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Activation of enteroendocrine membrane progesterone receptors promotes incretin secretion and improves glucose tolerance in mice.

Flock GB, Cao X, Maziarz M, Drucker DJ - Diabetes (2012)

Bottom Line: Glucagon-like peptide-1 (GLP-1) secretion is classically regulated by ingested nutrients.Furthermore, a cell-impermeable BSA-progesterone conjugate rapidly increased ERK1/2 phosphorylation and GLP-1 secretion.Enteral progesterone administration increased plasma levels of GLP-1, glucose-dependent insulinotropic polypeptide (GIP), and insulin, and improved oral glucose tolerance in an RU486-insensitve manner in mice: however, systemic progesterone exposure did not improve glucose homeostasis.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, Institute of Medical Sciences, Samuel Lunenfeld Research Institute, University of Toronto, Toronto, Ontario, Canada.

ABSTRACT
Glucagon-like peptide-1 (GLP-1) secretion is classically regulated by ingested nutrients. To identify novel molecular targets controlling incretin secretion, we analyzed enteroendocrine cell pathways important for hormone biosynthesis and secretion. We demonstrate that progesterone increases GLP-1 secretion and extracellular signal-related kinase 1/2 (ERK1/2) phosphorylation in enteroendocrine GLUTag cells via mechanisms sensitive to the mitogen-activated protein kinase inhibitor U0126. The stimulatory effects of progesterone (P4) or the synthetic progestin R5020 on ERK1/2 phosphorylation were independent of the classical progesterone receptor antagonist RU486. Furthermore, a cell-impermeable BSA-progesterone conjugate rapidly increased ERK1/2 phosphorylation and GLP-1 secretion. Knockdown of the membrane progesterone receptors Paqr5 or Paqr7 in GLUTag cells eliminated the stimulatory effect of R5020 and progesterone on GLP-1 secretion. Enteral progesterone administration increased plasma levels of GLP-1, glucose-dependent insulinotropic polypeptide (GIP), and insulin, and improved oral glucose tolerance in an RU486-insensitve manner in mice: however, systemic progesterone exposure did not improve glucose homeostasis. Unexpectedly, the glucoregulatory actions of enteral progesterone did not require classical incretin receptor signaling and were preserved in Glp1r(-/-) and Glp1r(-/-):Gipr(-/-) mice. Intestine-restricted activation of membrane progesterone receptors may represent a novel approach for stimulation of incretin hormone secretion and control of glucose homeostasis.

