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Reprogramming of pancreatic exocrine cells into cells resembling beta cells may provide a strategy for treating diabetes. Here we show that transient administration of epidermal growth factor and ciliary neurotrophic factor to adult mice with chronic hyperglycemia efficiently stimulates the conversion of terminally differentiated acinar cells to beta-like cells. Newly generated beta-like cells are epigenetically reprogrammed, functional and glucose responsive, and they reinstate normal glycemic control for up to 248 d. The regenerative process depends on Stat3 signaling and requires a threshold number of Neurogenin 3 (Ngn3)-expressing acinar cells. In contrast to previous work demonstrating in vivo conversion of acinar cells to beta-like cells by viral delivery of exogenous transcription factors, our approach achieves acinar-to-beta-cell reprogramming through transient cytokine exposure rather than genetic modification.

Ductal cells have been proposed as a source of adult β cell neogenesis, but this has remained controversial. By combining lineage tracing, 3D imaging, and single-cell RNA sequencing (scRNA-seq) approaches, we show that ductal cells contribute to the β cell population over time. Lineage tracing using the Neurogenin3 (Ngn3)-CreERT line identified ductal cells expressing the endocrine master transcription factor Ngn3 that were positive for the δ cell marker somatostatin and occasionally co-expressed insulin. The number of hormone-expressing ductal cells was increased in Akita+/- diabetic mice, and ngn3 heterozygosity accelerated diabetes onset. scRNA-seq of Ngn3 lineage-traced islet cells indicated that duct-derived somatostatin-expressing cells, some of which retained expression of ductal markers, gave rise to β cells. This study identified Ngn3-expressing ductal cells as a source of adult β cell neogenesis in homeostasis and diabetes, suggesting that this mechanism, in addition to β cell proliferation, maintains the adult islet β cell population.

Summary Generating an unlimited source of human insulin-producing cells is a prerequisite to advance β cell replacement therapy for diabetes. Here, we describe a 3D culture system that supports the expansion of adult human pancreatic tissue and the generation of a cell subpopulation with progenitor characteristics. These cells display high aldehyde dehydrogenase activity (ALDHhi), express pancreatic progenitors markers (PDX1, PTF1A, CPA1, and MYC), and can form new organoids in contrast to ALDHlo cells. Interestingly, gene expression profiling revealed that ALDHhi cells are closer to human fetal pancreatic tissue compared with adult pancreatic tissue. Endocrine lineage markers were detected upon in vitro differentiation. Engrafted organoids differentiated toward insulin-positive (INS+) cells, and circulating human C-peptide was detected upon glucose challenge 1 month after transplantation. Engrafted ALDHhi cells formed INS+ cells. We conclude that adult human pancreatic tissue has potential for expansion into 3D structures harboring progenitor cells with endocrine differentiation potential., Highlights • A 3D culture system can support the expansion of adult human pancreatic tissue • An ALDHhi cell subpopulation is identified in these organoids • ALDHhi cells, and not ALDHlo cells, are capable of forming new organoids • ALDHhi cells show endocrine differentiation potential, In the context of β cell replacement therapy for diabetes, de Koning and colleagues describe a 3D culture platform that supports ex vivo expansion of human pancreatic tissue as organoids. These organoids harbor a subpopulation of ALDHhi cells that display proliferative capacity and can differentiate to an endocrine fate.

A comparative analysis of mouse and human pancreatic development may reveal common mechanisms that control key steps as organ morphogenesis and cell proliferation and differentiation. More specifically, understanding beta cell development remains an issue, despite recent progress related to their generation from human embryonic and induced pluripotent stem cells. In this study, we use an integrated approach, including prospective isolation, organ culture, and characterization of intermediate stages, and report that cells from human and mouse fetal pancreas can be expanded in the long term and give rise to hollow duct-like structures in 3D cultures. The expanded cells express a combination of markers (E-cadherin, PDX1, NKX6-1, SOX9, and HNF1β) that reveals pancreatic progenitor identity. Proliferation of embryonic progenitors was stimulated by the Wnt agonist R-spondin1 (RSPO1), FGF10, and EGF. This combination of growth factors allowed maintaining human fetal pancreatic progenitors in culture for many passages, a finding not reported previously. Importantly, in the absence of EGF, proliferation was reduced, while endocrine differentiation was significantly enhanced. We conclude that modulation of EGF signaling affects in vitro expansion and differentiation of progenitors from embryonic pancreas of both mice and man.

Identifying pathways for beta cell generation is essential for cell therapy in diabetes. Here we investigate the potential of 17β-estradiol (E2) and estrogen receptor (ER) signaling for stimulating beta cell generation during embryonic development and in severely injured adult pancreas. Estradiol concentration, ER activity and number of ERα transcripts were enhanced in pancreas injured by partial duct ligation (PDL), along with nuclear localization of ERα in beta cells. PDL-induced proliferation of beta cells depended on aromatase activity. The activation of Neurogenin3 (Ngn3) gene expression and beta cell growth in PDL pancreas were impaired when ERα was turned off chemically or genetically (ERα(-/-)), while in situ delivery of E2 promoted beta cell formation. In embryonic pancreas, beta cell replication, number of Ngn3(+) progenitor cells and expression of key transcription factors of the endocrine lineage were decreased by ERα inactivation. The present study reveals that E2 and ERα signaling can drive betacell replication and formation in mouse pancreas.

Pancreas injury by partial duct ligation (PDL) activates a healing response, encompassing β-cell neogenesis and proliferation. Macrophages (MΦs) were recently shown to promote β-cell proliferation after PDL, but they remain poorly characterized. We assessed myeloid cell diversity and the factors driving myeloid cell dynamics following acute pancreas injury by PDL. In naive and sham-operated pancreas, the myeloid cell compartment consisted mainly of two distinct tissue-resident MΦ types, designated MHC-II(lo) and MHC-II(hi) MΦs, the latter being predominant. MHC-II(lo) and MHC-II(hi) pancreas MΦs differed at the molecular level, with MHC-II(lo) MΦs being more M2-activated. After PDL, there was an early surge of Ly6C(hi) monocyte infiltration in the pancreas, followed by a transient MHC-II(lo) MΦ peak and ultimately a restoration of the MHC-II(hi) MΦ-dominated steady-state equilibrium. These intricate MΦ dynamics in PDL pancreas depended on monocyte recruitment by C-C chemokine receptor 2 and macrophage-colony stimulating factor receptor as well as on macrophage-colony stimulating factor receptor-dependent local MΦ proliferation. Functionally, MHC-II(lo) MΦs were more angiogenic. We further demonstrated that, at least in C-C chemokine receptor 2-KO mice, tissue MΦs, rather than Ly6C(hi) monocyte-derived MΦs, contributed to β-cell proliferation. Together, our study fully characterizes the MΦ subsets in the pancreas and clarifies the complex dynamics of MΦs after PDL injury.

Inhibition of notch signaling is known to induce differentiation of endocrine cells in zebrafish and mouse. After performing an unbiased in vivo screen of ∼2,200 small molecules in zebrafish, we identified an inhibitor of Cdk5 (roscovitine), which potentiated the formation of β-cells along the intrapancreatic duct during concurrent inhibition of notch signaling. We confirmed and characterized the effect with a more selective Cdk5 inhibitor, (R)-DRF053, which specifically increased the number of duct-derived β-cells without affecting their proliferation. By duct-specific overexpression of the endogenous Cdk5 inhibitors Cdk5rap1 or Cdkal1 (which previously have been linked to diabetes in genome-wide association studies), as well as deleting cdk5, we validated the role of chemical Cdk5 inhibition in β-cell differentiation by genetic means. Moreover, the cdk5 mutant zebrafish displayed an increased number of β-cells independently of inhibition of notch signaling, in both the basal state and during β-cell regeneration. Importantly, the effect of Cdk5 inhibition to promote β-cell formation was conserved in mouse embryonic pancreatic explants, adult mice with pancreatic ductal ligation injury, and human induced pluripotent stem (iPS) cells. Thus, we have revealed a previously unknown role of Cdk5 as an endogenous suppressor of β-cell differentiation and thereby further highlighted its importance in diabetes.

