Matters arising: Insufficient evidence that pancreatic ß cells are derived from adult ductal Neurog3-expressing progenitors.

Understanding the origin of pancreatic ß cells has profound implications for regenerative therapies in diabetes. For over a century, it was widely held that adult pancreatic duct cells act as endocrine progenitors, but lineage-tracing experiments challenged this dogma. Gribben et al. recently used two existing lineage-tracing models and single-cell RNA sequencing to conclude that adult pancreatic ducts contain endocrine progenitors that differentiate to insulin-expressing ß cells at a physiologically important rate. We now offer an alternative interpretation of these experiments. Our data indicate that the two Cre lines that were used directly label adult islet somatostatin-producing ∂ cells, which precludes their use to assess whether ß cells originate from duct cells. Furthermore, many labeled ∂ cells, which have an elongated neuron-like shape, were likely misclassified as ß cells because insulin-somatostatin coimmunolocalizations were not used. We conclude that most evidence so far indicates that endocrine and exocrine lineage borders are rarely crossed in the adult pancreas.

Islet cell replacement and transplantation immunology in a mouse strain with inducible diabetes.

Improved models of experimental diabetes are needed to develop cell therapies for diabetes. Here, we introduce the B6 RIP-DTR mouse, a model of experimental diabetes in fully immunocompetent animals. These inbred mice harbor the H2b major histocompatibility complex (MHC), selectively express high affinity human diphtheria toxin receptor (DTR) in islet ß-cells, and are homozygous for the Ptprca (CD45.1) allele rather than wild-type Ptprcb (CD45.2). 100% of B6 RIP-DTR mice rapidly became diabetic after a single dose of diphtheria toxin, and this was reversed indefinitely after transplantation with islets from congenic C57BL/6 mice. By contrast, MHC-mismatched islets were rapidly rejected, and this allotransplant response was readily monitored via blood glucose and graft histology. In peripheral blood of B6 RIP-DTR with mixed hematopoietic chimerism, CD45.2 BALB/c donor blood immune cells were readily distinguished from host CD45.1 cells by flow cytometry. Reliable diabetes induction and other properties in B6 RIP-DTR mice provide an important new tool to advance transplant-based studies of islet replacement and immunomodulation to treat diabetes.

Generation of human islet cell type-specific identity genesets.

Generation of surrogate cells with stable functional identities is crucial for developing cell-based therapies. Efforts to produce insulin-secreting replacement cells to treat diabetes require reliable tools to assess islet cellular identity. Here, we conduct a thorough single-cell transcriptomics meta-analysis to identify robustly expressed markers used to build genesets describing the identity of human α-, ß-, γ- and δ-cells. These genesets define islet cellular identities better than previously published genesets. We show their efficacy to outline cell identity changes and unravel some of their underlying genetic mechanisms, whether during embryonic pancreas development or in experimental setups aiming at developing glucose-responsive insulin-secreting cells, such as pluripotent stem-cell differentiation or in adult islet cell reprogramming protocols. These islet cell type-specific genesets represent valuable tools that accurately benchmark gain and loss in islet cell identity traits.

Adult pancreatic islet endocrine cells emerge as fetal hormone-expressing cells.

The precise developmental dynamics of the pancreatic islet endocrine cell types, and their interrelation, are unknown. Some authors claim the persistence of islet cell differentiation from precursor cells after birth ("neogenesis"). Here, using four conditional cell lineage tracing ("pulse-and-chase") murine models, we describe the natural history of pancreatic islet cells, once they express a hormone gene, until late in life. Concerning the contribution of early-appearing embryonic hormone-expressing cells to the formation of islets, we report that adult islet cells emerge from embryonic hormone-expressing cells arising at different time points during development, without any evidence of postnatal neogenesis. We observe specific patterns of hormone gene activation and switching during islet morphogenesis, revealing that, within each cell type, cells have heterogeneous developmental trajectories. This likely applies to most maturating cells in the body, and explains the observed phenotypic variability within differentiated cell types. Such knowledge should help devising novel regenerative therapies.

Stage-specific transcriptomic changes in pancreatic α-cells after massive ß-cell loss.

