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Blood glucose homeostasis is the maintenance of blood glucose levels within a narrow range. The tight regulation of blood glucose levels is critical, as hypoglycemia can be acutely fatal and hyperglycemia leads to diabetes and its associated complications. Pancreatic islets play an essential role in maintaining normoglycemia. They are composed of insulin-secreting beta cells, glucagon-secreting alpha cells, and somatostatin-secreting delta cells, which coordinate to regulate blood glucose levels through paracrine interactions. Breakdown of these interactions contributes to the development of diabetes.This dissertation focuses on how beta, alpha, and delta cells are affected by changes in paracrine interactions and disease, with a focus on how delta cells contribute to the regulation of beta cells as well as alpha cells. Chapter 1 provides an overview of the beta, alpha and delta cells within the pancreatic islet and how their interactions shape glucose homeostasis, as well as a section on other known somatostatin-secreting cells. Chapter 2 focuses on UCN3 and its role as a marker but not a driver of beta cell maturity. Chapter 3 investigates how SST signaling affects gene expression in beta and alpha cells. Chapter 4 presents work on the contribution of delta cells and SST signaling to the glycemic set point and how this occurs through paracrine interaction with beta cells. Chapter 5 further discusses the role of delta cells in the control of blood glucose levels. Chapter 6 returns to the topic of beta cell and alpha cell gene expression and what changes occur in a model of obesity. Chapter 7 provides a summary of the work and potential future directions.

Aims/hypothesis Transcriptome analyses revealed insulin-gene-derived transcripts in non-beta endocrine islet cells. We studied alternative splicing of human INS mRNA in pancreatic islets. Methods Alternative splicing of insulin pre-mRNA was determined by PCR analysis performed on human islet RNA and single-cell RNA-seq analysis. Antisera were generated to detect insulin variants in human pancreatic tissue using immunohistochemistry, electron microscopy and single-cell western blot to confirm the expression of insulin variants. Cytotoxic T lymphocyte (CTL) activation was determined by MIP-1β release. Results We identified an alternatively spliced INS product. This variant encodes the complete insulin signal peptide and B chain and an alternative C-terminus that largely overlaps with a previously identified defective ribosomal product of INS. Immunohistochemical analysis revealed that the translation product of this INS-derived splice transcript was detectable in somatostatin-producing delta cells but not in beta cells; this was confirmed by light and electron microscopy. Expression of this alternatively spliced INS product activated preproinsulin-specific CTLs in vitro. The exclusive presence of this alternatively spliced INS product in delta cells may be explained by its clearance from beta cells by insulin-degrading enzyme capturing its insulin B chain fragment and a lack of insulin-degrading enzyme expression in delta cells. Conclusions/interpretation Our data demonstrate that delta cells can express an INS product derived from alternative splicing, containing both the diabetogenic insulin signal peptide and B chain, in their secretory granules. We propose that this alternative INS product may play a role in islet autoimmunity and pathology, as well as endocrine or paracrine function or islet development and endocrine destiny, and transdifferentiation between endocrine cells. INS promoter activity is not confined to beta cells and should be used with care when assigning beta cell identity and selectivity. Data availability The full EM dataset is available via www.nanotomy.org (for review: http://www.nanotomy.org/OA/Tienhoven2021SUB/6126-368/). Single-cell RNA-seq data was made available by Segerstolpe et al [13] and can be found at https://sandberglab.se/pancreas. The RNA and protein sequence of INS-splice was uploaded to GenBank (BankIt2546444 INS-splice OM489474). Graphical abstract

