G protein-coupled receptors (GPCRs) are cell-surface proteins that are targeted therapeutically for a range of disorders, including diabetes. Adhesion GPCRs (aGPCRs) are the second largest class of the GPCR superfamily and some members of this family have been implicated in appropriate organ development. However, the role of aGPCRs in endocrine pancreas specification is not yet known. Here, we systematically characterised expression of mRNAs encoding aGPCRs and their ligands in developing mouse and human pancreas using our own and publicly available single-cell RNA sequencing and spatial transcriptomics data, and we conducted qPCR analysis of aGPCR expression in human pancreas at different gestational stages. We then investigated the role of GPR56 (ADGRG1), the most abundant aGPCR in pancreatic endocrine progenitors, in islet development using Gpr56 null mice and their wildtype littermates. We demonstrated that aGPCRs are dynamically expressed during mouse and human pancreas development, with specific aGPCR mRNAs expressed in distinct endocrine, endothelial, mesenchymal, acinar, ductal, and immune cell clusters. aGPCR ligand mRNAs were mostly expressed by non-endocrine cells, and the most highly expressed receptor-ligand interacting mRNA pairs were those encoding GPR56 and COL3A1. Deletion of Gpr56 in neonatal mice was associated with an altered α-/β-/δ-cell ratio and reduced β-cell proliferation. Our data show that aGPCRs are expressed at key stages of human and mouse pancreas endocrine lineage decisions, and analysis of pancreases from Gpr56 knockout mice implicate this aGPCR in the development of a full complement of β-cells.

Introduction and Objective: The Wnt/β-catenin pathway is involved in pancreatic beta cell proliferation under compensatory hyperplasia conditions. In previous studies, we showed that there is an increase in Wnt3a and Wnt5b in hyperplastic islets from obese prediabetic mice fed a high-fat diet (HFD). In this work, we investigate the regulation of Wnt members in hyperplastic islets from obese (non-diabetic) male human donors and from two obese/diabetic mouse models. Methods: We performed RT-qPCR for analysis of Wnts and receptor targets in homogenates from human and mouse isolated islets. We used mice fed a HFD for 8 and 16 weeks (w) compared to control groups fed a standard rodent diet. Results: The results showed no regulation of Wnts in islets from obese vs healthy donors, but there was an upregulation of the mRNA level of Wnt target receptors: Fzd4, 3.9 ± 0.9, and Fzd9, 20 ± 3.3 (n=4 islet donors/group). Both groups of HFD mice become hyperglycemic, hyperinsulinemic, glucose intolerance, and insulin resistant relative to the CTL groups; the HFD mice also became obese, with all these changes being more pronounced in mice fed HFD 16w. RT-qPCR results showed a significant increase in Wnt3a (CTL 8w vs HFD 8w, 1.3-fold; p<0.05) and Wnt5b (CTL 8w vs HFD 8w, 4.7-fold; p<0.02), n=3-5 isolated islets pool from 9-10 mice/group, corroborating our previous work. However, in the HFD 16w group, this phenomenon was not observed (Wnt3a, CTL 16w vs HFD 16w; p=0.7)(Wnt5b, CTL 16w vs HFD; p=0.75; n=7 islets pool from 14 mice/group). Moreover, Wnt target receptor Fzd4 was significantly increased in both mice groups (CTL 8w vs HFD 8w, 2.5-fold; p<0.005)(CTL 16w vs HFD 16w, 1.4-fold; p<0.02). Conclusion: Our results showed that prolonged HFD exposure is not followed by further changes in Wnt3a and Wnt5b expression in mice. Yet, Wnt receptors can be upregulated, possibly to activate Wnt signaling, in both islets from obese humans and diabetic obese mice. These findings may support future studies aimed at maintaining beta cell mass. D. Maschio: None. J. Oliveira: None. C.B. Collares-Buzato: None. S. Persaud: None. H.C. Barbosa: None. São Paulo Research Foundation (FAPESP: 2023/11004-9, 2023/01572-0)

