Delta Cells Research Articles

PCGF1 is a prognostic biomarker and correlates with tumor immunity in gliomas.

BackgroundPolycomb group factor 1 (PCGF1) plays a vital role in the self-renewal of cancer stem cells (CSCs). However, the prognostic value and potential function of PCGF1 in glioma progression, especially in tumor immunity, remain unclear.MethodsThis study investigated PCGF1 expression in pan-cancers and glioma using The Cancer Genome Atlas Project data, and conducted a logistic regression analysis to analyze the association between PCGF1 expression and the clinicopathological features of glioma patients. Kaplan-Meier and Cox regression analyses were used to assess the prognostic roles of PCGF1. The functional analysis using gene ontology and Kyoto Encyclopedia of Genes and Genomes databases and a gene set enrichment analysis (GSEA) were conducted to examine the PCGF1-related biological processes and signaling pathways. Finally, the roles of PCGF1 in immune infiltration were analyzed by a single sample GSEA (ssGSEA).ResultsPCGF1 expression was upregulated in glioma tissues. The high expression of PCGF1 was significantly associated with an age >60 years (P<0.001), an increased World Health Organization grade (P<0.001), and wildtype isocitrate dehydrogenase (IDH) status (P<0.001), and predicted poor overall survival (OS) [hazards ratio (HR) =2.90, 95% confidence interval (CI): 2.25–3.74; P<0.001], the progression-free interval (PFI) (HR =1.99, 95% CI: 1.60–2.46; P<0.001), and disease specific survival (DSS) (HR =2.84, 95% CI: 2.18–3.71; P<0.001). The multivariate regression analysis confirmed that PCGF1 expression was an independent prognostic factor of OS (HR =1.581, 95% CI: 1.161–2.153; P=0.004). Based on the functional enrichment analysis, we identified the PCGF1-related differentially expressed genes (DEGs), and found that PCGF1 was involved in regulating many oncogenic signaling pathways, such as the phosphatidylinositol 3-kinase and protein kinase B pathway, the Janus kinase/signal transducers and activators of transcription (JKT-STAT) signaling pathway, notch signaling and integrin signaling pathway. In addition, we found that PCGF1 expression was positively associated with the abundance of Th2, but negatively associated with T follicular helper, T central memory, and T gamma delta cells. Additionally, PCGF1 participated in the regulation of numerous immune-related processes in glioma.ConclusionsPCGF1 is a promising prognostic biomarker, and as it governs cancer-related pathways and tumor immunity, it plays an important role in glioma development.

Oral mRNA delivery using capsule-mediated gastrointestinal tissue injections

<h2>Summary</h2> Nucleic acids are enabling a new generation of therapeutics and vaccines to treat and prevent a range of diseases. While these therapies have typically been limited to parenteral dosing, patients and clinicians prefer oral dosage forms. Furthermore, oral delivery enables local transfection of cells in the gastrointestinal tract not easily targeted via parenteral administration. To address these challenges, we synthesized and screened a library of branched hybrid poly(β-amino ester) mRNA nanoparticles for transfection efficiency; then we combined the highest performing formulations with ingestible milli-injector capsules capable of delivering formulations directly into gastric tissue. We validated the performance of formulations and devices in rodents and pigs, demonstrating protein translation in the delta, gastric, and parietal cells of the gastric mucosa, in addition to systemic uptake. We anticipate oral delivery of mRNA could facilitate rapid deployment of episodic interventions, such as vaccines, and support long-term therapies.

High-Mobility Group AT-Hook 1 Served as a Prognosis Biomarker and Associated with Immune Infiltrate in Hepatocellular Carcinoma.

