17-β Estradiol regulates proglucagon-derived peptide secretion in mouse and human α- and L cells

Abstract

Clinical and experimental data indicate a beneficial effect of estrogens on energy and glucose homeostasis associated with improved insulin sensitivity and positive effects on insulin secretion. The aim of the study was to investigate the impact of estrogens on proglucagon-producing cells, pancreatic α cells, and enteroendocrine L cells. The consequences of sexual hormone deprivation were evaluated in ovariectomized mice (ovx). Ovx mice exhibited impaired glucose tolerance during oral glucose tolerance tests (OGTT), which was associated with decreased GLP-1 intestinal and pancreatic secretion and content, an effect that was reversed by estradiol (E2) treatment. Indeed, E2 increased oral glucose-induced GLP-1 secretion in vivo and GLP-1 secretion from primary culture of mouse and human α cells through the activation of all 3 estrogen receptors (ERs), whereas E2-induced GLP-1 secretion from mouse and human intestinal explants occurred only by ERβ activation. Underlying the implication of ERβ, its selective agonist WAY20070 was able to restore glucose tolerance in ovx mice at least partly through plasma GLP-1 increase. We conclude that E2 directly controls both α- and L cells to increase GLP-1 secretion, in addition to its effects on insulin and glucagon secretion, highlighting the potential beneficial role of the estrogenic pathway and, more particularly, of ERβ agonists to prevent type 2 diabetes.

Keywords: Diabetes; Endocrinology; Homeostasis; Metabolism; Sex hormones.

Figure 1. Ovariectomized female mice develop glucose intolerance.

Plasma glucose levels during OGTT (2 g/kg) (A) and the related AUCs (B) in sham (gray bars, n = 12) and ovx (white bars, n = 13) mice, 1 week after the surgery. Plasma insulin (C), glucagon (n = 6 at least per group) (D), and GLP-1 levels 5 minutes after gavage (sham, n = 5; ovx, n = 3) (E) during the OGTT. One-way repeated ANOVA with Bonferonni post hoc test analyses were performed for glycemia (A), and 2-tailed Student’s t test statistical analyses were performed for the other results. *P ≤ 0.05 for sham vs. ovx mice.

Figure 2. Exogenous estradiol improves glucose tolerance during OGTT in ovx mice.

Plasma glucose levels during OGTT (2 g/kg) (A) and the related AUCs (B) in ovx+vehicle (black circles, white bars; n = 20) and ovx+E₂ (80 μg/kg/48 hours; red circles, red bars; n = 19) mice. Plasma insulin (ovx, n = 6; ovx+E₂, n = 6) (C), glucagon (ovx, n = 6; ovx+E₂, n = 6) (D), and GLP-1 levels 5 minutes after gavage (ovx, n = 5; ovx+E₂, n = 8) (E) during an OGTT. One-way repeated ANOVA with Bonferonni post hoc test analyses were performed for glycemia (A), and 2-tailed Student’s t test statistical analyses were performed for the other results. *P ≤ 0.05 for ovx+vehicle vs. ovx+E₂ mice.

Figure 3. In vivo effects of exogenous estradiol administration on the pancreas of ovx mice.

Insulin (A), glucagon (B), and GLP-1 (C) contents were evaluated from pancreases of ovx+vehicle (white bars, n = 4) and ovx+E₂ (80 μg/kg/48 hours; red bars, n = 4) mice. Insulin was measured in FACS-purified β cells (ovx, n = 4; ovx+E₂, n = 4) (D), glucagon (E), and GLP-1 (F) measured in FACS-purified α cells (ovx, n = 4; ovx+E₂, n = 4). Both α- (Venus⁺ cells) and β cell (Cherry⁺ cells) numbers were evaluated by FACS from ovx (n = 10) and ovx+E₂ (n = 11) dissociated pancreatic islets (G). Two-tailed Student’s t test statistical analyses. *P ≤ 0.05 for ovx+vehicle vs. ovx+E₂ mice.

Figure 4. Estradiol directly affects α- and β cells from ovx mice in vitro.

