Gfi1 Loss Protects against Two Models of Induced Diabetes.

Background: Although several approaches have revealed much about individual factors that regulate pancreatic development, we have yet to fully understand their complicated interplay during pancreas morphogenesis. Gfi1 is transcription factor specifically expressed in pancreatic acinar cells, whose role in pancreas cells fate identity and specification is still elusive. Methods: In order to gain further insight into the function of this factor in the pancreas, we generated animals deficient for Gfi1 specifically in the pancreas. Gfi1 conditional knockout animals were phenotypically characterized by immunohistochemistry, RT-qPCR, and RNA scope. To assess the role of Gfi1 in the pathogenesis of diabetes, we challenged Gfi1-deficient mice with two models of induced hyperglycemia: long-term high-fat/high-sugar feeding and streptozotocin injections. Results: Interestingly, mutant mice did not show any obvious deleterious phenotype. However, in depth analyses demonstrated a significant decrease in pancreatic amylase expression, leading to a diminution in intestinal carbohydrates processing and thus glucose absorption. In fact, Gfi1-deficient mice were found resistant to diet-induced hyperglycemia, appearing normoglycemic even after long-term high-fat/high-sugar diet. Another feature observed in mutant acinar cells was the misexpression of ghrelin, a hormone previously suggested to exhibit anti-apoptotic effects on ß-cells in vitro. Impressively, Gfi1 mutant mice were found to be resistant to the cytotoxic and diabetogenic effects of high-dose streptozotocin administrations, displaying a negligible loss of ß-cells and an imperturbable normoglycemia. Conclusions: Together, these results demonstrate that Gfi1 could turn to be extremely valuable for the development of new therapies and could thus open new research avenues in the context of diabetes research.

Single-cell RNA-seq with spike-in cells enables accurate quantification of cell-specific drug effects in pancreatic islets.

BACKGROUND: Single-cell RNA-seq (scRNA-seq) is emerging as a powerful tool to dissect cell-specific effects of drug treatment in complex tissues. This application requires high levels of precision, robustness, and quantitative accuracy-beyond those achievable with existing methods for mainly qualitative single-cell analysis. Here, we establish the use of standardized reference cells as spike-in controls for accurate and robust dissection of single-cell drug responses. RESULTS: We find that contamination by cell-free RNA can constitute up to 20% of reads in human primary tissue samples, and we show that the ensuing biases can be removed effectively using a novel bioinformatics algorithm. Applying our method to both human and mouse pancreatic islets treated ex vivo, we obtain an accurate and quantitative assessment of cell-specific drug effects on the transcriptome. We observe that FOXO inhibition induces dedifferentiation of both alpha and beta cells, while artemether treatment upregulates insulin and other beta cell marker genes in a subset of alpha cells. In beta cells, dedifferentiation and insulin repression upon artemether treatment occurs predominantly in mouse but not in human samples. CONCLUSIONS: This new method for quantitative, error-correcting, scRNA-seq data normalization using spike-in reference cells helps clarify complex cell-specific effects of pharmacological perturbations with single-cell resolution and high quantitative accuracy.

Role of ghrelin in pancreatic development and function.

Ghrelin is a gastric peptide with anabolic functions. It acutely stimulates growth hormone (GH) secretion from the anterior pituitary glands and modulates hypothalamic circuits that control food intake and energy expenditure. Besides its central activity, ghrelin is also involved in the regulation of pancreatic development and physiology. Particularly, several studies highlighted the ability of ghrelin to sustain ß-cell viability and proliferation. Furthermore, ghrelin seems to exert inhibitory effects on pancreatic acinar and endocrine secretory functions. Due to its pleiotropic activity on energy metabolism, ghrelin has become a topic of great interest for experimental research focused on type II diabetes and obesity. The aim of this review is to illustrate the complex and not fully understood interplay between ghrelin, pancreas and glucose homeostasis.

Neurog3 misexpression unravels mouse pancreatic ductal cell plasticity.