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P4 stimulates ERK1/2 phosphorylation and GLP-1 secretion independent of the classical PR in GLUTag cells. A: P4-stimulated ERK1/2 phosphorylation is not inhibited by RU486 in GLUTag cells. Western blot analysis of ERK1/2 phosphorylation in cells pretreated for 15 min with the PR antagonist RU486 (1 μmol) followed by treatment for 10 or 30 min with R5020 (20 nmol), P4 (20 nmol), and epidermal growth factor (EGF; 25 ng/mL, positive control for ERK1/2 phosphorylation). Anti-HSP90 was used to monitor loading and transfer conditions. B: Covalently bound BSA-P4 increases ERK1/2 phosphorylation. GLUTag cells were treated for 10 min with the progestin R5020 (20 nmol), P4 (20 nmol), or covalently bound BSA-P4 (20 nmol P4). Cells treated with V (ethanol) plus BSA-FA (20 nmol/L) were used as the basal control. Figure shows the densitometric analysis of ERK1/2 phosphorylation normalized to HSP90 in the same experiment and expressed relative to basal conditions. Results depict mean ± SD of two independent experiments each done in triplicate. C: BSA-P4 (20 nmol/L P4 equivalent) stimulates GLP-1 secretion in GLUTag cells. Cells were treated for 2 h with R5020 (20 nmol/L), P4 (20 nmol), or BSA-P4 (20 nmol/L of P4). Cells treated with V (ethanol) plus BSA-FA (20 nmol/L) were used as the basal control. Total GLP-1 secreted in the media (2 h incubation) (pg/mL) was normalized to total cell/protein content (μg). Results depict mean + SD of two independent experiments, each performed in quadruplicate and expressed relative to levels of GLP-1 secreted under basal conditions. B and C: ^P < 0.05, BSA-P4 vs. P4, *P < 0.05, **P < 0.01, ***P < 0.001 vs. basal conditions. D: BSA-P4 (20 nmol/L P4) does not transactivate the MMTV-Luc promoter. GLUTag cells transfected with MMTV-Luc were treated for 12 h with R5020 (20 nmol/L), BSA-P4 (20 nmol/L of P4), V, or BSA-FA (20 nmol/L). Results are expressed relative to the luciferase activity in V-treated cells and represent the mean + SD of a representative experiment done in triplicate. ***P < 0.001 relative to vehicle-treated cells. E: siRNA knockdown reduces levels of PR, Paqr5, and Paqr7 mRNA transcripts in GLUTag cells. siRNA was transfected as described in Research Design and Methods, and the efficiency of RNA knockdown was assessed by qPCR. Levels of mRNA transcripts after knockdown are expressed relative to the levels detected under basal conditions. *P < 0.05, **P < 0.01, ***P < 0.001 vs. basal. F: Relative GLP-1 secretion in cells transfected with the indicated siRNAs and treated for 2 h with R5020 (20 nmol/L), P4 (20 nmol/L), or V (ethanol). Total GLP-1 secreted in the media (pg/mL) was normalized to total cell protein content (μg). Results are expressed relative to levels of GLP-1 in cells treated with V and depict mean ± SD from three independent experiments each done in triplicate. *P < 0.05, ***P < 0.001 vs. basal, ###P < 0.001, small interfering PR (siPR) + P4 vs. basal + P4.
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Figure 3: P4 stimulates ERK1/2 phosphorylation and GLP-1 secretion independent of the classical PR in GLUTag cells. A: P4-stimulated ERK1/2 phosphorylation is not inhibited by RU486 in GLUTag cells. Western blot analysis of ERK1/2 phosphorylation in cells pretreated for 15 min with the PR antagonist RU486 (1 μmol) followed by treatment for 10 or 30 min with R5020 (20 nmol), P4 (20 nmol), and epidermal growth factor (EGF; 25 ng/mL, positive control for ERK1/2 phosphorylation). Anti-HSP90 was used to monitor loading and transfer conditions. B: Covalently bound BSA-P4 increases ERK1/2 phosphorylation. GLUTag cells were treated for 10 min with the progestin R5020 (20 nmol), P4 (20 nmol), or covalently bound BSA-P4 (20 nmol P4). Cells treated with V (ethanol) plus BSA-FA (20 nmol/L) were used as the basal control. Figure shows the densitometric analysis of ERK1/2 phosphorylation normalized to HSP90 in the same experiment and expressed relative to basal conditions. Results depict mean ± SD of two independent experiments each done in triplicate. C: BSA-P4 (20 nmol/L P4 equivalent) stimulates GLP-1 secretion in GLUTag cells. Cells were treated for 2 h with R5020 (20 nmol/L), P4 (20 nmol), or BSA-P4 (20 nmol/L of P4). Cells treated with V (ethanol) plus BSA-FA (20 nmol/L) were used as the basal control. Total GLP-1 secreted in the media (2 h incubation) (pg/mL) was normalized to total cell/protein content (μg). Results depict mean + SD of two independent experiments, each performed in quadruplicate and expressed relative to levels of GLP-1 secreted under basal conditions. B and C: ^P < 0.05, BSA-P4 vs. P4, *P < 0.05, **P < 0.01, ***P < 0.001 vs. basal conditions. D: BSA-P4 (20 nmol/L P4) does not transactivate the MMTV-Luc promoter. GLUTag cells transfected with MMTV-Luc were treated for 12 h with R5020 (20 nmol/L), BSA-P4 (20 nmol/L of P4), V, or BSA-FA (20 nmol/L). Results are expressed relative to the luciferase activity in V-treated cells and represent the mean + SD of a representative experiment done in triplicate. ***P < 0.001 relative to vehicle-treated cells. E: siRNA knockdown reduces levels of PR, Paqr5, and Paqr7 mRNA transcripts in GLUTag cells. siRNA was transfected as described in Research Design and Methods, and the efficiency of RNA knockdown was assessed by qPCR. Levels of mRNA transcripts after knockdown are expressed relative to the levels detected under basal conditions. *P < 0.05, **P < 0.01, ***P < 0.001 vs. basal. F: Relative GLP-1 secretion in cells transfected with the indicated siRNAs and treated for 2 h with R5020 (20 nmol/L), P4 (20 nmol/L), or V (ethanol). Total GLP-1 secreted in the media (pg/mL) was normalized to total cell protein content (μg). Results are expressed relative to levels of GLP-1 in cells treated with V and depict mean ± SD from three independent experiments each done in triplicate. *P < 0.05, ***P < 0.001 vs. basal, ###P < 0.001, small interfering PR (siPR) + P4 vs. basal + P4.