The endocrine pancreas comprises the islets of Langerhans, primarily consisting of beta cells, alpha cells and delta cells responsible for secretion of insulin, glucagon and somatostatin, respectively. A certain level of intra-islet communication is thought to exist, where the individual hormones may reach the other islet cells and regulate their secretion. Glucagon has been demonstrated to importantly regulate insulin secretion, while somatostatin powerfully inhibits both insulin and glucagon secretion. In this study we investigated how secretion of somatostatin is regulated by paracrine signalling from glucagon and insulin. Somatostatin secretion was measured from perfused mouse pancreases isolated from wild-type as well as diphtheria toxin-induced alpha cell knockdown, and global glucagon receptor knockout (Gcgr–/–) mice. We studied the effects of varying glucose concentrations together with infusions of arginine, glucagon, insulin and somatostatin, as well as infusions of antagonists of insulin, somatostatin and glucagon-like peptide 1 (GLP-1) receptors. A tonic inhibitory role of somatostatin was demonstrated with infusion of somatostatin receptor antagonists, which significantly increased glucagon secretion at low and high glucose, whereas insulin secretion was only increased at high glucose levels. Infusion of glucagon dose-dependently increased somatostatin secretion approximately twofold in control mice. Exogenous glucagon had no effect on somatostatin secretion in Gcgr–/– mice, and a reduced effect when combined with the GLP-1 receptor antagonist exendin 9-39. Diphtheria toxin-induced knockdown of glucagon producing cells led to reduced somatostatin secretion in response to 12 mmol/l glucose and arginine infusions. In Gcgr–/– mice (where glucagon levels are dramatically increased) overall somatostatin secretion was increased. However, infusion of exendin 9-39 in Gcgr–/– mice completely abolished somatostatin secretion in response to glucose and arginine. Neither insulin nor an insulin receptor antagonist (S961) had any effect on somatostatin secretion. Our findings demonstrate that somatostatin and glucagon secretion are linked in a reciprocal feedback cycle with somatostatin inhibiting glucagon secretion at low and high glucose levels, and glucagon stimulating somatostatin secretion via the glucagon and GLP-1 receptors.

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.

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 current study was designed to perform a transmission electron microscopic investigation focusing on the dark and light pancreatic acinar β-cells of young domesticated pig (Sus Suidae).This study depended on the fresh pancreatic specimens from 5 healthy young (2-month-old) pigs that were collected immediately after they were slaughtered at the abattoir of Abdelkader Alexandria, Egypt.In our findings, the acinar pancreas was formed of pyramidal pancreatic acinar cells with large spherical nuclei of condensed heterochromatin at the periphery and prominent eccentric nucleoli. Zymogen granules were observed at the apical region of the acinar cells, and they appear as electron dense bodies. Numerous mitochondria and Golgi complexes observed in the acinar cell cytoplasm. The electron dense acinar cells were joined by junctional complexes. The rough endoplasmic reticulum was more prominent in the electron-dense acinar cells than did electron-lucent acinar cells. There was no connective tissue capsule separate the acinar portion of pancreas from the pancreatic islets. The pancreatic islets mainly formed of β-cells. The irregular α-cells possess numerous small granules. The cytoplasmic β-cells granules were surrounded by hallow area and enclosed by a limiting membrane. Delta cells were generally polygonal in shape and found in clumps throughout the islet and they were also identified in between β-cells. Their granules were of moderate electron density and were generally smaller than β-cells' granules. The limiting membrane was tightly enclosed the delta cells granules and the hallow area around the granule were found similar to the granules of β-cells.

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.

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.

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 …

Background and objective: Reports indicate that Rheum ribes has antihyperglycemic effects. This study aimed to find out the hypoglycemic effects of aqueous extract of Rheum ribes on the pancreatic tissue of diabetic male rats and compare the results of both histological and biochemical findings with that of metformin. Methods: Thirty-two albino male rats were divided into four groups with eight animals for each group; 1st group as normal control, 2nd group as alloxan induced diabetics, 3rd group as diabetic treated with Rheum ribes, and 4th groups as diabetic treated with Metformin. The body weight and fasting blood glucose levels of all the rats were recorded at weekly intervals during the experimental period. Biochemical, histochemical, and immunohistochemical investigations were used. Results: Diabetic rats showed a decrease in serum amylase with marked elevation in blood glucose level. Histological examination of the pancreas from diabetic rats revealed an increase in the thickness of the basement membrane of the blood vessels and capillaries in islets of Langerhans, as well as decreased activity of Beta-cells and increase the activity of Alpha and Delta cells using both Periodic Acid Schiff stain and immunohistochemistry techniques. Administration of aqueous extract of Rheum ribes caused significant and dramatic changes in most of the parameters measured in this study, including an increase in serum amylase, and lowering the level of blood glucose, in comparison to the diabetic group. Conclusion: Daily administration of Rheum ribes significantly reduced blood glucose and caused a marked increase in the activity of Beta-cells, followed by a decrease in the activity of Alpha-cells. The results of tissue structures and serum biochemical estimation indicated that the plant extract has regeneration and repair effects.