BACKGROUND: Loss of pancreatic insulin-secreting ß-cells due to metabolic or autoimmune damage leads to the development of diabetes. The discovery that α-cells can be efficiently reprogrammed into insulin-secreting cells in mice and humans has opened promising avenues for innovative diabetes therapies. ß-cell loss triggers spontaneous reprogramming of only 1-2% of α-cells, limiting the extent of regeneration. Most α-cells are refractory to conversion and their global transcriptomic response to severe ß-cell loss as well as the mechanisms opposing their reprogramming into insulin producers are largely unknown. Here, we performed RNA-seq on FAC-sorted α-cells to characterize their global transcriptional responses at different time points after massive ß-cell ablation. RESULTS: Our results show that α-cells undergo stage-specific transcriptional changes 5- and 15-days post-diphtheria toxin (DT)-mediated ß-cell ablation. At 5 days, α-cells transiently upregulate various genes associated with interferon signaling and proliferation, including Interferon Induced Protein with Tetratricopeptide Repeats 3 (Ifit3). Subsequently, at 15 days post ß-cell ablation, α-cells undergo a transient downregulation of genes from several pathways including Insulin receptor, mTOR and MET signaling. CONCLUSIONS: The results presented here pinpoint novel markers discriminating α-cells at different stages after acute ß-cell loss, and highlight additional signaling pathways that are modulated in α-cells in this context.

Pancreatic Ppy-expressing γ-cells display mixed phenotypic traits and the adaptive plasticity to engage insulin production.

The cellular identity of pancreatic polypeptide (Ppy)-expressing γ-cells, one of the rarest pancreatic islet cell-type, remains elusive. Within islets, glucagon and somatostatin, released respectively from α- and δ-cells, modulate the secretion of insulin by ß-cells. Dysregulation of insulin production raises blood glucose levels, leading to diabetes onset. Here, we present the genetic signature of human and mouse γ-cells. Using different approaches, we identified a set of genes and pathways defining their functional identity. We found that the γ-cell population is heterogeneous, with subsets of cells producing another hormone in addition to Ppy. These bihormonal cells share identity markers typical of the other islet cell-types. In mice, Ppy gene inactivation or conditional γ-cell ablation did not alter glycemia nor body weight. Interestingly, upon ß-cell injury induction, γ-cells exhibited gene expression changes and some of them engaged insulin production, like α- and δ-cells. In conclusion, we provide a comprehensive characterization of γ-cells and highlight their plasticity and therapeutic potential.

KATP channel blockers control glucagon secretion by distinct mechanisms: A direct stimulation of α-cells involving a [Ca2+]c rise and an indirect inhibition mediated by somatostatin.

OBJECTIVE: Glucagon is secreted by pancreatic α-cells in response to hypoglycemia and its hyperglycemic effect helps to restore normal blood glucose. Insulin and somatostatin (SST) secretions from ß- and δ-cells, respectively, are stimulated by glucose by mechanisms involving an inhibition of their ATP-sensitive K+ (KATP) channels, leading to an increase in [Ca2+]c that triggers exocytosis. Drugs that close KATP channels, such as sulfonylureas, are used to stimulate insulin release in type 2 diabetic patients. α-cells also express KATP channels. However, the mechanisms by which sulfonylureas control glucagon secretion are still largely debated and were addressed in the present study. In particular, we studied the effects of KATP channel blockers on α-cell [Ca2+]c and glucagon secretion in the presence of a low (1 mM) or a high (15 mM) glucose concentration and evaluated the role of SST in these effects. METHODS: Using a transgenic mouse model expressing the Ca2+-sensitive fluorescent protein, GCaMP6f, specifically in α-cells, we measured [Ca2+]c in α-cells either dispersed or within whole islets (by confocal microscopy). By measuring [Ca2+]c in α-cells within islets and glucagon secretion using the same perifusion protocols, we tested whether glucagon secretion correlated with changes in [Ca2+]c in response to sulfonylureas. We studied the role of SST in the effects of sulfonylureas using multiple approaches including genetic ablation of SST, or application of SST-14 and SST receptor antagonists. RESULTS: Application of the sulfonylureas, tolbutamide, or gliclazide, to a medium containing 1 mM or 15 mM glucose increased [Ca2+]c in α-cells by a direct effect as in ß-cells. At low glucose, sulfonylureas inhibited glucagon secretion of islets despite the rise in α-cell [Ca2+]c that they triggered. This glucagonostatic effect was indirect and attributed to SST because, in the islets of SST-knockout mice, sulfonylureas induced a stimulation of glucagon secretion which correlated with an increase in α-cell [Ca2+]c. Experiments with exogenous SST-14 and SST receptor antagonists indicated that the glucagonostatic effect of sulfonylureas mainly resulted from an inhibition of the efficacy of cytosolic Ca2+ on exocytosis. Although SST-14 was also able to inhibit glucagon secretion by decreasing α-cell [Ca2+]c, no decrease in [Ca2+]c occurred during sulfonylurea application because it was largely counterbalanced by the direct stimulatory effect of these drugs on α-cell [Ca2+]c. At high glucose, i.e., in conditions where glucagon release was already low, sulfonylureas stimulated glucagon secretion because their direct stimulatory effect on α-cells exceeded the indirect effect by SST. Our results also indicated that, unexpectedly, SST-14 poorly decreased the efficacy of Ca2+ on exocytosis in ß-cells. CONCLUSIONS: Sulfonylureas exert two opposite actions on α-cells: a direct stimulation as in ß-cells and an indirect inhibition by SST. This suggests that any alteration of SST paracrine influence, as described in diabetes, will modify the effect of sulfonylureas on glucagon release. In addition, we suggest that δ-cells inhibit α-cells more efficiently than ß-cells.

