Pro-oxidant/antioxidant balance controls pancreatic ß-cell differentiation through the ERK1/2 pathway.

During embryogenesis, the intrauterine milieu affects cell proliferation, differentiation, and function by modifying gene expression in susceptible cells, such as the pancreatic ß-cells. In this limited energy environment, mitochondrial dysfunction can lead to overproduction of reactive oxygen species (ROS) and to a decline in ß-cell function. In opposition to this toxicity, ROS are also required for insulin secretion. Here we investigated the role of ROS in ß-cell development. Surprisingly, decreasing ROS production in vivo reduced ß-cell differentiation. Moreover, in cultures of pancreatic explants, progenitors were highly sensitive to ROS stimulation and responded by generating ß-cells. ROS enhanced ß-cell differentiation through modulation of ERK1/2 signaling. Gene transfer and pharmacological manipulations, which diminish cellular ROS levels, also interfered with normal ß-cell differentiation. This study highlights the role of the redox balance on ß-cell development and provides information that will be useful for improving ß-cell production from embryonic stem cells, a step in cell therapy for diabetes.

Placental antiangiogenic prolactin fragments are increased in human and rat maternal diabetes.

INTRODUCTION/OBJECTIVES: The role of the placenta in diabetic mothers on fetal development and programming is unknown. Prolactin (PRL) produced by decidual endometrial cells may have an impact. Although full-length PRL is angiogenic, the processed form by bone morphogenetic protein-1 (BMP-1) and/or cathepsin D (CTSD) is antiangiogenic. The objectives were to investigate the involvement of decidual PRL and its antiangiogenic fragments in placentas from type-1 diabetic women (T1D) and from pregnant diabetic rats with lower offspring weights than controls. METHODS: PRL, BMP-1, and CTSD gene expressions and PRL protein level were assessed in T1D placentas (n=8) at delivery and compared to controls (n=5). Wistar rats received, at day 7 of pregnancy, streptozotocin (STZ) (n=5) or nicotinamide (NCT) plus STZ (n=9) or vehicle (n=9). Placental whole-genome gene expression and PRL western blots were performed at birth. RESULTS: In human placentas, PRL (p<0.05) and BMP-1 (p<0.01) gene expressions were increased with a higher amount of cleaved PRL (p<0.05) in T1D than controls. In rats, diabetes was more pronounced in STZ than in NCT-STZ group with intra-uterine growth restriction. Decidual prolactin-related protein (Dprp) (p<0.01) and Bmp-1 (p<0.001) genes were up-regulated in both diabetic groups, with an increased cleaved PRL amount in the STZ (p<0.05) and NCT-STZ (p<0.05) groups compared to controls. No difference in CTSD gene expression was observed in rats or women. CONCLUSIONS: Alterations in the levels of the PRL family are associated with maternal diabetes in both rats and T1D women suggesting that placental changes in these hormones impact on fetal development.

Cell-based therapy of diabetes: what are the new sources of beta cells?

Diabetes affects 246 million people around the world. To date, no definitive cure has been discovered. Recent clinical trials have shed light on the possibility of successfully transplanting adult pancreatic islets into type 1 diabetic recipients. However, despite encouraging efforts to improve such protocols, the poor availability of pancreatic islets remains a limiting parameter for these transplantation programmes. In the present review, different strategies to obtain other sources of islet beta cells are discussed.

New insights into endocrine pancreatic development: the role of environmental factors.

The pancreas is a mixed gland that contains endocrine and exocrine components. Within the pancreatic islets, beta cells produce insulin and control the glycemia. Their deficiency leads to diabetes and several potential complications. In the last decade, numerous studies have focused on pancreas development. The objective was to characterize the cellular and molecular factors that control the differentiation of endocrine and exocrine cell types. Investigation of the role of transcription factors by using genetic approaches led to the discovery of key molecules that are expressed both in rodents and humans. Some of them are ubiquitous, and some others are specifically involved in endocrine or exocrine specification. In addition to these intrinsic factors, recent studies have focused on the role of environmental factors. In the present review, we describe the roles of nutrients and oxygen in the embryonic pancreas. Interestingly, these extrinsic parameters can interfere with beta-cell differentiation and function. Altogether, these data should help to generate beta cells in vitro and define strategies for a cell-based therapy of type 1 diabetes.

Beta-cell development: the role of intercellular signals.

Understanding in detail how pancreatic endocrine cells develop is important for many reasons. From a scientific point of view, elucidation of such a complex process is a major challenge. From a more applied point of view, this may help us to better understand and treat specific forms of diabetes. Although a variety of therapeutic approaches are well validated, no cure for diabetes is available. Many arguments indicate that the development of new strategies to cure diabetic patients will require precise understanding of the way beta-cells form during development. This is obvious for a future cell therapy using beta-cells produced from embryonic stem cells. This also holds true for therapeutic approaches based on regenerative medicine. In this review, we summarize our current knowledge concerning pancreatic development and focus on the role of extracellular signals implicated in beta-cell development from pancreatic progenitors.

