Chromatin accessibility differences between alpha, beta, and delta cells identifies common and cell type-specific enhancers.

BACKGROUND: High throughput sequencing has enabled the interrogation of the transcriptomic landscape of glucagon-secreting alpha cells, insulin-secreting beta cells, and somatostatin-secreting delta cells. These approaches have furthered our understanding of expression patterns that define healthy or diseased islet cell types and helped explicate some of the intricacies between major islet cell crosstalk and glucose regulation. All three endocrine cell types derive from a common pancreatic progenitor, yet alpha and beta cells have partially opposing functions, and delta cells modulate and control insulin and glucagon release. While gene expression signatures that define and maintain cellular identity have been widely explored, the underlying epigenetic components are incompletely characterized and understood. However, chromatin accessibility and remodeling is a dynamic attribute that plays a critical role to determine and maintain cellular identity. RESULTS: Here, we compare and contrast the chromatin landscape between mouse alpha, beta, and delta cells using ATAC-Seq to evaluate the significant differences in chromatin accessibility. The similarities and differences in chromatin accessibility between these related islet endocrine cells help define their fate in support of their distinct functional roles. We identify patterns that suggest that both alpha and delta cells are poised, but repressed, from becoming beta-like. We also identify patterns in differentially enriched chromatin that have transcription factor motifs preferentially associated with different regions of the genome. Finally, we not only confirm and visualize previously discovered common endocrine- and cell specific- enhancer regions across differentially enriched chromatin, but identify novel regions as well. We compiled our chromatin accessibility data in a freely accessible database of common endocrine- and cell specific-enhancer regions that can be navigated with minimal bioinformatics expertise. CONCLUSIONS: Both alpha and delta cells appear poised, but repressed, from becoming beta cells in murine pancreatic islets. These data broadly support earlier findings on the plasticity in identity of non-beta cells under certain circumstances. Furthermore, differential chromatin accessibility shows preferentially enriched distal-intergenic regions in beta cells, when compared to either alpha or delta cells.

Physiology labs during a pandemic: What did we learn?

This article captures a collective reflection on the successes and challenges we experienced when teaching physiology laboratories online during the COVID-19 pandemic. Physiology instructors from six institutions discussed their own efforts to redesign meaningful physiology laboratories that could be taught remotely, as the nation scrambled to respond to the sudden shift out of the classroom. Despite the complexity of this task, clear themes emerged as our courses transitioned to an online format in spring 2020 and were solidified in the fall of 2020. This article reflects on the history, features, benefits, and challenges of current laboratory teaching when applying a scientific teaching approach to facilitate the redesign process. We believe online networks like ours can facilitate information sharing, promote innovations, and provide support for instructors. The insights we gained through this collaboration will influence our thinking about the future of the physiology lab, whether online or in person.

Virgin ß-Cells at the Neogenic Niche Proliferate Normally and Mature Slowly.

Proliferation of pancreatic ß-cells has long been known to reach its peak in the neonatal stages and decline during adulthood. However, ß-cell proliferation has been studied under the assumption that all ß-cells constitute a single, homogenous population. It is unknown whether a subpopulation of ß-cells retains the capacity to proliferate at a higher rate and thus contributes disproportionately to the maintenance of mature ß-cell mass in adults. We therefore assessed the proliferative capacity and turnover potential of virgin ß-cells, a novel population of immature ß-cells found at the islet periphery. We demonstrate that virgin ß-cells can proliferate but do so at rates similar to those of mature ß-cells from the same islet under normal and challenged conditions. Virgin ß-cell proliferation rates also conform to the age-dependent decline previously reported for ß-cells at large. We further show that virgin ß-cells represent a long-lived, stable subpopulation of ß-cells with low turnover into mature ß-cells under healthy conditions. Our observations indicate that virgin ß-cells at the islet periphery can divide but do not contribute disproportionately to the maintenance of adult ß-cell mass.

Genetic deletion of Urocortin 3 does not prevent functional maturation of beta cells.