Both α- and β cells isolated from ovx mice were cultured with vehicle (white bars), or 17β-estradiol (E₂, 1 × 10^–8 mol/l) (red bars). Relative mRNA levels were quantified in sorted α cells (n = 11 in each group) (A). Glucagon (B) and GLP-1 (C) contents were assessed in sorted α cells treated for 48 hours with vehicle (white, n = 11) or with E₂, 1 × 10^–8 mol/l (red, n = 13). Contents are normalized to total protein. Ratios of GLP-1 to glucagon content of sorted α cells are represented in D. Glucagon (E) and GLP-1 (F) levels were quantified in supernatants relative to the contents of glucagon and GLP-1, respectively, after 6 hours of continuous release in complete medium of purified α cells (vehicle, n = 11; E₂, n = 13). Relative mRNA level quantification was assessed in sorted β cells (n = 12 in each group) (G), as well as insulin contents relative to total protein amount (H) and release relative to insulin content (I) (vehicle, n = 15; E₂, n = 15). Purified α- and β cells isolated from ovx mice were cultured in 2 separate drops, and only α cells were treated either with vehicle (white bars, n = 5) or 17β-estradiol (E₂, 1 × 10^–8 mol/l) (red bars, n = 5); both drops were then joined during the secretion tests. Insulin (J) and GLP-1 (K) levels were assessed in response to glucose and a GLP-1 receptor antagonist (Ex9-39). Two-tailed Student’s t test statistical analyses. *P ≤ 0.05 for vehicle- vs. E₂-treated cells.

Figure 5. Effects of estradiol on intestinal L cells of ovx mice in vivo and in vitro.

Relative Gcg and Pcsk1/3 mRNA levels (n = 6 animals in each group) (A) and GLP-1 cellular contents were measured from sorted intestinal L cells of ovx+vehicle (white bars, n = 5) and ovx+E₂ (red bars, n = 5) mice (B). L cell numbers (Venus⁺ cells) from the small intestine were also evaluated by FACS (n = 7 animals in each group) (C). GLP-1 levels were quantified in supernatants and contents from primary cultures of mixed intestinal cells (D) of ovx mice, cultured for 48 hours with vehicle DMSO (white bars, n = 11) or with 17β-estradiol (E₂, 1 × 10^–8 mol/l, red bars, n = 10) after 2 hours of continuous release in complete fresh medium. Two-tailed Student’s t test statistical analyses. *P ≤ 0.05 for ovx+vehicle vs. ovx+E₂ mice, and for DMSO- vs. E₂-treated mixed cells.

Figure 6. Estrogen receptors mediate estradiol effects in α- and L cells.

Sorted α- and L cells isolated from 13-week-old female mice ovariectomized for a week and GLUTag cells were studied for the expression of estrogen receptors (ERs) (A). GLP-1 releases during 2 hours in fresh medium were quantified in supernatants relative to contents of purified α cells (n = 7 experiments in each group) (B), of dissociated mixed cells from small intestine (jejunum/ileum) (n = 6 experiments in each group) (C), and of GLUTag cell cultures (n = 5 experiments in each groups) (D), cultured during 48h hours with vehicle (DMSO) or the several SERMs (E₂, PPT, WAY, and G1 at 1 × 10^–8 mol/l). One-way ANOVA with the Dunnett’s multiple comparison post test. *P ≤ 0.05 for vehicle- vs. E₂ agonist–treated cells.

Figure 7. Estrogen receptors mediate the PC2-to-PC1/3 switch in mouse α cells.

Sorted α cells isolated from 13-week-old female mice ovariectomized for a week were cultured during 48 hours with vehicle (DMSO), E₂, or SERMs (PPT, WAY, and G1 at 1 × 10^–8 mol/l). Expression of Gcg (A), Pcsk1/3 (B), and Pcsk2 (C) mRNA (n = 5 per group) was assessed. One-way ANOVA with the Dunnett’s multiple comparison post test was performed. *P ≤ 0.05 for vehicle- vs. E₂ agonist–treated cells.

Figure 8. ERβ activation improves glucose tolerance during OGTT in ovx mice.

Plasma glucose levels during OGTT (2 g/kg) (A) and the related AUC (B) in ovx+vehicle (black circles, white bars; n = 20) and ovx+WAY (100 mg/kg/48 hours; orange circles, orange bars; n = 13) mice are represented. Plasma insulin (ovx, n = 11; ovx+WAY, n = 6) (C), glucagon (ovx, n = 11; ovx+WAY, n = 6) (D), and GLP-1 levels 5 minutes after gavage (ovx, n = 9; ovx+WAY, n = 8) (E) during an OGTT. One-way repeated ANOVA with Bonferonni post hoc test analyses were performed for glycemia (A), and 2-tailed Student’s t test statistical analyses were performed for the other figures. *P ≤ 0.05 for ovx+vehicle vs. ovx+WAY mice.

Figure 9. Estradiol increases GLP-1 release from human α- and L cells in vitro.

GLP-1 (A) and glucagon (B) releases were quantified in supernatants relative to content of sorted human α cells (n = 4 experiments in each group). (C) Glucose-stimulated insulin secretion in Human isolated β cells (n = 4 in each group). (D) GLP-1 secretion of dissociated mixed cells from human small intestine (duodenum) (n = 3 experiments in each group). One-way ANOVA with the Dunnett’s multiple comparison post test. *P ≤ 0.05 for vehicle- vs. E₂ agonist–treated cells.