Directed differentiation of pancreatic δ cells from human pluripotent stem cells.

Dysfunction of pancreatic δ cells contributes to the etiology of diabetes. Despite their important role, human δ cells are scarce, limiting physiological studies and drug discovery targeting δ cells. To date, no directed δ-cell differentiation method has been established. Here, we demonstrate that fibroblast growth factor (FGF) 7 promotes pancreatic endoderm/progenitor differentiation, whereas FGF2 biases cells towards the pancreatic δ-cell lineage via FGF receptor 1. We develop a differentiation method to generate δ cells from human stem cells by combining FGF2 with FGF7, which synergistically directs pancreatic lineage differentiation and modulates the expression of transcription factors and SST activators during endoderm/endocrine precursor induction. These δ cells display mature RNA profiles and fine secretory granules, secrete somatostatin in response to various stimuli, and suppress insulin secretion from in vitro co-cultured ß cells and mouse ß cells upon transplantation. The generation of human pancreatic δ cells from stem cells in vitro would provide an unprecedented cell source for drug discovery and cell transplantation studies in diabetes.

Structural basis for delta cell paracrine regulation in pancreatic islets.

Little is known about the role of islet delta cells in regulating blood glucose homeostasis in vivo. Delta cells are important paracrine regulators of beta cell and alpha cell secretory activity, however the structural basis underlying this regulation has yet to be determined. Most delta cells are elongated and have a well-defined cell soma and a filopodia-like structure. Using in vivo optogenetics and high-speed Ca2+ imaging, we show that these filopodia are dynamic structures that contain a secretory machinery, enabling the delta cell to reach a large number of beta cells within the islet. This provides for efficient regulation of beta cell activity and is modulated by endogenous IGF-1/VEGF-A signaling. In pre-diabetes, delta cells undergo morphological changes that may be a compensation to maintain paracrine regulation of the beta cell. Our data provides an integrated picture of how delta cells can modulate beta cell activity under physiological conditions.

Signals in the pancreatic islet microenvironment influence ß-cell proliferation.

The progressive loss of pancreatic ß-cell mass that occurs in both type 1 and type 2 diabetes is a primary factor driving efforts to identify strategies for effectively increasing, enhancing or restoring ß-cell mass. While factors that seem to influence ß-cell proliferation in specific contexts have been described, reliable stimulation of human ß-cell proliferation has remained a challenge. Importantly, ß-cells exist in the context of a complex, integrated pancreatic islet microenvironment where they interact with other endocrine cells, vascular endothelial cells, extracellular matrix, neuronal projections and islet macrophages. This review highlights different components of the pancreatic microenvironment, and reviews what is known about how signaling that occurs between ß-cells and these other components influences ß-cell proliferation. Future efforts to further define the role of the pancreatic islet microenvironment on ß-cell proliferation may lead to the development of successful approaches to increase or restore ß-cell mass in diabetes.

A cullin 4B-RING E3 ligase complex fine-tunes pancreatic δ cell paracrine interactions.

Somatostatin secreted by pancreatic δ cells mediates important paracrine interactions in Langerhans islets, including maintenance of glucose metabolism through the control of reciprocal insulin and glucagon secretion. Disruption of this circuit contributes to the development of diabetes. However, the precise mechanisms that control somatostatin secretion from islets remain elusive. Here, we found that a super-complex comprising the cullin 4B-RING E3 ligase (CRL4B) and polycomb repressive complex 2 (PRC2) epigenetically regulates somatostatin secretion in islets. Constitutive ablation of CUL4B, the core component of the CRL4B-PRC2 complex, in δ cells impaired glucose tolerance and decreased insulin secretion through enhanced somatostatin release. Moreover, mechanistic studies showed that the CRL4B-PRC2 complex, under the control of the δ cell-specific transcription factor hematopoietically expressed homeobox (HHEX), determines the levels of intracellular calcium and cAMP through histone posttranslational modifications, thereby altering expression of the Cav1.2 calcium channel and adenylyl cyclase 6 (AC6) and modulating somatostatin secretion. In response to high glucose levels or urocortin 3 (UCN3) stimulation, increased expression of cullin 4B (CUL4B) and the PRC2 subunit histone-lysine N-methyltransferase EZH2 and reciprocal decreases in Cav1.2 and AC6 expression were found to regulate somatostatin secretion. Our results reveal an epigenetic regulatory mechanism of δ cell paracrine interactions in which CRL4B-PRC2 complexes, Cav1.2, and AC6 expression fine-tune somatostatin secretion and facilitate glucose homeostasis in pancreatic islets.

