Defining G protein-coupled receptor peptide ligand expressomes and signalomes in human and mouse islets
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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.
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.
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.
KeywordsGPCRs Islets of Langerhans Peptide ligands PYY Type 2 diabetes
G protein-coupled receptors (GPCRs) play important roles in regulating secretion of the islet-derived gluco-regulatory hormones insulin, glucagon and somatostatin, and more than one-third of all GPCRs expressed by human islets are activated by ligands that are either proteins or polypeptides . Many proteins and peptides that act as ligands of islet GPCRs are expressed by the islet cells themselves , while others are secreted from non-islet cell types and transported to the islet microenvironment by the systemic circulation. Examples of locally produced peptide ligands of islet GPCRs include glucagon and somatostatin, which act in a paracrine function to regulate secretion of islet hormones from neighbouring cells . In addition, collagen III and IV, which are components of the extracellular matrix that supports islet cells, interact with the islet-expressed GPCRs, GPR56 and GPR126 [3, 4, 5]. GPCR peptide ligands that are delivered to islets in the circulation include the incretin hormones GLP-1 and GIP, which are secreted by specialised enteroendocrine cells in the gastrointestinal tract , and it has recently been reported that the cardiomyocyte-derived atrial natriuretic peptide stimulates insulin secretion via activation of β-cell guanylate cyclase-coupled GC-A receptors .
It is clear that peptide and protein (collectively referred to as ‘peptides’) GPCR ligands are important regulators of islet function and the maintenance of glucose homeostasis . The majority of studies investigating the roles of islet-derived peptide ligands acting at GPCRs to regulate islet function have been carried out in vitro using mouse islets or in vivo using mouse models of metabolic disease. However, to date no systematic studies have been carried out to investigate how effectively observations made in these mouse models are translatable to the human setting. The notion that there might be significant differences in GPCR/peptide signalling pathways in mouse and man is supported by the observations that in some respects rodent and human islets show distinctive gene expression profiles [8, 9, 10, 11, 12]. In particular, we have recently reported that although the majority of GPCR mRNAs are common to mouse and human islets, some that are expressed by mouse islets are absent or expressed at only trace levels in human islets, and studies on these receptors in rodent islets will not be translatable to the human context . Moreover, mouse and human islets have distinct architecture such that mouse islet function relies mainly on homogenous cellular communications between β-cells, with a simple framework distribution of cell subpopulations, while human islets are formed by heterogeneous cell populations [13, 14]. The increased contacts between adjacent endocrine cell populations in human islets suggest that the intra-islet paracrine signalling is likely to be more complicated than in rodent islets, in terms of signalling molecules interacting with their respective receptors and triggering autocrine or paracrine regulatory circuits .
In the current study, mRNA expression of all peptide ligands of islet GPCRs has been quantified and used to generate “signalome” atlases mapping the pathways by which peptide ligands regulate human and mouse islet GPCR signalling. These atlases can be used to identify the extent to which GPCR peptide ligand gene expression and GPCR/peptide signalling pathways are common to mouse and human islets, which will facilitate the translation of experimental results obtained using mouse islets and mouse models of metabolic disease to the human islet context.
Materials and methods
TaqMan RT-PCR kits were from Thermo Fisher Scientific (Loughborough, UK) and QuantiTect SYBR Green qPCR kits with QuantiTect qPCR assays were from Qiagen Ltd. (Manchester, UK). The glucagon antibody was from Sigma-Aldrich (Dorset, UK), insulin antibody from DAKO UK Ltd. (Ely, UK) and the anti-PYY and somatostatin antibodies were from Abcam plc (Cambridge, UK). Anti-rabbit, anti-guinea pig, anti-rat and anti-mouse secondary antibodies were from Jackson ImmunoResearch (Suffolk, UK). [125I]-glucagon was from Millipore (UK) Limited (Watford, UK) and somatostatin EURIA was from Euro Diagnostica AB (Malmö, Sweden). All other chemicals were from Sigma-Aldrich or Thermo Fisher Scientific.