Transcription factors involved in pancreas development are expressed in paediatric solid pseudopapillary tumours.

AIMS: Solid pseudopapillary tumours (SPT) are rare pancreatic tumours, especially in children. The origin of this benign tumour remains unknown. Mutations of beta-catenin, a gene essential for pancreatic development, are constantly found, leading to delocalization of immunohistochemical signals from the cytoplasm to the nuclei of tumour cells. The aim was to report clinical and histological data of eight children with SPT and explore the immunohistochemical expression of pancreatic duodenal homeobox (PDX) 1 and Sox9, known to be crucial for pancreatic development and linked to the beta-catenin cascade. METHODS AND RESULTS: Eight children with features suggestive of SPT underwent surgical resection. Tumours displayed typical histological appearances. One was incompletely resected and recurred. Immunolabelling revealed nuclear location of beta-catenin in all cases and strong cytoplasmic but no nuclear expression of PDX1 or Sox9 in all but one case. CONCLUSIONS: The clinical behaviour of SPT in the paediatric population is similar to its adult counterpart. Complete surgical resection is essential. PDX1 and Sox9 proteins are exclusively expressed in the cytoplasmic compartment in SPT, suggesting overexpression of the corresponding genes linked to beta-catenin mutations. These findings favour the hypothesis that SPT originates from transformation of normally quiescent pancreatic stem cells.

Different mechanisms operating during different critical time-windows reduce rat fetal beta cell mass due to a maternal low-protein or low-energy diet.

AIMS/HYPOTHESIS: Adverse events during intra-uterine life may programme organ growth and favour disease later in life. In animals, protein or energy restriction during gestation alters the development of the endocrine pancreas, even though the duration of malnutrition is different. Here, we evaluate the specific effects of both diets during different periods of gestation and the mechanisms underlying the decreased beta cell mass. METHODS: Pregnant Wistar rats were fed either a low-protein or a low-energy diet during the last week of gestation or throughout gestation. Fetuses and their pancreases were analysed at days 15 and 21 of gestation. RESULTS: The low-energy diet reduced the beta cell mass from 21-day-old fetuses by 33 or 56% when administered during the last week or throughout gestation, respectively. Fetal corticosterone levels were increased. At 15 days of fetal age, the number of cells producing neurogenin 3 (NEUROG3) or pancreatic and duodenal homeobox gene 1 (PDX-1) was reduced. Neither islet vascularisation nor beta cell proliferation was affected. The low-protein diet, in contrast, was more efficient in decreasing the fetal beta cell mass when given during the last week of gestation (-53%) rather than throughout gestation (-33%). Beta cell proliferation was decreased by 50% by the low-protein diet, independently of its duration, and islet vascularisation was reduced. This diet did not affect NEUROG3- or PDX-1-positive cell numbers. CONCLUSION/INTERPRETATION: Although both diets reduced the fetal beta cell mass, the cellular mechanisms and the sensitivity windows were different. Early alteration of neogenesis due to elevated corticosterone levels is likely to be responsible for the decreased beta cell mass in low-energy fetuses, whereas impaired beta cell proliferation and islet vascularisation at later stages are implicated in low-protein fetuses.

Calsenilin is required for endocrine pancreas development in zebrafish.

Calsenilin/DREAM/Kchip3 is a neuronal calcium-binding protein. It is a multifunctional protein, mainly expressed in neural tissues and implicated in regulation of presenilin processing, repression of transcription, and modulation of A-type potassium channels. Here, we performed a search for new genes expressed during pancreatic development and have studied the spatiotemporal expression pattern and possible role of calsenilin in pancreatic development in zebrafish. We detected calsenilin transcripts in the pancreas from 21 somites to 39 hours postfertilization stages. Using double in situ hybridization, we found that the calsenilin gene was expressed in pancreatic endocrine cells. Loss-of-function experiments with anti-calsenilin morpholinos demonstrated that injected morphants have a significant decrease in the number of pancreatic endocrine cells. Furthermore, the knockdown of calsenilin leads to perturbation in islet morphogenesis, suggesting that calsenilin is required for early islet cell migration. Taken together, our results show that zebrafish calsenilin is involved in endocrine cell differentiation and morphogenesis within the pancreas.

Fibroblast growth factors 7 and 10 are expressed in the human embryonic pancreatic mesenchyme and promote the proliferation of embryonic pancreatic epithelial cells.