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

Objective Maintenance 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. Methods Glucose 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. Results Acute 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. Conclusions Inhibitory GPR120 signaling in δ cells contributes to both insulin and glucagon secretion in part by mitigating somatostatin release., Graphical abstract Image 1, Highlights • Gpr120 was enriched in mouse somatostatin-secreting δ cells. • GPR120 activation in mouse δ cells inhibited cAMP generation and somatostatin release. • GPR120 activation in mouse δ cells potentiated insulin and glucagon 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.

Direct modulation of pancreatic endocrine cell activity by autonomic innervation has been debated. To resolve this question, we established an in vivo imaging model which also allows chronic and acute neuromodulation. Starting at a stage when zebrafish islet architecture is reminiscent of that in adult rodents, we imaged calcium dynamics simultaneously in multiple islet cell types. We first find that activity coupling between beta cells increases upon glucose exposure. Surprisingly, glucose exposure also increases alpha-alpha, alpha-beta and beta-delta coordination. We further show that both chronic and acute loss of nerve activity diminish activity coupling, as observed upon gap junction depletion. Notably, chronic loss of innervation severely disrupts delta cell activity, suggesting that delta cells receive innervation which coordinates its output. Overall, these data show that innervation plays a vital role in the establishment and maintenance of homotypic and heterotypic cellular connectivity in pancreatic islets, a process critical for islet function.

Pancreatic beta cells are the only cell type in our body capable of producing and secreting insulin to instruct the insulin-sensitive cells and tissues of our bodies to absorb nutrients after a meal. Accurate control of insulin release is of critical importance; too little insulin leads to diabetes, while an excess of insulin can cause potentially fatal hypoglycaemia. Yet, the pancreas of most people will control insulin secretion safely and effectively over decades and in response to glucose excursions driven by tens of thousands of meals. Because we only become aware of the important contributions of the pancreas when it fails to maintain glucose homeostasis, it is easy to forget just how well insulin release from a healthy pancreas is matched to insulin need to ensure stable blood glucose levels. Beta cells achieve this feat by extensive crosstalk with the rest of the endocrine cell types in the islet, notably the glucagon-producing alpha cells and somatostatin-producing delta cells. Here I will review the important paracrine contributions that each of these cells makes to the stimulation and subsequent inhibition of insulin release in response to a transient nutrient stimulation, and make the case that a breakdown of this local crosstalk contributes to the pathophysiology of diabetes. Graphical abstract.

Background: Acid inhibition from chronic proton pump inhibitor use and a possible increase in gastrin can lead to changes in the regulation of hydrochloric acid production. However, it has not known whether such chronic use changes the presence of gastrin, delta, and enterochromaffin-like cells in the stomach or the relationship between gastrin and delta cells. Aim: To analyze the number of gastrin-producing gastrin cells, somatostatin-producing cells, and histamine-producing cells in patients who were chronic users of proton pump inhibitor, with or without related Helicobacter pylori infection. Methods: Biopsies from 105 patients, including 81 chronic proton pump inhibitor users (experimental group) and 24 controls, were processed immunohistochemically and subjected to counting of gastrin, delta, and enterochromaffin-like cells in high-magnification microscopic fields and in 10 glands. Results: Gastrin cell, delta cell, and enterochromaffin-like cells counts were similar across the groups and appeared to be unaffected by Helicobacter pylori infection. The ratio between gastrin cells and delta cells was higher in the chronic users of proton pump inhibitor group than in controls. Conclusion: Chronic users of proton pump inhibitor does not affect gastrin cell, delta cell, and enterochromaffin-like cell counts significantly, but may alter the ratio between gastrin cells and delta cells., Graphical Abstract IHC identifying G, D, and ECL cells

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 the 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 knockout 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 and circulating insulin and glucagon levels in vivo. Endogenous and/or pharmacological and 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 was 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 via mitigating somatostatin release.