BackgroundThe protein high-mobility group AT-hook 1 (HMGA1) has been demonstrated that modulated cellular proliferation, invasion, and apoptosis with a poor prognosis in miscellaneous carcinomas. However, the mechanism of circumstantial carcinogenesis and association with the immune microenvironment of HMGA1 in hepatocellular carcinoma (HCC) had not been extensively explored.MethodsThe gene expression, clinicopathological correlation, and prognosis analysis were performed in the data obtained from TCGA. The results were further validated by ICGC and GEO database and external validation cohort from Guangxi. The HMGA1 protein expression was further examined in the HPA database. Biological function analyses were conducted by GSEA, STRING database, and Coexpedia online tool. Using TIMER and CIBERSORT method, the relationship between immune infiltrate and HMGA1 was investigated.ResultsIn HCC, HMGA1 had much higher transcriptional and proteomic expression than in corresponding paraneoplastic tissue. Patients with high HMGA1 expression had a poor prognosis and unpromising clinicopathological features. High HMGA1 expression was closely related to the cell cycle, tumorigenesis, substance metabolism, and immune processes by regulating complex signaling pathways. Notably, HMGA1 may be associated with TP53 mutational carcinogenesis. Moreover, increased HMGA1 expression may lead to an increase in immune infiltration and a decrease in tumor purity in HCC. CIBERSORT analysis elucidated that the amount of B cell naive, B cell memory, T cells gamma delta, macrophages M2, and mast cell resting decreased when HMGA1 expression was high, whereas T cells follicular helper, macrophages M0, and Dendritic cells resting increased.ConclusionIn conclusions, HMGA1 is a potent prognostic biomarker and a sign of immune infiltration in HCC, which may be a potential immunotherapy target for HCC.

Regional melatonin and somatostatin dependent mechanisms in pancreatic incretory activity and in intestinal bacterial homeostasis.

For many years, it was generally accepted that melatonin and somatostatin have intracerebral sources of synthesis. Melatonin is produced in the pineal gland and somatostatin in the hypothalamus. However, there are very informative data regarding the extracerebral sources of melatonin and somatostatin synthesis in numerous literary sources published over the past 60 years. At the same time, the biological purpose of extracerebral somatostatin and melatonin has been insufficiently studied. This article presents the readers with an analysis of modern literature and the results of own researches on the biological role of melatonin and somatostatin produced in the pancreas and intestinal tract. Under experimental conditions on arginine-induced acute pancreatitis model in rats, structural and hormonal changes were studied in the pancreas, mucosa of the small and large intestines in regional pathological process using morphological, morphometric, immunomorphological and enzyme immunoassay methods. According to the morphological and morphometric analysis results, gross dystrophic changes in the pancreas and intestinal mucosa were observed at relatively early stages of the pathological process – in 2 and 24 hours after the arginine administration. Immunomorphological studies established that the content of melatonin-positive secretory cells noticeably decreased, especially in the areas of mucosal damage in this very period of the pathological process in pancreas, in the mucous membrane of the small and large intestines. At the same time, the content of somatotropin in blood serum and pancreas markedly increased during the indicated period of the experiment. Severe dystrophic processes on small intestinal mucosa, which are particularly expressed by inhibition of local melatonin-secreting cell function, apparently exclude the inhibitory effect of melatonin on somatostatin synthesis process in pancreas. It is not excluded that there is a directed activation of pancreatic delta cells responsible for the somatostatin synthesis at relatively early stages of “arginine pancreatitis”. Based on our own researches in context with available very informative literature data, we can conclude that extrapineal melatonin and extrahypothalamic somatostatin should be given an important role in the integrative activity of organ-digestive system – the pancreas and small intestine.

Identification of Immune-Related Key Genes in Ovarian Cancer Based on WGCNA.

Background: Ovarian cancer (OV) is a fatal gynecologic malignancy and has poor survival rate in women over the age of forty. In our study, we aimed to identify genes related to immune microenvironment regulations and explore genes associated with OV prognosis. Methods: The RNA-seq data of GDC TCGA Ovarian Cancer cohort of 376 patients was retrieved from website. Weighted gene co-expression network analysis (WGCNA) and ESTIMATE algorithm were applied to identify the key genes associated with the immune scores. The correlation between key genes and 22 immune cell types were estimated by using CIBERSORT algorithms. Results: WGCNA showed that the pink module was most correlated with the immune score. Seven of 14 key genes (FCRL3, IFNG, KCNA3, LY9, PLA2G2D, THEMIS, and TRAT1) were significantly associated with the OS of OV patients. Correlation analysis showed our key genes positively related to M1 macrophages, CD8 T cells, plasma cells, regulatory T (Treg) cells and activated memory CD4 T cells, and negatively related to naive CD4 T cells, M0 macrophages, activated dendritic cells (DCs) and memory B cells. Kaplan-Meier survival analysis showed that lower abundances of neutrophils and higher abundances of M1 macrophages, plasma cells, T cells gamma delta (γδT) cells and follicular helper T (Tfh) cells predicted better OV prognosis. Conclusion: Forteen key genes related to the immune infiltrating of OV were identified, and seven of them were significantly related to prognosis. These key genes have potential roles in tumor infiltrating immune cells differentiation and proliferation. This study provided potential prognostic markers and immunotherapy targets for OV.