CFTR is involved in the regulation of glucagon secretion in human and rodent alpha cells.

Glucagon is the main counterregulatory hormone in the body. Still, the mechanism involved in the regulation of glucagon secretion from pancreatic alpha cells remains elusive. Dysregulated glucagon secretion is common in patients with Cystic Fibrosis (CF) that develop CF related diabetes (CFRD). CF is caused by a mutation in the Cl- channel Cystic fibrosis transmembrane conductance regulator (CFTR), but whether CFTR is present in human alpha cells and regulate glucagon secretion has not been investigated in detail. Here, both human and mouse alpha cells showed CFTR protein expression, whereas CFTR was absent in somatostatin secreting delta cells. CFTR-current activity induced by cAMP was measured in single alpha cells. Glucagon secretion at different glucose levels and in the presence of forskolin was increased by CFTR-inhibition in human islets, whereas depolarization-induced glucagon secretion was unaffected. CFTR is suggested to mainly regulate the membrane potential through an intrinsic alpha cell effect, as supported by a mathematical model of alpha cell electrophysiology. In conclusion, CFTR channels are present in alpha cells and act as important negative regulators of cAMP-enhanced glucagon secretion through effects on alpha cell membrane potential. Our data support that loss-of-function mutations in CFTR contributes to dysregulated glucagon secretion in CFRD.

Revisiting the immunocytochemical detection of Neurogenin 3 expression in mouse and man.

During embryonic development, endocrine cells of the pancreas are specified from multipotent progenitors. The transcription factor Neurogenin 3 (NEUROG3) is critical for this development and it has been shown that all endocrine cells of the pancreas arise from endocrine progenitors expressing NEUROG3. A thorough understanding of the role of NEUROG3 during development, directed differentiation of pluripotent stem cells and in models of cellular reprogramming, will guide future efforts directed at finding novel sources of ß-cells for cell replacement therapies. In this article, we review the expression and function of NEUROG3 in both mouse and human and present the further characterization of a monoclonal antibody directed against NEUROG3. This antibody has been previously been used for detection of both mouse and human NEUROG3. However, our results suggest that the epitope recognized by this antibody is specific to mouse NEUROG3. Thus, we have also generated a monoclonal antibody specifically recognizing human NEUROG3 and present the characterization of this antibody here. Together, these antibodies will provide useful tools for future studies of NEUROG3 expression, and the data presented in this article suggest that recently described expression patterns of NEUROG3 in human foetal and adult pancreas should be re-examined.

Asymmetrical distribution of δ and PP cells in human pancreatic islets.

The aim of this study was to evaluate the location of PP and δ cells in relation to the vascularization within human pancreatic islets. To this end, pancreas sections were analysed by immunofluorescence using antibodies against endocrine islet and endothelial cells. Staining in different islet areas corresponding to islet cells adjacent or not to peripheral or central vascular channels was quantified by computerized morphometry. As results, α, PP and δ cells were preferentially found adjacent to vessels. In contrast to α cells, which were evenly distributed between islet periphery and intraislet vascular channels, PP and δ cells had asymmetric and opposite distributions: PP staining was higher and somatostatin staining was lower in the islet periphery than in the area around intraislet vascular channels. Additionally, frequencies of PP and δ cells were negatively correlated in the islets. No difference was observed between islets from the head and the tail of the pancreas, and from type 2 diabetic and non-diabetic donors. In conclusion, the distribution of δ cells differs from that of PP cells in human islets, suggesting that vessels at the periphery and at the centre of islets drain different hormonal cocktails.

Pax6 Inactivation in the Adult Pancreas Reveals Ghrelin as Endocrine Cell Maturation Marker.

The transcription factor Pax6 is an important regulator of development and cell differentiation in various organs. Thus, Pax6 was shown to promote neural development in the cerebral cortex and spinal cord, and to control pancreatic endocrine cell genesis. However, the role of Pax6 in distinct endocrine cells of the adult pancreas has not been addressed. We report the conditional inactivation of Pax6 in insulin and glucagon producing cells of the adult mouse pancreas. In the absence of Pax6, beta- and alpha-cells lose their molecular maturation characteristics. Our findings provide strong evidence that Pax6 is responsible for the maturation of beta-, and alpha-cells, but not of delta-, and PP-cells. Moreover, lineage-tracing experiments demonstrate that Pax6-deficient beta- and alpha-cells are shunted towards ghrelin marked cells, sustaining the idea that ghrelin may represent a marker for endocrine cell maturation.