Islet isolation and culture
Islets were isolated from 10 to 12 weeks old male mice of the inbred C57BL/6 (C57; Charles River) and outbred Crl:CD1 (ICR) mice (Charles River) strains by collagenase digestion of the exocrine pancreas . All animal procedures were carried out in accordance with the UK Home Office Animals (Scientific Procedures) Act 1986. Human islets were isolated from heart-beating non-diabetic donors as previously described , with appropriate ethical approval for research use. Isolated mouse and human islets were maintained in culture overnight (mouse: RPMI 1640; human: CMRL) at 37 °C, 5% CO2 before experimental use.
RNA extraction and quantitative real-time PCR
For enrichment of GPCR peptide ligand expression in mouse islets, the inverse of the above formula was used.
Construction of the human and mouse islet GPCR/peptide ligand signalome atlases
The pathways through which peptide ligands interacted with islet GPCRs were identified by manual searches in the PubMed, IUPHAR and Ingenuity databases and separate GPCR/peptide ligand pathways were constructed based on the mRNA expression of the peptide ligands and their receptors in human and mouse (ICR and C57) islets. The expression profiles of islet GPCRs that are activated by peptide ligands were extracted from our recent study that focused on defining expression of all GPCRs by human and mouse islets . Those GPCRs and GPCR peptide ligands that had confirmed islet mRNA expression at trace level or above were included in the human and mouse islet signalome atlases described in this paper. Peptide ligand genes that were absent in islets, but which are known to be expressed in other tissues, were not included in the atlases but GPCRs that were not expressed by human and mouse islets were included, to indicate that islet-derived peptide ligands have the potential to act at remote GPCRs.
Expression of PYY by cells within 5-μm sections of mouse and human pancreas was determined using a rabbit-derived antibody directed against PYY (1:50) and an anti-rabbit AlexaFluor® 488 secondary antibody (1:200). PYY localisation in islet cells was probed by co-staining with guinea pig anti-insulin (1:200), mouse anti-glucagon (1:100) and rat anti-somatostatin (1:50) antibodies and species-specific Alexa Fluor® 594 secondary antibodies (all at 1:200). Negative control sections were probed with AlexaFluor secondary antibodies in the absence of primary antibodies. Immunostained pancreas sections were visualised under a TE2000 fluorescent microscope (Nikon, Kanagawa, Japan) and analysed using Image J software (https://imagej.net/). The proportion of cells expressing PYY in each islet cell subpopulation was calculated by dividing the mean number of PYY-positive cells by the number of β-, α- or δ-cells per islet.
Quantification of insulin, glucagon and somatostatin secretion from mouse and human islets
Groups of ten mouse and human islets were incubated for 1 h in a physiological salt solution  in the absence or presence of the high affinity NPY1R antagonist, BIBO and insulin, glucagon and somatostatin in the supernatants were quantified by competitive radioimmunoassay, essentially as described elsewhere [23, 24, 25].
GraphPad Prism 5.0 (GraphPad Software, Inc.) was used for statistical analyses. Data are presented as mean ± SEM and were analysed using ANalysis Of VAriance (ANOVA). Statistical significance was set at p values of < 0.05 (*), < 0.01 (**), < 0.001 (***), considering p < 0.05 as statistically significant.