Objective: Type 1 diabetes (T1D) is characterised by destruction of pancreatic beta cells by islet-infiltrating cytotoxic lymphocytes, and elevated intra-islet secretion of pro-inflammatory cytokines. However, the underlying pathophysiological mechanisms remain incompletely understood. We hypothesise that abnormal elevation of islet NAD, via activation of NAMPT, plays a key role in driving islet autoimmune processes in T1D, and that conversely, NAMPT inhibition may be an attractive therapeutic approach for T1D. Methods: Islets were isolated from non-diabetic CD1 mice or obtained from human islet donors and exposed to pro-inflammatory cytokine cocktail (IL-1β, TNFα and IFNγ) +/- NAMPT inhibitor. Beta-cell function was determined by static glucose-stimulated insulin secretion. Islet apoptosis was determined by caspase 3/7 activity. Islet cell differential gene expression was assessed via bulk-RNA sequencing. Diabetes was induced in CD1 mice via multiple low dose streptozotocin (MLDS) injection. MLDS mice were then administered FK866 (10 mg/kg; IP) or saline equivalent for 16 days. Ambient blood glucose was measured every 2-3 days. Beta-cell function was assessed by measurement of serum insulin and c-peptide levels. Insulin content was assessed by flow cytometry. Intra-islet immune cell infiltration, as well as immune cell proliferation and cytokine production were assessed via flow cytometry. For migration assays, islets were isolated from BALB/c mice dispersed and reaggregated into reformed islets. CD8+ cells were isolated from splenocytes derived from NY8.3 NOD mice migration of immune cells was assessed using a transwell culture protocol. Results: NAMPT inhibition with FK866 or compound 17 protected mouse and human islets against cytokine-mediated beta cell dysfunction and death. RNAseq revealed that inhibiting NAMPT blocked pro-inflammatory cytokine-mediated gene expression linked to pro-inflammatory responses and leukocyte migration. FK866 improved glycaemic control and beta-cell function in MLDS mice. FK866 also reduced proportions of islet-residing TNFα producing CD4+T-cells and F4/80+macrophages, proliferation of spleen-derived CD4+ and CD8+T-cells, and proliferation of islet-derived CD4+T-cells and F4/80+macrophages. Finally, FK866 was able to block pro-inflammatory cytokine-mediated migration of cytotoxic CD8+T-cells into reformed islets. Conclusions: This data supports a key immunomodulatory role for NAMPT in islet autoimmunity. NAMPT inhibition may represent a novel therapeutic approach for T1D. ### Competing Interest Statement The authors have declared no competing interest.

Pancreatic alpha cells modulate beta cell function in a paracrine manner through the release of glucagon. However, the detailed molecular architecture underlying alpha-to-beta cell regulation remains poorly characterized. Here, we show that the glucagon-like peptide-1 receptor (GLP1R) is enriched as nanodomains on beta cell membranes that contact alpha cells, in keeping with increased single-molecule transcript expression. At low glucose, beta cells next to alpha cells directly sense micromolar glucagon release by pre-internalizing GLP1R. Pre-internalized GLP1R is associated with earlier beta cell Ca2+ responses to high glucose, which are then propagated across the islet. Beta cells adjacent to alpha cells are more secretory than beta cells next to other beta cells. Localized GLP1R signaling occurs in vitro and in vivo, is operative in the post-prandial state, and GLP1R contacts decrease between beta cells and alpha cells during metabolic stress. Thus, we detail a regulated pathway through which glucagon modulates insulin release.

Abstract Pancreatic islets are 3D micro-organs that maintain β-cell functionality through cell–cell and cell-matrix communication. While primary islets, the gold standard for in vitro models, have a short culture life of approximately 1–2 weeks, we developed a novel protocol that employs reformed islets following dispersion coupled with a fine-tuned culture environment. Reformed islets exhibit physiological characteristics similar to primary islets, enabling high-resolution imaging and repeated functional assessment. Unlike other in vitro platforms, reformed islets retain an immune population, allowing the study of interactions between β cells and resident and infiltrating immune cells. Analyses showed that reformed islets have a similar composition and cytoarchitecture to primary islets, including macrophages and T cells, and can secrete insulin in response to glucose. Reformed islets exhibited partial dedifferentiation compared to native islets but were otherwise transcriptionally similar. The reformed islets offer a useful platform for studying diabetes pathology and can recapitulate both T1DM and T2DM disease milieus, providing an advantage over other models, such as mouse and human β-cell lines, which lack the input of non-β-endocrine cells and immune cell crosstalk.