Profiling Calcium-Dependent Gene Expression and Identifying Functional Diversity in Human Islet Cell Types

All pancreatic endocrine cells depend on cytosolic calcium signaling to trigger the secretion of glucoregulatory hormones, and rapidly regulate transcription of genes important for islet function and the response to stimuli. In order to profile calcium-regulated genes in islet cell types, we performed single cell RNA sequencing on intact non-diabetic human islets (3 donors) exposed to conditions designed to acutely induce or inhibit intracellular calcium signaling for 1 hour: 1) a low glucose dose of 2.8 mmol/L, 2) stimulatory 25 mmol/L glucose and 40 mmol/L KCl, and 3) 25 mmol/L glucose, 40 mmol/L KCl, and 5 mmol/L inhibitory calcium chelator EGTA. After downstream filtering and quality control, we obtained a robust dataset of 68,650 cells, which included 59,373 alpha, beta, delta and PP cells. By comparing the transcriptomes from stimulatory and inhibitory conditions, we identified 38 calcium-regulated genes across the islet, including 15 specific to alpha cells and 10 specific to beta cells. We also observed distinct clusters expressing both insulin and glucagon, or insulin and somatostatin, and these clusters express a unique set of 14 calcium-regulated genes. In beta cell subtypes that expressed the greatest number of calcium-regulated genes, we identified the gene PCDH7 as a novel marker of beta cells with an enhanced glucose-stimulated insulin secretory function. We share this large human islet single cell transcriptome dataset as an accessible, user-friendly web tool to easily generate plots and query gene expression in single human islet cells.

Ordered hexagonal patterns via notch–delta signaling

Many developmental processes in biology utilize notch–delta signaling to construct an ordered pattern of cellular differentiation. This signaling modality is based on nearest-neighbor contact, as opposed to the more familiar mechanism driven by the release of diffusible ligands. Here, exploiting this ‘juxtacrine’ property, we present an exact treatment of the pattern formation problem via a system of nine coupled ordinary differential equations. The possible patterns that are realized for realistic parameters can be analyzed by considering a co-dimension 2 pitchfork bifurcation of this system. This analysis explains the observed prevalence of hexagonal patterns with high delta at their center, as opposed to those with central high notch levels (referred to as anti-hexagons). We show that outside this range of parameters, in particular for low cis-coupling, a novel kind of pattern is produced, where high delta cells have high notch as well. It also suggests that the biological system is only weakly first order, so that an additional mechanism is required to generate the observed defect-free patterns. We construct a simple strategy for producing such defect-free patterns.

Integrative bioinformatics and experimental analysis revealed down-regulated CDC42EP3 as a novel prognostic target for ovarian cancer and its roles in immune infiltration.

Ovarian cancer is a significant clinical challenge as no effective treatments are available to enhance patient survival. Recently, N6-methyladenosine (m6A) RNA modification has been demonstrated to play a pivotal role in tumorigenesis and progression. However, the roles of m6A target genes in ovarian cancer haven’t been clearly illustrated. In this study, we presented a comprehensive bioinformatics and in vitro analysis to evaluate the roles of m6A target genes. Cell division cycle 42 effector protein 3 (CDC42EP3), one probable m6A target gene, was identified to be down-regulated in ovarian cancer tissues and cells. Meanwhile, quantitative PCR (qPCR) and western blot were used to confirm the down-regulated CDC42EP3 in ovarian cancer cells A2780 and TOV112D. The biological function of CDC42EP3 in ovarian cancer was further validated with several algorithms, such as PrognoScan, K-M plotter, LinkedOmics and TISIDB. These findings indicated that lower expression of CDC42EP3 was correlated with poor prognosis in patients with ovarian cancer. In addition, CDC42EP3 expression was significantly associated with a diverse range of tumor-infiltrating immune cells, including natural killer cells (NK), T central memory cells (Tcm), T gamma delta cells (Tgd), etc. Taken together, this study uncovered the potential roles of m6A target gene CDC42EP3 in the regulation of immune microenvironment in the ovarian cancer, and identified CDC42EP3 as a novel prognostic target.