Expression of GPCR peptide ligand mRNAs in human and mouse islets
Comparative analysis of GPCR/peptide signalling pathways in human and mouse islets
Comparison of relative expression of GPCR peptide ligand and GPCR mRNAs in the signalome atlases led to the identification of species differences in mRNA expression, which can be used to predict the relative importance of particular pathways. For example, mRNAs encoding peptide YY (Pyy) and pancreatic polypeptide (Ppy), which are ligands at NPY receptors, were expressed at ~ 40,000-fold (Pyy) and ~ 400-fold higher levels in mouse islets than in human islets (Figs. 3, 4, Supplementary Figs. 1, 2). In addition, the prodynorphin (Pdyn) gene, which encodes several peptides of the dynorphin family that act as ligands at δ, μ and κ opioid receptors, was abundantly expressed in mouse islets but absent in human islets. Similarly, mRNA encoding the small secreted protein R-spondin, which acts as an agonist of the leucine-rich repeat GPCRs Lgr4, Lgr5 and Lgr6, was present at high levels in mouse islets but only found at trace levels in human islets. In contrast, our analysis indicated that human islets express high levels of mRNA encoding C1QL1, while mouse islets express very low levels of this mRNA. A similar pattern was observed for the CXCL8 gene that encodes the chemotactic peptide IL-8: CXCL8 is absent from the mouse genome and, therefore, was not present in mouse islet cells, but CXCL8 mRNA was abundantly expressed by human islets. As human islets do not express mRNAs encoding the CXCR1 and CXCR2 receptors, through which IL-8 signals, it is likely that this peptide may have an extra-islet role, perhaps in local inflammation, following its secretion from human islet cells. Overall, the signalomes depicted in Supplementary Fig. 2 allow us to predict that the C1QL1 signalling pathway may dominate in human islets, while the PYY, pancreatic polypeptide, dynorphin and R-spondin 4 signalling pathways are more important in regulating function of mouse islets.
PYY as a comparative example for ligand peptide GPCR expression in human and mouse islets
Peptides and small proteins are important regulators of islet function and glucose homeostasis, at least in part through interactions with GPCRs present both on islet cells and other organ systems . However, although human islets express 293 GPCRs, to date the GLP-1 receptor is the only islet GPCR that is currently a target for drugs used to treat type 2 diabetes [28, 29]. In addition to expression of numerous GPCRs, islets are thought to express mRNAs of > 100 genes encoding peptide and protein ligands of GPCRs , some of which may have potential as novel diabetes therapies. In the current study, we used qPCR with validated primers for GPCR peptide ligand mRNAs as a versatile yet cost effective and sensitive method for detection of low-abundant transcripts that are not easily detectable using low throughput antibody-based approaches . The data generated were used to construct three islet signalome atlases describing all known GPCR/peptide signalling pathways in human islets and islets from outbred ICR and inbred C57 mice, to allow identification of similarities and differences in GPCR peptide ligand expression between the two species and place these differences into a pharmacological perspective.
In total, the signalome atlases indicated the existence of 418 signalling pathways through which human- and mouse islet-derived peptides could interact with GPCRs, approximately 20% of which were conserved between species. An additional 50% of all identified pathways shared a large number of components between human islets and islets from the two mouse strains, but were not identical. About 4% of all GPCR/peptide signalling pathways were only found in mouse islets, whereas 20% were unique to human islets. The majority of the pathways identified constitute endogenous islet signalling in which the mRNAs encoding both the peptide ligand and the corresponding cognate GPCR were expressed by islet cells. Although many of these GPCRs are also expressed in other cell types and organ systems, it is likely that the islet-derived peptides will have local effects on the islet GPCRs before being transported by the systemic circulation to other cells expressing the receptors.
When considering genes encoding specific GPCR peptide ligands, it was found that glucagon and islet amyloid polypeptide (Iapp) mRNAs showed the highest expression in human and mouse islets, respectively. These observations are consistent with human islets having a greater proportion of α-cells than mouse islets [13, 14] and with Iapp being synthesised and co-stored with insulin in β-cells , which constitute approximately 80% of the endocrine cells of mouse islets [13, 14]. While mouse and human islets express very high levels of preproinsulin mRNA this was not quantified in the current study as insulin is an agonist at a tyrosine kinase receptor rather than a GPCR. The peptide ligand mRNA expression profile of ICR mouse islets was closer than that of C57 mouse islets to the mRNA expression of human islets, most likely as a consequence of normal genetic variation being retained better in outbred ICR mice than in the more homogenous inbred C57 mice. Interestingly, we found that several genes encoding ‘classical’ islet peptides, such as peptide YY (PPY), pancreatic polypeptide (PPY), vasoactive intestinal polypeptide (VIP) and urocortin 3 (UCN3) showed considerably higher expression in mouse than human islets. In particular, the gene encoding PYY was ~ 40,000-fold more abundant in mouse islets. Similarly, mRNAs encoding R-spondin 4 (RSPO4) and dynorphin peptides (PDYN) were expressed at very high levels in mouse islets, but were either absent or present only at trace levels in human islets. In contrast, mRNAs encoding the small secreted protein C1QL1 and the chemokine IL-8 were enriched in human islets.