Ultrafast pulse shaping modulates perceived visual brightness in living animals.

Vision is usually assumed to be sensitive to the light intensity and spectrum but not to its spectral phase. However, experiments performed on retinal proteins in solution showed that the first step of vision consists in an ultrafast photoisomerization that can be coherently controlled by shaping the phase of femtosecond laser pulses, especially in the multiphoton interaction regime. The link between these experiments in solution and the biological process allowing vision was not demonstrated. Here, we measure the electric signals fired from the retina of living mice upon femtosecond multipulse and single-pulse light stimulation. Our results show that the electrophysiological signaling is sensitive to the manipulation of the light excitation on a femtosecond time scale. The mechanism relies on multiple interactions with the light pulses close to the conical intersection, like pump-dump (photoisomerization interruption) and pump-repump (reverse isomerization) processes. This interpretation is supported both experimentally and by dynamics simulations.

SGLT2 is not expressed in pancreatic α- and ß-cells, and its inhibition does not directly affect glucagon and insulin secretion in rodents and humans.

OBJECTIVE: Sodium-glucose cotransporter 2 (SGLT2) inhibitors (SGLT2i), or gliflozins, are anti-diabetic drugs that lower glycemia by promoting glucosuria, but they also stimulate endogenous glucose and ketone body production. The likely causes of these metabolic responses are increased blood glucagon levels, and decreased blood insulin levels, but the mechanisms involved are hotly debated. This study verified whether or not SGLT2i affect glucagon and insulin secretion by a direct action on islet cells in three species, using multiple approaches. METHODS: We tested the in vivo effects of two selective SGLT2i (dapagliflozin, empagliflozin) and a SGLT1/2i (sotagliflozin) on various biological parameters (glucosuria, glycemia, glucagonemia, insulinemia) in mice. mRNA expression of SGLT2 and other glucose transporters was assessed in rat, mouse, and human FACS-purified α- and ß-cells, and by analysis of two human islet cell transcriptomic datasets. Immunodetection of SGLT2 in pancreatic tissues was performed with a validated antibody. The effects of dapagliflozin, empagliflozin, and sotagliflozin on glucagon and insulin secretion were assessed using isolated rat, mouse and human islets and the in situ perfused mouse pancreas. Finally, we tested the long-term effect of SGLT2i on glucagon gene expression. RESULTS: SGLT2 inhibition in mice increased the plasma glucagon/insulin ratio in the fasted state, an effect correlated with a decline in glycemia. Gene expression analyses and immunodetections showed no SGLT2 mRNA or protein expression in rodent and human islet cells, but moderate SGLT1 mRNA expression in human α-cells. However, functional experiments on rat, mouse, and human (29 donors) islets and the in situ perfused mouse pancreas did not identify any direct effect of dapagliflozin, empagliflozin or sotagliflozin on glucagon and insulin secretion. SGLT2i did not affect glucagon gene expression in rat and human islets. CONCLUSIONS: The data indicate that the SGLT2i-induced increase of the plasma glucagon/insulin ratio in vivo does not result from a direct action of the gliflozins on islet cells.