The Human and Mouse Islet Peptidome: Effects of Obesity and Type 2 Diabetes, and Assessment of Intraislet Production of Glucagon-like Peptide-1.

To characterize the impact of metabolic disease on the peptidome of human and mouse pancreatic islets, LC-MS was used to analyze extracts of human and mouse islets, purified mouse alpha, beta, and delta cells, supernatants from mouse islet incubations, and plasma from patients with type 2 diabetes. Islets were obtained from healthy and type 2 diabetic human donors, and mice on chow or high fat diet. All major islet hormones were detected in lysed islets as well as numerous peptides from vesicular proteins including granins and processing enzymes. Glucose-dependent insulinotropic peptide (GIP) was not detectable. High fat diet modestly increased islet content of proinsulin-derived peptides in mice. Human diabetic islets contained increased content of proglucagon-derived peptides at the expense of insulin, but no evident prohormone processing defects. Diabetic plasma, however, contained increased ratios of proinsulin and des-31,32-proinsulin to insulin. Active GLP-1 was detectable in human and mouse islets but 100–1000-fold less abundant than glucagon. LC-MS offers advantages over antibody-based approaches for identifying exact peptide sequences, and revealed a shift toward islet insulin production in high fat fed mice, and toward proglucagon production in type 2 diabetes, with no evidence of systematic defective prohormone processing.

Necrostatin-1 Supplementation to Islet Tissue Culture Enhances the In-Vitro Development and Graft Function of Young Porcine Islets.

Pre-weaned porcine islets (PPIs) represent an unlimited source for islet transplantation but are functionally immature. We previously showed that necrostatin-1 (Nec-1) immediately after islet isolation enhanced the in vitro development of PPIs. Here, we examined the impact of Nec-1 on the in vivo function of PPIs after transplantation in diabetic mice. PPIs were isolated from pancreata of 8–15-day-old, pre-weaned pigs and cultured in media alone, or supplemented with Nec-1 (100 µM) on day 0 or on day 3 of culture (n = 5 for each group). On day 7, islet recovery, viability, oxygen consumption rate, insulin content, cellular composition, insulin secretion capacity, and transplant outcomes were evaluated. While islet viability and oxygen consumption rate remained high throughout 7-day tissue culture, Nec-1 supplementation on day 3 significantly improved islet recovery, insulin content, endocrine composition, GLUT2 expression, differentiation potential, proliferation capacity of endocrine cells, and insulin secretion. Adding Nec-1 on day 3 of tissue culture enhanced the islet recovery, proportion of delta cells, beta-cell differentiation and proliferation, and stimulation index. In vivo, this leads to shorter times to normoglycemia, better glycemic control, and higher circulating insulin. Our findings identify the novel time-dependent effects of Nec-1 supplementation on porcine islet quantity and quality prior to transplantation.

Single cell transcriptomics reveal trans-differentiation of pancreatic beta cells following inactivation of the TFIID subunit Taf4

Regulation of gene expression involves a complex and dynamic dialogue between transcription factors, chromatin remodelling and modification complexes and the basal transcription machinery. To address the function of the Taf4 subunit of general transcription factor TFIID in the regulation of insulin signalling, it was inactivated in adult murine pancreatic beta cells. Taf4 inactivation impacted the expression of critical genes involved in beta-cell function leading to increased glycaemia, lowered plasma insulin levels and defective glucose-stimulated insulin secretion. One week after Taf4-loss, single-cell RNA-seq revealed cells with mixed beta cell, alpha and/or delta cell identities as well as a beta cell population trans-differentiating into alpha-like cells. Computational analysis of single-cell RNA-seq defines how known critical beta cell and alpha cell determinants may act in combination with additional transcription factors and the NuRF chromatin remodelling complex to promote beta cell trans-differentiation.

High Coexpression of the Ghrelin and LEAP2 Receptor GHSR With Pancreatic Polypeptide in Mouse and Human Islets.