Lgr5 marks adult stem cells in multiple adult organs and is a receptor for the Wnt-agonistic R-spondins (RSPOs). Intestinal, stomach and liver Lgr5+ stem cells grow in 3D cultures to form ever-expanding organoids, which resemble the tissues of origin. Wnt signalling is inactive and Lgr5 is not expressed under physiological conditions in the adult pancreas. However, we now report that the Wnt pathway is robustly activated upon injury by partial duct ligation (PDL), concomitant with the appearance of Lgr5 expression in regenerating pancreatic ducts. In vitro, duct fragments from mouse pancreas initiate Lgr5 expression in RSPO1-based cultures, and develop into budding cyst-like structures (organoids) that expand five-fold weekly for >40 weeks. Single isolated duct cells can also be cultured into pancreatic organoids, containing Lgr5 stem/progenitor cells that can be clonally expanded. Clonal pancreas organoids can be induced to differentiate into duct as well as endocrine cells upon transplantation, thus proving their bi-potentiality., Unlimited in vitro expansion of adult bi-potent pancreas progenitors through the Lgr5/R-spondin axis The establishment of conditions for long-term culture and expansion of adult, bi-potent pancreas progenitors may facilitate novel and tailored therapeutic approaches.

International audience; The recent discovery that genetically modified α cells can regenerate and convert into β-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-β-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 β-like cells, solely upon sustained GABA exposure. Importantly, these neo-generated β-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 β-like cell counts, suggestive of α-to-β-like cell conversion processes also in humans. This newly discovered GABA-induced α cell-mediated β-like cell neogenesis could therefore represent an unprecedented hope toward improved therapies for diabetes.

Partial pancreatic duct ligation (PDL) of mouse pancreas induces a doubling of the β-cell mass mainly through proliferation of pre-existing and newly formed β-cells. The molecular mechanism governing this process is still largely unknown. Given the inflammatory nature of PDL and inflammation-induced signaling via the signal transducer and activator of transcription 3 (STAT3), the activation and the role of STAT3 in PDL-induced β-cell proliferation were investigated. Duct ligation stimulates the expression of several cytokines that can act as ligands inducing STAT3 signaling and phosphorylation in β-cells. β-Cell cycling increased by conditional β-cell-specific Stat3 knockout and decreased by STAT3 activation through administration of interleukin-6. In addition, the level of DNA damage in β-cells of PDL pancreas increased after deletion of Stat3. These data indicate a role for STAT3 in maintaining a steady state in the β-cell, by modulating its cell cycle and protection from DNA damage.

AIMS/HYPOTHESIS: Efficient stimulation of cycling activity in cultured beta cells would allow the design of new strategies for cell therapy in diabetes. Neural crest stem cells (NCSCs) play a role in beta cell development and maturation and increase the beta cell number in co-transplants. The mechanism behind NCSC-induced beta cell proliferation and the functional capacity of the new beta cells is not known. METHODS: We developed a new in vitro co-culture system that enables the dissection of the elements that control the cellular interactions that lead to NCSC-dependent increase in islet beta cells. RESULTS: Mouse NCSCs were cultured in vitro, first in medium that stimulated their proliferation, then under conditions that supported their differentiation. When mouse islet cells were cultured together with the NCSCs, more than 35% of the beta cells showed cycle activity. This labelling index is more than tenfold higher than control islets cultured without NCSCs. Beta cells that proliferated under these culture conditions were fully glucose responsive in terms of insulin secretion. NCSCs also induced beta cell proliferation in islets isolated from 1-year-old mice, but not in dissociated islet cells isolated from human donor pancreas tissue. To stimulate beta cell proliferation, NCSCs need to be in intimate contact with the beta cells. CONCLUSIONS/INTERPRETATION: Culture of islet cells in contact with NCSCs induces highly efficient beta cell proliferation. The reported culture system is an excellent platform for further dissection of the minimal set of factors needed to drive this process and explore its potential for translation to diabetes therapy.

AIMS/HYPOTHESIS: Prolonged exposure of rat beta cells to the insulin secretagogue glibenclamide has been found to induce a sustained increase in basal insulin synthesis. This effect was calcium-dependent and localised in cells that had been degranulated by the drug. Since it was blocked by the translation inhibitor cycloheximide, we examined whether sustained exposure to glibenclamide activates translational factors by calcium-dependent signalling pathways. METHODS: Purified rat beta cells were cultured with and without glibenclamide in the presence or absence of inhibitors of calcium-dependent signalling pathways before measurement of basal and stimulated protein and insulin synthesis, and assessment of abundance of (phosphorylated) translation factors. RESULTS: A 24 h exposure to glibenclamide induced activation of four translation factors, i.e. phosphorylation of eukaryotic initiation factor (eIF) 4e binding protein 1 and ribosomal protein S6 (rpS6), and dephosphorylation of eIF-2alpha and eukaryotic elongation factor 2. The rise in phospho-rpS6 intensity was localised to a subpopulation of beta cells with low insulin content. This activation of translational factors and the associated elevation of insulin synthesis were completely blocked by the calcium channel blocker verapamil and partially blocked by the mammalian target of rapamycin (mTOR) inhibitor rapamycin, the protein kinase A (PKA) inhibitor Rp-8-Br-cAMPs and the mitogen-activated protein kinase/ extracellular signal-regulated kinase kinase (MEK) inhibitor U0126; a combination of inhibitors exhibited additive effects. CONCLUSIONS/INTERPRETATION: Prolonged exposure to glibenclamide activates protein translation in pancreatic beta cells through the calcium-regulated mTOR, PKA and MEK signalling pathways. The observed intercellular differences in translation activation are proposed as underlying mechanism for functional heterogeneity in the pancreatic beta cell population.

Transcription factor mediated lineage reprogramming of human pancreatic exocrine tissue could conceivably provide an unlimited supply of islets for transplantation in the treatment of diabetes. Exocrine tissue can be efficiently reprogrammed to islet-like cells using a cocktail of transcription factors: Pdx1, Ngn3, MafA and Pax4 in combination with growth factors. We show here that overexpression of exogenous Pax4 in combination with suppression of the endogenous transcription factor ARX considerably enhances the production of functional insulin-secreting β-like cells with concomitant suppression of α-cells. The efficiency was further increased by culture on laminin-coated plates in media containing low glucose concentrations. Immunocytochemistry revealed that reprogrammed cultures were composed of ~45% islet-like clusters comprising >80% monohormonal insulin+ cells. The resultant β-like cells expressed insulin protein levels at ~15-30% of that in adult human islets, efficiently processed proinsulin and packaged insulin into secretory granules, exhibited glucose responsive insulin secretion, and had an immediate and prolonged effect in normalising blood glucose levels upon transplantation into diabetic mice. We estimate that approximately 3 billion of these cells would have an immediate therapeutic effect following engraftment in type 1 diabetes patients and that one pancreas would provide sufficient tissue for numerous transplants.

