.)

FIG. 3.

The HIF1α signaling pathway is induced by hypoxia in the pancreas. A: Protein extracts from pancreata cultured in collagen for 1 day at 21 or 80% O₂ were analyzed by Western blot to quantify HIF1α expression and β-actin (loading control). B: Pancreata were cultured for 1 day at 21 or 80% O₂ and pimonidazole was added in the culture medium during the last 2 h of the culture. HIF1α-positive cells were detected by immunohistochemistry (in brown). On serial sections, hypoxia was analyzed using anti-Mab1 antibodies (in green). Note the topographical relationship between HIF activation and pimonidazole adduct formation. Bar: 50 μm. C: Expression of HIF1α target genes Pgk1, LdhA, Glut1, Vegf, and Redd1 was analyzed by qPCR in pancreata cultured in collagen for 1 day at 21% () or 80% (■) O₂. Gene expression is presented as a percentage of the highest sample. Each point represents the mean ± SEM of three individual pools. *P < 0.05; **P < 0.01. (A high-quality digital representation of this figure is available in the online issue.)

FIG. 4.

DMOG, an inhibitor of prolyl hydroxylases, stabilizes HIF1α and represses β-cell differentiation. A: Pancreata at E13.5 were cultured at the air-medium interface for 1 day at 21 or 3% O₂, and HIF1α was analyzed by Western blot. B: Real-time PCR quantification of Ngn3 mRNA in pancreata cultured for 1 day at 21% (■) or 3% () O₂. Gene expression is presented as a percentage of the highest sample. Each point represents the mean ± SEM of three individual pools. *P < 0.05. C: Pancreata were cultured for 7 days at the air-medium interface at 21 or 3% O₂. Immunohistochemistry was used to detect β- and acinar cells using antibodies directed against insulin (in red) and carboxypeptidase A (in green). Next, the absolute surface occupied by insulin-positive cells was quantified. Black and gray bars represent the surface of insulin-positive cells in pancreata cultured at 21 and 3% O₂, respectively. **P < 0.01. D: Pancreata at E13.5 were cultured at the air-medium interface for 1 day in the presence or absence of 1 or 2 mmol/l DMOG, and HIF1α was analyzed by Western blot. E: Real-time PCR quantification of Ngn3 mRNA in pancreata cultured for 1 day with 0 (■), 1 (), or 2 (□) mmol/l DMOG at 21% O₂. **P < 0.01. F: Pancreata cultured in the presence or absence of 1 mmol/l DMOG for 7 days were analyzed by immunohistochemistry. Acinar cells were detected using anti–carboxypeptidase A antibody (in green) and β-cells using anti-insulin antibody (in red). Next, the absolute surface occupied by insulin-positive cells was quantified. Gray and black bars represent the surface of insulin-positive cells in pancreata cultured at 21% O₂ with or without DMOG, respectively. ***P < 0.001. (A high-quality digital representation of this figure is available in the online issue.)

FIG. 5.

Regulation of Hes1 expression by hypoxia. A: Real-time PCR quantification of Hes1 mRNA in pancreata cultured at the air-medium interface with 21% (■) or 3% () O₂ for 1 day. *P < 0.05. B: Real-time PCR quantification of Hes1 mRNA in pancreata cultured at the air-medium interface with 0 (■), 1 (), or 2 (□) mmol/l DMOG for 1 day. **P < 0.01. C: Detection of Ngn3 transcripts by in situ hybridization in pancreata cultured for 1 day at 21% O₂, 21%O₂ with DMOG, or 3% O₂ with or without γ-secretase inhibitor treatment. Epithelium is circled in black in each panel. The absolute number of Ngn3-positive cells was quantified in each condition in the presence (■) or absence (□) of γ-secretase inhibitor treatment. *P < 0.05; **P < 0.01. (A high-quality digital representation of this figure is available in the online issue.)

FIG. 6.

HIF1α protein expression during pancreatic development in vivo. A: Immunohistochemical analysis of HIF1α protein expression (in brown) in pancreata at E11.5, E12.5, E16.5, E20.5, and adult phases. In the left panel, epithelium is circled in black. In the right panel, a pancreatic islet is circled in black. Antibodies against PDX1 (in green) and insulin (INS) (in red) were used on serial sections to visualize progenitors and β-cells, respectively. B: NGN3 expression is repressed in the pancreas of embryos exposed to hypoxia. Pregnant rats were maintained with 21 or 8% O₂ for 24 h. Animal weight did not differ between the hypoxic and normoxic groups. The pancreata were harvested at E14.5 and analyzed by immunohistochemistry and qPCR. Detection of NGN3 (brown; **P < 0.01). Expression of HIF1α target genes, Pgk1, LdhA, Glut-1, Vegf-A, and Redd1 in pancreata in hypoxia (□) or normoxia (■). *P < 0.05; ***P < 0.001. (A high-quality digital representation of this figure is available in the online issue.)