Combined inhibition of menin-MLL interaction and TGF-ß signaling induces replication of human pancreatic beta cells.

Both type 1 and type 2 diabetes are associated with hyperglycemia and loss of functional beta cell mass. Inducing proliferation of preexisting beta cells is an approach to increase the numbers of beta cells. In this study, we examined a panel of selected small molecules for their proliferation-inducing effects on human pancreatic beta cells. Our results demonstrated that a small molecule inhibitor of the menin-MLL interaction (MI-2) and small molecule inhibitors of TGF-ß signaling (SB431542, LY2157299, or LY364947) synergistically increased ex vivo replication of human beta cells. We showed that this increased proliferation did not affect insulin production, as a pivotal indication of beta cell function. We further provided evidence which suggested that menin-MLL and TGF-ß inhibition cooperated through downregulation of cell cycle inhibitors CDKN1A, CDKN1B, and CDKN2C. Our findings might provide a new option for extending the pharmacological repertoire for induction of beta cell proliferation as a potential therapeutic approach for diabetes.

Tissue repair brakes: A common paradigm in the biology of regeneration.

To date, most attention on tissue regeneration has focused on the exploration of positive cues promoting or allowing the engagement of natural cellular restoration upon injury. In contrast, the signals fostering cell identity maintenance in the vertebrate body have been poorly investigated; yet they are crucial, for their counteraction could become a powerful method to induce and modulate regeneration. Here we review the mechanisms inhibiting pro-regenerative spontaneous adaptive cell responses in different model organisms and organs. The pharmacological or genetic/epigenetic modulation of such regenerative brakes could release a dormant but innate adaptive competence of certain cell types and therefore boost tissue regeneration in different situations.

Cell Heterogeneity and Paracrine Interactions in Human Islet Function: A Perspective Focused in ß-Cell Regeneration Strategies.

The ß-cell regeneration field has shown a strong knowledge boost in the last 10 years. Pluripotent stem cell differentiation and direct reprogramming from other adult cell types are becoming more tangible long-term diabetes therapies. Newly generated ß-like-cells consistently show hallmarks of native ß-cells and can restore normoglycemia in diabetic mice in virtually all recent studies. Nonetheless, these cells still show important compromises in insulin secretion, cell metabolism, electrical activity, and overall survival, perhaps due to a lack of signal integration from other islet cells. Mounting data suggest that diabetes is not only a ß-cell disease, as the other islet cell types also contribute to its physiopathology. Here, we present an update on the most recent studies of islet cell heterogeneity and paracrine interactions in the context of restoring an integrated islet function to improve ß-cell replacement therapies.

Diabetes relief in mice by glucose-sensing insulin-secreting human α-cells.

Cell-identity switches, in which terminally differentiated cells are converted into different cell types when stressed, represent a widespread regenerative strategy in animals, yet they are poorly documented in mammals. In mice, some glucagon-producing pancreatic α-cells and somatostatin-producing δ-cells become insulin-expressing cells after the ablation of insulin-secreting ß-cells, thus promoting diabetes recovery. Whether human islets also display this plasticity, especially in diabetic conditions, remains unknown. Here we show that islet non-ß-cells, namely α-cells and pancreatic polypeptide (PPY)-producing γ-cells, obtained from deceased non-diabetic or diabetic human donors, can be lineage-traced and reprogrammed by the transcription factors PDX1 and MAFA to produce and secrete insulin in response to glucose. When transplanted into diabetic mice, converted human α-cells reverse diabetes and continue to produce insulin even after six months. Notably, insulin-producing α-cells maintain expression of α-cell markers, as seen by deep transcriptomic and proteomic characterization. These observations provide conceptual evidence and a molecular framework for a mechanistic understanding of in situ cell plasticity as a treatment for diabetes and other degenerative diseases.

A Map of Human Type 1 Diabetes Progression by Imaging Mass Cytometry.

Type 1 diabetes (T1D) results from the autoimmune destruction of insulin-producing ß cells. A comprehensive picture of the changes during T1D development is lacking due to limited sample availability, inability to sample longitudinally, and the paucity of technologies enabling comprehensive tissue profiling. Here, we analyzed 1,581 islets from 12 human donors, including eight with T1D, using imaging mass cytometry (IMC). IMC enabled simultaneous measurement of 35 biomarkers with single-cell and spatial resolution. We performed pseudotime analysis of islets through T1D progression from snapshot data to reconstruct the evolution of ß cell loss and insulitis. Our analyses revealed that ß cell destruction is preceded by a ß cell marker loss and by recruitment of cytotoxic and helper T cells. The approaches described herein demonstrate the value of IMC for improving our understanding of T1D pathogenesis, and our data lay the foundation for hypothesis generation and follow-on experiments.

