Using single-cell multi-omics screening of human fetal pancreas to identify novel players in human beta cell development.

Islet transplantation from organ donors can considerably improve glucose homeostasis and well-being in individuals with type 1 diabetes, where the beta cells are destroyed by the autoimmune attack, but there are insufficient donor islets to make this a widespread therapy. Strategies are therefore being developed to generate unlimited amounts of insulin-producing beta cells from pluripotent stem cells, with the aim that they will be transplanted to treat diabetes. Whilst much progress has been made in recent years in the directed differentiation of pluripotent stem cells to beta-like cells, essential gaps still exist in generating stem cell-derived beta cells that are fully functional in vitro. This short review provides details of recent multi-'omics' studies of the human fetal pancreas, which are revealing granular information on the various cell types in the developing pancreas. It is anticipated that this fine mapping of the pancreatic cells at single-cell resolution will provide additional insights that can be utilised to reproducibly produce human beta cells in vitro that have the functional characteristics of beta cells within native human islets.

The selective serotonin reuptake inhibitor fluoxetine has direct effects on beta cells, promoting insulin secretion and increasing beta-cell mass.

AIM: This study investigated whether therapeutically relevant concentrations of fluoxetine, which have been shown to reduce plasma glucose and glycated haemoglobin independent of changes in food intake and body weight, regulate beta-cell function and improve glucose homeostasis. METHODS: Cell viability, insulin secretion, beta-cell proliferation and apoptosis were assessed after exposure of MIN6 beta cells or isolated mouse and human islets to 0.1, 1 or 10 µmol/L fluoxetine. The effect of fluoxetine (10 mg/kg body weight) administration on glucose homeostasis and islet function was also examined in ob/ob mice. RESULTS: Exposure of MIN6 cells and mouse islets to 0.1 and 1 µmol/L fluoxetine for 72 hours did not compromise cell viability but 10 µmol/L fluoxetine significantly increased Trypan blue uptake. The dose of 1 µmol/L fluoxetine significantly increased beta-cell proliferation and protected islet cells from cytokine-induced apoptosis. In addition, 1 µmol/L fluoxetine induced rapid and reversible potentiation of glucose-stimulated insulin secretion from islets isolated from mice, and from lean and obese human donors. Finally, intraperitoneal administration of fluoxetine to ob/ob mice over 14 days improved glucose tolerance and resulted in significant increases in beta-cell proliferation and enhanced insulin secretory capacity. CONCLUSIONS: These data are consistent with a role for fluoxetine in regulating glucose homeostasis through direct effects on beta cells. Fluoxetine thus demonstrates promise as a preferential antidepressant for patients with concomitant occurrence of depression and diabetes.

CXCL14 Inhibits Insulin Secretion Independently of CXCR4 or CXCR7 Receptor Activation or cAMP Inhibition.

BACKGROUND/AIMS: CXCL14, a secreted chemokine peptide that promotes obesity-induced insulin resistance, is expressed by islets, but its effects on islet function are unknown. The aim of this study was to determine the role of CXCL14 in ß-cells and investigate how it transduces these effects. METHODS: Cxcl14 and Cxc-receptor mRNA expression was quantified by qPCR and CXCL14 expression in the pancreas was determined by immunohistochemistry. The putative function of CXCL14 at CXCR4 and CXCR7 receptors was determined by ß-arrestin recruitment assays. The effects of CXCL14 on glucose-stimulated insulin secretion, cAMP production, glucose-6-phosphate accumulation, ATP generation, apoptosis and proliferation were determined using standard techniques. RESULTS: CXCL14 was present in mouse islets, where it was mainly localised to islet δ-cells. Cxc-receptor mRNA profiling indicated that Cxcr4 and Cxcr7 are the most abundant family members in islets, but CXCL14 did not promote ß-arrestin recruitment at CXCR4 or CXCR7 or antagonise CXCL12 activation of these receptors. CXCL14 induced a concentration-dependent inhibition of glucose-stimulated insulin secretion, which was not coupled to Gαi signalling. However, CXCL14 inhibited glucose-6-phosphate generation and ATP production in mouse islets. CONCLUSION: CXCL14 is expressed by islet δ-cells where it may have paracrine effects to inhibit insulin secretion in a CXCR4/CXCR7-independent manner through reductions in ß-cell ATP levels. These observations, together with the previously reported association of CXCL14 with obesity and impaired glucose homeostasis, suggest that inhibition of CXCL14 signalling could be explored to treat type 2 diabetes.

The adhesion receptor GPR56 is activated by extracellular matrix collagen III to improve ß-cell function.

