Pharmacological blockade of the EP3 prostaglandin E2 receptor in the setting of type 2 diabetes enhances β-cell proliferation and identity and relieves oxidative damage

Abstract

Objective: Type 2 diabetes is characterized by hyperglycemia and inflammation. Prostaglandin E₂, which signals through four G protein-coupled receptors (EP1-4), is a mediator of inflammation and is upregulated in diabetes. We have shown previously that EP3 receptor blockade promotes β-cell proliferation and survival in isolated mouse and human islets ex vivo. Here, we analyzed whether systemic EP3 blockade could enhance β-cell mass and identity in the setting of type 2 diabetes using mice with a spontaneous mutation in the leptin receptor (Lepr^db).

Methods: Four- or six-week-old, db/+, and db/db male mice were treated with an EP3 antagonist daily for two weeks. Pancreata were analyzed for α-cell and β-cell proliferation and β-cell mass. Islets were isolated for transcriptomic analysis. Selected gene expression changes were validated by immunolabeling of the pancreatic tissue sections.

Results: EP3 blockade increased β-cell mass in db/db mice through enhanced β-cell proliferation. Importantly, there were no effects on α-cell proliferation. EP3 blockade reversed the changes in islet gene expression associated with the db/db phenotype and restored the islet architecture. Expression of the GLP-1 receptor was slightly increased by EP3 antagonist treatment in db/db mice. In addition, the transcription factor nuclear factor E2-related factor 2 (Nrf2) and downstream targets were increased in islets from db/db mice in response to treatment with an EP3 antagonist. The markers of oxidative stress were decreased.

Conclusions: The current study suggests that EP3 blockade promotes β-cell mass expansion in db/db mice. The beneficial effects of EP3 blockade may be mediated through Nrf2, which has recently emerged as a key mediator in the protection against cellular oxidative damage.

Keywords: Beta cell proliferation; Mouse model; Nrf2; Prostaglandin E(2); Type 2 diabetes.

Figure 1

Systemic EP3 blockade promotes β-cell mass expansion in db/db mice. (A) Representative images of insulin-labeled (brown) pancreatic sections from vehicle (veh)- or DG-041 (DG)-treated db/+ and db/db mice treated from 6 to 8 weeks of age. (B) β-cell mass from 4 to 6 weeks of age and (C) 6–8 weeks of age. Empty squares, db/+ veh. Filled squares, db/+ DG. Empty triangles, db/db veh. Filled triangles, db/db DG. ∗versus vehicle. Two asterisks indicate p < 0.01. Data were analyzed using Student's t-test and Bonferroni post hoc analysis. (Scale bar, 100 μm)

Figure 2

β-cell proliferation is increased in db/db mice in response to EP3 blockade. (A) Representative images of pancreatic sections obtained from vehicle (veh)- or DG-041 (DG)-treated db/+ or db/db mice treated from 4 to 6 weeks of age and immunolabelled for Ki67 (red) and insulin (green) and stained with DAPI (blue). β-cell proliferation was quantified by the percentage of Ki67-expressing insulin+ cells. (B) Summarized data for vehicle- and DG-treated db/+ or db/db mice treated from 4 to 6 weeks of age. (C) Summarized data for vehicle- and DG-treated db/+ or db/db mice treated from 6 to 8 weeks of age. Yellow arrows indicate Ki67/insulin dual-positive cells. Empty squares, db/+ veh. Filled squares, db/+ DG. Empty triangles, db/db veh. Filled triangles, db/db DG. Data were analyzed using Student's t-test and Bonferroni post hoc analysis. (Scale bar, 100 μm)

Figure 3

Genotype-dependent changes in gene expression in db/+ and db/db mice. (A) Principal component analysis based on gene expression from RNA sequencing for each of three biologic replicates for vehicle- or DG-041-treated db/+ or db/db mouse islets. Mice were treated from 6 to 8 weeks of age. db/+ veh2 was determined to be an outlier and was removed from further analysis. (B) Heatmap depicting alterations in the expression of 42 selected islet markers in vehicle-treated db/+ or db/db mouse islets. (C) Heatmap depicting alterations in the expression of 42 selected islet markers in vehicle-treated or DG-041-treated db/+ mouse islets. (D) Heatmap depicting alterations in the expression of 42 selected islet markers in vehicle-treated or DG-041-treated db/db mouse islets. For all heatmaps, red indicates high expression and blue indicates low expression. Samples are grouped by genotype or treatment.

