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.

SUMMARYPancreatic 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

This study demonstrates that rat islet ␤ cells constitutively express an apoptotic program which is activated when mRNA or protein synthesis is blocked. Apoptotic ␤ cells were detectable by electron microscopy after treatment with actinomycin D or cycloheximide. With a fluorescence microscopic assay both agents were found to increase the number of apoptotic ␤ cells dose-and time-dependently, up to 70% after 1 wk of culture; virtually no apoptotic ␤ cells occurred in control preparations or in conditions leading to primary necrosis. Thus, survival of ␤ cells seems dependent on synthesis of proteins which suppress an endogenous suicide program. This mechanism explains earlier observed effects of glucose on survival of cultured ␤ cells. Glucose is known to dose-dependently increase the percentage of ␤ cells in active biosynthesis and the percentage that survives during culture. It is now demonstrated that the glucose-induced survival of ␤ cells cultured for 1 wk results from a dose-dependent reduction in the percentage of ␤ cells dying in apoptosis (49% at 3 mM glucose, 40% at 6 mM, 9% at 10 mM). Thus, intercellular differences in glucose sensitivity appear responsible for the heterogeneity in ␤ cell sensitivity to apoptotic conditions. These data indicate that glucose promotes

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.

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.

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.

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 reinstate normal glycemic control for up to 248 days. 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

The aim of this work was to study the e¡ect 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. ß