To determine whether glucocorticoids are involved in pancreas development, glucocorticoid treatment of rat pancreatic buds in vitro was combined with the analysis of transgenic mice lacking the glucocorticoid receptor (GR) in specific pancreatic cells. In vitro treatment of embryonic pancreata with dexamethasone, a glucocorticoid agonist, induced a decrease of insulin-expressing cell numbers and a doubling of acinar cell area, indicating that glucocorticoids favored acinar differentiation; in line with this, expression of Pdx-1, Pax-6, and Nkx6.1 was downregulated, whereas the mRNA levels of Ptf1-p48 and Hes-1 were increased. The selective inactivation of the GR gene in insulin-expressing ␤-cells in mice (using a RIP-Cre transgene) had no measurable consequences on ␤-or ␣-cell mass, whereas the absence of GR in the expression domain of Pdx-1 (Pdx-Cre transgene) led to a twofold increased ␤-cell mass, with increased islet numbers and size but normal ␣-cell mass in adults. These results demonstrate that glucocorticoids play an important role in pancreatic ␤-cell lineage, acting before hormone gene expression onset and possibly also modulating the balance between endocrine and exocrine cell differentiation. Diabetes 53:2322-2329, 2004 I ncreasing evidence from epidemiological studies led to the concept of the early-life origins of adult diseases, suggesting that late-onset disorders such as type 2 diabetes, glucose intolerance, or hypertension may be programmed by nutritional inadequacy in utero (1-5). Excess glucocorticoids also retard fetal growth, and overexposure to these hormones during intrauterine life has been shown to play a role in fetal programming in both humans (6) and rodents (7). The link between glucocorticoid overexposure in utero and the occurrence of metabolic diseases in adulthood has been well documented in rats. Maternal treatment with dexamethasone (DEX), a glucocorticoid agonist, induces in the offspring growth retardation at birth as well as hyperglycemia and increased systolic blood pressure at adult age (8 -10).Similarly, inhibition of the placental 11␤-hydroxysteroid dehydrogenase type 2, the enzyme that protects the fetus from maternal glucocorticoids, induces intrauterine growth retardation as well as glucose intolerance and hypertension in adults (11).We have previously shown that maternal general food restriction during late pregnancy decreased the ␤-cell mass of newborn rats (12). This reduction was irreversible and persisted in adults despite restoration of normal nutrition from weaning (13), ultimately leading to impaired glucose tolerance associated with aging (14) or pregnancy (15). These results sustain the notion that some of the late alterations observed in humans born with intrauterine growth retardation may result from altered ␤-cell development in utero, as initially suggested by Hales and Barker (2). Additionally, we have recently demonstrated that maternal general food restriction in the rat induced a rise in both maternal and fetal corticosterone levels, which in turn w...

dPancreas development is initiated by the specification and expansion of a small group of endodermal cells. Several transcription factors are crucial for progenitor maintenance and expansion, but their interactions and the downstream targets mediating their activity are poorly understood. Among those factors, PTF1a, a basic helix-loop-helix (bHLH) transcription factor which controls pancreas exocrine cell differentiation, maintenance, and functionality, is also needed for the early specification of pancreas progenitors. We used RNA profiling and chromatin immunoprecipitation (ChIP) sequencing to identify a set of targets in pancreas progenitors. We demonstrate that Mnx1, a gene that is absolutely required in pancreas progenitors, is a major direct target of PTF1a and is regulated by a distant enhancer element. Pdx1, Nkx6.1, and Onecut1 are also direct PTF1a targets whose expression is promoted by PTF1a. These proteins, most of which were previously shown to be necessary for pancreas bud maintenance or formation, form a transcription factor network that allows the maintenance of pancreas progenitors. In addition, we identify Bmp7, Nr5a2, RhoV, and P2rx1 as new targets of PTF1a in pancreas progenitors.

