Mouse islet-derived stellate cells are similar to, but distinct from, mesenchymal stromal cells and influence the beta cell function.

AIMS: Evidence is accumulating of the therapeutic benefits of mesenchymal stromal cells (MSCs) in diabetes-related conditions. We have identified a novel population of stromal cells within islets of Langerhans - islet stellate cells (ISCs) - which have a similar morphology to MSCs. In this study we characterize mouse ISCs and compare their morphology and function to MSCs to determine whether ISCs may also have therapeutic potential in diabetes. METHODS: ISCs isolated from mouse islets were compared to mouse bone marrow MSCs by analysis of cell morphology; expression of cell-surface markers and extracellular matrix (ECM) components; proliferation; apoptosis; paracrine activity; and differentiation into adipocytes, chondrocytes and osteocytes. We also assessed the effects of co-culture with ISCs or MSCs on the insulin secretory capacity of islet beta cells. RESULTS: Although morphological similar, ISCs were functionally distinct from MSCs. Thus, ISCs were less proliferative and more apoptotic; they had different expression levels of important paracrine factors; and they were less efficient at differentiation down multiple lineages. Co-culture of mouse islets with ISCs enhanced glucose induced insulin secretion more effectively than co-culture with MSCs. CONCLUSIONS: ISCs are a specific sub-type of islet-derived stromal cells that possess biological behaviors distinct from MSCs. The enhanced beneficial effects of ISCs on islet beta cell function suggests that they may offer a therapeutic target for enhancing beta cell functional survival in diabetes.

In vitro profiling and functional assessments of the anti-diabetic capacity of phenolic-rich extracts of Bulbine natalensis and Bulbine frutescens.

AIMS: Bulbine natalensis (BN) and Bulbine frutescens (BF) are recommended in South African traditional medicine to treat diabetes, but their modes of action are unknown. This study assessed the phenolic acid profiles, mineral composition and in vitro functional effects of BN and BF to better understand their glucose-lowering capabilities. METHODS: Phenolic acid and mineral composition of BN and BF methanolic extracts were determined by HPLC and inductively coupled plasma optical emission spectroscopy respectively. Antioxidant capacity was assessed by potassium ferricyanide reducing power and 2,2-diphenyl-2-picrylhydrazyl radical scavenging assays, and inhibition of alpha-amylase, alpha-glucosidase, pancreatic lipase and DPP4 was evaluated by standard enzyme assays. The effects of BN and BF extracts on insulin secretion were investigated using static incubations of isolated mouse islets and molecular docking analysis was used to identify interactions of BN and BF with partners that could mediate stimulatory effects on insulin secretion. RESULTS: Methanolic extracts of BN and BF contained high concentrations of protocatechuic and gallic acids, and high levels of Zn, Mn and Cr. The extracts inhibited alpha-glucosidase, alpha-amylase, pancreatic lipase and DPP4 activities, and they also inhibited free radical generation. Both extracts significantly potentiated glucose-stimulated insulin secretion without significantly affecting basal insulin secretion or islet cell viability. Protocatechuic acid, the most abundant phenolic acid in the extracts, showed high affinity for PKA, PKC, DPP4 and CaMK II in the docking analysis. CONCLUSIONS: BN and BF have multiple beneficial effects on glucoregulatory pathways and they, or their derivatives, could be developed to treat type 2 diabetes.

Effects of miR-33 Deficiency on Metabolic and Cardiovascular Diseases: Implications for Therapeutic Intervention.

MicroRNAs (miRNAs) are small noncoding RNAs that post-transcriptionally inhibit gene expression. These small molecules are involved in several biological conditions such as inflammation, cell growth and proliferation, and regulation of energy metabolism. In the context of metabolic and cardiovascular diseases, miR-33 is of particular interest as it has been implicated in the regulation of lipid and glucose metabolism. This miRNA is located in introns harboured in the genes encoding sterol regulatory element-binding protein (SREBP)-1 and SREBP-2, which are key transcription factors involved in lipid biosynthesis and cholesterol efflux. This review outlines the role of miR-33 in a range of metabolic and cardiovascular pathologies, such as dyslipidaemia, nonalcoholic fatty liver disease (NAFLD), obesity, diabetes, atherosclerosis, and abdominal aortic aneurysm (AAA), and it provides discussion about the effectiveness of miR-33 deficiency as a possible therapeutic strategy to prevent the development of these diseases.