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.

AIM:To investigate the effect of G protein-coupled receptor 55 (GPR55) deletion on glucose homeostasis and islet function following diet-induced obesity. METHODS:GPR55-/- and wild-type (WT) mice were fed ad libitum either standard chow (SC) or a high-fat diet (HFD) for 20 weeks. Glucose and insulin tolerance tests were performed at 9/10 and 19/20 weeks of dietary intervention. Insulin secretion in vivo and dynamic insulin secretion following perifusion of isolated islets were also determined, as were islet caspase-3/7 activities and β-cell 5-bromo-20-deoxyuridine (BrdU) incorporation. RESULTS:GPR55-/- mice fed a HFD were more susceptible to diet-induced obesity and were more glucose intolerant and insulin resistant than WT mice maintained on a HFD. Islets isolated from HFD-fed GPR55-/- mice showed impaired glucose- and pcacahorbol 12-myristate 13-acetate-stimulated insulin secretion, and they also displayed increased cytokine-induced apoptosis. While there was a 5.6 ± 1.6-fold increase in β-cell BrdU incorporation in the pancreases of WT mice fed a HFD, this compensatory increase in β-cell proliferation in response to the HFD was attenuated in GPR55-/- mice. CONCLUSIONS:Under conditions of diet-induced obesity, GPR55-/- mice show impaired glucose handling, which is associated with reduced insulin secretory capacity, increased islet cell apoptosis and insufficient compensatory increases in β-cell proliferation. These observations support that GPR55 plays an important role in positively regulating islet function.

Islets are approximately spherical, with an average diameter of 100–200 micrometer, and a healthy human pancreas may contain up to a million individual islets, each having its own complex anatomy, blood supply, and innervation. Advances in high-throughput functional and molecular phenotyping have demonstrated that human islets show considerable molecular, anatomical, and functional heterogeneity, which may be important in the islet dysfunction associated with the development of type 2 diabetes. Islets are well supplied by autonomic nerve fibres and terminals containing the classic neurotransmitters acetylcholine and norepinephrine, along with a variety of biologically active neuropeptides. The insulin secretory granule has a typical appearance in electron micrographs, with a wide space between the crystalline electronopaque core and its limiting membrane. The microtubular network is in a process of continual remodelling and the dynamic turnover of tubulin, rather than the total number of microtubules, is an important regulator of insulin secretion.

AIMS:To investigate the effects of the selective serotonin reuptake inhibitors (SSRIs) sertraline and paroxetine at therapeutically relevant concentrations on beta-cell mass and function. METHODS:Viability was quantified in mouse insulinoma (MIN6) beta cells and mouse islets after 48-h exposure to sertraline (1-10 μM) or paroxetine (0.01-1 μM) using the Trypan blue exclusion test. The effects of therapeutic concentrations of these SSRIs on insulin secretion were determined by static incubation and perifusion experiments, while islet apoptosis was investigated by Caspase-Glo 3/7 assay, TUNEL staining and quantitative PCR analysis. Finally, proliferation of MIN6 and mouse islet beta cells was assessed by bromodeoxyuridine (BrdU) enzyme-linked immunosorbent assay and immunofluorescence. RESULTS:Sertraline (0.1-1 μM) and paroxetine (0.01-0.1 μM) were well tolerated by MIN6 beta cells and islets, whereas 10 μM sertraline and 1 μM paroxetine were cytotoxic. Exposure to 1 μM sertraline and 0.1 μM paroxetine significantly potentiated glucose-stimulated insulin secretion from mouse and human islets. Moreover, they showed protective effects against cytokine- and palmitate-induced apoptosis of islets, they downregulated cytokine-induced Stat1 and Traf1 mRNA expression, and they significantly increased proliferation of mouse beta cells. CONCLUSIONS:Our data demonstrate that sertraline and paroxetine act directly on beta cells to enhance glucose-stimulated insulin secretion and stimulate beta-cell mass expansion by increasing proliferation and decreasing apoptosis. These drugs are therefore likely to be appropriate for treating depression in people with type 2 diabetes.