There is great interest in generating functionally mature beta cells from stem cells, as loss of functional beta cell mass contributes to the pathophysiology of diabetes. Identifying markers of beta cell maturity is therefore very helpful for distinguishing stem cells that have been successfully differentiated into fully mature beta cells from stem cells that did not. Urocortin 3 (UCN3) is a peptide hormone whose expression is associated with the acquisition of functional maturity in beta cells. The onset of its expression occurs after other beta cell maturity markers are already expressed and its loss marks the beginning of beta cell dedifferentiation. Its expression pattern is therefore tightly correlated with beta cell maturity. While this makes UCN3 an excellent marker of beta cell maturity, it is not established whether UCN3 is required for beta cell maturation. Here, we compared gene expression and function of beta cells from Ucn3-null mice relative to WT mice to determine whether beta cells are functionally mature in the absence of UCN3. Our results show that genetic deletion of Ucn3 does not cause a loss of beta cell maturity or an increase in beta cell dedifferentiation. Furthermore, virgin beta cells, first identified as insulin-expressing, UCN3-negative beta cells, can still be detected at the islet periphery in Ucn3-null mice. Beta cells from Ucn3-null mice also exhibit normal calcium response when exposed to high glucose. Collectively, these observations indicate that UCN3 is an excellent mature beta cell marker that is nevertheless not necessary for beta cell maturation.

Author Correction: CRISPR/Cas9 microinjection in oocytes disables pancreas development in sheep.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Mosaicism diminishes the value of pre-implantation embryo biopsies for detecting CRISPR/Cas9 induced mutations in sheep.

The production of knock-out (KO) livestock models is both expensive and time consuming due to their long gestational interval and low number of offspring. One alternative to increase efficiency is performing a genetic screening to select pre-implantation embryos that have incorporated the desired mutation. Here we report the use of sheep embryo biopsies for detecting CRISPR/Cas9-induced mutations targeting the gene PDX1 prior to embryo transfer. PDX1 is a critical gene for pancreas development and the target gene required for the creation of pancreatogenesis-disabled sheep. We evaluated the viability of biopsied embryos in vitro and in vivo, and we determined the mutation efficiency using PCR combined with gel electrophoresis and digital droplet PCR (ddPCR). Next, we determined the presence of mosaicism in ~ 50% of the recovered fetuses employing a clonal sequencing methodology. While the use of biopsies did not compromise embryo viability, the presence of mosaicism diminished the diagnostic value of the technique. If mosaicism could be overcome, pre-implantation embryo biopsies for mutation screening represents a powerful approach that will streamline the creation of KO animals.

The Difference δ-Cells Make in Glucose Control.

The role of beta and α-cells to glucose control are established, but the physiological role of δ-cells is poorly understood. Delta-cells are ideally positioned within pancreatic islets to modulate insulin and glucagon secretion at their source. We review the evidence for a negative feedback loop between delta and ß-cells that determines the blood glucose set point and suggest that local δ-cell-mediated feedback stabilizes glycemic control.

Evidence for a Neogenic Niche at the Periphery of Pancreatic Islets.

We recently discovered a novel subset of beta cells that resemble immature beta cells during pancreas development. We named these "virgin" beta cells as they do not stem from existing mature beta cells. Virgin beta cells are found exclusively at the islet periphery in areas that we therefore designated as the "neogenic niche." As beta cells are our only source of insulin, their loss leads to diabetes. Islets also contain glucagon-producing alpha cells and somatostatin-producing delta cells, that are important for glucose homeostasis and form a mantle surrounding the beta cell core. This 3D architecture is important and determines access to blood flow and innervation. We propose that the distinctive islet architecture may also play an important, but hitherto unappreciated role in generation of new endocrine cells, including beta cells. We discuss several predictions to further test the contribution of the neogenic niche to beta cell regeneration.

Artemether Does Not Turn α Cells into ß Cells.

Pancreatic α cells retain considerable plasticity and can, under the right circumstances, transdifferentiate into functionally mature ß cells. In search of a targetable mechanistic basis, a recent paper suggested that the widely used anti-malaria drug artemether suppresses the α cell transcription factor Arx to promote transdifferentiation into ß cells. However, key initial experiments in this paper were carried out in islet cell lines, and most subsequent validation experiments implied transdifferentiation without direct demonstration of α to ß cell conversion. Indeed, we find no evidence that artemether promotes transdifferentiation of primary α cells into ß cells. Moreover, artemether reduces Ins2 expression in primary ß cells >100-fold, suppresses glucose uptake, and abrogates ß cell calcium responses and insulin secretion in response to glucose. Our observations suggest that artemether induces general islet endocrine cell dedifferentiation and call into question the utility of artemisinins to promote α to ß cell transdifferentiation in treating diabetes.

CRISPR/Cas9 microinjection in oocytes disables pancreas development in sheep.