Identification of Potential Plant-Derived Pancreatic Beta-Cell-Directed Agents Using New Custom-Designed Screening Method: Gymnema sylvestre as an Example.

BACKGROUND: Folk medicines are attractive therapeutic agents for treating type 2 diabetes mellitus (T2DM). Most plant extracts that have been suggested to restore ß-cells function were tested in vivo. Some only have been tested in vitro to determine whether they have a direct effect on ß-cells islets of Langerhans. Currently, there are no defined criteria for screening of ß-cell-directed plant-based remedies as potential antidiabetic agents. SUMMARY: In this review, we have identified certain criteria/characteristics that can be used to generate a "screening portfolio" to identify plant extracts as potential ß-cell-directed agents for the treatment of T2DM. To validate our screening method, we studied the potential therapeutic efficacy of a Gymnema sylvestre (GS) extract using the screening criteria detailed in the review. Six criteria have been identified and validated using OSA®, a GS extract. By using this screening method, we show that OSA® fulfilled most of the criteria identified for an effective ß-cell-directed antidiabetic therapy, being an effective insulin-releasing agent at nontoxic concentrations; maintaining ß-cell insulin content by stimulating a concomitant increase in insulin gene transcription; maintaining ß-cell mass by protecting against apoptosis; and being effective at maintaining normoglycemia in vivo in a mouse model and a human cohort with T2DM. KEY MESSAGES: The present review has highlighted the importance of having a screening portfolio for plant extracts that have potential antidiabetic effects in the treatment of T2DM. We propose that this screening method should be adopted for future studies to identify new ß-cell-directed antidiabetic plant derived agents.

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.

Alterations in mouse visceral adipose tissue mRNA expression of islet G-protein-coupled receptor ligands in obesity.

BACKGROUND: Adipose tissue mass expansion in obesity leads to alterations in expression and secretion of adipokines, some of which may alter islet function by binding to G-protein-coupled receptors (GPCRs) expressed by islets. We have therefore quantified expression of mRNAs encoding islet GPCR ligands in visceral adipose tissue retrieved from lean and diet-induced obese mice to determine alterations in islet GPCR ligand mRNAs in obesity. METHODS: Epididymal adipose tissue was retrieved from C57BL/6 mice that had been maintained on a control-fat diet (10% fat) or high-fat diet (60% fat) for 16 weeks and RT-qPCR was used to quantify mRNAs encoding ligands for islet GPCRs. RESULTS: Of the 155 genes that encode ligands for islet GPCRs, 45 and 40 were expressed in visceral adipose tissue retrieved from lean and obese mice respectively. The remaining mRNAs were either expressed at trace level (0.0001% to 0.001% relative to Actb expression) or absent (<0.0001%). Obesity was associated with significant alterations in GPCR ligand mRNA expression in visceral adipose tissue, some of which encode for peptides with established effects on islet function (e.g. neuropeptide Y), or for GPCR ligands that have not previously been investigated for their effects on islets (e.g. (C-C motif) ligand 4; Ccl4). CONCLUSION: Mouse visceral adipose tissue showed significant alterations in expression of mRNAs encoding islet GPCR ligands in obesity. Our data point to ligands of interest for future research on adipose-islet crosstalk via secreted ligands acting at islet GPCRs. Such research may identify islet GPCRs with therapeutic potential for T2D.

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.

The cannabinoid ligands SR141716A and AM251 enhance human and mouse islet function via GPR55-independent signalling.