The observation that PYY mRNA expression was considerably higher in mouse islets than in human islets led us to determine whether this 36-amino acid peptide that binds to the neuropeptide Y family of GPCRs (NPY1R, NPY2R, NPY4R, NPY5R) to regulate food intake and energy homeostasis  has differential functional effects in mouse and human islets. Fluorescence immunohistochemistry indicated higher PYY immunoreactivity in mouse than human islets, and co-staining with antibodies against islet hormones demonstrated that it was mainly localised to mouse and human islet α-cells, with lesser expression in β- and δ-cells. The main source of PYY is from enteroendocrine L-cells in the gastrointestinal tract following food intake, and it is rapidly cleaved N-terminally such that the major circulating form is PYY3–36 [31, 32]. This truncated peptide preferentially activates NPY2R, which is absent from mouse and human islets [1, 11, 33], so it is likely that islet-derived, full-length PYY (PYY1–36), which has high affinity for NPY1R, may exert local effects following its release from mouse islets.
We used l-arginine, an amino acid that induces islet endocrine cell exocytosis, to maximise PYY release from islets then blocked action of locally secreted PYY with the selective NPY1R antagonist BIBO. PYY1–36 is reported to inhibit insulin secretion through NPY1R [31, 32, 34, 35, 36, 37] so it was expected that exposure of arginine-stimulated mouse islets to BIBO would have enhanced insulin release, as has been observed in experiments where this antagonist blocked the inhibitory effects of exogenous PYY . However, neither 100 nM nor 1μM BIBO significantly affected insulin secretion, suggesting that local PYY concentrations were not sufficiently high to reduce arginine-induced insulin release. In contrast, we found that BIBO caused a concentration-dependent potentiation of arginine-stimulated glucagon release from mouse islets, consistent with the reported effect of PYY to inhibit glucagon secretion . As expected, given the lower levels of PYY in human islets, BIBO did not increase glucagon release in the human islet experiments. There have been no previous reports on the effects of PYY on somatostatin secretion, but our data suggest that locally released PYY acts at δ-cell NPY1R to inhibit somatostatin release, which is antagonised by BIBO. Again, this effect was not observed in human islets, which contain less PYY. Overall, these data point to a role for intra-islet signalling by PYY in mouse but not human islets.
In summary, in this study we have systematically compared the expression of all known human GPCR peptide ligand mRNAs with their mouse orthologues in human and mouse islets, and created detailed islet GPCR/peptide ligand signalome atlases to illustrate the similarities and differences in GPCR peptide ligand expression and signalling pathways. We have used the signalomes to predict a more important role for islet-derived PYY in mouse than human islets, and confirmed this by functional studies with an NPY1R antagonist. The data presented here will allow researchers to focus on GPCR peptide ligands that are present in human islets but not in mouse, perhaps leading to development of novel diabetes therapeutics.
This study was supported by grants from the EFSD/Boehringer-Ingelheim Research Programme (to SA and SP) and a Diabetes UK RD Lawrence Fellowship to SA (11/0004172).
The study was designed by SA, AS and SJP. Data were collected and analysed by PA, SA, RH, IRM, BL and IMA. GCH and MZ provided human islets of Langerhans. The article was drafted by SA, PA and SJP. All authors revised the article critically for intellectual content and gave their approval of this version to be published. PA, SA and SJP take responsibility for the contents of the article. We are grateful to the relatives of organ donors for human pancreases for research.
Compliance with ethical standards
Conflict of interest
All authors declare that they have no competing interests.
- 2.Jones PM, Persaud SJ (2017) Islet function and insulin secretion textbook of diabetes, 5th edn. Wiley, ChichesterGoogle Scholar
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