One of the ultimate goals of regenerative medicine is the generation of patient-specific organs from pluripotent stem cells (PSCs). Sheep are potential hosts for growing human organs through the technique of blastocyst complementation. We report here the creation of pancreatogenesis-disabled sheep by oocyte microinjection of CRISPR/Cas9 targeting PDX1, a critical gene for pancreas development. We compared the efficiency of target mutations after microinjecting the CRISPR/Cas9 system in metaphase II (MII) oocytes and zygote stage embryos. MII oocyte microinjection reduced lysis, improved blastocyst rate, increased the number of targeted bi-allelic mutations, and resulted in similar degree of mosaicism when compared to zygote microinjection. While the use of a single sgRNA was efficient at inducing mutated fetuses, the lack of complete gene inactivation resulted in animals with an intact pancreas. When using a dual sgRNA system, we achieved complete PDX1 disruption. This PDX1-/- fetus lacked a pancreas and provides the basis for the production of gene-edited sheep as a host for interspecies organ generation. In the future, combining gene editing with CRISPR/Cas9 and PSCs complementation could result in a powerful approach for human organ generation.

Identification of STS-1 as a novel ShcA-binding protein.

ShcA is a cytoplasmic signaling protein that supports signal transduction by receptor protein-tyrosine kinases by providing auxiliary tyrosine phosphorylation sites that engage additional signaling proteins. The principal binding partner for tyrosine phosphorylation sites on ShcA is Grb2. In the current study, we have used phosphotyrosine-containing peptides to isolate and identify STS-1 as a novel ShcA-binding protein. Our results further show that the interaction between STS-1 and ShcA is regulated in response to EGF receptor activation.

Virgin Beta Cells Persist throughout Life at a Neogenic Niche within Pancreatic Islets.

Postnatal maintenance or regeneration of pancreatic beta cells is considered to occur exclusively via the replication of existing beta cells, but clinically meaningful restoration of human beta cell mass by proliferation has never been achieved. We discovered a population of immature beta cells that is present throughout life and forms from non-beta precursors at a specialized micro-environment or "neogenic niche" at the islet periphery. These cells express insulin, but lack other key beta cell markers, and are transcriptionally immature, incapable of sensing glucose, and unable to support calcium influx. They constitute an intermediate stage in the transdifferentiation of alpha cells to cells that are functionally indistinguishable from conventional beta cells. We thus identified a lifelong source of new beta cells at a specialized site within healthy islets. By comparing co-existing immature and mature beta cells within healthy islets, we stand to learn how to mature insulin-expressing cells into functional beta cells.

Comprehensive alpha, beta and delta cell transcriptomes reveal that ghrelin selectively activates delta cells and promotes somatostatin release from pancreatic islets.

OBJECTIVE: Complex local crosstalk amongst endocrine cells within the islet ensures tight coordination of their endocrine output. This is illustrated by the recent demonstration that the negative feedback control by delta cells within pancreatic islets determines the homeostatic set-point for plasma glucose during mouse postnatal development. However, the close association of islet endocrine cells that facilitates paracrine crosstalk also complicates the distinction between effects mediated directly on beta cells from indirect effects mediated via local intermediates, such as somatostatin from delta cells. METHODS: To resolve this problem, we generated reporter mice that allow collection of pure pancreatic delta cells along with alpha and beta cells from the same islets and generated comprehensive transcriptomes for each islet endocrine cell type. These transcriptomes afford an unparalleled view of the receptors expressed by delta, alpha and beta cells, and allow the prediction of which signal targets which endocrine cell type with great accuracy. RESULTS: From these transcriptomes, we discovered that the ghrelin receptor is expressed exclusively by delta cells within the islet, which was confirmed by fluorescent in situ hybridization and qPCR. Indeed, ghrelin increases intracellular calcium in delta cells in intact mouse islets, measured by GCaMP6 and robustly potentiates glucose-stimulated somatostatin secretion on mouse and human islets in both static and perfusion assays. In contrast, des-acyl-ghrelin at the same dose had no effect on somatostatin secretion and did not block the actions of ghrelin. CONCLUSIONS: These results offer a straightforward explanation for the well-known insulinostatic actions of ghrelin. Rather than engaging beta cells directly, ghrelin engages delta cells to promote local inhibitory feedback that attenuates insulin release. These findings illustrate the power of our approach to resolve some of the long-standing conundrums with regard to the rich feedback that occurs within the islet that is integral to islet physiology and therefore highly relevant to diabetes.

Urocortin3 mediates somatostatin-dependent negative feedback control of insulin secretion.