AIMS: Endocannabinoids are lipid mediators involved in the regulation of glucose homeostasis. They interact with the canonical cannabinoid receptors CB1 and CB2, and it is now apparent that some cannabinoid receptor ligands are also agonists at GPR55. Thus, CB1 antagonists such as SR141716A, also known as rimonabant, and AM251 act as GPR55 agonists in some cell types. The complex pharmacological properties of cannabinoids make it difficult to fully identify the relative importance of CB1 and GPR55 in the functional effects of SR141716A, and AM251. Here, we determine whether SR141716A and AM251 regulation of mouse and human islet function is through their action as GPR55 agonists. METHODS: Islets isolated from Gpr55+/+ and Gpr55-/- mice and human donors were incubated in the absence or presence of 10 µM SR141716A or AM251, concentrations that are known to activate GPR55. Insulin secretion, cAMP, IP1, apoptosis and ß-cell proliferation were quantified by standard techniques. RESULTS: Our results provide the first evidence that SR141716A and AM251 are not GPR55 agonists in islets, as their effects are maintained in islets isolated from Gpr55-/- mice. Their signalling through Gq-coupled cascades to induce insulin secretion and human ß-cell proliferation, and protect against apoptosis in vitro, indicate that they have direct beneficial effects on islet function. CONCLUSION: These observations may be useful in directing development of peripherally restricted novel therapeutics that are structurally related to SR141716A and AM251, and which potentiate glucose-induced insulin secretion and stimulate ß-cell proliferation.

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 Association Between Selective Serotonin Reuptake Inhibitors and Glycemia: A Systematic Review and Meta-Analysis of Randomized Controlled Trials.

OBJECTIVE: Individual studies have reported conflicting effects of selective serotonin reuptake inhibitors (SSRIs) on glycemia. We systematically reviewed the effects of SSRIs on glycemia and whether metabolic and psychological factors moderated these effects. METHODS: We systematically searched for placebo-controlled randomized controlled trials investigating the effect of SSRIs on glycemia (fasting blood glucose or HbA1c) as a primary or secondary outcome. Random effects meta-analysis was conducted to compute an overall treatment effect. Meta-regression tested whether depression, type 2 diabetes, insulin resistance, treatment duration, and weight loss moderated treatment effects. RESULTS: Sixteen randomized controlled trials (n = 835) were included and glycemia was usually a secondary outcome. Overall, SSRIs improved glycemia versus placebo (pooled effect size (ES) = -0.34, 95% confidence interval (CI) = -0.48 to -0.21; p < .001, I = 0%). Individually, fluoxetine (ES = -0.29, 95% CI = -0.54 to -0.05; p = .018) and escitalopram/citalopram (ES = -0.33, 95% CI = -0.59 to -0.07; p = .012) outperformed placebo, but paroxetine (ES = -0.19, 95% CI = -0.58 to 0.19; p = .33) did not. Results were similar in populations selected for depression as those not. Across studies, baseline insulin resistance (p = .46), treatment duration (p = .47), diabetes status (p = .41), and weight loss (p = .93) did not moderate changes. Heterogeneity for all analyses was nonsignificant. CONCLUSIONS: SSRIs seem to have an association with improvement in glycemia, which is not moderated by depression status, diabetes status, or change in weight across studies. Future powered trials with longer treatment duration are needed to confirm these findings. REGISTRATION: PROSPERO ID: CRD4201809239.

Short chain fatty acids stimulate insulin secretion and reduce apoptosis in mouse and human islets in vitro: Role of free fatty acid receptor 2.

AIMS: To evaluate the role of free fatty acid receptor 2 (FFAR2)/G-protein coupled receptor 43 in mediating the effects of the short chain fatty acids (SCFAs) sodium acetate (SA) and sodium propionate (SP) on islet function in vitro, and to identify the intracellular signalling pathways used in SCFA-induced potentiation of glucose-induced insulin secretion. MATERIALS AND METHODS: Islets of Langerhans were isolated from wild-type and FFAR2-/- mice and from human donors without diabetes. The effects of SA and SP on dynamic insulin secretion from perifused islets were quantified by radioimmunoassay, signalling downstream of SCFAs was profiled by single-cell calcium microfluorimetry, and measurement of cAMP was performed using a fluorescence assay. Islet apoptosis was induced by exposure to cytokines or sodium palmitate, and the effects of SA and SP in regulating islet apoptosis were assessed by quantification of caspase 3/7 activities. RESULTS: Deletion of FFAR2 did not affect islet morphology or insulin content. SA and SP reversibly potentiated insulin secretion from mouse islets in a FFAR2-dependent manner. SCFA-induced potentiation of insulin secretion was coupled to Gq activation of phospholipase C and protein kinase C, with no evidence of Gi-mediated signalling. SA and SP protected human and mouse islets from apoptosis, and these pro-survival properties were dependent on islet expression of FFAR2. CONCLUSION: Our results indicate that FFAR2 directly mediates both the stimulatory effects of SA and SP on insulin secretion and their protection against islet apoptosis. We have also shown that SCFA coupling in islets occurs via Gq-coupled intracellular signalling.