Islets represent an important site of direct action of the hormone ghrelin, with expression of the ghrelin receptor (growth hormone secretagogue receptor; GHSR) having been localized variably to alpha cells, beta cells, and/or somatostatin (SST)-secreting delta cells. To our knowledge, GHSR expression by pancreatic polypeptide (PP)-expressing gamma cells has not been specifically investigated. Here, histochemical analyses of Ghsr-IRES-Cre × Cre-dependent ROSA26-yellow fluorescent protein (YFP) reporter mice showed 85% of GHSR-expressing islet cells coexpress PP, 50% coexpress SST, and 47% coexpress PP + SST. Analysis of single-cell transcriptomic data from mouse pancreas revealed 95% of Ghsr-expressing cells coexpress Ppy, 100% coexpress Sst, and 95% coexpress Ppy + Sst. This expression was restricted to gamma-cell and delta-cell clusters. Analysis of several single-cell human pancreatic transcriptome data sets revealed 59% of GHSR-expressing cells coexpress PPY, 95% coexpress SST, and 57% coexpress PPY + SST. This expression was prominent in delta-cell and beta-cell clusters, also occurring in other clusters including gamma cells and alpha cells. GHSR expression levels were upregulated by type 2 diabetes mellitus in beta cells. In mice, plasma PP positively correlated with fat mass and with plasma levels of the endogenous GHSR antagonist/inverse agonist LEAP2. Plasma PP also elevated on LEAP2 and synthetic GHSR antagonist administration. These data suggest that in addition to delta cells, beta cells, and alpha cells, PP-expressing pancreatic cells likely represent important direct targets for LEAP2 and/or ghrelin both in mice and humans.

1245-P: Characteristic Distribution of GRP78 in Islets and Intraislet Ductal Cells of Diabetic C414A-CRY1 Transgenic Mice

Our preceding studies demonstrated that cysteine414 (zinc-binding site of mCRY1) - alanine mutant mCRY1 transgenic mice (TG mice) exhibit diabetes characterized by the reduction of β-cell proliferation caused by senescence-associated secretory phenotype (SASP)-like characters of β-cells. Not only pancreatic intraepithelial neoplasias (PanINs) with pancreatic duct glands (PDGs) but also intra-islet ducts emerged age-dependently in TG mice (Okano S. et al., 2019). Reportedly GRP78 (also called BiP) is highly expressed in pancreatic ductal adenocarcinoma (PDAC), and in lesions of acinar-to-ductal metaplasia (ADM) which is known as precursors of PanIN. As for the islet, GRP78 has been intensively studied with regard to the unfolded protein response (UPR) in the β cells. However, the details of in vivo distribution of GRP78, particularly in endocrine cells outside β cells, are still remain elusive. In this study, we conducted staining experiments for GRP78 in the pancreatic sections of well-matured mice to explore the relation between endocrine cells and intra-islet ducts. The results showed that the expression of GRP78 was higher in PanINs and PDGs than in normal ductal cells in TG mice, suggesting that GRP78 plays important roles in the maintenance of the atypical ductal lesions. In both wild-type and TG mice, GRP78 was observed in the whole islet. The expression of GRP78 was higher in β cells than in alpha and delta cells in both mice. Also, GRP78 was highly expressed in primary intra-islet ducts. Some of the GRP78 high-expressing primary intra-islet ductal cells were co-expressed with chromogranin A, which is known as an endocrine marker. Taken together, our results suggest that the direct trans-differentiation of endocrine cells (possibly β cells) to ductal cells is involved in the generation of the intra-islet ductal cells. Disclosure S. Okano: None. A. Yasui: None. S. Kanno: None. K. Satoh: None. M. Igarashi: None. O. Nakajima: None. Funding Japan Society for the Promotion of Science (19K07498); Tohoku University

Do sodium-glucose co-transporter-2 inhibitors increase plasma glucagon by direct actions on the alpha cell? And does the increase matter for the associated increase in endogenous glucose production?