Objective: While expression of the ghrelin receptor, GHSR (growth hormone secretagogue receptor), by pancreatic islets has been investigated, data regarding the specific islet cell types that express GHSR are inconsistent and incomplete. For instance, transcriptomic studies indicate marked GHSR expression by somatostatin (SST)-secreting delta cells vs. little to no GHSR expression by alpha cells and beta cells, which contrasts studies suggesting direct actions of ghrelin on GHSRs expressed by alpha cells and beta cells. Also, to our knowledge, GHSR expression by pancreatic polypeptide (PP) cells has not previously been directly investigated. The aims of this study were to determine GHSR expression within the four principal islet endocrine cell types and the effects of ghrelin on circulating PP. Methods: Ghsr-IRES-Cre mice, which express Cre recombinase directed by the GHSR promoter, crossed to Cre-dependent ROSA26-YFP reporter mice were used to histologically identify GHSR expression in islets. Double and triple labeling for YFP-immunoreactivity (IR) with insulin-IR, glucagon-IR, SST-IR, and/or PP-IR was performed to determine GHSR co-expression with islet hormones. Pharmacological manipulations were used to examine in vivo effects of acyl-ghrelin on plasma PP. Results: Eighty-five % of YFP-IR cells expressed PP-IR, 50% expressed SST-IR, and none expressed insulin-IR or glucagon-IR. GHSR antagonist raised plasma PP in overnight-fasted C57BL/6N mice. However, acyl-ghrelin administration had no effect on plasma PP in C57BL/6N mice. Conclusions: These results suggest that within mouse pancreatic islets, GHSR expression occurs most abundantly in PP cells and that pharmacologic blockade of ghrelin action raises plasma PP. Disclosure D. Gupta: None. B.K. Mani: None. K. Shankar: None. S. Osborne-Lawrence: None. N. Metzger: None. J.M. Zigman: Stock/Shareholder; Self; Medtronic. Funding Diana and Richard C. Strauss Professorship in Biomedical Research; Mr. and Mrs. Bruce G. Brookshire Professorship in Medicine; Kent and Jodi Foster Distinguished Chair in Endocrinology; University of Texas Southwestern Medical Center (to J.M.Z.)

We selectively inactivated the Taf4 subunit of general transcription factor TFIID in adult murine pancreatic beta cells (BCs). Taf4 inactivation rapidly diminishes expression of critical genes involved in BC function leading to increased glycaemia, lowered plasma insulin levels, defective glucose-stimulated insulin secretion and in the longer term reduced BC mass through apoptosis of a subpopulation of BCs. Nevertheless, glycaemia and blood insulin levels are stabilised after 11 weeks with mutant animals showing long term survival. Bulk RNA-seq and ATAC-seq together with single cell RNA-seq on isolated Langerhans islets show that Taf4 loss leads to a remodelling of chromatin accessibility and gene expression not only in targeted BCs, but also alpha and delta cells. One week after Taf4-loss, cells with mixed BC, alpha and/or delta cell identities were observed as well as a BC population trans-differentiating into alpha-like cells. Computational analysis defines how known critical BC and alpha cell determinants may act in combination with additional transcription factors and the NuRF chromatin remodelling complex to promote BC trans-differentiation.

The cellular distribution of a novel porcine peptide, diazepam-binding inhibitor (DBI), was immunohistochemically mapped in the human and porcine gastrointestinal tract and pancreas. Using a rabbit antiserum, raised against porcine DBI, immunoreactive epithelial cells were found in the gastric antrum and duodenal mucosa and in the parenchymal cells of the islets of Langerhans of both species. The immunoreactivity could not be absorbed by high concentrations of insulin, somatostatin (SS-14, SS-28), glucagon, or pancreatic polypeptide but was abolished by porcine DBI. Using semithin, consecutive sections, the immunoreactive intestinal and islet cells were found to be identical to the somatostatin-producing D cells. Since it has been shown that porcine DBI interferes with the secretion of insulin, the peptide may act as a modulator of islet hormone release, possibly in a paracrine manner.