C3aR and C5aR1 act as key regulators of human and mouse ß-cell function.

AIMS: Complement components 3 and 5 (C3 and C5) play essential roles in the complement system, generating C3a and C5a peptides that are best known as chemotactic and inflammatory factors. In this study we characterised islet expression of C3 and C5 complement components, and the impact of C3aR and C5aR1 activation on islet function and viability. MATERIALS AND METHODS: Human and mouse islet mRNAs encoding key elements of the complement system were quantified by qPCR and distribution of C3 and C5 proteins was determined by immunohistochemistry. Activation of C3aR and C5aR1 was determined using DiscoverX beta-arrestin assays. Insulin secretion from human and mouse islets was measured by radioimmunoassay, and intracellular calcium ([Ca2+]i), ATP generation and apoptosis were assessed by standard techniques. RESULTS: C3 and C5 proteins and C3aR and C5aR1 were expressed by human and mouse islets, and C3 and C5 were mainly localised to ß- and α-cells. Conditioned media from islets exposed for 1 h to 5.5 and 20 mM glucose stimulated C3aR and C5aR1-driven beta-arrestin recruitment. Activation of C3aR and C5aR1 potentiated glucose-induced insulin secretion from human and mouse islets, increased [Ca2+]i and ATP generation, and protected islets against apoptosis induced by a pro-apoptotic cytokine cocktail or palmitate. CONCLUSIONS: Our observations demonstrate a functional link between activation of components of the innate immune system and improved ß-cell function, suggesting that low-level chronic inflammation may improve glucose homeostasis through direct effects on ß-cells.

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.

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

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.

The Placental Secretome: Identifying Potential Cross-Talk Between Placenta and Islet ß-Cells.

BACKGROUND/AIMS: Insulin-secreting islet ß-cells adapt to the insulin resistance associated with pregnancy by increasing functional ß-cell mass, but the placental signals involved in this process are not well defined. In the current study, we analysed expression of G-protein coupled receptor (GPCR) mRNAs in mouse islets and islet GPCR ligand mRNAs in placenta during pregnancy to generate an atlas of potential interactions between the placenta and ß-cells to inform future functional studies of islet adaptive responses to pregnancy. METHODS: Quantative RT-PCR arrays were used to measure mRNA expression levels of: (i) 342 GPCRs in islets from non-pregnant mice, and in islets isolated from mice on gestational days 12 and 18; (ii) 126 islet GPCR ligands in mouse placenta at gestational days 12 and 18. RESULTS: At gestational day 12, a time of rapid expansion of the ß-cell mass, 189 islet GPCR mRNAs were quantifiable, while 79 of the 126 known islet GPCR ligand mRNAs were detectable in placental extracts. Approximately half of the quantifiable placental GPCR ligand genes were of unknown function in ß-cells. The expression of some islet GPCR and placental ligand mRNAs varied during pregnancy, with altered expression of both GPCR and ligand mRNAs by gestational day 18. CONCLUSION: The current study has revealed numerous potential routes for interaction between the placenta and islets, and offers an atlas to inform further functional studies of their roles in adaptive responses to pregnancy, and in the regulation of the ß-cell mass.

Identifying Signalling Pathways Regulated by GPRC5B in ß-Cells by CRISPR-Cas9-Mediated Genome Editing.