Sodium-glucose co-transporter-2 inhibitors (SGLT2is) lower blood glucose and are used for treatment of type 2 diabetes. However, SGLT2is have been associated with increases in endogenous glucose production (EGP) by mechanisms that have been proposed to result from SGLT2i-mediated increases in circulating glucagon concentrations, but the relative importance of this effect is debated, and mechanisms possibly coupling SGLT2is to increased plasma glucagon are unclear. A direct effect on alpha-cell activity has been proposed, but data on alpha-cell SGLT2 expression are inconsistent, and studies investigating the direct effects of SGLT2 inhibition on glucagon secretion are conflicting. By contrast, alpha-cell sodium-glucose co-transporter-1 (SGLT1) expression has been found more consistently and appears to be more prominent, pointing to an underappreciated role for this transporter. Nevertheless, the selectivity of most SGLT2is does not support interference with SGLT1 during therapy. Paracrine effects mediated by secretion of glucagonotropic/static molecules from beta and/or delta cells have also been suggested to be involved in SGLT2i-induced increase in plasma glucagon, but studies are few and arrive at different conclusions. It is also possible that the effect on glucagon is secondary to drug-induced increases in urinary glucose excretion and lowering of blood glucose, as shown in experiments with glucose clamping where SGLT2i-associated increases in plasma glucagon are prevented. However, regardless of the mechanisms involved, the current balance of evidence does not support that SGLT2 plays a crucial role for alpha-cell physiology or that SGLT2i-induced glucagon secretion is important for the associated increased EGP, particularly because the increase in EGP occurs before any rise in plasma glucagon.

516 UROCORTIN 3 EXPRESSION IN HUMAN ENTEROENDOCRINE CELLS AND CORRELATION WITH PLASMA GLP1 LEVELS IN HUMAN OBESITY

Introduction Enteroendocrine cells (EECs) are hormone secretory cells sparsely distributed throughout the GI tract, and implicated in obesity. Intestinal L-type EECs secrete Glucagon-Like peptide 1(GLP-1) in response to a meal to promote satiety and maintain glucose homeostasis. Abnormal post prandial satiety (APS) is a phenotypic subset of obesity characterized by blunted post-prandial levels of GLP-1. Intestinal D-type EECs release somatostatin (SST) to inhibit GLP-1 release from L cells via the STT-receptor. In the pancreas, somatostatin release from delta cell is stimulated by pancreatic urocortin-3 from the beta cells. It is unknown whether urocortin-3 is expressed in intestinal EEC cells. Thus, we aimed to study the role of UCN3 in intestinal L, and D- type EECs, and its association with GLP-1 secretion in human obesity. Methods We studied a total of 34 participants, 17 healthy controls (age 38±13y, BMI 23±2kg/m2, 59% female), and 17 with obesity (age 39±9y, BMI 37±5kg/m2, 65% female). Obesity was sub-grouped into: Abnormal postprandial satiety (APS, low postprandial GLP-1, n=6) and normal post prandial satiety (NPS, normal postprandial GLP-1, n=11). Human colonic biopsies from participants were obtained by sigmoidoscopy and preserved for scRNA-seq (CryoStorCS10), gene expression (RNAlater), or microscopy studies (10%NBF). Single-cell transcriptomics was completed using the Chromium 10X Genomics platform followed by Illumina HiSeq4000 sequencing. Blood was collected from participants at fasting, and postprandial at 15, 45, and 90 minutes. Immunofluorescence (IF) confocal microscopy was used to detect protein expression of UCN3 in L-type EECs in the gut mucosa. UCN3 mRNA expression was determined by RT-qPCR. Blood GLP-1 levels were determined by ELISA. Results Our transcriptomic investigation of EECs in human obesity revealed expression of UCN3 in Chromogranin A-positive EECs, and GLP-1 positive L-type EECs, previously not known to be expressed in EECs (Fig1A). Protein expression of UCN3 in GLP-1 positive EEC was confirmed using immunofluorescence in human ileum and colon (Fig 1B). Colonic UCN3 mRNA expression was positively associated with BMI (r=0.43, p Conclusions These findings suggest that intestinal UCN3 is overexpressed in obesity, and further overexpressed in APS obesity phenotype. This study introduces a new pathophysiological underpinning of obesity where negative feedback mechanisms of SST-mediated GLP-1 inhibition are exaggerated, and may potentially promote blunted postprandial GLP-1 levels and decreased satiety in obesity. Download : Download high-res image (100KB) Download : Download full-size image Figure 1 . Expression of Urocortin 3 (UCN3) in human enteroendocrine cells (EECs). (A) Ratio of cell population clusters of the human colonic mucosa in either all EECs or just GCG-expressing L-Type EECs, expressing UCN3 of 10 patients (n=4 lean, n=6 obesityall). (B) Double immunoflurescent staining of the human intestinal mucosa demonstrating expression of UCN3 (Red) in a subset of GLP-1 (Green) L-type EECs. Red bar indicates 20µm. Download : Download high-res image (95KB) Download : Download full-size image Figure 2 . Urocortin 3 (UCN3) Associations with BMI and GLP-1 Release. (A) Association of colonic UCN3 mRNA expression (A) to BMI (kg/m2). Relative Human colonic mucosal mRNA expression of UCN3 comparing (B) lean and all-obesity; and (C) lean and obesity with and without APS. Association of intestinal UCN3 expression with (D) plasma concentrations of GLP-1 measured 45 and (C) resulting association of GLP-1 area under the curve (AUC). Significance testing used a linear regression for A,D, E, and a two-tailed student t-test compared between all groupings; # p