The peptide hormone urocortin3 (Ucn3) is abundantly expressed by mature beta cells, yet its physiological role is unknown. Here we demonstrate that Ucn3 is stored and co-released with insulin and potentiates glucose-stimulated somatostatin secretion via cognate receptors on delta cells. Further, we found that islets lacking endogenous Ucn3 have fewer delta cells, reduced somatostatin content, impaired somatostatin secretion, and exaggerated insulin release, and that these defects are rectified by treatment with synthetic Ucn3 in vitro. Our observations indicate that the paracrine actions of Ucn3 activate a negative feedback loop that promotes somatostatin release to ensure the timely reduction of insulin secretion upon normalization of plasma glucose. Moreover, Ucn3 is markedly depleted from beta cells in mouse and macaque models of diabetes and in human diabetic islets. This suggests that Ucn3 is a key contributor to stable glycemic control, whose reduction during diabetes aggravates glycemic volatility and contributes to the pathophysiology of this disease.

Role of transcription factors in the transdifferentiation of pancreatic islet cells.

The α and ß cells act in concert to maintain blood glucose. The α cells release glucagon in response to low levels of glucose to stimulate glycogenolysis in the liver. In contrast, ß cells release insulin in response to elevated levels of glucose to stimulate peripheral glucose disposal. Despite these opposing roles in glucose homeostasis, α and ß cells are derived from a common progenitor and share many proteins important for glucose sensing and hormone secretion. Results from recent work have underlined these similarities between the two cell types by revealing that ß-to-α as well as α-to-ß transdifferentiation can take place under certain experimental circumstances. These exciting findings highlight unexpected plasticity of adult islets and offer hope of novel therapeutic paths to replenish ß cells in diabetes. In this review, we focus on the transcription factor networks that establish and maintain pancreatic endocrine cell identity and how they may be perturbed to facilitate transdifferentiation.

Data-driven synthesis of proteolysis-resistant peptide hormones.

Peptide hormones are key physiological regulators, and many would make terrific drugs; however, the therapeutic use of peptides is limited by poor metabolism including rapid proteolysis. To develop novel proteolysis-resistant peptide hormone analogs, we utilize a strategy that relies on data from simple mass spectrometry experiments to guide the chemical synthesis of proteolysis-resistant analogs (i.e., data-driven synthesis). Application of this strategy to oxyntomodulin (OXM), a peptide hormone that stimulates insulin secretion from islets and lowers blood glucose in vivo, defined the OXM cleavage site in serum, and this information was used to synthesize a proteolysis-resistant OXM analog (prOXM). prOXM and OXM have similar activity in binding and glucose stimulated-insulin secretion assays. Furthermore, prOXM is also active in vivo. prOXM reduces basal glucose levels and improves glucose tolerance in mice. The discovery of prOXM suggests that proteolysis-resistant variants of other important peptide hormones can also be found using this strategy to increase the number of candidate therapeutic peptides.

CRFR1 activation protects against cytokine-induced ß-cell death.

During the development of diabetes ß-cells are exposed to elevated concentrations of proinflammatory cytokines, TNFα and IL1ß, which in vitro induce ß-cell death. The class B G-protein-coupled receptors (GPCRs): corticotropin-releasing factor receptor 1 (CRFR1) and CRFR2 are expressed in pancreatic islets. As downstream signaling by other class B GPCRs can protect against cytokine-induced ß-cell apoptosis, we evaluated the protective potential of CRFR activation in ß-cells in a pro-inflammatory setting. CRFR1/CRFR2 ligands activated AKT and CRFR1 signaling and reduced apoptosis in human islets. In rat and mouse insulin-secreting cell lines (INS-1 and MIN6), CRFR1 agonists upregulated insulin receptor substrate 2 (IRS2) expression, increased AKT activation, counteracted the cytokine-mediated decrease in BAD phosphorylation, and inhibited apoptosis. The anti-apoptotic signaling was dependent on prolonged exposure to corticotropin-releasing factor family peptides and followed PKA-mediated IRS2 upregulation. This indicates that CRFR signaling counteracts proinflammatory cytokine-mediated apoptotic pathways through upregulation of survival signaling in ß-cells. Interestingly, CRFR signaling also counteracted basal apoptosis in both cultured INS-1 cells and intact human islets.

Maturation of stem cell-derived beta-cells guided by the expression of urocortin 3.