BACKGROUND/AIMS: CRISPR-Cas9, a RNA-guided targeted genome editing tool, has revolutionized genetic engineering by offering the ability to precisely modify DNA. GPRC5B is an orphan receptor belonging to the group C family of G protein-coupled receptors (GPCRs). In this study, we analysed the functional roles of the Gprc5b receptor in MIN6 ß-cells using CRISPR-Cas9 and transient over-expression of Gprc5b. METHODS: The optimal transfection reagent for use in MIN6 ß-cells was determined by analysing efficiency of GFP plasmid delivery by cell sorting. A MIN6 ß-cell line in which Gprc5b expression was knocked down (Gprc5b KD) was generated using CRISPR-Cas9 technology. Gprc5b receptor mRNA expression, proliferation, apoptosis, Cignal 45-Pathway Reporter Array signalling and western blot assays were carried out using Gpcr5b KD MIN6 ß-cells that had been transiently transfected with different concentrations of mouse Gprc5b plasmid to over-express Gprc5b. RESULTS: JetPRIME® was the best candidate for MIN6 ß-cell transfection, providing approximately 30% transfection efficiency. CRISPR-Cas9 technology targeting Gprc5b led to stable knock-down of this receptor in MIN6 ß-cells and its re-expression induced proliferation and potentiated cytokine- and palmitate-induced apoptosis. The Cignal 45 Reporter analysis indicated Gprc5b-dependent regulation of apoptotic and proliferative pathways, and western blotting confirmed activation of signalling via TGF-ß and IFNγ. CONCLUSION: This study provides evidence of CRISPR-Cas9 technology being used to down-regulate Gprc5b expression in MIN6 ß-cells. This strategy allowed us to identify signalling pathways linking GPRC5B receptor expression to ß-cell proliferation and apoptosis.

Mesenchymal stromal cell secretory factors induce sustained improvements in islet function pre- and post-transplantation.

BACKGROUND AIMS: Mesenchymal stromal cells (MSCs) enhance islet function both in vitro and in vivo, at least in part by secreting ligands that activate islet G-protein coupled receptors (GPCRs). We assessed whether pre-treatment with a defined "cocktail" of MSC-secreted GPCR ligands enhances islet functional survival in vitro and improves the outcomes of islet transplantation in an experimental model of diabetes. METHODS: Isolated islets were cultured for 48 h with ANXA1, SDF-1 or C3a, alone or in combination. Glucose-stimulated insulin secretion (GSIS) and cytokine-induced apoptosis were measured immediately after the 48 h culture period and at 24 h or 72 h following removal of the ligands from the culture media. Islets were syngeneically transplanted underneath the kidney capsule of streptozotocin-induced diabetic C57BL/6 mice and blood glucose levels monitored for 28 days. RESULTS: Pre-culturing islets with a cocktail of ANXA1/SDF-1/C3a potentiated GSIS and protected islet cells from cytokine-induced apoptosis in vitro. These effects were maintained for up to 72 h after the removal of the factors from the culture medium, suggesting a sustained protection of islet graft functional survival during the immediate post-transplantation period. Islets pre-treated with the cocktail of MSC secretory factors were more effective in reducing blood glucose in diabetic mice, consistent with their improved functional survival in vivo. DISCUSSION: Pre-culturing islets with a cocktail of MSC secretory products offers a well-defined, cell-free approach to improve clinical islet transplantation outcomes while avoiding many of the safety, regulatory and logistical hurdles of incorporating MSCs into transplantation protocols.

Islet neuropeptide Y receptors are functionally conserved and novel targets for the preservation of beta-cell mass.