Single-cell chromatin accessibility identifies pancreatic islet cell type- and state-specific regulatory programs of diabetes risk.

Single nucleus ATAC-seq (snATAC-seq) creates new opportunities to dissect cell type-specific mechanisms of complex diseases. As pancreatic islets are central to type 2 diabetes (T2D), we profiled 15.3k islet cells using combinatorial barcoding snATAC-seq and identified 12 clusters, including multiple alpha, beta and delta cell states. We cataloged 228,873 accessible chromatin sites and identified transcription factors underlying lineage- and state-specific regulation. We observed state-specific enrichment of fasting glucose and T2D GWAS for beta cells as well as enrichment for other endocrine cell types. At T2D signals localized to islet accessible chromatin, we prioritized variants with predicted regulatory function and co-accessibility with target genes. A causal T2D variant rs231361 at the KCNQ1 locus had predicted effects on a beta cell enhancer co-accessible with INS, and genome editing in embryonic stem cell-derived beta cells affected INS levels. Together our findings demonstrate the power of single cell epigenomics for interpreting complex disease genetics.

Free fatty acid receptor 4 inhibitory signaling in delta cells regulates islet hormone secretion in mice

ObjectiveMaintenance of glucose homeostasis requires the precise regulation of hormone secretion from the endocrine pancreas. Free fatty acid receptor 4 (FFAR4/GPR120) is a G protein-coupled receptor whose activation in islets of Langerhans promotes insulin and glucagon secretion and inhibits somatostatin secretion. However, the contribution of individual islet cell types (α, β, and δ cells) to the insulinotropic and glucagonotropic effects of GPR120 remains unclear. As gpr120 mRNA is enriched in somatostatin-secreting δ cells, we hypothesized that GPR120 activation stimulates insulin and glucagon secretion via inhibition of somatostatin release. MethodsGlucose tolerance tests were performed in mice after administration of selective GPR120 agonist Compound A. Insulin, glucagon, and somatostatin secretion were measured in static incubations of isolated mouse islets in response to endogenous (ω-3 polyunsaturated fatty acids) and/or pharmacological (Compound A and AZ-13581837) GPR120 agonists. The effect of Compound A on hormone secretion was tested further in islets isolated from mice with global or somatostatin cell-specific knock-out of gpr120. Gpr120 expression was assessed in pancreatic sections by RNA in situ hybridization. Cyclic AMP (cAMP) and calcium dynamics in response to pharmacological GPR120 agonists were measured specifically in α, β, and δ cells in intact islets using cAMPER and GCaMP6 reporter mice, respectively. ResultsAcute exposure to Compound A increased glucose tolerance, circulating insulin, and glucagon levels in vivo. Endogenous and/or pharmacological GPR120 agonists reduced somatostatin secretion in isolated islets and concomitantly demonstrated dose-dependent potentiation of glucose-stimulated insulin secretion and arginine-stimulated glucagon secretion. Gpr120 was enriched in δ cells. Pharmacological GPR120 agonists reduced cAMP and calcium levels in δ cells but increased these signals in α and β cells. Compound A-mediated inhibition of somatostatin secretion was insensitive to pertussis toxin. The effect of Compound A on hormone secretion was completely absent in islets from mice with either global or somatostatin cell-specific deletion of gpr120 and partially reduced upon blockade of somatostatin receptor signaling by cyclosomatostatin. ConclusionsInhibitory GPR120 signaling in δ cells contributes to both insulin and glucagon secretion in part by mitigating somatostatin release.