Figure 4

Effects of EP3 blockade on gene expression and islet architecture in db/db mice. (A) Gene ontology analysis of cell adhesion genes differentially expressed in DG-041-treated vs vehicle-treated db/db mice (black bars) and db/db vehicle vs db/+ vehicle (gray bars). The log2 fold change is plotted along the x-axis. (B) Summary of select differentially expressed collagen (Col), ephrin (Eph), and integrin (Itg) genes in DG-041-treated vs vehicle-treated db/db mice (black bars) and db/db vehicle vs db/+ vehicle (gray bars). The log2 fold change is plotted along the x-axis. (C) β/α-cell ratio in vehicle (−)- or DG-041 (+)-treated db/+ and db/db mice. β/α-cell ratio was determined by quantifying the total insulin + cells divided by the total glucagon + cells. Each point represents one mouse. (D) Representative images of the pancreatic sections showing the distribution of insulin+ (green) and glucagon+ (white) cells in db/db mice with and without DG-041 treatment. DAPI (blue). Empty squares, db/+ veh. Filled squares, db/+ DG-041. Empty triangles, db/db veh. Filled triangles, db/db DG-041. ∗versus veh-treated db/+, ^#versus veh-treated db/db. One symbol indicates p < 0.05; three symbols indicate p < 0.001. Mice were treated from 6 to 8 weeks of age for 14 days. All summarized data were analyzed using ANOVA or Student's t-test and Bonferroni post hoc analysis. (Scale bar, 100 μm).

Figure 5

Effects of EP3 blockade on GLP-1R protein expression in db/+ and db/db mice. Paraffin-embedded sections from pancreata isolated from the indicated genotype and treatment groups were immunolabeled with antibodies directed against insulin (Ins; green), glucagon (Gcg; purple), or GLP-1R (Glp-1r; red). Nuclei were stained with DAPI (blue). Mice were treated from 6 to 8 weeks of age for 14 days. (Scale bar, 20 μm)

Figure 6

EP3 blockade activates the Nrf2 antioxidant pathway. (A) Paraffin-embedded sections from pancreata isolated from the indicated genotype and treatment groups were immunolabeled with antibodies directed against insulin (Ins; white), glucagon (Gcg; green), or Nrf2 (red). Nuclei were stained with DAPI (blue). Depicted are representative images of three individual mice in each group. (A) Quantification of nuclear Nrf2 in β-cells. One asterisk indicates p < 0.05. (B) Heatmap depicting alterations in the expression of Nrf2(Nfe2l2) and selected Nrf2 target genes between vehicle-treated db/+ and db/db mice. (C) Heatmap depicting alterations in the expression of Nrf2(Nfe2l2) and selected target genes between vehicle-treated and DG-041-treated db/db mice. Mice were treated from 6 to 8 weeks of age. Red indicates high expression; blue indicates low expression. (Scale bar, 20 μm)

Figure 7

Effects of EP3 blockade on DNA damage in db/+ and db/db mice. (A) Paraffin-embedded sections from pancreata isolated from the indicated genotype and treatment groups were immunolabeled with antibodies directed against insulin (Ins; white), glucagon (Gcg; red), or 8OHdG (green). Nuclei were stained with DAPI (blue). Mice were treated from 6 to 8 weeks of age for 14 days. (Scale bar, 20 μm) (B) Quantification of nuclear 8OHdG in β-cells. Two asterisks indicate p < 0.01.