Altered Phenotype of ß-Cells and Other Pancreatic Cell Lineages in Patients With Diffuse Congenital Hyperinsulinism in Infancy Caused by Mutations in the ATP-Sensitive K-Channel.

Diffuse congenital hyperinsulinism in infancy (CHI-D) arises from mutations inactivating the KATP channel; however, the phenotype is difficult to explain from electrophysiology alone. Here we studied wider abnormalities in the ß-cell and other pancreatic lineages. Islets were disorganized in CHI-D compared with controls. PAX4 and ARX expression was decreased. A tendency toward increased NKX2.2 expression was consistent with its detection in two-thirds of CHI-D δ-cell nuclei, similar to the fetal pancreas, and implied immature δ-cell function. CHI-D δ-cells also comprised 10% of cells displaying nucleomegaly. In CHI-D, increased proliferation was most elevated in duct (5- to 11-fold) and acinar (7- to 47-fold) lineages. Increased ß-cell proliferation observed in some cases was offset by an increase in apoptosis; this is in keeping with no difference in INSULIN expression or surface area stained for insulin between CHI-D and control pancreas. However, nuclear localization of CDK6 and P27 was markedly enhanced in CHI-D ß-cells compared with cytoplasmic localization in control cells. These combined data support normal ß-cell mass in CHI-D, but with G1/S molecules positioned in favor of cell cycle progression. New molecular abnormalities in δ-cells and marked proliferative increases in other pancreatic lineages indicate CHI-D is not solely a ß-cell disorder.

Loss of Fbw7 reprograms adult pancreatic ductal cells into α, δ, and ß cells.

The adult pancreas is capable of limited regeneration after injury but has no defined stem cell population. The cell types and molecular signals that govern the production of new pancreatic tissue are not well understood. Here, we show that inactivation of the SCF-type E3 ubiquitin ligase substrate recognition component Fbw7 induces pancreatic ductal cells to reprogram into α, δ, and ß cells. Loss of Fbw7 stabilized the transcription factor Ngn3, a key regulator of endocrine cell differentiation. The induced ß cells resemble islet ß cells in morphology and histology, express genes essential for ß cell function, and release insulin after glucose challenge. Thus, loss of Fbw7 appears to reawaken an endocrine developmental differentiation program in adult pancreatic ductal cells. Our study highlights the plasticity of seemingly differentiated adult cells, identifies Fbw7 as a master regulator of cell fate decisions in the pancreas, and reveals adult pancreatic duct cells as a latent multipotent cell type.

Diabetes recovery by age-dependent conversion of pancreatic δ-cells into insulin producers.

Total or near-total loss of insulin-producing ß-cells occurs in type 1 diabetes. Restoration of insulin production in type 1 diabetes is thus a major medical challenge. We previously observed in mice in which ß-cells are completely ablated that the pancreas reconstitutes new insulin-producing cells in the absence of autoimmunity. The process involves the contribution of islet non-ß-cells; specifically, glucagon-producing α-cells begin producing insulin by a process of reprogramming (transdifferentiation) without proliferation. Here we show the influence of age on ß-cell reconstitution from heterologous islet cells after near-total ß-cell loss in mice. We found that senescence does not alter α-cell plasticity: α-cells can reprogram to produce insulin from puberty through to adulthood, and also in aged individuals, even a long time after ß-cell loss. In contrast, before puberty there is no detectable α-cell conversion, although ß-cell reconstitution after injury is more efficient, always leading to diabetes recovery. This process occurs through a newly discovered mechanism: the spontaneous en masse reprogramming of somatostatin-producing δ-cells. The juveniles display 'somatostatin-to-insulin' δ-cell conversion, involving dedifferentiation, proliferation and re-expression of islet developmental regulators. This juvenile adaptability relies, at least in part, upon the combined action of FoxO1 and downstream effectors. Restoration of insulin producing-cells from non-ß-cell origins is thus enabled throughout life via δ- or α-cell spontaneous reprogramming. A landscape with multiple intra-islet cell interconversion events is emerging, offering new perspectives for therapy.

The influence of the different morphological changes on gastric mucosa on somatostatin cell number in antrum mucosa and serum somatostatin.