In the context of type 1 diabetes research and the development of insulin-producing ß-cell replacement strategies, whether pancreatic ductal cells retain their developmental capability to adopt an endocrine cell identity remains debated, most likely due to the diversity of models employed to induce pancreatic regeneration. In this work, rather than injuring the pancreas, we developed a mouse model allowing the inducible misexpression of the proendocrine gene Neurog3 in ductal cells in vivo. These animals developed a progressive islet hypertrophy attributed to a proportional increase in all endocrine cell populations. Lineage tracing experiments indicated a continuous neo-generation of endocrine cells exhibiting a ductal ontogeny. Interestingly, the resulting supplementary ß-like cells were found to be functional. Based on these findings, we suggest that ductal cells could represent a renewable source of new ß-like cells and that strategies aiming at controlling the expression of Neurog3, or of its molecular targets/co-factors, may pave new avenues for the improved treatments of diabetes.

Ectopic expression of Pax4 in pancreatic δ cells results in ß-like cell neogenesis.

The recent demonstration that pancreatic α cells can be continuously regenerated and converted into ß-like cells upon ectopic expression of Pax4 opened new avenues of research in the endocrine cell differentiation and diabetes fields. To determine whether such plasticity was also shared by δ cells, we generated and characterized transgenic animals that express Pax4 specifically in somatostatin-expressing cells. We demonstrate that the ectopic expression of Pax4 in δ cells is sufficient to induce their conversion into functional ß-like cells. Importantly, this conversion induces compensatory mechanisms involving the reactivation of endocrine developmental processes that result in dramatic ß-like cell hyperplasia. Importantly, these ß-like cells are functional and can partly reverse the consequences of chemically induced diabetes.

GABA signaling stimulates α-cell-mediated ß-like cell neogenesis.

Diabetes is a chronic and progressing disease, the number of patients increasing exponentially, especially in industrialized countries. Regenerating lost insulin-producing cells would represent a promising therapeutic alternative for most diabetic patients. To this end, using the mouse as a model, we reported that GABA, a food supplement, could induce insulin-producing beta-like cell neogenesis offering an attractive and innovative approach for diabetes therapeutics.

ß-Cell Replacement Strategies: The Increasing Need for a "ß-Cell Dogma".

Type 1 diabetes is an auto-immune disease resulting in the loss of pancreatic ß-cells and, consequently, in chronic hyperglycemia. Insulin supplementation allows diabetic patients to control their glycaemia quite efficiently, but treated patients still display an overall shortened life expectancy and an altered quality of life as compared to their healthy counterparts. In this context and due to the ever increasing number of diabetics, establishing alternative therapies has become a crucial research goal. Most current efforts therefore aim at generating fully functional insulin-secreting ß-like cells using multiple approaches. In this review, we screened the literature published since 2011 and inventoried the selected markers used to characterize insulin-secreting cells generated by in vitro differentiation of stem/precursor cells or by means of in vivo transdifferentiation. By listing these features, we noted important discrepancies when comparing the different approaches for the initial characterization of insulin-producing cells as true ß-cells. Considering the recent advances achieved in this field of research, the necessity to establish strict guidelines has become a subject of crucial importance, especially should one contemplate the next step, which is the transplantation of in vitro or ex vivo generated insulin-secreting cells in type 1 diabetic patients.

Long-Term GABA Administration Induces Alpha Cell-Mediated Beta-like Cell Neogenesis.

The recent discovery that genetically modified α cells can regenerate and convert into ß-like cells in vivo holds great promise for diabetes research. However, to eventually translate these findings to human, it is crucial to discover compounds with similar activities. Herein, we report the identification of GABA as an inducer of α-to-ß-like cell conversion in vivo. This conversion induces α cell replacement mechanisms through the mobilization of duct-lining precursor cells that adopt an α cell identity prior to being converted into ß-like cells, solely upon sustained GABA exposure. Importantly, these neo-generated ß-like cells are functional and can repeatedly reverse chemically induced diabetes in vivo. Similarly, the treatment of transplanted human islets with GABA results in a loss of α cells and a concomitant increase in ß-like cell counts, suggestive of α-to-ß-like cell conversion processes also in humans. This newly discovered GABA-induced α cell-mediated ß-like cell neogenesis could therefore represent an unprecedented hope toward improved therapies for diabetes.

Artemisinins Target GABAA Receptor Signaling and Impair α Cell Identity.