AIMS: G-protein coupled receptor 56 (GPR56) is the most abundant islet-expressed G-protein coupled receptor, suggesting a potential role in islet function. This study evaluated islet expression of GPR56 and its endogenous ligand collagen III, and their effects on ß-cell function. METHODS: GPR56 and collagen III expression in mouse and human pancreas sections was determined by fluorescence immunohistochemistry. Effects of collagen III on ß-cell proliferation, apoptosis, intracellular calcium ([Ca2+]i) and insulin secretion were determined by cellular BrdU incorporation, caspase 3/7 activities, microfluorimetry and radioimmunoassay, respectively. The role of GPR56 in islet vascularisation and innervation was evaluated by immunohistochemical staining for CD31 and TUJ1, respectively, in pancreases from wildtype (WT) and Gpr56-/- mice, and the requirement of GPR56 for normal glucose homeostasis was determined by glucose tolerance tests in WT and Gpr56-/- mice. RESULTS: Immunostaining of mouse and human pancreases revealed that GPR56 was expressed by islet ß-cells while collagen III was confined to the peri-islet basement membrane and islet capillaries. Collagen III protected ß-cells from cytokine-induced apoptosis, triggered increases in [Ca2+]i and potentiated glucose-induced insulin secretion from WT islets but not from Gpr56-/- islets. Deletion of GPR56 did not affect glucose-induced insulin secretion in vitro and it did not impair glucose tolerance in adult mice. GPR56 was not required for normal islet vascularisation or innervation. CONCLUSION: We have demonstrated that collagen III improves islet function by increasing insulin secretion and protecting against apoptosis. Our data suggest that collagen III may be effective in optimising islet function to improve islet transplantation outcomes, and GPR56 may be a target for the treatment of type 2 diabetes.

SNAP-tag-enabled super-resolution imaging reveals constitutive and agonist-dependent trafficking of GPR56 in pancreatic ß-cells.

OBJECTIVE: Members of the adhesion G protein-coupled receptor (aGPCR) subfamily are important actors in metabolic processes, with GPR56 (ADGRG1) emerging as a possible target for type 2 diabetes therapy. GPR56 can be activated by collagen III, its endogenous ligand, and by a synthetic seven amino-acid peptide (TYFAVLM; P7) contained within the GPR56 Stachel sequence. However, the mechanisms regulating GPR56 trafficking dynamics and agonist activities are not yet clear. METHODS: Here, we introduced SNAPf-tag into the N-terminal segment of GPR56 to monitor GPR56 cellular activity in situ. Confocal and super-resolution microscopy were used to investigate the trafficking pattern of GPR56 in native MIN6 ß-cells and in MIN6 ß-cells where GPR56 had been deleted by CRISPR-Cas9 gene editing. Insulin secretion, changes in intracellular calcium, and ß-cell apoptosis were determined by radioimmunoassay, single-cell calcium microfluorimetry, and measuring caspase 3/7 activities, respectively, in MIN6 ß-cells and human islets. RESULTS: SNAP-tag labelling indicated that GPR56 predominantly underwent constitutive internalisation in the absence of an exogenous agonist, unlike GLP-1R. Collagen III further stimulated GPR56 internalisation, whereas P7 was without significant effect. The overexpression of GPR56 in MIN6 ß-cells did not affect insulin secretion. However, it was associated with reduced ß-cell apoptosis, while the deletion of GPR56 made MIN6 ß-cells more susceptible to cytokine-induced apoptosis. P7 induced a rapid increase in the intracellular calcium in MIN6 ß-cells (in a GPR56-dependent manner) and human islets, and it also caused a sustained and reversible increase in insulin secretion from human islets. Collagen III protected human islets from cytokine-induced apoptosis, while P7 was without significant effect. CONCLUSIONS: These data indicate that GPR56 exhibits both agonist-dependent and -independent trafficking in ß-cells and suggest that while GPR56 undergoes constitutive signalling, it can also respond to its ligands when required. We have also identified that constitutive and agonist-dependent GPR56 activation is coupled to protect ß-cells against apoptosis, offering a potential therapeutic target to maintain ß-cell mass in type 2 diabetes.

Assessing Mouse Islet Function.

Islets of Langerhans are clusters of endocrine cells embedded within the exocrine pancreas. Islets constitute only approximately 1-2% of the total pancreas mass in all species, so methods have been developed to digest the pancreas and purify islets from the surrounding acinar cells. This chapter provides detailed protocols for isolation of mouse islets and their in vitro functional characterization in terms of assessments of islet viability, hormone content and secretion, second messenger generation and ß-cell proliferation.

Adhesion G-protein coupled receptors: Implications for metabolic function.

Adhesion G-protein coupled receptors (aGPCRs) are emerging as important actors in energy homeostasis. Recent biochemical and functional studies using transgenic mice indicate that aGPCRs play important roles in endocrine and metabolic functions including ß-cell differentiation, insulin secretion, adipogenesis and whole body fuel homeostasis. Most aGPCRs are orphans, for which endogenous ligands have not yet been identified, and many of the endogenous ligands of the already de-orphanised aGPCRs are components of the extracellular matrix (ECM). In this review we focus on aGPCR expression in metabolically active tissues, their activation by ECM proteins, and current knowledge of their potential roles in islet development, insulin secretion, adipogenesis and muscle function.

Identifying novel therapeutic targets for diabetes through improved understanding of islet adhesion receptors.

Adhesion receptors are transmembrane proteins that mediate cell-cell and cell-matrix communications. In addition to their adhesive role in maintaining islet architecture, they are also important for promoting islet cell survival, proliferation and secretory function. Their capacity for improving ß-cell mass and insulin secretion suggest that they may be suitable targets for pharmacological intervention, and their interactions with extracellular matrix proteins hold promise in improving islet transplantation outcomes. In this review, we have focused on integrins, cadherins and adhesion GPCRs, and highlight recent advances in their roles in islet function and discuss whether they could be targeted for diabetes therapy.