In vitro cultured pancreatic acinar cells rapidly differentiate. Low temperature exposure prevents this process and improves the efficiency of acinar cell labelling with adenovirus vectors. This may help in tracing β-cell neogenesis from human pancreatic acinar cells., The regenerative medicine field is expanding with great successes in laboratory and preclinical settings. Pancreatic acinar cells in diabetic mice were recently converted into β-cells by treatment with ciliary neurotrophic factor (CNTF) and epidermal growth factor (EGF). This suggests that human acinar cells might become a cornerstone for diabetes cell therapy in the future, if they can also be converted into glucose-responsive insulin-producing cells. Presently, studying pancreatic acinar cell biology in vitro is limited by their high plasticity, as they rapidly lose their phenotype and spontaneously transdifferentiate to a duct-like phenotype in culture. We questioned whether human pancreatic acinar cell phenotype could be preserved in vitro by physico-chemical manipulations and whether this could be valuable in the study of β-cell neogenesis. We found that culture at low temperature (4°C) resulted in the maintenance of morphological and molecular acinar cell characteristics. Specifically, chilled acinar cells did not form the spherical clusters observed in controls (culture at 37°C), and they maintained high levels of acinar-specific transcripts and proteins. Five-day chilled acinar cells still transdifferentiated into duct-like cells upon transfer to 37°C. Moreover, adenoviral-mediated gene transfer evidenced an active Amylase promoter in the 7-day chilled acinar cells, and transduction performed in chilled conditions improved acinar cell labelling. Together, our findings indicate the maintenance of human pancreatic acinar cell phenotype at low temperature and the possibility to efficiently label acinar cells, which opens new perspectives for the study of human acinar-to-β-cell transdifferentiation.

Rodent acinar cells exhibit a remarkable plasticity as they can transdifferentiate to duct-, hepatocyte- and islet β-like cells. We evaluated whether exocrine cells from adult human pancreas can similarly respond to proendocrine stimuli. Exocrine cells from adult human pancreas were transduced directly with lentiviruses expressing activated MAPK (mitogen-activated protein kinase) and STAT3 (signal transducer and activator of transcription 3) and cultured as monolayers or as 3D structures. Expression of STAT3 and MAPK in human exocrine cells activated expression of the proendocrine factor neurogenin 3 in 50% to 80% of transduced exocrine cells. However, the number of insulin-positive cells increased only in the exocrine cells grown initially in suspension before 3D culture. Lineage tracing identified human acinar cells as the source of Ngn3- and insulin-expressing cells. Long-term engraftment into immunocompromised mice increased the efficiency of reprogramming to insulin-positive cells. Our data demonstrate that exocrine cells from human pancreas can be reprogrammed to transplantable insulin-producing cells that acquire functionality. Given the large number of exocrine cells in a donor pancreas, this approach presents a novel strategy to expand cell therapy in type 1 diabetes.

We have recently shown that conditions known to activate AMP-activated protein kinase (AMPK) in primary beta-cells can trigger their apoptosis. The present study demonstrates that this is also the case in the MIN6 beta-cell line, which was used to investigate the underlying mechanism. Sustained activation of AMPK was induced by culture with the adenosine analogue AICA-riboside or at low glucose concentrations. Both conditions induced a sequential activation of AMPK, c-Jun-N-terminal kinase (JNK) and caspase-3. The effects of AMPK on JNK activation and apoptosis were demonstrated by adenoviral expression of constitutively active AMPK, a condition which reproduced the earlier-described AMPK-dependent effects on pyruvate kinase and acetyl-coA-carboxylase. The effects of JNK activation on apoptosis were demonstrated by the observations that (i). its inhibition by dicumarol prevented caspase-3 activation and apoptosis, (ii). adenoviral expression of the JNK-interacting scaffold protein JIP-1/IB-1 increased AICA-riboside-induced JNK activation and apoptosis. In primary beta-cells, AMPK activation was also found to activate JNK, involving primarily the JNK 2 (p54) isoform. It is concluded that prolonged stimulation of AMPK can induce apoptosis of insulin-producing cells through an activation pathway that involves JNK, and subsequently, caspase-3.

Writs arc important signaling molecules involved in many normal developmental processes in the human body as well as some forms of cancer. Nineteen Wnt genes arc found in the human genome, as well as 10 Wnt receptor genes called Frizzled. Two coreceptors called LRP 5 and 6 are critical for Wnt signal transduction. The interaction of the Wnts with the receptors is regulated by two classes of extracellular Wnt or LRP binding proteins called sFRP and Dickkopf (DKK), which modulate Writ signaling. We have examined the expression of all Wnt family members both in the exocrine portion and in isolated islets of adult human pancreas. RT-PCR analysis of the 1-day cultured exocrine pellet fraction from the islet isolation procedure showed that Writ 2, 2b, 3. 4, 5a, 5b, 7a, 7b, 14, and 15 were detectable. All 10 Frizzled (Frz) receptors were expressed but only Frizzled 1. 2. 4 5, and 6 strongly. RT-PCR performed on purified human islets revealed that Wnt 2b, 3, 4, 5a, 7b, 10a, and 14 and Frz 4, 5, and 6 were the most highly expressed. DKK 1, 3, and 4 as well as sFRP 1, 4, and 5 were expressed in the exocrine fraction. sFRP 2 and 3 were detectable but only at low levels. In situ hybridization for Frz 1-7 showed that expression colocalized with the islets of Langerhans. Together the data suggest that active Wnt signaling occurs in adult pancreas and is probably important for physiological functions.

A replenishable source of insulin-producing cells has the potential to cure type 1 diabetes. Attempts to culture and expand pancreatic β-cells in vitro have resulted in their transition from insulin-producing epithelial cells to mesenchymal stromal cells (MSCs) with high proliferative capacity but devoid of any hormone production. The aim of this study was to determine whether the transcription factor Krüppel-like factor 4 (KLF4), could induce a mesenchymal-to-epithelial transition (MET) of the cultured cells. Islet-enriched pancreatic cells, allowed to dedifferentiate and expand in adherent cell culture, were transduced with an adenovirus containing KLF4 (Ad-Klf4). Cells were subsequently analysed for changes in cell morphology by light microscopy, and for the presence of epithelial and pancreatic markers by immunocytochemistry and quantitative RT/PCR. Infection with Ad-Klf4 resulted in morphological changes, down-regulation of mesenchymal markers, and re-expression of both epithelial and pancreatic cell markers including insulin and transcription factors specific to β-cells. This effect was further enhanced by culturing cells in suspension. However, the effects of Ad-KLf4 were transient and this was shown to be due to increased apoptosis in Klf4-expressing cells. Klf4 has been recently identified as a pioneer factor with the ability to modulate the structure of chromatin and enhance reprogramming/transdifferentiation. Our results show that Klf4 may have a role in the redifferentiation of expanded pancreatic cells in culture, but before this can be achieved the off-target effects that result in increased apoptosis would need to be overcome.

The presence of pancreatic duct cells in clinical islet grafts may affect long-term metabolic success. Human pancreatic duct cells express factors that may exert both protective and damaging effects on islet cells in the graft. Here, we studied the potential of commonly used immunosuppressive drugs in islet transplantation - sirolimus, tacrolimus and mycophenolate mofetil (MMF) - to influence the inflammatory and angiogenic capacity of human pancreatic duct cells in addition to their proliferation and reprogramming abilities. Our data show that the expression of specific pro-inflammatory cytokines by the human pancreatic duct cells was either unaltered or inhibited by the immunosuppressants studied, especially tacrolimus and MMF, whereas expression of chemotactic and angiogenic factors was unaffected. Although none of the immunosuppressants directly led to duct cell death, MMF prevented duct cell proliferation and sirolimus inhibited Ngn-3-mediated duct-to-(neuro)endocrine cell reprogramming. Our data indicate that the immunosuppressant tacrolimus was the least aggressive on the angiogenic, proliferative, and reprogramming potential of human pancreatic duct cells, while it was most powerful in inhibiting inflammatory cytokines, which may influence the outcome of islet transplantation. ispartof: Cell Transplantation vol:24 issue:8 pages:1585-98 ispartof: location:United States status: published

Macrophages are classically considered detrimental for pancreatic β-cell survival and function, thereby contributing to β-cell failure in both type 1 (T1D) and 2 (T2D) diabetes mellitus. In addition, adipose tissue macrophages negatively influence peripheral insulin signaling and promote obesity-induced insulin resistance in T2D. In contrast, recent data unexpectedly uncovered that macrophages are not only able to protect β cells during pancreatitis but also to orchestrate β-cell proliferation and regeneration after β-cell injury. Moreover, by altering their activation state, macrophages are able to improve insulin resistance in murine models of T2D. This review will elaborate on current insights in macrophage heterogeneity and on the evolving role of pancreas macrophages during organogenesis, tissue injury, and repair. Additional identification of macrophage subtypes and of their secreted factors might ultimately translate into novel therapeutic strategies for both T1D and T2D. Significance Diabetes mellitus is a pandemic disease, characterized by severe acute and chronic complications. Macrophages have long been considered prime suspects in the pathogenesis of both type 1 and 2 diabetes mellitus. In this concise review, current insights in macrophage heterogeneity and on the, as yet, underappreciated role of alternatively activated macrophages in insulin sensing and β-cell development/repair are reported. Further identification of macrophage subtypes and of their secreted factors might ultimately translate into novel therapeutic strategies for diabetes mellitus.