Mentions: The induction of ERK1/2 phosphorylation by R5020 and the inhibition of R5020-stimulated GLP-1 secretion by U0126 raised the possibility of a nongenomic mechanism of action. Consistent with this possibility, both R5020 and P4 rapidly increased ERK1/2 phosphorylation in GLUTag cells in the presence of the classical PR antagonist RU486 (Fig. 3A). Furthermore, we detected expression of mRNA transcripts for membrane PRs, specifically the progestin and adipoQ receptor family member V (Paqr5) and the progestin and adipoQ receptor family member VII (Paqr7) in RNA from murine jejunum and colon, as well as in GLUTag cells (Supplementary Fig. 1). To examine the importance of membrane-localized progesterone in GLUTag cells, we used BSA-P4, a P4-albumin conjugate (26). BSA-P4 rapidly increased ERK1/2 phosphorylation (Fig. 3B) and GLP-1 secretion (Fig. 3C) from GLUTag cells to levels comparable to or modestly greater than that seen with R5020 or P4. In contrast, BSA-P4, unlike R5020, had no effect on the activity of MMTV-Luc in transfected GLUTag cells (Fig. 3D).


Activation of enteroendocrine membrane progesterone receptors promotes incretin secretion and improves glucose tolerance in mice.

Flock GB, Cao X, Maziarz M, Drucker DJ - Diabetes (2012)

