Mark Van de Casteele

β Cells Can Be Generated from Endogenous Progenitors in Injured Adult Mouse Pancreas

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 todays 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.

Recapitulation of embryonic neuroendocrine differentiation in adult human pancreatic duct cells expressing neurogenin 3.

Regulatory proteins have been identified in embryonic development of the endocrine pancreas. It is unknown whether these factors can also play a role in the formation of pancreatic endocrine cells from postnatal nonendocrine cells. The present study demonstrates that adult human pancreatic duct cells can be converted into insulin-expressing cells after ectopic, adenovirus-mediated expression of the class B basic helix-loop-helix factor neurogenin 3 (ngn3), which is a critical factor in embryogenesis of the mouse endocrine pancreas. Infection with adenovirus ngn3 (Adngn3) induced gene and/or protein expression of NeuroD/β2, Pax4, Nkx2.2, Pax6, and Nkx6.1, all known to be essential for β-cell differentiation in mouse embryos. Expression of ngn3 in adult human duct cells induced Notch ligands Dll1 and Dll4 and neuroendocrine- and β-cell–specific markers: it increased the percentage of synaptophysin- and insulin-positive cells 15-fold in ngn3-infected versus control cells. Infection with NeuroD/β2 (a downstream target of ngn3) induced similar effects. These data indicate that the Delta-Notch pathway, which controls embryonic development of the mouse endocrine pancreas, can also operate in adult human duct cells driving them to a neuroendocrine phenotype with the formation of insulin-expressing cells.

Sustained activation of AMP-activated protein kinase induces c-Jun N-terminal kinase activation and apoptosis in liver cells

The aim of this work was to study the effect of a sustained activation of AMP‐activated protein kinase (AMPK) on liver cell survival. AMPK activation was achieved by incubating FTO2B cells with AICA‐riboside, which is transformed into ZMP, an AMP analogue, or by adenoviral transfection of hepatocytes with a constitutively active form of AMPK. Prolonged AMPK activation triggered apoptosis and activated c‐Jun N‐terminal kinase (JNK) and caspase‐3. Experiments with iodotubercidin, dicoumarol and z‐VAD‐fmk, which inhibited AMPK, JNK and caspase activation, respectively, supported the notion that prolonged AMPK activation in liver cells induces apoptosis through an activation pathway that involves JNK and caspase‐3.

IA1 is NGN3-dependent and essential for differentiation of the endocrine pancreas

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.

Spatiotemporal patterns of multipotentiality in Ptf1a -expressing cells during pancreas organogenesis and injury-induced facultative restoration

Pancreatic multipotent progenitor cells (MPCs) produce acinar, endocrine and duct cells during organogenesis, but their existence and location in the mature organ remain contentious. We used inducible lineage-tracing from the MPC-instructive gene Ptf1a to define systematically in mice the switch of Ptf1a+ MPCs to unipotent proacinar competence during the secondary transition, their rapid decline during organogenesis, and absence from the mature organ. Between E11.5 and E15.5, we describe tip epithelium heterogeneity, suggesting that putative Ptf1a+Sox9+Hnf1β+ MPCs are intermingled with Ptf1aHISox9LO proacinar progenitors. In the adult, pancreatic duct ligation (PDL) caused facultative reactivation of multipotency factors (Sox9 and Hnf1β) in Ptf1a+ acini, which undergo rapid reprogramming to duct cells and longer-term reprogramming to endocrine cells, including insulin+ β-cells that are mature by the criteria of producing Pdx1HI, Nkx6.1+ and MafA+. These Ptf1a lineage-derived endocrine/β-cells are likely formed via Ck19+/Hnf1β+/Sox9+ ductal and Ngn3+ endocrine progenitor intermediates. Acinar to endocrine/β-cell transdifferentiation was enhanced by combining PDL with pharmacological elimination of pre-existing β-cells. Thus, we show that acinar cells, without exogenously introduced factors, can regain aspects of embryonic multipotentiality under injury, and convert into mature β-cells.

Transient cytokine treatment induces acinar cell reprogramming and regenerates functional beta cell mass in diabetic mice

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.

Derepression of Polycomb targets during pancreatic organogenesis allows insulin-producing beta-cells to adopt a neural gene activity program.

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.

Plasticity of Adult Human Pancreatic Duct Cells by Neurogenin3-Mediated Reprogramming

Aims/Hypothesis Duct 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. Methods The 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. Results Ngn3 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/Interpretation The 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.

Akt activation protects pancreatic beta cells from AMPK-mediated death through stimulation of mTOR.

Sustained activation of AMP-activated protein kinase (AMPK) induces apoptosis in several cell types. In pancreatic beta cells this occurs under glucose limitation, or in the presence of the pharmacological AMPK activator 5-aminoimidazole-4-carboxamide-riboside (AICAR). It is unknown whether Akt activation can counteract AMPK-mediated apoptosis, nor whether mTOR activation downstream of Akt mediates any survival signal in these conditions. We report that expression of a constitutively active form of Akt increases mTOR activity and prevents apoptosis upon AMPK activation. Akt-mediated survival was inhibited by rapamycin. Expression of a constitutively active form of the mTOR target ribosomal protein S6 kinase (S6K) or of translation factor eIF4E reduced apoptosis by glucose limitation, and co-expression of S6K and eIF4E protected beta cells to the same extent as active Akt. The protective effects of active Akt and S6K were associated with increased cellular protein synthesis activity. It is concluded that Akt stimulation of mTOR and subsequent activation of the targets by which mTOR affects protein translation are required and sufficient mechanisms for Akt-mediated survival of beta cells undergoing sustained AMPK activation.

Specificity in Beta Cell Expression of l-3-Hydroxyacyl-CoA Dehydrogenase, Short Chain, and Potential Role in Down-regulating Insulin Release

A loss-of-function mutation of the mitochondrial β-oxidation enzyme l-3-hydroxyacyl-CoA dehydrogenase, short chain (HADHSC), has been associated with hyperinsulinemic hypoglycemia in man. It is still unclear whether loss of glucose homeostasis in these patients (partly) results from a dysregulation of beta cells. This study examines HADHSC expression in purified rat beta cells and investigates whether its selective suppression elevates insulin release. Beta cells expressed the highest levels of HADHSC mRNA and protein of all examined tissues, including those with high rates of mitochondrial β-oxidation. On the other hand, beta cells expressed relatively low levels of other β-oxidation enzymes (acyl-CoA dehydrogenase short, medium, and long chain and acetyl-coenzyme A acyltransferase 2). HADHSC expression was sequence-specifically silenced by RNA interference, and the effects were examined on glucose-stimulated insulin secretion following 48–72 h of suppression. In both rat beta cells and in the beta cell line INS1 832-13, HADHSC silencing resulted in elevated insulin release at low and at high glucose concentrations, which appeared not to be caused by increased rates of glucose metabolism or an inhibition in fatty acid oxidation. These data indicate that the normal beta cell phenotype is characterized by a high expression of HADHSC and a low expression of other β-oxidation enzymes. Down-regulation of HADHSC causes an elevated secretory activity suggesting that this enzyme protects against inappropriately high insulin levels and hypoglycemia.