Type 1 diabetes is characterized by the destruction of pancreatic ß cells, and generating new insulin-producing cells from other cell types is a major aim of regenerative medicine. One promising approach is transdifferentiation of developmentally related pancreatic cell types, including glucagon-producing α cells. In a genetic model, loss of the master regulatory transcription factor Arx is sufficient to induce the conversion of α cells to functional ß-like cells. Here, we identify artemisinins as small molecules that functionally repress Arx by causing its translocation to the cytoplasm. We show that the protein gephyrin is the mammalian target of these antimalarial drugs and that the mechanism of action of these molecules depends on the enhancement of GABAA receptor signaling. Our results in zebrafish, rodents, and primary human pancreatic islets identify gephyrin as a druggable target for the regeneration of pancreatic ß cell mass from α cells.

Pax4 acts as a key player in pancreas development and plasticity.

The embryonic development of the pancreas is orchestrated by a complex and coordinated transcription factor network. Neurogenin3 (Neurog3) initiates the endocrine program by activating the expression of additional transcription factors driving survival, proliferation, maturation and lineage allocation of endocrine precursors. Among the direct targets of Neurog3, Pax4 appears as one of the key regulators of ß-cell specification. Indeed, mice lacking Pax4 die a few days postpartum, as they develop severe hyperglycemia due to the absence of mature pancreatic ß-cells. Pax4 also directly regulates the expression of Arx, a gene that plays a crucial role in α-cell specification. Comparative analysis of Pax4 and Arx mutants, as well as Arx/Pax4 double mutants, showed that islet subtype destiny is mainly directed by cross-repression of the Pax4 and Arx factors. Importantly, the ectopic expression of Pax4 in α-cells was found sufficient to induce their neogenesis and conversion into ß-like cells, not only during development but also in adult rodents. Therefore, differentiated endocrine α-cells can be considered as a putative source for insulin-producing ß-like cells. These findings have clearly widened our understanding regarding pancreatic development, but they also open new research avenues in the context of diabetes research.

From pancreas morphogenesis to ß-cell regeneration.

Type 1 diabetes is a metabolic disease resulting in the selective loss of pancreatic insulin-producing ß-cells and affecting millions of people worldwide. The side effects of diabetes are varied and include cardiovascular, neuropathologic, and kidney diseases. Despite the most recent advances in diabetes care, patients suffering from type 1 diabetes still display a shortened life expectancy compared to their healthy counterparts. In an effort to improve ß-cell-replacement therapies, numerous approaches are currently being pursued, most of these aiming at finding ways to differentiate stem/progenitor cells into ß-like cells by mimicking embryonic development. Unfortunately, these efforts have hitherto not allowed the generation of fully functional ß-cells. This chapter summarizes recent findings, allowing a better insight into the molecular mechanisms underlying the genesis of ß-cells during the course of pancreatic morphogenesis. Furthermore, a focus is made on new research avenues concerning the conversion of pre-existing pancreatic cells into ß-like cells, such approaches holding great promise for the development of type 1 diabetes therapies.

The inactivation of Arx in pancreatic α-cells triggers their neogenesis and conversion into functional ß-like cells.

Recently, it was demonstrated that pancreatic new-born glucagon-producing cells can regenerate and convert into insulin-producing ß-like cells through the ectopic expression of a single gene, Pax4. Here, combining conditional loss-of-function and lineage tracing approaches, we show that the selective inhibition of the Arx gene in α-cells is sufficient to promote the conversion of adult α-cells into ß-like cells at any age. Interestingly, this conversion induces the continuous mobilization of duct-lining precursor cells to adopt an endocrine cell fate, the glucagon(+) cells thereby generated being subsequently converted into ß-like cells upon Arx inhibition. Of interest, through the generation and analysis of Arx and Pax4 conditional double-mutants, we provide evidence that Pax4 is dispensable for these regeneration processes, indicating that Arx represents the main trigger of α-cell-mediated ß-like cell neogenesis. Importantly, the loss of Arx in α-cells is sufficient to regenerate a functional ß-cell mass and thereby reverse diabetes following toxin-induced ß-cell depletion. Our data therefore suggest that strategies aiming at inhibiting the expression of Arx, or its molecular targets/co-factors, may pave new avenues for the treatment of diabetes.