Somatostatin secretion from islet delta cells is important in maintaining low glycemic variability (GV) by providing negative feedback to beta cells and inhibiting insulin secretion. Capromorelin is a ghrelin-receptor agonist that activates the growth hormone secretagogue receptor on delta cells. We hypothesized that in cats, capromorelin administration will result in decreased GV at the expense of reduced insulin secretion and glucose tolerance. Seven healthy cats were treated with capromorelin from days 1-30. After the first day, fasting blood glucose increased (+13 ± 3 mg/dL, P0.0001), insulin decreased (+128 ± 122 ng/dL, P = 0.03), and glucagon was unchanged. Blood glucose was increased throughout an intravenous glucose tolerance test on day 1 with blunting of first-phase insulin response ([FPIR] 4,931 ± 2,597 ng/L/15 min) compared with day -3 (17,437 ± 8,302 ng/L/15 min, P = 0.004). On day 30, FPIR was still blunted (9,993 ± 4,285 ng/L/15 min, P = 0.045), but glucose tolerance returned to baseline. Mean interstitial glucose was increased (+19 ± 6 mg/dL, P = 0.03) on days 2-4 but returned to baseline by days 27-29 (P = 0.3). On days 2-4, GV was increased (SD = 9.7 ± 3.2) compared with baseline (SD = 5.0 ± 1.1, P = 0.02) and returned to baseline on days 27-29 (SD = 6.1 ± 1.1, P = 0.16). In summary, capromorelin caused a decline in insulin secretion and glycemic control and an increase in glucose variability early in the course of treatment, but these effects diminished toward the end of 30 d of treatment.

BACKGROUND: Necroptosis has been demonstrated to be a primary mechanism of islet cell death. This study evaluated whether the supplementation of necrostatin-1 (Nec-1), a potent inhibitor of necroptosis, to islet culture media could improve the recovery, maturation, and function of pre-weaned porcine islets (PPIs).METHODS: PPIs were isolated from pre-weaned Yorkshire piglets (8-15 days old) and either cultured in control islet culture media (n = 6) or supplemented with Nec-1 (100 µM, n = 5). On days 3 and 7 of culture, islets were assessed for recovery, insulin content, viability, cellular composition, GLUT2 expression in beta cells, differentiation of pancreatic endocrine progenitor cells, function, and oxygen consumption rate.RESULTS: Nec-1 supplementation induced a 2-fold increase in the insulin content of PPIs on day 7 of culture. When compared to untreated islets, Nec-1 treatment doubled the beta- and alpha-cell composition and accelerated the development of delta cells. Additionally, beta cells of Nec-1-treated islets had a significant upregulation in GLUT2 expression. The enhanced development of major endocrine cells and GLUT2 expression after Nec-1 treatment subsequently led to a significant increase in the amount of insulin secreted in response to in vitro glucose challenge. Islet recovery, viability, and oxygen consumption rate were unaffected by Nec-1.CONCLUSION: This study underlines the importance of necroptosis in islet cell death after isolation and demonstrates the novel effects of Nec-1 to increase islet insulin content, enhance pancreatic endocrine cell development, facilitate GLUT2 upregulation in beta cells, and augment insulin secretion. Nec-1 supplementation to culture media significantly improves islet quality prior to xenotransplantation.

Islets of Langerhans, found in the pancreas, are microorgans essential for glucose homeostasis within the body. Many cells are found with an islet, such as beta cells (~70%), alpha cells (~20%), delta cells (~5%), F cells (~4%), and epsilon cells (1%), each with its own unique function. To better understand the roles of these cells and how cell communication alters their function, several techniques have been established such as islet isolation and beta cell dispersion. Here we describe how to isolate primary rodent islets, disperse pancreatic islets, measure intracellular calcium, and use immunofluorescent staining to distinguish beta cells and alpha cells.