P4 stimulates ERK1/2 phosphorylation and GLP-1 secretion independent of the classical PR in GLUTag cells. A: P4-stimulated ERK1/2 phosphorylation is not inhibited by RU486 in GLUTag cells. Western blot analysis of ERK1/2 phosphorylation in cells pretreated for 15 min with the PR antagonist RU486 (1 μmol) followed by treatment for 10 or 30 min with R5020 (20 nmol), P4 (20 nmol), and epidermal growth factor (EGF; 25 ng/mL, positive control for ERK1/2 phosphorylation). Anti-HSP90 was used to monitor loading and transfer conditions. B: Covalently bound BSA-P4 increases ERK1/2 phosphorylation. GLUTag cells were treated for 10 min with the progestin R5020 (20 nmol), P4 (20 nmol), or covalently bound BSA-P4 (20 nmol P4). Cells treated with V (ethanol) plus BSA-FA (20 nmol/L) were used as the basal control. Figure shows the densitometric analysis of ERK1/2 phosphorylation normalized to HSP90 in the same experiment and expressed relative to basal conditions. Results depict mean ± SD of two independent experiments each done in triplicate. C: BSA-P4 (20 nmol/L P4 equivalent) stimulates GLP-1 secretion in GLUTag cells. Cells were treated for 2 h with R5020 (20 nmol/L), P4 (20 nmol), or BSA-P4 (20 nmol/L of P4). Cells treated with V (ethanol) plus BSA-FA (20 nmol/L) were used as the basal control. Total GLP-1 secreted in the media (2 h incubation) (pg/mL) was normalized to total cell/protein content (μg). Results depict mean + SD of two independent experiments, each performed in quadruplicate and expressed relative to levels of GLP-1 secreted under basal conditions. B and C: ^P < 0.05, BSA-P4 vs. P4, *P < 0.05, **P < 0.01, ***P < 0.001 vs. basal conditions. D: BSA-P4 (20 nmol/L P4) does not transactivate the MMTV-Luc promoter. GLUTag cells transfected with MMTV-Luc were treated for 12 h with R5020 (20 nmol/L), BSA-P4 (20 nmol/L of P4), V, or BSA-FA (20 nmol/L). Results are expressed relative to the luciferase activity in V-treated cells and represent the mean + SD of a representative experiment done in triplicate. ***P < 0.001 relative to vehicle-treated cells. E: siRNA knockdown reduces levels of PR, Paqr5, and Paqr7 mRNA transcripts in GLUTag cells. siRNA was transfected as described in Research Design and Methods, and the efficiency of RNA knockdown was assessed by qPCR. Levels of mRNA transcripts after knockdown are expressed relative to the levels detected under basal conditions. *P < 0.05, **P < 0.01, ***P < 0.001 vs. basal. F: Relative GLP-1 secretion in cells transfected with the indicated siRNAs and treated for 2 h with R5020 (20 nmol/L), P4 (20 nmol/L), or V (ethanol). Total GLP-1 secreted in the media (pg/mL) was normalized to total cell protein content (μg). Results are expressed relative to levels of GLP-1 in cells treated with V and depict mean ± SD from three independent experiments each done in triplicate. *P < 0.05, ***P < 0.001 vs. basal, ###P < 0.001, small interfering PR (siPR) + P4 vs. basal + P4.
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Figure 3: P4 stimulates ERK1/2 phosphorylation and GLP-1 secretion independent of the classical PR in GLUTag cells. A: P4-stimulated ERK1/2 phosphorylation is not inhibited by RU486 in GLUTag cells. Western blot analysis of ERK1/2 phosphorylation in cells pretreated for 15 min with the PR antagonist RU486 (1 μmol) followed by treatment for 10 or 30 min with R5020 (20 nmol), P4 (20 nmol), and epidermal growth factor (EGF; 25 ng/mL, positive control for ERK1/2 phosphorylation). Anti-HSP90 was used to monitor loading and transfer conditions. B: Covalently bound BSA-P4 increases ERK1/2 phosphorylation. GLUTag cells were treated for 10 min with the progestin R5020 (20 nmol), P4 (20 nmol), or covalently bound BSA-P4 (20 nmol P4). Cells treated with V (ethanol) plus BSA-FA (20 nmol/L) were used as the basal control. Figure shows the densitometric analysis of ERK1/2 phosphorylation normalized to HSP90 in the same experiment and expressed relative to basal conditions. Results depict mean ± SD of two independent experiments each done in triplicate. C: BSA-P4 (20 nmol/L P4 equivalent) stimulates GLP-1 secretion in GLUTag cells. Cells were treated for 2 h with R5020 (20 nmol/L), P4 (20 nmol), or BSA-P4 (20 nmol/L of P4). Cells treated with V (ethanol) plus BSA-FA (20 nmol/L) were used as the basal control. Total GLP-1 secreted in the media (2 h incubation) (pg/mL) was normalized to total cell/protein content (μg). Results depict mean + SD of two independent experiments, each performed in quadruplicate and expressed relative to levels of GLP-1 secreted under basal conditions. B and C: ^P < 0.05, BSA-P4 vs. P4, *P < 0.05, **P < 0.01, ***P < 0.001 vs. basal conditions. D: BSA-P4 (20 nmol/L P4) does not transactivate the MMTV-Luc promoter. GLUTag cells transfected with MMTV-Luc were treated for 12 h with R5020 (20 nmol/L), BSA-P4 (20 nmol/L of P4), V, or BSA-FA (20 nmol/L). Results are expressed relative to the luciferase activity in V-treated cells and represent the mean + SD of a representative experiment done in triplicate. ***P < 0.001 relative to vehicle-treated cells. E: siRNA knockdown reduces levels of PR, Paqr5, and Paqr7 mRNA transcripts in GLUTag cells. siRNA was transfected as described in Research Design and Methods, and the efficiency of RNA knockdown was assessed by qPCR. Levels of mRNA transcripts after knockdown are expressed relative to the levels detected under basal conditions. *P < 0.05, **P < 0.01, ***P < 0.001 vs. basal. F: Relative GLP-1 secretion in cells transfected with the indicated siRNAs and treated for 2 h with R5020 (20 nmol/L), P4 (20 nmol/L), or V (ethanol). Total GLP-1 secreted in the media (pg/mL) was normalized to total cell protein content (μg). Results are expressed relative to levels of GLP-1 in cells treated with V and depict mean ± SD from three independent experiments each done in triplicate. *P < 0.05, ***P < 0.001 vs. basal, ###P < 0.001, small interfering PR (siPR) + P4 vs. basal + P4.
Mentions: The induction of ERK1/2 phosphorylation by R5020 and the inhibition of R5020-stimulated GLP-1 secretion by U0126 raised the possibility of a nongenomic mechanism of action. Consistent with this possibility, both R5020 and P4 rapidly increased ERK1/2 phosphorylation in GLUTag cells in the presence of the classical PR antagonist RU486 (Fig. 3A). Furthermore, we detected expression of mRNA transcripts for membrane PRs, specifically the progestin and adipoQ receptor family member V (Paqr5) and the progestin and adipoQ receptor family member VII (Paqr7) in RNA from murine jejunum and colon, as well as in GLUTag cells (Supplementary Fig. 1). To examine the importance of membrane-localized progesterone in GLUTag cells, we used BSA-P4, a P4-albumin conjugate (26). BSA-P4 rapidly increased ERK1/2 phosphorylation (Fig. 3B) and GLP-1 secretion (Fig. 3C) from GLUTag cells to levels comparable to or modestly greater than that seen with R5020 or P4. In contrast, BSA-P4, unlike R5020, had no effect on the activity of MMTV-Luc in transfected GLUTag cells (Fig. 3D).