Understanding the mechanisms triggering hepatogenic differentiation of stem/progenitor cells would be useful for studying postnatal liver regeneration and development of liver cell therapies. Many evidences support the involvement of Sox9 transcription factor in liver development. Here, we investigate the possibility of liver mesenchymal stem/progenitor cells to constitutively express Sox9 by using reverse transcription-quantitative polymerase chain reaction, immunocytochemistry, and western blotting. The involvement of Sox9 in hepatogenic differentiation was assessed by following its expression at different steps of the process, evaluating the impact of its altered expression, and analyzing its expression in human liver disease specimen. Liver mesenchymal stem/progenitor cells constitutively express Sox9 at both the mRNA and protein levels. Upon hepatogenic differentiation, Sox9 expression is downregulated mainly in the maturation step after oncostatin M treatment. Induction of Sox9 expression using transforming growth factor beta is accompanied with a decrease of the quality of hepatogenic differentiation. Blunting Sox9 expression using specific ShRNA clearly alters the levels of several hepatic markers, an effect confirmed in HepG2 cells. In human liver disease specimen, Sox9 expression is enhanced at both the mRNA and protein levels compared with healthy donors. The current data demonstrate that Sox9 may play a pivotal role in hepatocyte lineage development, including adult liver mesenchymal stem/progenitor cells. Further studies on the identification of pathways regulated by or regulating Sox9 will certainly gain insight into the molecular networks controlling hepatogenic differentiation.

Pancreatic endocrine progenitors obtained from human embryonic stem cells (hESCs) represent a promising source to develop cell-based therapies for diabetes. Although endocrine pancreas progenitor cells have been isolated from mouse pancreata on the basis of Ngn3 expression, human endocrine progenitors have not been isolated yet. As substantial differences exist between human and murine pancreas biology, we investigated whether it is possible to isolate pancreatic endocrine progenitors from differentiating hESC cultures by lineage tracing of NGN3. We targeted the 3' end of NGN3 using zinc finger nuclease-mediated homologous recombination to allow selection of NGN3eGFP(+) cells without disrupting the coding sequence of the gene. Isolated NGN3eGFP(+) cells express PDX1, NKX6.1, and chromogranin A and differentiate in vivo toward insulin, glucagon, and somatostatin single hormone-expressing cells but not to ductal or exocrine pancreatic cells or other endodermal, mesodermal, or ectodermal lineages. This confirms that NGN3(+) cells represent pancreatic endocrine progenitors in humans. In addition, this hESC reporter line constitutes a unique tool that may aid in gaining insight into the developmental mechanisms underlying fate choices in human pancreas and in developing cell-based therapies. ispartof: Stem Cells Translational Medicine vol:3 issue:4 pages:489-499 ispartof: location:England status: published

Aims/hypothesis. In adult pancreatic regeneration models exocrine acini are found to transdifferentiate to duct-like complexes. This has also been associated with the formation of new endocrine islet cells. We aimed to establish an in vitro model in which this transdifferentiation process is characterised and can be modulated.¶Methods. Purified rat pancreatic acini were cultured in suspension. Differentiation was analysed by immunocytochemistry, electron microscopy, western blotting and RT-PCR.¶Results. During culture acinar cells directly transdifferentiated without dividing, the cells lost their acinar phenotype and started to express cytokeratins 20 and 7 and fetal liver kinase-1 (Flk-1) receptors for vascular endothelial growth factor. Expression of the acinar pancreatic exocrine transcription factor (PTF-1) remained and the pancreatic duodenal homeobox-containing transcription factor (PDX-1) was induced. When transdifferentiation was completed, the cells started to express protein gene product 9.5, a pan-neuroendocrine marker. By combining these features, the transdifferentiated cells show similar characteristics to precursor cells during active beta-cell neogenesis. We were able to modulate the differentiation state by addition of nicotinamide or sodium butyrate, agents which are known to stimulate endocrine differentiation in other models.¶Conclusion/interpretation. Here, we present an in vitro system in which the cellular differentiation of putative pancreatic endocrine precursor cells and their PDX-1 expression can be modulated, thereby providing a possible model for the study of beta-cell transdifferentiation. [Diabetologia (2000) 43: 907–914]

Transgenic or tumoral pancreatic islet beta cells with enhanced expression of low K(m) hexokinases (HK) exhibit a leftward shift of the normal dose-response curve for glucose-induced insulin release. Furthermore, HK catalyzes roughly 50% of total glucose phosphorylation measured in extracts from freshly isolated rodent islets, suggesting that HK participates in the process of glucose sensing in beta cells. We previously observed that HK activity represents 20% of total glucose phosphorylation in purified rat beta cell preparations and that HK is not homogenously distributed over these cells. The present study provides several arguments for the idea that HK detected in freshly isolated rat islets or islet cell preparations originates mainly from contaminating exocrine cells. First, reverse transcriptase-polymerase chain reaction using isoform-specific primers allowed detection of hexokinase I and IV mRNA in rat beta cells, whereas the messenger levels encoding the hexokinase II and III isoforms were undetectably low. However, immunoblots indicated that hexokinase I protein was 10-fold more abundant in freshly isolated islets and flow-sorted exocrine cells than in purified rat beta cell preparations. Second, comparison of HK activity in the different pancreatic cell types resulted in 15-25-fold higher values in exocrine than in endocrine cells (acinar cells: 21 +/- 3 pmol of glucose 6-phosphate formed/h/ng of DNA; duct cells: 30 +/- 8 pmol/h/ng of DNA; islet beta cells: 1.2 +/- 0.2 pmol/h/ng DNA; alpha cells: 0.9 +/- 0.4 pmol/h/ng of DNA). Since freshly purified beta cell preparations contain 3 +/- 1% exocrine cells, at least 50% of their HK activity can be accounted for by exocrine contamination. Third, after 5 days of culture of purified islet beta cells, both HK activity and the proportion of exocrine cells decreased by more than 1 order of magnitude, while the ratio of glucokinase over hexokinase activity increased more than 10-fold. Finally, preincubating the cells with 50 mmol/liter 2-deoxyglucose did not affect glucose stimulation of insulin biosynthesis and release. In conclusion, the observation that pancreatic exocrine cells are responsible for a major part of HK activity in islet cell preparations cautions against the use of HK measurements in islet extracts in the study of these enzymes in glucose sensing by pancreatic beta cells.