Objectives Elevated plasma glucagon is an early symptom of diabetes, occurring in subjects with impaired glucose regulation. Here, we explored alpha-cell function in female mice fed a high-fat diet (HFD). Methods Female mice expressing the Ca2+ indicator GCaMP3 specifically in alpha-cells were fed a high-fat or control (CTL) diet. We then conducted in vivo phenotyping of these mice, as well as experiments on isolated (ex vivo) islets and in the in situ perfused pancreas. Results In HFD-fed mice, fed plasma glucagon levels were increased and glucagon secretion from isolated islets and in the perfused mouse pancreas was also elevated. In mice fed a CTL diet, increasing glucose reduced intracellular Ca2+ ([Ca2+]i) oscillation frequency and amplitude. This effect was also observed in HFD mice; however, both the frequency and amplitude of the [Ca2+]i oscillations were higher than those in CTL alpha-cells. Given that alpha-cells are under strong paracrine control from neighbouring somatostatin-secreting delta-cells, we hypothesised that this elevation of alpha-cell output was due to a lack of somatostatin (SST) secretion. Indeed, SST secretion in isolated islets from HFD-fed mice was reduced but exogenous SST also failed to suppress glucagon secretion and [Ca2+]i activity from HFD alpha-cells, in contrast to observations in CTL mice. Conclusions These findings suggest that reduced delta-cell function, combined with intrinsic changes in alpha-cells including sensitivity to somatostatin, accounts for the hyperglucagonaemia in mice fed a HFD., Highlights • HFD feeding causes hyperglucagonaemia in vivo. • Glucagon is inadequately suppressed by glucose in HFD-fed mice. • Alpha-cell [Ca2+]i oscillations and glucagon output are elevated ex vivo in response to HFD feeding. • SST secretion from HFD islets is reduced. • Alpha-cells from HFD-fed mice become ‘resistant’ to SST.

Made available in DSpace on 2019-10-06T16:02:14Z (GMT). No. of bitstreams: 0 Previous issue date: 2018-12-01 Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Aims: The objective of this study was to assess the mechanisms underlying pancreatic islet adaptation in diabetic mothers and their pups. Additionally, the influence of pancreatic adaptations on maternal reproductive performance was also investigated. Main methods: Wistar rats were injected with streptozotocin for diabetes induction. At adulthood (3 months), all animals underwent an oral glucose tolerance test (OGTT) for glucose assessment as an inclusion criterion. Following, the animals were mated. At day 18 of pregnancy, the mothers were killed for blood collect ion to determine fasting insulin and glucagon concentrations. The pancreas was removed and processed for the immunohistochemical analysis of insulin, glucagon, somatostatin, Ki-67 and PDX-1, superoxide dismutase 1 (SOD-1), glutathione peroxidase (GSH-Px) and malondialdehyde (MDA). The pregnant uterus was also collected for the evaluation of embryofetal loss. Key findings: The diabetic rats showed increased glucose, serum glucagon and insulin concentrations, and embryofetal loss rates. They also showed a reduction in pancreatic islets area and percentage of cells stained for insulin, increased the percentage of non-β cells (alpha e delta cells) stained for Ki-67, glucagon, and somatostatin. Moreover, the cells stained for somatostatin were spread across the islets and showed stronger staining for MDA and weaker staining for GSH-Px. Significance: Diabetes leads to adaptive responses from the endocrine pancreas in pregnancy that especially involves non-β cells, modifying the mantle-core structure. Nonetheless, these adaptations are not enough for glucose homeostasis and affect the maternal environment, which in turn impairs fetal development. Laboratory of Experimental Research on Gynecology and Obstetrics Gynecology Obstetrics and Mastology Post Graduate Course Botucatu Medical School Univ Estadual Paulista_Unesp DeVry Ruy Barbosa School (DeVry Brazil Group) Laboratory of System Physiology and Reproductive Toxicology Institute of Biological and Health Sciences Federal University of Mato Grosso (UFMT) Department of Morphology Botucatu Bioscience Institute Univ Estadual Paulista_Unesp Biomedical Research Centre Universidad de Valparaíso Laboratory of Experimental Research on Gynecology and Obstetrics Gynecology Obstetrics and Mastology Post Graduate Course Botucatu Medical School Univ Estadual Paulista_Unesp Department of Morphology Botucatu Bioscience Institute Univ Estadual Paulista_Unesp FAPESP: 2011/18519-7

