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.

A regulatory path associated with X-linked intellectual disability and epilepsy links KDM5C to the polyalanine expansions in ARX.

Intellectual disability (ID) and epilepsy often occur together and have a dramatic impact on the development and quality of life of the affected children. Polyalanine (polyA)-expansion-encoding mutations of aristaless-related homeobox (ARX) cause a spectrum of X-linked ID (XLID) diseases and chronic epilepsy, including infantile spasms. We show that lysine-specific demethylase 5C (KDM5C), a gene known to be mutated in XLID-affected children and involved in chromatin remodeling, is directly regulated by ARX through the binding in a conserved noncoding element. We have studied altered ARX carrying various polyA elongations in individuals with XLID and/or epilepsy. The changes in polyA repeats cause hypomorphic ARX alterations, which exhibit a decreased trans-activity and reduced, but not abolished, binding to the KDM5C regulatory region. The altered functioning of the mutants tested is likely to correlate with the severity of XLID and/or epilepsy. By quantitative RT-PCR, we observed a dramatic Kdm5c mRNA downregulation in murine Arx-knockout embryonic and neural stem cells. Such Kdm5c mRNA diminution led to a severe decrease in the KDM5C content during in vitro neuronal differentiation, which inversely correlated with an increase in H3K4me3 signal. We established that ARX polyA alterations damage the regulation of KDM5C expression, and we propose a potential ARX-dependent path acting via chromatin remodeling.

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.

Cell-based treatments for diabetes.

In Type 1 diabetes mellitus the insulin-secreting beta-cells in pancreatic islets of Langerhans are selectively destroyed by autoimmune assault. Because diabetes is caused by the loss of a single cell type it is amenable to treatment by cell replacement therapy. Advances in islet transplantation procedures have demonstrated that people with Type 1 diabetes can be cured by human islet transplantation, but the severely limited availability of donor islets has restricted the widespread application of this approach, and driven the search for substitute transplant tissues. Recent experimental studies suggest that three separate sources of tissue show therapeutic potential--xenografts from other species, tissue stem cells and embryonic stem cells. Of these, xenografts are closest to clinical application but there are still major obstacles to be overcome. Insulin-expressing cells have been derived from a number of different stem cell populations but embryonic stem cells offer the major advantage of being able, in principle, to provide the vast numbers of cells required for transplantation therapy.

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.

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.

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.

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.

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.

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.

The homeodomain-containing transcription factors Arx and Pax4 control enteroendocrine subtype specification in mice.

Intestinal hormones are key regulators of digestion and energy homeostasis secreted by rare enteroendocrine cells. These cells produce over ten different hormones including GLP-1 and GIP peptides known to promote insulin secretion. To date, the molecular mechanisms controlling the specification of the various enteroendocrine subtypes from multipotent Neurog3(+) endocrine progenitor cells, as well as their number, remain largely unknown. In contrast, in the embryonic pancreas, the opposite activities of Arx and Pax4 homeodomain transcription factors promote islet progenitor cells towards the different endocrine cell fates. In this study, we thus investigated the role of Arx and Pax4 in enteroendocrine subtype specification. The small intestine and colon of Arx- and Pax4-deficient mice were analyzed using histological, molecular, and lineage tracing approaches. We show that Arx is expressed in endocrine progenitors (Neurog3(+)) and in early differentiating (ChromograninA(-)) GLP-1-, GIP-, CCK-, Sct- Gastrin- and Ghrelin-producing cells. We noted a dramatic reduction or a complete loss of all these enteroendocrine cell types in Arx mutants. Serotonin- and Somatostatin-secreting cells do not express Arx and, accordingly, the differentiation of Serotonin cells was not affected in Arx mutants. However, the number of Somatostatin-expressing D-cells is increased as Arx-deficient progenitor cells are redirected to the D-cell lineage. In Pax4-deficient mice, the differentiation of Serotonin and Somatostatin cells is impaired, as well as of GIP and Gastrin cells. In contrast, the number of GLP-1 producing L-cells is increased concomitantly with an upregulation of Arx. Thus, while Arx and Pax4 are necessary for the development of L- and D-cells respectively, they conversely restrict D- and L-cells fates suggesting antagonistic functions in D/L cell allocation. In conclusion, these finding demonstrate that, downstream of Neurog3, the specification of a subset of enteroendocrine subtypes relies on both Arx and Pax4, while others depend only on Arx or Pax4.