Defining G protein-coupled receptor peptide ligand expressomes and signalomes in human and mouse islets

Abstract

Introduction: Islets synthesise and secrete numerous peptides, some of which are known to be important regulators of islet function and glucose homeostasis. In this study, we quantified mRNAs encoding all peptide ligands of islet G protein-coupled receptors (GPCRs) in isolated human and mouse islets and carried out in vitro islet hormone secretion studies to provide functional confirmation for the species-specific role of peptide YY (PYY) in mouse islets.

Materials and methods: GPCR peptide ligand mRNAs in human and mouse islets were quantified by quantitative real-time PCR relative to the reference genes ACTB, GAPDH, PPIA, TBP and TFRC. The pathways connecting GPCR peptide ligands with their receptors were identified by manual searches in the PubMed, IUPHAR and Ingenuity databases. Distribution of PYY protein in mouse and human islets was determined by immunohistochemistry. Insulin, glucagon and somatostatin secretion from islets was measured by radioimmunoassay.

Results: We have quantified GPCR peptide ligand mRNA expression in human and mouse islets and created specific signalomes mapping the pathways by which islet peptide ligands regulate human and mouse GPCR signalling. We also identified species-specific islet expression of several GPCR ligands. In particular, PYY mRNA levels were ~ 40,000-fold higher in mouse than human islets, suggesting a more important role of locally secreted Pyy in mouse islets. This was confirmed by IHC and functional experiments measuring insulin, glucagon and somatostatin secretion.

Discussion: The detailed human and mouse islet GPCR peptide ligand atlases will allow accurate translation of mouse islet functional studies for the identification of GPCR/peptide signalling pathways relevant for human physiology, which may lead to novel treatment modalities of diabetes and metabolic disease.

Keywords: GPCRs; Islets of Langerhans; PYY; Peptide ligands; Type 2 diabetes.

Fig. 1

Summary of the mean relative expression of the ten most abundant GPCR peptide ligand genes in human islets (a) and ICR (b) and C57 (c) mouse islets. Data for each peptide are presented as mean % of the mRNA expression of all GPCR peptide ligands in each islet type

Fig. 2

Mean relative mRNA expression of GPCR peptide ligand genes in human and ICR and C57 mouse islets relative to the reference genes Actb, Gapdh, Ppia, Tbp and Tfrc. Insert panels A and B in b, c represent enlarged areas of the scatter plots, added to allow visualisation of individual mRNAs. a Data for 147 GPCR peptide ligand genes from non-pooled biological replicates from islets isolated from four C57 and four ICR mice. b Data for 159 human and 147 mouse GPCR peptide ligand genes from non-pooled biological replicates from four C57 and four human islet preparations. c Data for 159 human and 147 mouse GPCR peptide ligand genes from non-pooled biological replicates from four ICR and four human islet preparations

Fig. 2

Mean relative mRNA expression of GPCR peptide ligand genes in human and ICR and C57 mouse islets relative to the reference genes Actb, Gapdh, Ppia, Tbp and Tfrc. Insert panels A and B in b, c represent enlarged areas of the scatter plots, added to allow visualisation of individual mRNAs. a Data for 147 GPCR peptide ligand genes from non-pooled biological replicates from islets isolated from four C57 and four ICR mice. b Data for 159 human and 147 mouse GPCR peptide ligand genes from non-pooled biological replicates from four C57 and four human islet preparations. c Data for 159 human and 147 mouse GPCR peptide ligand genes from non-pooled biological replicates from four ICR and four human islet preparations

Fig. 3

Species- and strain-specific enrichment of GPCR peptide ligand genes. Data are presented as mean + SEM relative to Actb, Gapdh, Ppia, Tbp and Tfrc for four human islet donors and four ICR and four C57 mouse islet preparations. T denotes trace expression and A denotes absent expression. a Expression of 42 GPCR peptide ligand genes that are expressed above trace level in human islets, but either absent or present only at trace level in either ICR or C57 mouse islets. b Expression of 21 GPCR peptide ligand genes that are expressed above trace level in ICR or C57 mouse islets, but either absent or present only at trace level in human islets

Fig. 4

Peptide ligands expressed above trace level in human and mouse islets. Data in a are presented as mean + SEM relative to Actb, Gapdh, Ppia, Tbp and Tfrc and data in b, c are ratios of the mean expression relative to Actb, Gapdh, Ppia, Tbp and Tfrc. Data were generated using non-pooled islet preparations from four ICR mice, four C57 mice and four human donors. a Expression of 43 GPCR ligand genes that are expressed above trace level in mouse (ICR and C57) and human islets. b Species enrichment of GPCR peptide ligand genes present above trace level in mouse islets. c Species enrichment of GPCR peptide ligand genes present above trace level in human islets

Fig. 4

Peptide ligands expressed above trace level in human and mouse islets. Data in a are presented as mean + SEM relative to Actb, Gapdh, Ppia, Tbp and Tfrc and data in b, c are ratios of the mean expression relative to Actb, Gapdh, Ppia, Tbp and Tfrc. Data were generated using non-pooled islet preparations from four ICR mice, four C57 mice and four human donors. a Expression of 43 GPCR ligand genes that are expressed above trace level in mouse (ICR and C57) and human islets. b Species enrichment of GPCR peptide ligand genes present above trace level in mouse islets. c Species enrichment of GPCR peptide ligand genes present above trace level in human islets

Fig. 5

Summary of intra- and extra-islet peptide/GPCR signalling pathways in human islets (a) and in ICR (b) and C57 (c) mouse islets. Data are presented as % of the pathways that occur within islets (intra-islet), where the ligands are released from islets to act at non-islet GPCRs (extra-islet), or where the peptide ligands have been proposed to interact with as yet unidentified GPCRs (undefined)

Fig. 6

Expression of PYY protein in mouse and human pancreas sections. Co-localisation of PYY (green) with the islet hormones insulin (A–C), glucagon (D–F) and somatostatin (G–I) (red) in mouse (a) and human (c) islets. The data in b, d indicate quantification of the percentage of mouse (b) and human (d) islet endocrine cells that express PYY. ^&&p < 0.01, ***^,###p < 0.001. n = 7–9 islets

Fig. 6

Expression of PYY protein in mouse and human pancreas sections. Co-localisation of PYY (green) with the islet hormones insulin (A–C), glucagon (D–F) and somatostatin (G–I) (red) in mouse (a) and human (c) islets. The data in b, d indicate quantification of the percentage of mouse (b) and human (d) islet endocrine cells that express PYY. ^&&p < 0.01, ***^,###p < 0.001. n = 7–9 islets

Fig. 7

Effect of BIBO 3304 trifluoroacetate on insulin (a, d), glucagon (b, e) and somatostatin (c, f) secretion from mouse (a–c) and human (d–f) islets. Data are shown as mean + SEM insulin, glucagon and somatostatin release per islet per hour in static incubations, n = 8 for all hormones in both mouse and human islets. *p < 0.05, **p < 0.01; ***p < 0.001