Experimentally induced injury is an established strategy for studying mechanisms of tissue remodeling with the final goal of developing new regenerative therapies. Under normal physiological conditions, proliferation and differentiation of progenitor cells, including even canonical stem cell−like activity, can be stimulated in tissues, such as brain and liver, that have a low cellular turnover rate (1,2). The presence of stem/progenitor cells in the pancreas could be relevant to normal homeostatic maintenance of various cell types in this organ, such as endocrine hormone−expressing cells, enzyme-secreting acinar cells, and the less secretory exocrine duct cells. Further, pancreatic stem/progenitor cells may be a possible source for replenishing cells destroyed by autoimmune disease or other stressors. We speculate that proliferative progenitors might be isolated, expanded, and differentiated in vitro to alleviate the donor scarcity in human islet transplantation and may therefore be developed as a therapy for diabetes. However, the existence and exact location of adult stem- or progenitor-like cells that can give rise to functional β-cells is highly controversial. This Perspective focuses on findings from a severe insult model (partial duct ligation [PDL]) with a long history (3). PDL received renewed attention when a 2008 study combined it with genetic reporter strategies now possible in mice to try to identify and isolate cells acting as β-cell progenitors (4). In vivo β-cell neogenesis under PDL was recently substantiated (5,6). Because the outcomes from this technique appear to vary across laboratories, we summarize and discuss some of the reported discrepancies to help identify current limitations and pitfalls of this model as well as opportunities for forward progress. The mechanisms leading to replacement of the endogenous β-cell pool have been studied under several cell ablation paradigms to test for the existence and type of cells in the adult mouse pancreas that are …

Recently, it was demonstrated that pancreatic new-born glucagon-producing cells can regenerate and convert into insulin-producing β-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 β-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 β-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 β-like cell neogenesis. Importantly, the loss of Arx in α-cells is sufficient to regenerate a functional β-cell mass and thereby reverse diabetes following toxin-induced β-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., Author Summary Type 1 diabetes is a condition that results from the loss of insulin-producing β-cells. Despite current therapies, diabetic patients are prone to vascular complications. Using the mouse as a model, we previously found that pancreatic glucagon-expressing cells can be regenerated and converted into β-like cells by the forced expression of a single gene, Pax4. Here, we generated transgenic mice allowing both the permanent labeling of α-cells and the inactivation of Arx solely in this cell subtype. Our results indicate that, upon Arx inactivation, α-cells can be continuously regenerated from duct-lining precursors and converted into β-like cells. Importantly, the additional loss of Pax4 does not impact these processes, suggesting that Arx is the main trigger of α-cell-mediated β-like cell neogenesis. Most interestingly, upon chemical induction of diabetes/β-cell loss, while control animals die or remain severely hyperglycemic, a normalization of the glycemia, a clear regeneration of the β-like cell mass, and an extended lifespan are noted in animals with the conditional inactivation of Arx. 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.

International audience; It was recently demonstrated that embryonic glucagon-producing cells in the pancreas can regenerate and convert into insulin-producing β-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 β-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-β-like cell conversion, pancreatic duct-lining precursor cells are continuously mobilized, re-express the developmental gene Ngn3, and successively adopt a glucagon(+) and a β-like cell identity through a mechanism involving the reawakening of the epithelial-to-mesenchymal transition. Importantly, these processes can repeatedly regenerate the whole β cell mass and thereby reverse several rounds of toxin-induced diabetes, providing perspectives to design therapeutic regenerative strategies.

AIMS/HYPOTHESIS: Long-term labelling of mice with halogenated thymidine analogues is an established method for quantifying the contribution of beta cell proliferation to in vivo beta cell mass expansion in (re)generation models. The method is believed to give accurate information on the accrued number of cycling beta cells over a period of time. Multiple thymidine analogue labelling is applied for evaluating the duration of postmitotic quiescence in beta cells. We hypothesise, however, that long-term labelling by thymidine analogues hampers beta cell proliferation. METHODS: Thymidine analogues were administered for 7-14 days via the i.p. route to neonatal mice, or via drinking water to young mice with normal pancreases or adult mice with injured pancreases. The proliferation of insulin-positive cells was assessed by their expression of the proliferation markers Ki67 or phosphorylated histone H3 and by their incorporation of nucleotide analogues. RESULTS: In the mouse models of beta cell proliferation investigated herein, long-term administration of thymidine analogues decreased the percentage of Ki67(+) and phosphorylated histone H3(+) beta cells as compared with administration of normal drinking water. Proliferation was restored by washout of the analogue. Labelling with one analogue decreased the subsequent incorporation of another analogue by beta cells. CONCLUSIONS/INTERPRETATION: Long-term labelling with halogenated thymidine analogues is a biased method that underestimates the proliferation and re-division potential of mouse beta cells.

Because of the lack of tissue available for islet transplantation, new sources of β-cells have been sought for the treatment of type 1 diabetes. The aim of this study was to determine whether the human exocrine-enriched fraction from the islet isolation procedure could be reprogrammed to provide additional islet tissue for transplantation. The exocrine-enriched cells rapidly dedifferentiated in culture and grew as a mesenchymal monolayer. Genetic lineage tracing confirmed that these mesenchymal cells arose, in part, through a process of epithelial-to-mesenchymal transitioning (EMT). A protocol was developed whereby transduction of these mesenchymal cells with adenoviruses containing Pdx1, Ngn3, MafA, and Pax4 generated a population of cells that were enriched in glucagon-secreting α-like cells. Transdifferentiation or reprogramming toward insulin-secreting β-cells was enhanced, however, when using unpassaged cells in combination with inhibition of EMT by inclusion of Rho-associated kinase (ROCK) and transforming growth factor-β1 inhibitors. Resultant cells were able to secrete insulin in response to glucose and on transplantation were able to normalize blood glucose levels in streptozotocin diabetic NOD/SCID mice. In conclusion, reprogramming of human exocrine-enriched tissue can be best achieved using fresh material under conditions whereby EMT is inhibited, rather than allowing the culture to expand as a mesenchymal monolayer.

Aims/hypothesisDuct cells isolated from adult human pancreas can be reprogrammed to express islet beta cell genes by adenoviral transduction of the developmental transcription factor neurogenin3 (Ngn3). In this study we aimed to fully characterize the extent of this reprogramming and intended to improve it.MethodsThe extent of the Ngn3-mediated duct-to-endocrine cell reprogramming was measured employing genome wide mRNA profiling. By modulation of the Delta-Notch signaling or addition of pancreatic endocrine transcription factors Myt1, MafA and Pdx1 we intended to improve the reprogramming.ResultsNgn3 stimulates duct cells to express a focused set of genes that are characteristic for islet endocrine cells and/or neural tissues. This neuro-endocrine shift however, is incomplete with less than 10% of full duct-to-endocrine reprogramming achieved. Transduction of exogenous Ngn3 activates endogenous Ngn3 suggesting auto-activation of this gene. Furthermore, pancreatic endocrine reprogramming of human duct cells can be moderately enhanced by inhibition of Delta-Notch signaling as well as by co-expressing the transcription factor Myt1, but not MafA and Pdx1.Conclusions/interpretationThe results provide further insight into the plasticity of adult human duct cells and suggest measurable routes to enhance Ngn3-mediated in vitro reprogramming protocols for regenerative beta cell therapy in diabetes.