[Reprogramming pancreatic cells to ß cells]. / Reprogrammation des cellules pancréatiques en cellules ß

Type 1 diabetes (T1DM) is a common metabolic disorder affecting an ever-increasing number of patients worldwide. T1DM is caused by the selective destruction of pancreatic insulin-producing ß-cells by the immune system. Such loss results in chronic hyperglycemia and could induce a number of cardio-vascular complications. Despite the classical insulin-based therapy, compared to healthy people, patients with T1DM display a shortened life expectancy due to the treatment's inability to strictly regulate glycemic levels. An alternative therapy involves pancreatic islet transplantation but the shortage of donors and the required immuno-suppressive treatments limit the widespread use of this approach. Therefore, the search of new approaches to generate functional ß-cells is of growing interest. In this review, we describe several novel strategies aiming at the conversion of diverse pancreatic cells into ß-cells, such as acinar, ductal, and endocrine cells. Clearly, such promising results could open new research avenues in the context of type 1 diabetes research.

Adult duct-lining cells can reprogram into ß-like cells able to counter repeated cycles of toxin-induced diabetes.

It was recently demonstrated that embryonic glucagon-producing cells in the pancreas can regenerate and convert into insulin-producing ß-like cells through the constitutive/ectopic expression of the Pax4 gene. However, whether α cells in adult mice display the same plasticity is unknown. Similarly, the mechanisms underlying such reprogramming remain unclear. We now demonstrate that the misexpression of Pax4 in glucagon(+) cells age-independently induces their conversion into ß-like cells and their glucagon shortage-mediated replacement, resulting in islet hypertrophy and in an unexpected islet neogenesis. Combining several lineage-tracing approaches, we show that, upon Pax4-mediated α-to-ß-like cell conversion, pancreatic duct-lining precursor cells are continuously mobilized, re-express the developmental gene Ngn3, and successively adopt a glucagon(+) and a ß-like cell identity through a mechanism involving the reawakening of the epithelial-to-mesenchymal transition. Importantly, these processes can repeatedly regenerate the whole ß cell mass and thereby reverse several rounds of toxin-induced diabetes, providing perspectives to design therapeutic regenerative strategies.

From pancreatic islet formation to beta-cell regeneration.

Diabetes mellitus represents a major healthcare burden and, due to the increasing prevalence of type I diabetes and the complications arising from current treatments, other alternative therapies must be found. Type I diabetes arises as a result of a cell-mediated autoimmune destruction of insulin producing pancreatic ß-cells. Thus, a cell replacement therapy would be appropriate, using either in vitro or in vivo cell differentiation/reprogramming from different cell sources. Increasing our understanding of the molecular mechanisms controlling endocrine cell specification during pancreas morphogenesis and gaining further insight into the complex transcriptional network and signaling pathways governing ß-cell development should facilitate efforts to achieve this ultimate goal, that is to regenerate insulin-producing ß-cells. This review will therefore describe briefly the genetic program underlying mouse pancreas development and present new insights regarding ß-cell regeneration.

Confluence switch signaling regulates ECM composition and the plasmin proteolytic cascade in keratinocytes.

In culture, cell confluence generates signals that commit actively growing keratinocytes to exit the cell cycle and differentiate to form a stratified epithelium. Using a comparative proteomic approach, we studied this 'confluence switch' and identified a new pathway triggered by cell confluence that regulates basement membrane (BM) protein composition by suppressing the uPA-uPAR-plasmin pathway. Indeed, confluence triggers adherens junction maturation and enhances TGF-ß and activin A activity, resulting in increased deposition of PAI-1 and perlecan in the BM. Extracellular matrix (ECM)-accumulated PAI-1 suppresses the uPA-uPAR-plasmin pathway and further enhances perlecan deposition by inhibiting its plasmin-dependent proteolysis. We show that perlecan deposition in the ECM strengthens cell adhesion, inhibits keratinocyte motility and promotes additional accumulation of PAI-1 in the ECM at confluence. In agreement, during wound-healing, perlecan concentrates at the wound-margin, where BM matures to stabilize keratinocyte adhesion. Our results demonstrate that confluence-dependent signaling orchestrates not only growth inhibition and differentiation, but also controls ECM proteolysis and BM formation. These data suggest that uncontrolled integration of confluence-dependent signaling, might favor skin disorders, including tumorigenesis, not only by promoting cell hyperproliferation, but also by altering protease activity and deposition of ECM components.