Pancreatic endocrine islets are vital for glucose homeostasis. However, the islet developmental trajectory and its regulatory network are not well understood. To define the features of these specification and differentiation processes, we isolated individual islet cells from TgBAC(neurod1:EGFP) transgenic zebrafish and analyzed islet developmental dynamics across four different embryonic stages using a single-cell RNA-seq strategy. We identified proliferative endocrine progenitors, which could be further categorized by different cell cycle phases with the G1/S subpopulation displaying a distinct differentiation potential. We identified endocrine precursors, a heterogeneous intermediate-state population consisting of lineage-primed alpha, beta and delta cells that were characterized by the expression of lineage-specific transcription factors and relatively low expression of terminally differentiation markers. The terminally differentiated alpha, beta, and delta cells displayed stage-dependent differentiation states, which were related to their functional maturation. Our data unveiled distinct states, events and molecular features during the islet developmental transition, and provided resources to comprehensively understand the lineage hierarchy of islet development at the single-cell level.

Background: Knowledge of normal development pattern of various endocrine cells of the human pancreas is of paramount importance, due to its implication during replacement therapies in the treatment of diabetes mellitus. Development related literature of fetal pancreas is scanty due to ethical and technical difficulties to obtain fetuses. Aims and Objectives: Present study was conducted on 49 fetuses from gestational weeks 10 to 40 to obtain the sequential appearance and arrangement of islets of langerhans cells of pancreas. Material and Methods: Forty nine fetuses were collected in formal saline immediately after abortion, medical termination of pregnancy or premature delivery. The fetal pancreas was processed for paraffin blocks. Sections were taken and stained with Haematoxylin and Eosin as well as Gomori’s modified Aldehyde Fuchsin stain and observed under light microscope. Results: Islets were initially a small number of cells with Alpha cells, which were seen at 10th week of gestation. Beta cells were identified in the islets of fetuses around 16 weeks of gestation. Delta cells were observed from 17 weeks onwards and islets appeared as a group from the beginning of 19th week. Conclusion: In the present study we observed a sequential development of Alpha, Beta and Delta cells and subsequent grouping of the islets. The exact gestational time of appearance of above islet cells and their encapsulation can add to the existing knowledge data base for successful transplant of pancreas in patients of insulin dependent diabetes mellitus. Asian Journal of Medical Sciences Vol.9(5) 2018 67-72

This work aimed to discuss the histological, histochemical and ultrastructure of the ostrich pancreas. Specimens were collected from eight adult apparent healthy male ostriches. The specimens immediately immersed in 10 % neutral buffered formalin and Bouin,s solution. Tissue sections were prepared and stained for further examination. The histological examination demonstrated that the ostrich pancreas surrounded by a thick connective tissue capsule which gave septa to the parenchyma and divided it into numerous pancreatic lobules. The parenchyma composed of exocrine and endocrine parts. The exocrine part formed by acini, the acinar cell was large pyramidal, with a large rounded vesicular nucleus. Its apical part had fine deeply stained zymogenic granules. The endocrine portion was formed by beta, alpha and mixed islets. Ultrastructurally, the acinar cells forming the pancreatic acini appeared pyramidal having a basally large vesicular nucleus with large spherical and electron dense nucleoli. Its zymogene granules were spherical and electron dense. The beta cells were large ovoid or polygonal contained electron dense secretory granules with the presence of halo zoon between its limited membrane and the content. Alpha cells are elongated or polygonal contain electron dense spherical secretory granules with ill distinct space between the limited membrane and its content. Delta cells appeared oval or triangular having spherical electron dense granules its limited membrane closely adhere to its content. There were a different histochemical reaction to acid phosphatase, alkaline phosphatase, ATPase and succinic dehydrogenase in the acinar cells and in the pancreatic islets.