Ethnopharmacological relevance: Ayurvedic medicine has been used in the treatment of diabetes mellitus for centuries. In Arabia and some areas of Africa, Commiphora myrrha (CM) has been extensively used as a plantbased remedy. We have previously shown that an aqueous CM resin solution directly stimulates insulin secretion from MIN6 cells, a mouse 13-cell line, and isolated mouse and human islets. However, the signaling pathways involved in CM-induced insulin secretion are completely unknown. Insulin secretion is normally triggered by elevations in intracellular Ca2+ ([Ca2+]i) through voltage gated Ca2+ channels (VGCC) and activation of protein kinases. Protein and lipid kinases such as protein kinase A (PKA), Ca2+-calmodulin dependent protein kinase II (CaMKII), phosphoinositide 3-kinases (PI3Ks), protein kinase C (PKC) and mitogen-activated protein kinase (MAPK), specifically extracellular signal-regulated kinases (ERK1/2), may be involved in receptor-operated insulin secretion. Therefore, we hypothesized that CM may induce insulin secretion by modulating the activity of VGCC and/or one or more of the above kinases. Aim of the study: To investigate the possible molecular mechanism of action of CM-induced insulin secretion. The effects of aqueous CM resin extract on [Ca2+]i and protein kinase activation from 13-cells were examined. Methods: The effect of aqueous CM resin solution on [Ca2+]i was assessed using Ca2+ microfluorimetry. The involvement of VGCC in CM-induced insulin secretion was investigated using static and perifusion insulin secretion experiments in the presence of either EGTA, a Ca2+ chelator, or nifedipine, a blocker of VGCC. The involvement of kinase activation in the stimulatory effect of CM on insulin secretion was examined by using static and perifusion insulin secretion experiments in the presence of known pharmacological inhibitors and/or downregulation of specific kinases. The effects of CM on phosphorylation of PKC zeta and ERK1/2 were also assessed using the WesTM capillary-based protein electrophoresis. Results: Ca2+ microfluorimetry measurements showed that exposing MIN6 cells to CM (0.5-2 mg/mL) was not associated with changes in [Ca2+]i. Similarly, incubating MIN6 cells and mouse islets with EGTA and nifedipine, respectively, did not attenuate the insulin secretion induced by CM. However, incubating mouse and human islets with CM in the presence of staurosporine, a non-selective protein kinase inhibitor, completely blocked the effect of CM on insulin secretion. Exposing mouse islets to CM in the presence of H89, KN62 and LY294002, inhibitors of PKA, CaMKII and PI3K, respectively, did not reduce CM-induced insulin secretion. However, incubating mouse and human islets with CM in the presence of Ro 31-8220, a pan-PKC inhibitor, diminished insulin secretion stimulated by CM, whereas inhibiting the action of typical PKC (with Go6976) and PLC13 (with U73122) did not affect CM-stimulated insulin secretion. Similarly, downregulating typical and novel PKC by chronic exposure of mouse islets to phorbol 12-myristate 13-acetate (PMA) was also not associated with a decrease in the stimulatory effect of CM on insulin secretion. Interestingly, CM-induced insulin secretion from mouse islets was inhibited in the presence of the PKC zeta inhibitor ZIP and a MAPK inhibitor PD 98059. In addition, WesTM capillary-based protein electrophoresis indicated that expression of the phosphorylated forms of PKC zeta and ERK1/2, a MAPK, was significantly increased following exposure of INS-1832/13 cells, a rat insulinoma cell line, to CM. Conclusions: Our data indicate that CM directly stimulates insulin secretion through activating known down-stream effectors of insulin-stimulus secretion coupling. Indeed, the increase in insulin secretion seen with CM is independent of changes in [Ca2+]i and does not involve activation of VGCC. Instead, the CM stimulatory effect on insulin secretion is completely dependent on protein kinase activation. Our findings indicate that CM could induce insulin exocytosis by stimulating the phosphorylation and activation of PKC zeta, which in turn phosphor-ylates and activates ERK1/2.

