Exploring the insulin secretory properties of the PGD2-GPR44/DP2 axis in vitro and in a randomized phase-1 trial of type 2 diabetes patients

Stanko Skrtic, Björn Tyrberg, Malin Broberg, Hans Ericsson, Volker Schnecke, Magnus Kjaer, Marcus Hompesch, Eva-Marie Andersson, Erik Ryberg, Alexander Aivazidis, Charlotte Wennberg Huldt, Lars Löfgren, Linda Morrow, Joanna Parkinson, Tina Rydén-Bergsten, Elaine Watkins, Maria Sörhede Winzell, Stanko Skrtic, Björn Tyrberg, Malin Broberg, Hans Ericsson, Volker Schnecke, Magnus Kjaer, Marcus Hompesch, Eva-Marie Andersson, Erik Ryberg, Alexander Aivazidis, Charlotte Wennberg Huldt, Lars Löfgren, Linda Morrow, Joanna Parkinson, Tina Rydén-Bergsten, Elaine Watkins, Maria Sörhede Winzell

Abstract

Aims/hypothesis: GPR44 (DP2, PTGDR2, CRTh2) is the receptor for the pro-inflammatory mediator prostaglandin D2 (PGD2) and it is enriched in human islets. In rodent islets, PGD2 is produced in response to glucose, suggesting that the PGD2-GPR44/DP2 axis may play a role in human islet function during hyperglycemia. Consequently, the aim of this work was to elucidate the insulinotropic role of GPR44 antagonism in vitro in human beta-cells and in type 2 diabetes (T2DM) patients.

Methods: We determined the drive on PGD2 secretion by glucose and IL-1beta, as well as, the impact on insulin secretion by pharmacological GPR44/DP2 antagonism (AZD1981) in human islets and beta-cells in vitro. To test if metabolic control would be improved by antagonizing a hyperglycemia-driven increased PGD2 tone, we performed a proof-of-mechanism study in 20 T2DM patients (average 54 years, HbA1c 9.4%, BMI 31.6 kg/m2). The randomized, double-blind, placebo-controlled cross-over study consisted of two three-day treatment periods (AZD1981 or placebo) separated by a three-day wash-out period. Mixed meal tolerance test (MMTT) and intravenous graded glucose infusion (GGI) was performed at start and end of each treatment period. Assessment of AZD1981 pharmacokinetics, glucose, insulin, C-peptide, glucagon, GLP-1, and PGD2 pathway biomarkers were performed.

Results: We found (1) that PGD2 is produced in human islet in response to high glucose or IL-1beta, but likely by stellate cells rather than endocrine cells; (2) that PGD2 suppresses both glucose and GLP-1 induced insulin secretion in vitro; and (3) that the GPR44/DP2 antagonist (AZD1981) in human beta-cells normalizes insulin secretion. However, AZD1981 had no impact on neither glucose nor incretin dependent insulin secretion in humans (GGI AUC C-peptide 1-2h and MMTT AUC Glucose 0-4h LS mean ratios vs placebo of 0.94 (80% CI of 0.90-0.98, p = 0.12) and 0.99 (90% CI of 0.94-1.05, p = 0.45), despite reaching the expected antagonist exposure.

Conclusion/interpretation: Pharmacological inhibition of the PGD2-GPR44/DP2 axis has no major impact on the modulation of acute insulin secretion in T2DM patients.

Trial registration: ClinicalTrials.gov NCT02367066.

Conflict of interest statement

All authors apart from MH, LM and EW are employees or shareholders of AstraZeneca AB. MH, LM, and EW received funding as a contract research organization by AstraZeneca for the clinical study. EW is affiliated with ProSciento Inc. There are no patents, products in development, or marketed products to declare. This does not alter our adherence to all the PLOS ONE policies on sharing data and materials.

Figures

Fig 1. Study flow chart according to…
Fig 1. Study flow chart according to CONSORT 2010 guidelines.
The design of a human Proof of Mechanism study to test the Mechanism of Action hypothesis. The phase 1 study was designed as a randomised, double-blind, placebo-controlled, multiple-dose, cross-over study, consisting of a run-in, two treatment periods separated by a wash-out period and a follow-up for each subject. The subjects were T2DM patients on metformin (n = 20).
Fig 2. Tissue expression of GPR44/DP2 and…
Fig 2. Tissue expression of GPR44/DP2 and PGD2 related genes and characterization of pancreatic stellate cells.
PTGDR2 (GPR44/DP2) mRNA expression in human beta-cells (Healthy, n = 197; T2DM, n = 112) (a) and pancreatic stellate cells (Healthy, n = 23; T2DM, n = 31) (b). Heat map of stellate cell signature gene expression sub-classified as quiescent, immune activated (imm) and standard activated (stand) stellate cells according to the description in the main text (c). Quantification of stellate cell subpopulations in healthy and T2D donor human islets (d). Expression of GPR44/DP2 and PGD2 related genes in subclasses of stellate cells (e). Data are presented as mean ± SEM (a-b), total number of cells (d), and individual cells (e). *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.
Fig 3. Human islet gene expression and…
Fig 3. Human islet gene expression and regulation by high glucose with and without IL-1beta.
Expression of PTGDR2 (GPR44/DP2) (a), PTGDR1 (DP1) (b), PTGDS (L-PGDS) (c), PTGS1 (COX-1) (d) and PTGS2 (COX-2) (e). In vitro human islet PGD2 secretion (f). Data are presented as mean ± SEM from 5 different human islet donors, where each condition was evaluated in quadruplicates. Glc = glucose. *p<0.05, **p<0.01, ****p<0.0001.
Fig 4. Effect of PGD 2 on…
Fig 4. Effect of PGD2 on insulin secretion and in vitro pharmacology of AZD1981 in the human beta-cell line EndoC- betaH1.
Dose response curve for the PGD2 analogue (15(R)-15-methyl-PGD2) on insulin secretion, from which the EC50 was determined (a). The in vitro potency of the GPR44/DP2 antagonist AZD1981 was determined using DMR (IC50 = 4.9±1nM) (b), cAMP (IC50 = 21±8nM) (c) and by glucose-stimulated insulin secretion (IC50 = 13±2nM) (d). The assays were performed in the human beta cell line EndoC-betaH1 with addition of 150 pM 15(R)-15-methyl-PGD2. Insulin secretion in EndoC-betaH1 cells was determined with and without addition of 150pM 15(R)-15-methyl-PGD2 at 11.1 mM glucose and 100nM exendin-4 (GLP-1 receptor agonist). 100nM of AZD1981 was added to restore GSIS. The effect of PGD2 on human islet insulin secretion was studied in islets from three donors where each incubation condition was run with 5 islets in 6 replicates (f). Data are presented as mean ± SEM.*p<0.05, **p<0.01***p<0.001.
Fig 5. Hypothesis for the endogenous PGD…
Fig 5. Hypothesis for the endogenous PGD2-GPR44/DP2 axis mechanism of action in islets.
Glucose and IL-1beta induced PTGS2 (COX-2) expression in stellate cells which stimulates the production of PGD2 synthesis. PGD2 is secreted and may act in a paracrine fashion on GPR44/DP2 on beta-cells, inhibiting cAMP and thereby reducing GSIS.
Fig 6. Mixed Meal Tolerance Test in…
Fig 6. Mixed Meal Tolerance Test in T2DM patients.
Glucose assessments at baseline (a), and after 3 days treatment (b) or Insulin Secretion Rate (ISR) at baseline (c) and after 3 days treatment (d). Circles connected with dotted lines represent placebo and squares connected with full lines represent AZD1981. Data are presented as mean ± SD.

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