The aim of the study was to identify surface bio-markers and corresponding antibody tools that can be used for the imaging and immunoisolation of the pancreatic beta cell and its progenitors. This may prove essential to obtain therapeutic grade human beta cells via stem cell differentiation. Using bioinformatics-driven data mining, we generated a gene list encoding putative plasma membrane proteins specifically expressed at distinct stages of the developing pancreas and islet beta cells. In situ hybridisation and immunohistochemistry were used to further prioritise and identify candidates. In the developing pancreas seizure related 6 homologue like (SEZ6L2), low density lipoprotein receptor-related protein 11 (LRP11), dispatched homologue 2 (Drosophila) (DISP2) and solute carrier family 30 (zinc transporter), member 8 (SLC30A8) were found to be expressed in early islet cells, whereas discoidin domain receptor tyrosine kinase 1 (DDR1) and delta/notch-like EGF repeat containing (DNER) were expressed in early pancreatic progenitors. The expression pattern of DDR1 overlaps with the early pancreatic and duodenal homeobox 1 (PDX1)(+)/NK6 homeobox 1 (NKX6-1)(+) multipotent progenitor cells from embryonic day 11, whereas DNER expression in part overlaps with neurogenin 3 (NEUROG3)(+) cells. In the adult pancreas SEZ6L2, LRP11, DISP2 and SLC30A8, but also FXYD domain containing ion transport regulator 2 (FXYD2), tetraspanin 7 (TSPAN7) and transmembrane protein 27 (TMEM27), retain an islet-specific expression, whereas DDR1 is undetectable. In contrast, DNER is expressed at low levels in peripheral mouse and human islet cells. Re-expression of DDR1 and upregulation of DNER is observed in duct-ligated pancreas. Antibodies to DNER and DISP2 have been successfully used in cell sorting. Extracellular epitopes of SEZ6L2, LRP11, DISP2, DDR1 and DNER have been identified as useful tags by applying specific antibodies to visualise pancreatic cell types at specific stages of development. Furthermore, antibodies recognising DISP2 and DNER are suitable for FACS-mediated cell purification.

The epigenome changes that underlie cellular differentiation in developing organisms are poorly understood. To gain insights into how pancreatic beta-cells are programmed, we profiled key histone methylations and transcripts in embryonic stem cells, multipotent progenitors of the nascent embryonic pancreas, purified beta-cells, and 10 differentiated tissues. We report that despite their endodermal origin, beta-cells show a transcriptional and active chromatin signature that is most similar to ectoderm-derived neural tissues. In contrast, the beta-cell signature of trimethylated H3K27, a mark of Polycomb-mediated repression, clusters with pancreatic progenitors, acinar cells and liver, consistent with the epigenetic transmission of this mark from endoderm progenitors to their differentiated cellular progeny. We also identified two H3K27 methylation events that arise in the beta-cell lineage after the pancreatic progenitor stage. One is a wave of cell-selective de novo H3K27 trimethylation in non-CpG island genes. Another is the loss of bivalent and H3K27me3-repressed chromatin in a core program of neural developmental regulators that enables a convergence of the gene activity state of beta-cells with that of neural cells. These findings reveal a dynamic regulation of Polycomb repression programs that shape the identity of differentiated beta-cells.

Background & Aims Animal studies have indicated that pancreatic exocrine acinar cells have phenotypic plasticity. In rodents, acinar cells can differentiate into ductal precursors that can be converted to pancreatic ductal adenocarcinoma or insulin-producing endocrine cells. However, little is known about human acinar cell plasticity. We developed nongenetic and genetic lineage tracing methods to study the fate of human acinar cells in culture. Methods Human exocrine tissue was obtained from organ donors, dissociated, and cultured. Cell proliferation and survival were measured, and cell phenotypes were analyzed by immunocytochemistry. Nongenetic tracing methods were developed based on selective binding and uptake by acinar cells of a labeled lectin ( Ulex europaeus agglutinin 1). Genetic tracing methods were developed based on adenoviral introduction of a Cre-lox reporter system, controlled by the amylase promoter. Results Both tracing methods showed that human acinar cells can transdifferentiate into cells that express specific ductal markers, such as cytokeratin 19, hepatocyte nuclear factor 1β, SOX9, CD133, carbonic anhydrase II, and cystic fibrosis transmembrane conductance regulator. Within 1 week of culture, all surviving acinar cells had acquired a ductal phenotype. This transdifferentiation was decreased by inhibiting mitogen-activated protein kinase signaling. Conclusions Human acinar cells have plasticity similar to that described in rodent cells. These results might be used to develop therapeutic strategies for patients with diabetes or pancreatic cancer.

The pancreas is composed of two main compartments consisting of endocrine and exocrine tissues. The majority of the organ is exocrine and responsible for the synthesis of digestive enzymes and for their transport via an intricate ductal system into the duodenum. The endocrine tissue represents less than 2% of the organ and is organized into functional units called islets of Langerhans, comprising alpha-, beta-, delta-, epsilon- and PP-cells, producing the hormones glucagon, insulin, somatostatin, ghrelin and pancreatic polypeptide (PP), respectively. Insulin-producing beta-cells play a central role in the control of the glucose homeostasis. Accordingly, absolute or relative deficiency in beta-cells may ultimately lead to type 1 and/or type 2 diabetes, respectively. One major goal of diabetes research is therefore to understand the molecular mechanisms controlling the development of beta-cells during pancreas morphogenesis, but also those underlying the regeneration of adult injured pancreas, and assess their significance for future cell-based therapy. In this review, we will therefore present new insights into beta-cell development with focus on beta-cell regeneration.

BACKGROUND & AIMS: Exocrine acinar cells in the pancreas are highly differentiated cells that retain a remarkable degree of plasticity. After isolation and an initial phase of dedifferentiation in vitro, rodent acinar cells can convert to endocrine beta-cells when cultured in the presence of appropriate factors. The mechanisms regulating this phenotypic conversion are largely unknown. METHODS: Using rat acinar cell cultures, we studied the role of Notch signaling in a model of acinar-to-beta-cell conversion. RESULTS: We report a novel lectin-based cell labeling method to demonstrate the acinar origin of newly formed insulin-expressing beta-cells. This method allows for specific tracing of the acinar cells. We demonstrate that growth factor-induced conversion of adult acinar cells to beta-cells is negatively regulated by Notch1 signaling. Activated Notch1 signaling prevents the reexpression of the proendocrine transcription factor Neurogenin-3, the key regulator of endocrine development in the embryonic pancreas. Interfering with Notch1 signaling allows modulating the acinar cell susceptibility to the differentiation-inducing factors. Its inhibition significantly improves beta-cell neoformation with approximately 30% of acinar cells that convert to beta-cells. The newly formed beta-cells mature when transplanted ectopically and are capable of restoring normal blood glycemia in diabetic recipients. CONCLUSIONS: We report for the first time an efficient way to reprogram one third of the acinar cells to beta-cells by adult cell type conversion. This could find application in cell replacement therapy of type 1 diabetes, provided that it can be translated from rodent to human models.

We have previously reported that the loss of Arx and/or Pax4 gene activity leads to a shift in the fate of the different endocrine cell subtypes in the mouse pancreas, without affecting the total endocrine cell numbers. Here, we conditionally and ectopically express Pax4 using different cell-specific promoters and demonstrate that Pax4 forces endocrine precursor cells, as well as mature alpha cells, to adopt a beta cell destiny. This results in a glucagon deficiency that provokes a compensatory and continuous glucagon+ cell neogenesis requiring the re-expression of the proendocrine gene Ngn3. However, the newly formed alpha cells fail to correct the hypoglucagonemia since they subsequently acquire a beta cell phenotype upon Pax4 ectopic expression. Notably, this cycle of neogenesis and redifferentiation caused by ectopic expression of Pax4 in alpha cells is capable of restoring a functional beta cell mass and curing diabetes in animals that have been chemically depleted of beta cells.

BackgroundIn both humans and rodents, glucose homeostasis is controlled by micro-organs called islets of Langerhans composed of beta cells, associated with other endocrine cell types. Most of our understanding of islet cell differentiation and morphogenesis is derived from rodent developmental studies. However, little is known about human islet formation. The lack of adequate experimental models has restricted the study of human pancreatic development to the histological analysis of different stages of pancreatic development. Our objective was to develop a new experimental model to (i) transfer genes into developing human pancreatic cells and (ii) validate gene transfer by defining the clonality of developing human islets.Methods and findingsIn this study, a unique model was developed combining ex vivo organogenesis from human fetal pancreatic tissue and cell type-specific lentivirus-mediated gene transfer. Human pancreatic progenitors were transduced with lentiviruses expressing GFP under the control of an insulin promoter and grafted to severe combined immunodeficient mice, allowing human beta cell differentiation and islet morphogenesis. By performing gene transfer at low multiplicity of infection, we created a chimeric graft with a subpopulation of human beta cells expressing GFP and found both GFP-positive and GFP-negative beta cells within single islets.ConclusionThe detection of both labeled and unlabeled beta cells in single islets demonstrates that beta cells present in a human islet are derived from multiple progenitors thus providing the first dynamic analysis of human islet formation during development. This human transgenic-like tool can be widely used to elucidate dynamic genetic processes in human tissue formation.