Type 1 diabetes (T1D) is a devastating disease precipitated by an autoimmune response directed at the insulin-producing beta-cells of the pancreas for which no cure exists. Stem cell-derived beta-cells show great promise for a cure as they have the potential to supply unlimited numbers of cells that could be derived from a patient's own cells, thus eliminating the need for immunosuppression. Current in vitro protocols for the differentiation of stem cell-derived beta-cells can successfully generate pancreatic endoderm cells. In diabetic rodents, such cells can differentiate further along the beta-cell lineage until they are eventually capable of restoring normoglycemia. While these observations demonstrate that stem cell-derived pancreatic endoderm has the potential to differentiate into mature, glucose-responsive beta-cells, the signals that direct differentiation and maturation from pancreatic endoderm onwards remain poorly understood. In this review, we analyze the sequence of events that culminates in the formation of beta-cells during embryonic development. and summarize how current protocols to generate beta-cells have sought to capitalize on this ontogenic template. We place particular emphasis on the current challenges and opportunities which occur in the later stages of beta-cell differentiation and maturation of transplantable stem cell-derived beta-cells. Another focus is on the question how the use of recently identified maturation markers such as urocortin 3 can be instrumental in guiding these efforts.

The transcriptional landscape of mouse beta cells compared to human beta cells reveals notable species differences in long non-coding RNA and protein-coding gene expression.

BACKGROUND: Insulin producing beta cell and glucagon producing alpha cells are colocalized in pancreatic islets in an arrangement that facilitates the coordinated release of the two principal hormones that regulate glucose homeostasis and prevent both hypoglycemia and diabetes. However, this intricate organization has also complicated the determination of the cellular source(s) of the expression of genes that are detected in the islet. This reflects a significant gap in our understanding of mouse islet physiology, which reduces the effectiveness by which mice model human islet disease. RESULTS: To overcome this challenge, we generated a bitransgenic reporter mouse that faithfully labels all beta and alpha cells in mouse islets to enable FACS-based purification and the generation of comprehensive transcriptomes of both populations. This facilitates systematic comparison across thousands of genes between the two major endocrine cell types of the islets of Langerhans whose principal hormones are of cardinal importance for glucose homeostasis. Our data leveraged against similar data for human beta cells reveal a core common beta cell transcriptome of 9900+ genes. Against the backdrop of overall similar beta cell transcriptomes, we describe marked differences in the repertoire of receptors and long non-coding RNAs between mouse and human beta cells. CONCLUSIONS: The comprehensive mouse alpha and beta cell transcriptomes complemented by the comparison of the global (dis)similarities between mouse and human beta cells represent invaluable resources to boost the accuracy by which rodent models offer guidance in finding cures for human diabetes.

Urocortin 3 marks mature human primary and embryonic stem cell-derived pancreatic alpha and beta cells.

The peptide hormone Urocortin 3 (Ucn 3) is abundantly and exclusively expressed in mouse pancreatic beta cells where it regulates insulin secretion. Here we demonstrate that Ucn 3 first appears at embryonic day (E) 17.5 and, from approximately postnatal day (p) 7 and onwards throughout adult life, becomes a unifying and exclusive feature of mouse beta cells. These observations identify Ucn 3 as a potential beta cell maturation marker. To determine whether Ucn 3 is similarly restricted to beta cells in humans, we conducted comprehensive immunohistochemistry and gene expression experiments on macaque and human pancreas and sorted primary human islet cells. This revealed that Ucn 3 is not restricted to the beta cell lineage in primates, but is also expressed in alpha cells. To substantiate these findings, we analyzed human embryonic stem cell (hESC)-derived pancreatic endoderm that differentiates into mature endocrine cells upon engraftment in mice. Ucn 3 expression in hESC-derived grafts increased robustly upon differentiation into mature endocrine cells and localized to both alpha and beta cells. Collectively, these observations confirm that Ucn 3 is expressed in adult beta cells in both mouse and human and appears late in beta cell differentiation. Expression of Pdx1, Nkx6.1 and PC1/3 in hESC-derived Ucn 3(+) beta cells supports this. However, the expression of Ucn 3 in primary and hESC-derived alpha cells demonstrates that human Ucn 3 is not exclusive to the beta cell lineage but is a general marker for both the alpha and beta cell lineages. Ucn 3(+) hESC-derived alpha cells do not express Nkx6.1, Pdx1 or PC1/3 in agreement with the presence of a separate population of Ucn 3(+) alpha cells. Our study highlights important species differences in Ucn 3 expression, which have implications for its utility as a marker to identify mature beta cells in (re)programming strategies.