The aim of our paper was to investigate the influence of the different morphological changes on gastric mucosa on somatostatin D-cell number in antral mucosa and serum Somatostatin. We analyzed according to Sydney classification to what extent the severity of gastritis affect the observed hormonal values. somatostatin D-cell number in antral mucosa and serum Somatostatin values were compared between three groups of patients; mild, moderate and severe chronic gastritis. The average number of somatostatin cell in biopsy sample of antrum mucosa was 30.41 +/- 35.38 (N = 17) in the case of middle form, 18.69 +/- 26.65 (N = 56) in moderate and in severe case of chronic gastritis 5.23 +/- 5.93 (N = 7) cells in mm2 of mucosa. The level of somatostatin in the serum of middle form gastritis were 26.43 +/- 28.76, moderate 19.95 +/- 35.93 and severe 17.88 +/- 17.66 pg/mL. In order to determine the number of somatostatin cells in antrum mucosa and serum somatostatin with present morphological changes of mucosa, it might helpful to exclude the patients with non-ulcer dyspepsia, but with the higher risk of premalignant and malignant changes.

Glucagon deficiency reduces hepatic glucose production and improves glucose tolerance in adult mice.

The major role of glucagon is to promote hepatic gluconeogenesis and glycogenolysis to raise blood glucose levels during hypoglycemic conditions. Several animal models have been established to examine the in vivo function of glucagon in the liver through attenuation of glucagon via glucagon receptor knockout animals and pharmacological interventions. To investigate the consequences of glucagon loss to hepatic glucose production and glucose homeostasis, we derived mice with a pancreas specific ablation of the alpha-cell transcription factor, Arx, resulting in a complete loss of the glucagon-producing pancreatic alpha-cell. Using this model, we found that glucagon is not required for the general health of mice but is essential for total hepatic glucose production. Our data clarifies the importance of glucagon during the regulation of fasting and postprandial glucose homeostasis.

Cellular origins of adult human islet in vitro dedifferentiation.

Cultured human islets can be dedifferentiated to duct-like structures composed mainly of cytokeratin+ and nestin+ cells. Given that these structures possess the potential to redifferentiate into islet-like structures, we sought to elucidate their specific cellular origins. Adenoviral vectors were engineered for beta-, alpha-, delta- or PP-cell-specific GFP expression. A double-stranded system was designed whereby cultures were infected with two vectors: one expressed GFP behind the cumate-inducible promoter sequence, and the other expressed the requisite transactivator behind the human insulin, glucagon, somatostatin or pancreatic polypeptide promoter. This system labels hormone+ cells in the islet in a cell-specific manner, allowing these cells to be tracked during the course of transformation from islet to duct-like structure. Post-infection, islets were cultured to induce dedifferentiation. Fluorescence microscopy demonstrated that alpha-, delta- and PP-cells contributed equally to the cytokeratin+ population, with minimal beta-cell contribution, whereas the converse was true for nestin+ cells. Complementary targeted cell ablation studies, using streptozotocin or similar adenoviral expression of the Bax (Bcl2-associated X protein) toxigene, validated these findings and suggested a redundancy between alpha-, delta- and PP-cells with respect to cytokeratin+ cell derivation. These results call into question the traditional understanding of islet cells as being terminally differentiated and provide support for the concept of adult islet morphogenetic plasticity.

Gene expression heterogeneity in human islet endocrine cells in vitro: the insulin signalling cascade.

AIMS/HYPOTHESIS: Insulin secretion is a highly regulated mechanism involving a complex insulin-dependent network of communication between alpha, beta and delta cells. However, whereas the role of insulin in beta cells has been well documented, very little is known about its role in alpha and delta cells. Having recently demonstrated heterogeneity of insulin receptor (INSR) isoform expression in these three endocrine cell types, our current study aimed to characterise the expression pattern of the multiple isoforms involved in the insulin signal transduction cascade in human alpha, beta and delta cells in vitro. MATERIALS AND METHODS: cDNA samples prepared from single human islet cells were subjected to nested PCRs. RESULTS: Of 706 cells analysed, 15% were alpha cells, 28% beta cells, 8% delta cells and 46% non-endocrine cells. Profiling of expression of the insulin signalling cascade elements showed a heterogeneity between islet cell types, although at least one member of each protein family was expressed in the three populations of endocrine cells. Thus, the mRNAs coding for INSR-B, phosphoinositide-dependent protein kinase-1 and the human homologue of v-akt murine thymoma viral oncogene homologue 1 (AKT1) could not be detected in alpha cells, but were expressed by beta and delta cells. In addition, while the insulin receptor substrates IRS1 and IRS2, phosphoinositide-3-kinase, catalytic, beta polypeptide (PIK3CB) and AKT2 were expressed with relatively low frequencies in alpha and delta cells (<17% for IRS1, IRS2, PIK3CB; <25% for AKT2), their frequencies of expression in beta cells were 50, 33, 33 and 100%, respectively. CONCLUSIONS/INTERPRETATION: Our results suggest that insulin signalling cascade elements in human alpha, beta and delta cells have distinct expression patterns.