Pancreatic islet-autonomous insulin and smoothened-mediated signalling modulate identity changes of glucagon+ α-cells.

The mechanisms that restrict regeneration and maintain cell identity following injury are poorly characterized in higher vertebrates. Following ß-cell loss, 1-2% of the glucagon-producing α-cells spontaneously engage in insulin production in mice. Here we explore the mechanisms inhibiting α-cell plasticity. We show that adaptive α-cell identity changes are constrained by intra-islet insulin- and Smoothened-mediated signalling, among others. The combination of ß-cell loss or insulin-signalling inhibition, with Smoothened inactivation in α- or δ-cells, stimulates insulin production in more α-cells. These findings suggest that the removal of constitutive 'brake signals' is crucial to neutralize the refractoriness to adaptive cell-fate changes. It appears that the maintenance of cell identity is an active process mediated by repressive signals, which are released by neighbouring cells and curb an intrinsic trend of differentiated cells to change.

ß Cell-Specific Deletion of the IL-1 Receptor Antagonist Impairs ß Cell Proliferation and Insulin Secretion.

Interleukin-1 receptor antagonist (IL-1Ra) is elevated in the circulation during obesity and type 2 diabetes (T2D) but is decreased in islets from patients with T2D. The protective role of local IL-1Ra was investigated in pancreatic islet ß cell (ßIL-1Ra)-specific versus myeloid-cell (myeloIL-1Ra)-specific IL-1Ra knockout (KO) mice. Deletion of IL-1Ra in ß cells, but not in myeloid cells, resulted in diminished islet IL-1Ra expression. Myeloid cells were not the main source of circulating IL-1Ra in obesity. ßIL-1Ra KO mice had impaired insulin secretion, reduced ß cell proliferation, and decreased expression of islet proliferation genes, along with impaired glucose tolerance. The key cell-cycle regulator E2F1 partly reversed IL-1ß-mediated inhibition of potassium channel Kir6.2 expression and rescued impaired insulin secretion in IL-1Ra knockout islets. Our findings provide evidence for the importance of ß cell-derived IL-1Ra for the local defense of ß cells to maintain normal function and proliferation.

Kinetics of functional beta cell mass decay in a diphtheria toxin receptor mouse model of diabetes.

Functional beta cell mass is an essential biomarker for the diagnosis and staging of diabetes. It has however proven technically challenging to study this parameter during diabetes progression. Here we have detailed the kinetics of the rapid decline in functional beta cell mass in the RIP-DTR mouse, a model of hyperglycemia resulting from diphtheria toxin induced beta cell ablation. A novel combination of imaging modalities was employed to study the pattern of beta cell destruction. Optical projection tomography of the pancreas and longitudinal in vivo confocal microscopy of islets transplanted into the anterior chamber of the eye allowed to investigate kinetics and tomographic location of beta cell mass decay in individual islets as well as at the entire islet population level. The correlation between beta cell mass and function was determined by complementary in vivo and ex vivo characterizations, demonstrating that beta cell function and glucose tolerance were impaired within the first two days following treatment when more than 50% of beta cell mass was remaining. Our results illustrate the importance of acquiring quantitative functional and morphological parameters to assess the functional status of the endocrine pancreas.

Insulin and Glucagon: Partners for Life.

In August 2016, several leaders in glucagon biology gathered for the European Association for the Study of Diabetes Hagedorn Workshop in Oxford, England. A key point of discussion focused on the need for basal insulin to allow for the therapeutic benefit of glucagon blockade in the treatment of diabetes. Among the most enlightening experimental results presented were findings from studies in which glucagon receptor-deficient mice were administered streptozotocin to destroy pancreatic ß cells or had undergone diphtheria toxin-induced ß cell ablation. This article summarizes key features of the discussion as a consensus was reached. Agents that antagonize glucagon may be of great benefit for the treatment of diabetes; however, sufficient levels of basal insulin are required for their therapeutic efficacy.