Objective Single-cell RNA sequencing studies have revealed that the type-2 diabetes associated two-pore domain K+ (K2P) channel TALK-1 is abundantly expressed in somatostatin-secreting δ-cells. However, a physiological role for TALK-1 in δ-cells remains unknown. We previously determined that in β-cells, K+ flux through endoplasmic reticulum (ER)-localized TALK-1 channels enhances ER Ca2+ leak, modulating Ca2+ handling and insulin secretion. As glucose amplification of islet somatostatin release relies on Ca2+-induced Ca2+ release (CICR) from the δ-cell ER, we investigated whether TALK-1 modulates δ-cell Ca2+ handling and somatostatin secretion. Methods To define the functions of islet δ-cell TALK-1 channels, we generated control and TALK-1 channel-deficient (TALK-1 KO) mice expressing fluorescent reporters specifically in δ- and α-cells to facilitate cell type identification. Using immunofluorescence, patch clamp electrophysiology, Ca2+ imaging, and hormone secretion assays, we assessed how TALK-1 channel activity impacts δ- and α-cell function. Results TALK-1 channels are expressed in both mouse and human δ-cells, where they modulate glucose-stimulated changes in cytosolic Ca2+ and somatostatin secretion. Measurement of cytosolic Ca2+ levels in response to membrane potential depolarization revealed enhanced CICR in TALK-1 KO δ-cells that could be abolished by depleting ER Ca2+ with sarco/endoplasmic reticulum Ca2+ ATPase (SERCA) inhibitors. Consistent with elevated somatostatin inhibitory tone, we observed significantly reduced glucagon secretion and α-cell Ca2+ oscillations in TALK-1 KO islets, and found that blockade of α-cell somatostatin signaling with a somatostatin receptor 2 (SSTR2) antagonist restored glucagon secretion in TALK-1 KO islets. Conclusions These data indicate that TALK-1 reduces δ-cell cytosolic Ca2+ elevations and somatostatin release by limiting δ-cell CICR, modulating the intraislet paracrine signaling mechanisms that control glucagon secretion., Highlights • The two-pore domain K+ channel TALK-1 is expressed in islet and gastric δ-cells. • TALK-1 channels set δ-cell ER Ca2+ stores, regulating CICR and somatostatin release. • TALK-1 control of δ-cell function tunes α-cell Ca2+ handling and glucagon secretion. • Glucose enhances δ-cell Ca2+ signals and coordinates δ-cell function with β-cells.

Cells of the pancreatic islets produce several molecules including insulin (beta cells), glucagon (alpha cells), somatostatin (delta cells), pancreatic polypeptide (PP cells), ghrelin (epsilon cells), serotonin (enterochromaffin cells), gastrin (G cells) and small granules of unknown content secreted by the P/D1 cells. Secretion mechanism of some of these molecules is still poorly understood. However, Cathepsin L is shown to regulate insulin exocytosis in beta cells and activate the trypsinogen produced by the pancreatic serous acini cells into trypsin. The structure of the propeptide region of Cathepsin L is homologous to Cytotoxic T-lymphocyte antigen-2 alpha (CTLA-2 alpha) which is also shown to exhibit selective inhibitory activities against Cathepsin L. It was thought that if CTLA-2 alpha was expressed in the pancreas; then, it would be an important regulator of protease activation and insulin secretion. The purpose of this study was, therefore, to examine by immunohistochemistry the cellular localization and distribution pattern of CTLA-2 alpha in the pancreas. Results showed that strong immunoreactivity was specifically detected in the pancreatic islets (endocrine pancreas) but not in the exocrine pancreas and pancreatic stroma. Immunostaining was further performed to investigate more on localization of Cathepsin L in the pancreas. Strong immunoreactivity for Cathepsin L was detected in the pancreatic islets, serous cells and the pancreas duct system. These findings suggest that CTLA-2 alpha may be involved in the proteolytic processing and secretion of insulin through regulation of Cathepsin L and that the regulated inhibition of Cathepsin L may have therapeutic potential for type 1 diabetes.