AIMS: Two unmet therapeutic strategies for diabetes treatment are prevention of beta-cell death and stimulation of beta-cell replication. Our aim was to characterize the role of neuropeptide Y receptors in the control of beta-cell mass. MATERIALS AND METHODS: We used endogenous and selective agonists of the NPY receptor system to explore its role in the prevention of beta-cell apoptosis and proliferation in islets isolated from both mouse and human donors. We further explored the intra-cellular signalling cascades involved, using chemical inhibitors of key signalling pathways. As proof of principle we designed a long-acting analogue of [Leu31 Pro34 ]-NPY, an agonist of the islet-expressed Y receptors, to determine if targeting this system could preserve beta-cell mass in vivo. RESULTS: Our data reveal that NPY Y1, 4 and 5 receptor activation engages a generalized and powerful anti-apoptotic pathway that protects mouse and human islets from damage. These anti-apoptotic effects were dependent on stimulating a Gαi-PLC-PKC signalling cascade, which prevented cytokine-induced NFkB signalling. NPY receptor activation functionally protected islets by restoring glucose responsiveness following chemically induced injury in both species. NPY receptor activation attenuated beta-cell apoptosis, preserved functional beta-cell mass and attenuated the hyperglycaemic phenotype in a low-dose streptozotocin model of diabetes. CONCLUSION: Taken together, our observations identify the islet Y receptors as promising targets for the preservation of beta-cell mass. As such, targeting these receptors could help to maintain beta-cell mass in both type 1 and type 2 diabetes, and may also be useful for improving islet transplantation outcomes.

LH-21 and abnormal cannabidiol improve ß-cell function in isolated human and mouse islets through GPR55-dependent and -independent signalling.

AIMS: To examine the effects of Abn-CBD (GPR55 agonist) and LH-21 (CB1 antagonist) on human and mouse islet function, and to determine signalling via GPR55 using islets from GPR55-/- mice. MATERIALS AND METHODS: Islets isolated from human organ donors and mice were incubated in the absence or presence of Abn-CBD or LH-21, and insulin secretion, [Ca2+ ]i, cAMP, apoptosis, ß-cell proliferation and CREB and AKT phosphorylation were examined using standard techniques. RESULTS: Abn-CBD potentiated glucose-stimulated insulin secretion and elevated [Ca2+ ]i in human islets and islets from both GPR55+/+ and GPR55-/- mice. LH-21 also increased insulin secretion and [Ca2+ ]i in human islets and GPR55+/+ mouse islets, but concentrations of LH-21 up to 0.1 µM were ineffective in islets from GPR55-/- mice. Neither ligand affected basal insulin secretion or islet cAMP levels. Abn-CBD and LH-21 reduced cytokine-induced apoptosis in human islets and GPR55+/+ mouse islets, and these effects were suppressed after GPR55 deletion. They also increased ß-cell proliferation: the effects of Abn-CBD were preserved in islets from GPR55-/- mice, while those of LH-21 were abolished. Abn-CBD and LH-21 increased AKT phosphorylation in mouse and human islets. CONCLUSIONS: This study showed that Abn-CBD and LH-21 improve human and mouse islet ß-cell function and viability. Use of islets from GPR55-/- mice suggests that designation of Abn-CBD and LH-21 as a GPR55 agonist and a CB1 antagonist, should be revised.

Dynamic Profiling of Insulin Secretion and ATP Generation in Isolated Human and Mouse Islets Reveals Differential Glucose Sensitivity.

BACKGROUND/AIMS: Rodent islets are often used for basic science research but they do not always recapitulate signalling events in human islets. This study evaluated the glucose-dependent responses of human and mouse islets in terms of dynamic insulin secretion, metabolic coupling and the role of glucose transporters. METHODS: Glucose-induced insulin secretion from isolated mouse and human islets was profiled by perifusion and islet ATP levels were measured by chemoluminescence assay. Glucose transporter expression was determined by qPCR and western blotting. RESULTS: Human islets show a left-shifted glucose concentration-insulin secretion profile compared to mouse islets. These data are consistent with glucose transporter expression, with human islets expressing mainly GLUT1 and GLUT3, and GLUT2 being the predominant transporter in mouse islets. Using the GLUT1 inhibitor STF-31 we unveiled an important role for GLUT1 for differences in glucose-induced insulin secretion profiles observed between the two species. CONCLUSION: The high affinity of GLUT1/3 for glucose reflects the left-shifted glucose-induced insulin secretory response of human islets and the impairment of insulin secretion from human islets after STF-31 treatment indicates an important role for GLUT1 in human islet stimulus-secretion coupling. Our data provide further insight into key differences between insulin secretion regulation in mouse and human islets.