Search

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.

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., 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.

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., 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.

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.

AIMS/HYPOTHESIS: Prolonged exposure of beta cells to low glucose concentrations triggers their apoptosis and is known to activate AMP-activated protein kinase (AMPK) in beta cell lines. We examined whether prolonged activation of AMPK can trigger apoptosis in rodent beta cells. METHODS: Primary beta cells were FACS-purified from rats, and from wild-type and AMPK(alpha2)-deficient mice. AMPK activation in beta cells was induced by the adenosine analog AICA-riboside and detected by immunoblotting using a phosphospecific antibody. Apoptosis of rodent beta cells was monitored by FACS analysis of beta cell DNA content, by direct counting of apoptotic cells using fluorescence microscopy, or by measurement of their caspase-3 activity. RESULTS: Dose-dependent and time-dependent apoptosis of the cells, concommittant with an activation of caspase-3, were suppressed by the caspase inhibitors zVAD-fmk and zDEVD-fmk. Apoptosis induction by AICA-riboside was also prevented by adding the MAPK-inhibitor SB203580 which blocked the AICA-riboside-induced phosphorylation of AMPK. Beta cells isolated from AMPK-(alpha2)-deficient mice were resistant against AICA-riboside induced apoptosis. CONCLUSION/INTERPRETATION: Sustained activation of AMPK by AICA-riboside can trigger a caspase-dependent apoptosis of pancreatic beta cells.

Type 1 diabetes mellitus results from an autoimmune destruction of pancreatic beta-cells. Cytokines, such as interleukin-1 beta 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-1 beta + 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 (AdI kappa B((SA)2)) containing an inhibitor of NF-kappa B alpha (I kappa 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., Journal Article, Research Support, Non-U.S. Gov't, info:eu-repo/semantics/published

Cytokine-induced beta-cell death is an important event in the pathogenesis of type 1 diabetes. The transcription factor nuclear factor-kappaB (NF-kappaB) is activated by interleukin-1beta (IL-1beta), and its activity promotes the expression of several beta-cell genes, including pro- and anti-apoptotic genes. To elucidate the role of cytokine (IL-1beta + gamma-interferon [IFN-gamma])-induced expression of NF-kappaB in beta-cell apoptosis, rat beta-cells were infected with the recombinant adenovirus AdIkappaB((SA)2), which contained a nondegradable mutant form of inhibitory kappaB (IkappaB((SA)2), with S32A and S36A) that locks NF-kappaB in a cytosolic protein complex, preventing its nuclear action. Expression of IkappaB((SA)2) inhibited cytokine-stimulated nuclear translocation and DNA-binding of NF-kappaB. Cytokine-induced gene expression of several NF-kappaB targets, namely inducible nitric oxide synthase, Fas, and manganese superoxide dismutase, was prevented by AdIkappaB((SA)2), as established by reverse transcriptase-polymerase chain reaction, protein blot, and measurement of nitrite in the medium. Finally, beta-cell survival after IL-1beta + IFN-gamma treatment was significantly improved by IkappaB((SA)2) expression, mostly through inhibition of the apoptotic pathway. Based on these findings, we conclude that NF-kappaB activation, under in vitro conditions, has primarily a pro-apoptotic function in beta-cells., Journal Article, Research Support, Non-U.S. Gov't, info:eu-repo/semantics/published

Rat beta-cells differ in their individual rates of glucose-induced insulin biosynthesis and release. This functional heterogeneity has been correlated with intercellular differences in metabolic redox responsiveness to glucose. The present study compares glucose metabolism in two beta-celt subpopulations that have been separated on the basis of the presence (high responsive) or absence (low responsive) of a metabolic redox shift at 7.5 mM glucose. Mean rates of glucose utilization and glucose oxidation in high responsive beta-cells were 2- to 4-fold higher than in low responsive beta-cells, whereas their leucine and glutamine oxidation was only 10-50% higher. This heterogeneity in glucose metabolism cannot be attributed to differences in GLUT2 mRNA levels or in glucose transport. In both cell subpopulations, the rates of glucose transport (13-19 pmol/min/10(3) beta-cells) were at least 50-fold higher than corresponding rates of glucose utilization. On the other hand, rates of glucose phosphorylation (0.3-0.7 pmol/min/10(3) beta-cells) ranged within those of total glucose utilization (0.2-0.4 pmol/min/10(3) beta-cells). High responsive beta-cells exhibited a 60% higher glucokinase activity than low responsive beta-cells and their glucokinase mRNA level was 100% higher. Furthermore, glucose phosphorylation via low K(m) hexokinase was detected only in the high responsive beta-cell subpopulation. Heterogeneity in glucose sensitivity among pancreatic beta-cells can therefore be explained by intercellular differences in glucose phosphorylation rather than in glucose transport.