Diabetic MedicineVolume 40, Issue 12 e15239 EDITORIAL Basic science lays the foundation to success Claire Hills, Corresponding Author Claire Hills [email protected] University of Lincoln, Lincoln, UK Correspondence Claire Hills, University of Lincoln, Lincoln, UK. Email: [email protected]Search for more papers by this authorShanta Persaud, Shanta Persaud King's College London, Lincoln, UKSearch for more papers by this author Claire Hills, Corresponding Author Claire Hills [email protected] University of Lincoln, Lincoln, UK Correspondence Claire Hills, University of Lincoln, Lincoln, UK. Email: [email protected]Search for more papers by this authorShanta Persaud, Shanta Persaud King's College London, Lincoln, UKSearch for more papers by this author First published: 10 November 2023 https://doi.org/10.1111/dme.15239Read the full textAboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onEmailFacebookTwitterLinkedInRedditWechat No abstract is available for this article. REFERENCES 1Reimann F. Dorothy Hodgkin lecture 2023: The enteroendocrine system-sensors in your guts. Diabet Med. 2023; 40:e15212. doi:10.1111/dme.15212 10.1111/dme.15212 Google Scholar 2Tiwari M, Mcilroy GD. From scarcity to solutions: therapeutic strategies to restore adipose tissue functionality in rare disorders of lipodystrophy. Diabet Med. 2023; 40:e15214. doi:10.1111/dme.15214 10.1111/dme.15214 PubMedGoogle Scholar 3Sridhar A, Khan D, Moffett CR. The impact of diabetes and obesity on fertility and the potential role of gut hormones as treatment. Diabet Med. 2023; 40:e15230. doi:10.1111/dme.15230 10.1111/dme.15230 Google Scholar 4Leete P. Type 1 diabetes in the pancreas: a histological perspective. Diabet Med. 2023; 40:e15228. doi:10.1111/dme.15228 10.1111/dme.15228 Web of Science®Google Scholar 5Rückert AK, Ast J, Hasib A, et al. Fine-tuned photochromic sulfonylureas for optical control of beta cell Ca2+ fluxes. Diabet Med. 2023; 40:e15220. doi:10.1111/dme.15220 10.1111/dme.15220 Google Scholar 6Ahmed A, Justo S, Yaghootkar H. Genetic scores associated with favourable and unfavourable adiposity have consistent effect on metabolic profile and disease risk across diverse ethnic groups. Diabet Med. 2023; 40:e15213. doi:10.1111/dme.15213 10.1111/dme.15213 Google Scholar 7Hirani D, Alabdulkader S, Miras AD, Salem V. What can functional brain imaging teach us about remission of type 2 diabetes? Diabet Med. 2023; 40:e15235. doi:10.1111/dme.15235 10.1111/dme.15235 Google Scholar 8Abraham A, Yaghootkar H. Identifying obesity subtypes: a review of studies utilising clinical biomarkers and genetic data. Diabet Med. 2023; 40:e15226. doi:10.1111/dme.15226 10.1111/dme.15226 Google Scholar 9Clavelo-Farrow C, Thomas P. The role of candidate transport proteins in β-cell long-chain fatty acid uptake: where are we now? Diabet Med. 2023; 40:e15198. doi:10.1111/dme.15198 10.1111/dme.15198 Google Scholar 10Walker SL, Noble J, Thomson A, et al. Ultrasound-guided hepatic portal vein injection is not a reproducible technique for delivery of cell therapies to the liver in mice. Diabet Med. 2023; 40:e15192.doi:10.1111/dme.15192 10.1111/dme.15192 Web of Science®Google Scholar 11Hong TW, Caxaria S, Daniels Gatward LF, et al. Mesenchymal stromal cell secretory molecules improve the functional survival of human islets. Diabet Med. 2023; 40:e15227. doi:10.1111/dme.15227 10.1111/dme.15227 Google Scholar Volume40, Issue12Special Issue: Basic Science Special Issue 2023December 2023e15239 ReferencesRelatedInformation