A comparative analysis of G-protein- coupled receptor and peptide ligand expression within human and mouse islets of Langerhans, and its application in exploring the role of CXCL14 in islet function

摘要

G protein-coupled receptors (GPCRs) are a diverse super family of seven transmembrane spanning proteins whose primary function is to initiate the activation of intracellular signalling pathways following stimulation by extracellular stimuli, which include photons, amines, lipids, ions, peptides and proteins. Due to the ubiquitous expression of GPCRs throughout various tissues, they are implicated in the regulation of a variety of diverse physiological processes, such as secretion of the blood glucose controlling hormones insulin, glucagon and somatostatin from islets. As a result, GPCRs are being identified as therapeutic targets for the treatment of type-2 diabetes. Despite the large array of potential GPCR targets available, only a handful of GPCRs have proven to be successful clinical targets, which may partially be due to the lack of availability of suitable translational models that reflect the human GPCR landscape. The aim of the experiments described in this thesis was to compare the mRNA expression profiles of all GPCRs (the GPCRome) and all GPCR peptide ligands (the Secretome) in human and mouse islets in order to determine the suitability of using mouse islets as a translational model for predicting the role of islet GPCRs and GPCR peptide ligands in human islet function. In addition, some experiments demonstrate how the GPCRome and Secretome data were used to assess the role of CXCL14 in islet function. Quantitative real-time PCR (qPCR) was used to compare the mRNA expression profiles of 376 GPCRs and 159 GPCR peptide ligands in human islets with their orthologous counterparts in islets isolated from two strains of mice (outbred ICR mice and inbred C57/BL6 mice). A reasonable degree of similarity in GPCR mRNA expression between human islets and islets from each mouse strain was found (r2 = 0.360 vs. ICR; r2 = 0.304 vs C57), with a highly similar expression profile observed between the ICR and the C57 mouse strains (r2 = 0.946). Regression analysis of GPCR peptide ligand mRNA expression revealed that human islets exhibit a reasonable degree of similarity compared to islets of both mouse strains (r2 = 0.245 vs. ICR; r2 = 0.225 vs. C57) with a highly similar expression profile observed between the two mouse strains (r2 = 0.968). In the process of quantifying the GPCR peptide ligand mRNA expression profiles, it was revealed that the orphan chemokine CXCL14 is expressed by both mouse and human islets. Studies have shown that CXCL14 knockout mice are protected from hyperglycaemia and hyperinsulinemia and they have improved insulin sensitivity. However, CXCL14’s role in islet function has yet to be explored. The experiments described in this thesis demonstrate that CXCL14 inhibits insulin secretion from mouse islets and the MIN6 mouse β-cell line by a mechanism that is not transduced through a Gαi-mediated reduction in intracellular cAMP, but is likely to occur through an inhibition of glucose uptake or glucokinase activity. Further experiments designed to elucidate the target involved in CXCL14 function revealed that CXCL14 is neither an agonist nor an antagonist for the CXCR7 receptor and the putative CXCR4 receptor, and thus these receptors are not responsible for mediating CXCL14 function. In summary, the experiments described in this thesis reveal that human and mouse islets exhibit some degree of similarity in GPCR and GPCR peptide ligand mRNA expression, but the suitability of using mouse islets as surrogates for predicting human islet physiology is receptor/receptor family specific. This thesis also reveals how the GPCRome and Secretome data can be employed to investigate the role of particular ligands, such as CXCL14, and potential GPCRs responsible for mediating the actions of such ligands in islet function.