Bottom Line: Glucagon-like peptide-1 (GLP-1) secretion is classically regulated by ingested nutrients.Furthermore, a cell-impermeable BSA-progesterone conjugate rapidly increased ERK1/2 phosphorylation and GLP-1 secretion.Enteral progesterone administration increased plasma levels of GLP-1, glucose-dependent insulinotropic polypeptide (GIP), and insulin, and improved oral glucose tolerance in an RU486-insensitve manner in mice: however, systemic progesterone exposure did not improve glucose homeostasis.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, Institute of Medical Sciences, Samuel Lunenfeld Research Institute, University of Toronto, Toronto, Ontario, Canada.

ABSTRACT
Glucagon-like peptide-1 (GLP-1) secretion is classically regulated by ingested nutrients. To identify novel molecular targets controlling incretin secretion, we analyzed enteroendocrine cell pathways important for hormone biosynthesis and secretion. We demonstrate that progesterone increases GLP-1 secretion and extracellular signal-related kinase 1/2 (ERK1/2) phosphorylation in enteroendocrine GLUTag cells via mechanisms sensitive to the mitogen-activated protein kinase inhibitor U0126. The stimulatory effects of progesterone (P4) or the synthetic progestin R5020 on ERK1/2 phosphorylation were independent of the classical progesterone receptor antagonist RU486. Furthermore, a cell-impermeable BSA-progesterone conjugate rapidly increased ERK1/2 phosphorylation and GLP-1 secretion. Knockdown of the membrane progesterone receptors Paqr5 or Paqr7 in GLUTag cells eliminated the stimulatory effect of R5020 and progesterone on GLP-1 secretion. Enteral progesterone administration increased plasma levels of GLP-1, glucose-dependent insulinotropic polypeptide (GIP), and insulin, and improved oral glucose tolerance in an RU486-insensitve manner in mice: however, systemic progesterone exposure did not improve glucose homeostasis. Unexpectedly, the glucoregulatory actions of enteral progesterone did not require classical incretin receptor signaling and were preserved in Glp1r(-/-) and Glp1r(-/-):Gipr(-/-) mice. Intestine-restricted activation of membrane progesterone receptors may represent a novel approach for stimulation of incretin hormone secretion and control of glucose homeostasis.

Show MeSH
Related in: MedlinePlus