Low birth weight is strongly predictive of hypertension, cardiovascular diseases, obesity, insulin resistance and diabetes. The mechanisms by which fetal undernutrition and, hence, low birth weight increase the risk of developing these diseases are unclear. To investigate the hypothesis of a primary defect in beta-cell development, we designed a rat model of undernutrition, involving an overall reduction in maternal food intake. In this model, fetuses with intrauterine growth retardation have a decreased beta-cell mass, which persists into adulthood and ultimately causes glucose intolerance, thereby mimicking features of the metabolic syndrome. Maternal undernutrition causes elevations in glucocorticoid concentrations, which, in turn, cause a reduction in beta-cell mass in the fetus. Our data also suggest a key role of glucocorticoids when nutrient supply is normal. By combining in-vitro studies with in-vivo investigations in mice lacking the glucocorticoid receptor in the whole organism or in specific pancreatic cell populations, we have shown that the glucocorticoid receptor is critical for ensuring pancreatic architecture and survival, as well as for beta-cell mass expansion during a critical developmental window. Glucocorticoids act on precursor cells before the onset of hormone gene expression and are likely to programme beta-cell differentiation by modifying the balance of specific transcription factors, mostly Pdx-1. Glucocorticoids should therefore be considered as important hormones in pancreatic development, in situations of both normal nutrition and undernutrition. To investigate whether this is also the case in human pancreatic development, we studied the expression of the glucocorticoid receptor and that of the transcription factor Pdx-1 on pancreatic specimens from very early to late stages of development of the human embryo. In terms of beta-cell ontogeny, expression of the glucocorticoid receptor in the pancreas coincides with that of the transcription factor Pdx-1 in beta cells. These results are consistent with a possible role for glucocorticoids during human pancreatic development.

Aims/hypothesis Beta cell development is sensitive to glucocorticoid levels. Although direct effects of glucocorticoids on pancreatic precursors have been shown to control beta cell mass expansion, indirect effects of these hormones on pancreatic development remain unexplored. This issue was addressed in mice lacking the glucocorticoid receptor (GR) in the whole organism. Materials and Methods The pancreatic phenotype of GR null/null mice was studied at fetal ages (embryonic day [E]) E15.5 and E18 by immunohistochemistry and beta cell fraction measurements. To distinguish between direct and indirect effects, mutant E15.5 fetal pancreata were grafted under the kidney capsule of immunodeficient mice and analysed after 1 week. Results E18 GR null/null fetuses had smaller digestive tracts and tiny pancreata. Massive pancreatic disorganisation and apoptosis were observed despite the presence of all cell types. E15.5 GR null/null mutants were indistinguishable from wild-type regarding pancreatic size, tissue structure and organisation, beta cell fraction and production of exocrine transcription factor Ptf1a, neurogenin 3 and Pdx-1. Grafting E15.5 GR null/null pancreata into a GR-expressing environment rescued the increased apoptosis and mature islets were observed, suggesting that GR null/null pancreatic cell death can be attributed to indirect effects of glucocorticoids on this tissue. Heterozygous GR +/null mutants with reduced GR numbers showed no apoptosis but increased beta cell fraction at E18 and the adult age, strengthening the importance of an accurate GR dosage on beta cell mass expansion. Conclusions/interpretation Our results provide evidence for GR involvement in pancreatic tissue organisation and survival through indirect effects. GR does not appear necessary for early phases, but its accurate dosage is critical to modulate beta cell mass expansion at later fetal stages, presumably through direct effects.

The absence of the transcriptional repressor RE-1 Silencing Transcription Factor (REST) in insulin-secreting beta cells is a major cue for the specific expression of a large number of genes. These REST target genes were largely ascribed to a function of neurotransmission in a neuronal context, whereas their role in pancreatic beta cells has been poorly explored. To identify their functional significance, we have generated transgenic mice expressing REST in beta cells (RIP-REST mice), and previously discovered that REST target genes are essential to insulin exocytosis. Herein we characterized a novel line of RIP-REST mice featuring diabetes. In diabetic RIP-REST mice, high levels of REST were associated with postnatal beta cell apoptosis, which resulted in gradual beta cell loss and sustained hyperglycemia in adults. Moreover, adenoviral REST transduction in INS-1E cells led to increased cell death under control conditions, and sensitized cells to death induced by cytokines. Screening for REST target genes identified several anti-apoptotic genes bearing the binding motif RE-1 that were downregulated upon REST expression in INS-1E cells, including Gjd2, Mapk8ip1, Irs2, Ptprn, and Cdk5r2. Decreased levels of Cdk5r2 in beta cells of RIP-REST mice further confirmed that it is controlled by REST, in vivo. Using siRNA-mediated knock-down in INS-1E cells, we showed that Cdk5r2 protects beta cells against cytokines and palmitate-induced apoptosis. Together, these data document that a set of REST target genes, including Cdk5r2, is important for beta cell survival.