Success of cell-replacement therapy for diabetes will largely depend on the establishment of alternative sources of pancreatic islet grafts. Embryonic stem (ES) cell differentiation toward pancreatic insulin-producing cells offers such perspectives, but there are still many challenges to overcome. Our previous studies suggested that the limited amount of insulin-positive cells derived from ES cells is related to the activation of pancreas inhibitory signals. To confirm this hypothesis, we report here that exposure of mouse embryonic pancreas explants to soluble factors from embryoid bodies (EBs) inhibits growth, morphogenesis, and endocrine and exocrine differentiation as evaluated by explant size and mRNA and protein expression. Sonic Hedgehog (Shh), an established pancreas repressor both at early and late developmental stages, was produced and secreted by EBs, and participated in the inhibitory effect by inducing its target Gli1 in the explants. Inhibition of Hedgehog pathway rescued the differentiation of Insulin-positive cells in the explants. In contrast to pancreatic cells, hepatic progenitors exposed to EB-conditioned medium showed improved differentiation of albumin-positive cells. In a model system of ES cell differentiation in vitro, we found that definitive endoderm induction by serum removal or activin A treatment further increased Hedgehog production and activity in EBs. Concomitantly, downregulation of the pancreas marker Pdx1 was recorded in activin-treated EBs, a phenomenon that was prevented by antagonizing Hedgehog signaling with Hedgehog interacting protein. These data strongly suggest that Hedgehog production in EBs limits pancreatic fate acquisition and forms a major obstacle in the specification of pancreatic cells from ES-derived definitive endoderm. Disclosure of potential conflicts of interest is found at the end of this article.

AMP-activated protein kinase influences cellular metabolism, glucose-regulated gene expression, and insulin secretion of pancreatic beta cells. Its sustained activation by culture at low glucose concentrations or in the presence of 5-aminoimidazole-4-carboxamide riboside (AICAR) was shown to trigger apoptosis in beta cells. This study shows that both low glucose- and AICAR-induced apoptosis are associated with increased formation of mitochondrial superoxide-derived radicals and decreased mitochondrial activity. Mitochondrial dysfunction was reflected by an increased oxidized state of the mitochondrial flavins (FMN/FAD) but not of NAD(P)H. It was accompanied by suppression of glucose oxidation and glucose-induced insulin secretion, while palmitate oxidation appeared unaffected. When the cellular accumulation of superoxide-derived radicals was quenched by the ROS scavengers vitamin E, N-acetylcysteine, or the SOD-mimetic compound MnTBAP, apoptosis was significantly inhibited. Both low glucose and AICAR also elevated the expression of BH3-domain-only Bcl-2 antagonists, and induced caspase-3 activation, causing caspase-dependent truncation of Bcl-2. Overexpression of recombinant human Bcl-2 prevented caspase-3 activation, endogenous Bcl-2 processing, and apoptosis, but did not attenuate oxygen radical formation, AMPK activation, or JNK phosphorylation. We conclude that apoptosis by prolonged AMPK activation in beta cells results from enhanced production of mitochondria-derived oxygen radicals and onset of the intrinsic mitochondrial apoptosis pathway, followed by caspase activation and Bcl-2 cleavage which may amplify the death signal.

The basic helix-loop-helix protein Neurogenin3 specifies precursor cells of the endocrine pancreas during embryonic development, and is thought to be absent postnatally. We have studied Ngn3 expression during in vitro generation of beta-cells from adult rat exocrine pancreas tissue treated with epidermal growth factor and leukaemia inhibitory factor. This treatment induced a transient expression of both Ngn3 and its upstream activator hepatocyte nuclear factor 6. Inhibition of EGF and LIF signalling by pharmacological antagonists of the JAK2/STAT3 pathway, or knockdown of Ngn3 by RNA interference prevented the generation of new insulin-positive cells. This study demonstrates that in vitro growth factor stimulation can induce recapitulation of an embryonic endocrine differentiation pathway in adult dedifferentiated exocrine cells. This could prove to be important for understanding the mechanism of beta-cell regeneration and for therapeutic ex vivo neogenesis of beta cells.

Neurogenin 3 (NEUROG3), a basic helix-loop-helix transcription factor that is needed for endocrine cell development in the embryonic pancreas, has been shown to induce transdifferentiation of duct cells from adult pancreas towards a neuro-endocrine phenotype. Our study explored the endocrine transdifferentiation potential of NEUROG3 in neonatal pancreatic precursor cells. A replication-deficient adenovirus expressing Neurog3 and green fluorescent protein (GFP) (Ad-NEUROG3) was used to infect neonatal pig pancreatic cell preparations enriched for endocrine islet and cytokeratin-positive precursor cells. GFP-positive cells were sorted using flow cytometry on days 3 and 8 after infection and characterised at the transcript and protein level. For in vivo experiments, the total population of Ad-NEUROG3-infected pancreatic cells was transplanted, then later removed for determination of graft hormone content and immunohistochemistry. Among the GFP-positive cells, the fraction of precursor cells decreased by more than 85% at day 8 after infection, while the fraction of glucagon-positive cells increased 2.5-fold and the beta cell number remained the same. Transplantation of the Ad-NEUROG3-infected pancreatic cell preparation failed to reverse streptozotocin-induced hyperglycaemia, while non-infected cells and a control cell preparation infected with replication-deficient adenovirus expressing only GFP were able to do so. At day 109 after transplantation, kidneys grafted with Ad-NEUROG3-infected pancreatic cells contained significantly decreased insulin and increased glucagon levels. Abundant glucagon-immunopositive cells were seen in Ad-NEUROG3-infected grafts, which were virtually devoid of proliferating insulin-positive cells. In summary, adenoviral delivery of NEUROG3 to pancreatic precursor cells from neonatal pig pancreas promotes alpha cell differentiation in vitro and in vivo.

Prolonged culture in low-glucose concentrations (

Type 1 diabetes mellitus results from an autoimmune destruction of pancreatic beta -cells. Cytokines, such as interleukin-1beta and interferon-gamma , are putative mediators of immune-induced beta -cell death and, under in vitro conditions, cause beta -cell apoptosis. We have recently shown that interleukin-1beta + interferon-gamma modifies the expression of >200 genes in beta -cells. Several of these genes are putative targets for the transcription factor nuclear factor-kappa B (NF-kappa B), and in subsequent experiments we showed that NF-kappa B activation is mostly pro-apoptotic in beta -cells. To identify cytokine-induced and NF-kappa B-regulated genes in primary rat beta -cells, we presently combined two experimental approaches: 1) blocking of NF-kappa B activation in cytokine-exposed beta -cells by a recombinant adenovirus (AdIkappa B(SA)2) containing an inhibitor of NF-kappa B alpha (Ikappa Bac) super-repressor (S32A/S36A) and 2) study of gene expression by microarray analysis. We identified 66 cytokine-modified and NF-kappa B-regulated genes in beta -cells. Cytokine-induced NF-kappa B activation decreased Pdx-1 and increased c-Myc expression. This, together with NF-kappa B-dependent inhibition of Glut-2, pro-hormone convertase-1, and Isl-1 expression, probably contributes to the loss of differentiated beta -cell functions. NF-kappa B also regulates several genes encoding for chemokines and cytokines in beta -cells. The present data suggest that NF-kappa B is a key "switch regulator" of transcription factors and gene networks controlling cytokine-induced beta -cell dysfunction and death.