Biosynthetic Activity Differs Between Islet Cell Types and in Beta Cells Is Modulated by Glucose and Not by Secretion.

A correct biosynthetic activity is thought to be essential for the long-term function and survival of islet cells in culture and possibly also after islet transplantation. Compared to the secretory activity, biosynthetic activity has been poorly studied in pancreatic islet cells. Here we aimed to assess biosynthetic activity at the single cell level to investigate if protein synthesis is dependent on secretagogues and increased as a consequence of hormonal secretion. Biosynthetic activity in rat islet cells was studied at the single cell level using O-propargyl-puromycin (OPP) that incorporates into newly translated proteins and chemically ligates to a fluorescent dye by “click” reaction. Heterogeneous biosynthetic activity was observed between the four islet cell types, with delta cells showing the higher relative protein biosynthesis. Beta cells protein biosynthesis was increased in response to glucose while 3-isobutyl-1-methylxanthine and phorbol-12-myristate-13-acetate, 2 drugs known to stimulate insulin secretion, had no similar effect on protein biosynthesis. However, after several hours of secretion, protein biosynthesis remained high even when cells were challenged to basal conditions. These results suggest that mechanisms regulating secretion and biosynthesis in islet cells are different, with glucose directly triggering beta cells protein biosynthesis, independently of insulin secretion. Furthermore, this OPP labeling approach is a promising method to identify newly synthesized proteins under various physiological and pathological conditions.

There Is Something About Insulin Granules.

Insulin secretory granules of pancreatic β-cells are complex intracellular organelles comprising many proteins with different catalytic activities and messenger functions (1). These subcellular organelles have a distinctive morphology in electron micrographs as a sphere of around 200–300 nm in diameter comprising a crystalline core of insulin surrounded by a halo of less dense material in a phospholipid bilayer envelope (Fig. 1) (2). The granule is more than just a hormone repository in the cell. It is the site of proteolytic conversion of proinsulin into insulin. This dynamic structure is also involved in intercellular transport and communication through the secretion of other biologically active molecules. Around 10% of the granule population turns over every hour during active secretion. An important contribution to our understanding of the regulation of insulin secretion has come from studies of the granules, and in particular the elucidation of the structure and function of many granule proteins (1). Figure 1 Multitasking by insulin granules. Insulin secretory granules (right) are unique features of β-cells (green; left) in pancreatic islets. Light organelles represent immature granules, whereas mature granules are dark and dense spheres containing insulin crystals, which are distinct from somatostatin granules in delta cells (blue). Native proteins as well as posttranscriptionally or posttranslationally modified proteins and sphingolipids have now been identified in these characteristic organelles that act as autoantigenic targets or immune modulators of islet-specific autoreactive CD4 and CD8 T cells. (Electron microscopy graph kindly shared by Dr. Ben Giepmans [2].) But there is something else …

Purification of pancreatic endocrine subsets reveals increased iron metabolism in beta-cells

Abstract Objectives The main endocrine cell types in pancreatic islets are alpha, beta, and delta cells. Although these cell types have distinct roles in the regulation of glucose homeostasis, inadequate purification methods preclude the study of cell type-specific effects. We developed a reliable approach that enables simultaneous sorting of live alpha, beta, and delta cells from mouse islets for downstream analyses. Methods We developed an antibody panel against cell surface antigens to enable isolation of highly purified endocrine subsets from mouse islets based on the specific differential expression of CD71 on beta cells and CD24 on delta cells. We rigorously demonstrated the reliability and validity of our approach using bulk and single cell qPCR, immunocytochemistry, reporter mice, and transcriptomics. Results Pancreatic alpha, beta, and delta cells can be separated based on beta cell-specific CD71 surface expression and high expression of CD24 on delta cells. We applied our new sorting strategy to demonstrate that CD71, which is the transferrin receptor mediating the uptake of transferrin-bound iron, is upregulated in beta cells during early postnatal weeks. We found that beta cells express higher levels of several other genes implicated in iron metabolism and iron deprivation significantly impaired beta cell function. In human beta cells, CD71 is similarly required for iron uptake and CD71 surface expression is regulated in a glucose-dependent manner. Conclusions This study provides a novel and efficient purification method for murine alpha, beta, and delta cells, identifies for the first time CD71 as a postnatal beta cell-specific marker, and demonstrates a central role of iron metabolism in beta cell function.