Aims/hypothesis. The Ca2+/calmodulin-dependent protein kinase II (CaMK II) is highly expressed in pancreatic islets and associated with insulin secretion vesicles. The suppression of CaMK II disturbs insulin secretion and insulin gene expression. There are four isoforms of CaMK II, α to δ, that are expressed from different genes in mammals. Our aim was to identify the isoforms of CaMK II expressed in human beta cells by molecular cloning from a human insulinoma cDNA library and to assess its distribution in humans.¶Methods. The previously unknown complete coding sequences of human CaMK IIβ and the kinase domain of CaMK IIδ were cloned from a human insulinoma cDNA library. Quantitative determination of CaMK II isoform mRNA was carried out in several tissues and beta cells purified by fluorescence activated cell sorting and compared to the housekeeping enzyme pyruvate dehydrogenase.¶Results. We found CaMK IIβ occurred in three splice variants and was highly expressed in endocrine tissues such as adrenals, pituitary and beta cells. Liver showed moderate expression but adipose tissue or lymphocytes had very low levels of CaMK IIβ-mRNA. In human beta cells CaMK IIβ and δ were expressed equally with pyruvate dehydrogenase whereas tenfold lower expression of CaMK IIγ and no expression of CaMK IIα were found.¶Conclusion/interpretation. Although CaMK IIδ is ubiquitously expressed, CaMK IIβ shows preferential expression in neuroendocrine tissues. In comparison with the expression of a key regulatory enzyme in glucose oxidation, pyruvate dehydrogenase, two of the four CaM kinases investigated are expressed at equally high levels, which supports an important role in beta-cell physiology. These results provide the basis for exploring the pathophysiological relevance of CaMK IIβ in human diabetes. [Diabetologia (2000) 43: 465–473]

ADP-ribosylation factor (ARF) 6 has been shown to play a role in vesicular transport; however, the expression and subcellular localization of the endogenous protein have not been clearly delineated. In this study, an ARF6-specific monoclonal antibody was raised and used to examine the subcellular distribution and expression of ARF6 in various tissues and during the differentiation of several well characterized cell types. We found that ARF6 localizes in both the cytosol and membranes of all tissues and cells tested. Moreover, ARF6 in 3T3-L1 adipocytes is principally localized on the plasma membrane, but substantial amounts are detected in the cytosolic and intracellular membrane fractions. We observed an increased expression of ARF6 during the differentiation of B lymphocytes to plasmocytes. However, the expression of ARF6 decreased during adipogenesis and monocyte differentiation. In contrast, the expression of other ARFs, detected by the monoclonal antibody 1D9, did not significantly change during differentiation of the aforementioned cell types. Taken together, our results indicate that ARF6 is a broadly expressed, differentially regulated GTPase that is present in cytoplasm and on both cell-surface and intracellular membranes and whose functions may include tissue-specific effects on vesicular trafficking during cellular differentiation.

Glucosamine, which enters the hexosamine pathway downstream of the rate-limiting step, has been routinely used to mimic the insulin resistance caused by high glucose and insulin. We investigated the effect of glucosamine on insulin-stimulated glucose transport in 3T3-L1 adipocytes. The Delta-insulin (insulin-stimulated minus basal) value for 2-deoxyglucose uptake was dramatically inhibited with increasing concentrations of glucosamine with an ED50 of 1.95 mM. Subcellular fractionation experiments demonstrated that reduction in insulin-stimulated 2-deoxyglucose uptake by glucosamine was due to an inhibition of translocation of both Glut 1 and Glut 4 from the low density microsomes (LDM) to the plasma membrane. Analysis of the insulin signaling cascade revealed that glucosamine impaired insulin receptor autophosphorylation, insulin receptor substrate (IRS-1) phosphorylation, IRS-1-associated PI 3-kinase activity in the LDM, and AKT-1 activation by insulin. Measurement of intracellular ATP demonstrated that the effects of glucosamine were highly correlated with its ability to reduce ATP levels. Reduction of intracellular ATP using azide inhibited Glut 1 and Glut 4 translocation from the LDM to the plasma membrane, insulin receptor autophosphorylation, and IRS-1 tyrosine phosphorylation. Additionally, both the reduction in intracellular ATP and the effects on insulin action caused by glucosamine could be prevented by the addition of inosine, which served as an alternative energy source in the medium. We conclude that direct administration of glucosamine can rapidly lower cellular ATP levels and affect insulin action in fat cells by mechanisms independent of increased intracellular UDP-N-acetylhexosamines and that increased metabolism of glucose via the hexosamine pathway may not represent the mechanism of glucose toxicity in fat cells.

We previously showed that injury by partial duct ligation (PDL) in adult mouse pancreas activates Neurogenin 3 (Ngn3)(+) progenitor cells that can differentiate to beta cells ex vivo. Here we evaluate the role of Ngn3(+) cells in beta cell expansion in situ. PDL not only induced doubling of the beta cell volume but also increased the total number of islets. beta cells proliferated without extended delay (the so-called 'refractory' period), their proliferation potential was highest in small islets, and 86% of the beta cell expansion was attributable to proliferation of pre-existing beta cells. At sufficiently high Ngn3 expression level, upto 14% of all beta cells and 40% of small islet beta cells derived from non-beta cells. Moreover, beta cell proliferation was blunted by a selective ablation of Ngn3(+) cells but not by conditional knockout of Ngn3 in pre-existing beta cells supporting a key role for Ngn3(+) insulin(-) cells in beta cell proliferation and expansion. We conclude that Ngn3(+) cell-dependent proliferation of pre-existing and newly-formed beta cells as well as reprogramming of non-beta cells contribute to in vivo beta cell expansion in the injured pancreas of adult mice.

Novel strategies in diabetes therapy would obviously benefit from the use of beta (beta) cell stem/progenitor cells. However, whether or not adult beta cell progenitors exist is one of the most controversial issues in today's diabetes research. Guided by the expression of Neurogenin 3 (Ngn3), the earliest islet cell-specific transcription factor in embryonic development, we show that beta cell progenitors can be activated in injured adult mouse pancreas and are located in the ductal lining. Differentiation of the adult progenitors is Ngn3 dependent and gives rise to all islet cell types, including glucose responsive beta cells that subsequently proliferate, both in situ and when cultured in embryonic pancreas explants. Multipotent progenitor cells thus exist in the pancreas of adult mice and can be activated cell autonomously to increase the functional beta cell mass by differentiation and proliferation rather than by self-duplication of pre-existing beta cells only.

Neurogenin 3 (Ngn3) is key for endocrine cell specification in the embryonic pancreas and induction of a neuroendocrine cell differentiation program by misexpression in adult pancreatic duct cells. We identify the gene encoding IA1, a zinc-finger transcription factor, as a direct target of Ngn3 and show that it forms a novel branch in the Ngn3-dependent endocrinogenic transcription factor network. During embryonic development of the pancreas, IA1 and Ngn3 exhibit nearly identical spatio-temporal expression patterns. However, embryos lacking Ngn3 fail to express IA1 in the pancreas. Upon ectopic expression in adult pancreatic duct cells Ngn3 binds to chromatin in the IA1 promoter region and activates transcription. Consistent with this direct effect, IA1 expression is normal in embryos mutant for NeuroD1, Arx, Pax4 and Pax6, regulators operating downstream of Ngn3. IA1 is an effector of Ngn3 function as inhibition of IA1 expression in embryonic pancreas decreases the formation of insulin- and glucagon-positive cells by 40%, while its ectopic expression amplifies neuroendocrine cell differentiation by Ngn3 in adult duct cells. IA1 is therefore a novel Ngn3-regulated factor required for normal differentiation of pancreatic endocrine cells.