AIMS/HYPOTHESIS: The fibroblast growth factor (FGF) family consists of 22 members. In rodents, several FGFs are expressed in the pancreas, where they participate in epithelial-mesenchymal interactions. Our objective was to describe the pattern of expression of FGFs in the human embryonic pancreas and to analyse their effect on pancreas development. METHODS: The expression of FGFs was analysed by RT-PCR. To investigate the cell types expressing FGF7 and FGF10, we separated epithelial from mesenchymal cells using immunomagnetic beads linked to E-cadherin antibodies and performed real-time PCR. The effect of FGF7 and FGF10 on proliferation of human embryonic pancreatic epithelial cells was evaluated in vitro by measuring BrdU incorporation. RESULTS: We found that different FGFs are expressed in the human embryonic pancreas, and we focused on FGF7 and FGF10. We defined a new approach to separating epithelial cells (containing the pancreatic progenitor cells) from mesenchymal cells. This allowed us to demonstrate that human embryonic pancreatic mesenchymal cells express both FGF7 and FGF10. We next demonstrated that FGF7 and FGF10 were able to induce the proliferation of the epithelial cells in vitro. CONCLUSION/INTERPRETATION: These findings indicate that it is now possible to efficiently separate human embryonic pancreatic epithelial from mesenchymal cells, an important step to characterize and expand progenitor cells. This method allowed us to demonstrate that human embryonic pancreatic mesenchyme expresses FGF7 and FGF10 that act on epithelial cells to activate their proliferation. Such growth factors could thus be used to expand human embryonic pancreatic epithelial cells.

Differential expression and imprinting status of Ins1 and Ins2 genes in extraembryonic tissues of laboratory mice.

There are two functional insulin genes in the mouse genome. The Ins2 gene is imprinted and expressed monoallelically from the paternal allele in the yolk sac. In the present study we have re-examined the imprinting status of Ins1. We found that Ins1 is not expressed in the yolk sac of several laboratory mouse strains. The asynchrony of replication at the wild type locus was significantly lower than at imprinted loci and was more similar to non-imprinted loci. Finally, we have taken the advantage of the Ins1(neo) allele created by homologous recombination to examine the allelic usage at this locus. We observed that the neo gene inserted at the Ins1 locus was expressed from both the paternally and the maternally transmitted allele. Therefore, the Ins1 gene does not share any of the basic properties of imprinted genes. On the basis of these data, we concluded that Ins1 locus is unlikely to be imprinted in common laboratory mice.

Increased islet cell proliferation, decreased apoptosis, and greater vascularization leading to beta-cell hyperplasia in mutant mice lacking insulin.

The targeted disruption of the two nonallelic insulin genes in mouse was reported previously to result in intrauterine growth retardation, severe diabetes immediately after suckling, and death within 48 h of birth. We have further used these animals to investigate the morphology and cell biology of the endocrine pancreas in late gestation and at birth when insulin is absent throughout development. Pancreatic beta-cells were identified by detecting the activity of the LacZ gene inserted at the Ins2 locus. A significant increase in the mean area of the islets was found at embryonic d 18.5 (E18.5) and in the newborn in Ins1-/-, Ins2-/- animals compared with Ins1-/-, Ins2+/- and wild-type controls, whereas the blood glucose levels were unaltered. The individual size of the beta-cells in the insulin-deficient fetuses was similar to controls, suggesting that the relative increase in islet size was due to an increase in cell number. Immunohistochemistry for proliferating cell nuclear antigen within the pancreatic ductal epithelium showed no differences in labeling index between insulin-deficient and control mice, and no change in the number of beta-cells associated with ducts, but the relative size distribution of the islets was altered so that fewer islets under 5,000 microm(2) and more islets greater than 10,000 microm(2) were present in Ins1-/-, Ins2-/- animals. This suggests that the greater mean islet size seen in insulin-deficient animals represented an enlargement of formed islets and was not associated with an increase in islet neogenesis. The proportional contribution of alpha- and beta-cells to the islets was not altered. This was supported by an increase in the number of cells containing immunoreactive proliferating cell nuclear antigen in both islet alpha- and beta-cells at E18.5 in insulin-deficient mice, and a significantly lower incidence of apoptotic cells, as determined by molecular histochemistry using the terminal deoxynucleotidyl transferase-mediated deoxy-UTP nick end labeling reaction. The density of blood vessels within sections of whole pancreas, or within islets, was determined by immunohistochemistry for the endothelial cell marker CD31 and was found to be increased 2-fold in insulin-deficient mice compared with controls at E18.5. However, no changes were found in the steady-state expression of mRNAs encoding vascular endothelial growth factor, its receptor Flk-1, IGF-I or -II, the IGF-I and insulin receptors, or insulin receptor substrates-1 or -2 in pancreata from Ins1-/-, Ins2-/- mice compared with Ins1-/-, Ins2+/- controls. Thus, we conclude that the relative hyperplasia of the islets in late gestation in the insulin-deficient mice was due to an increased islet cell proliferation coupled with a reduced apoptosis, which may be related to an increased vascularization of the pancreas.

Compensatory responses in mice carrying a null mutation for Ins1 or Ins2.

Intrauterine growth retardation and postnatal acute diabetes result from insulin deficiency in double homozygous null mutants for Ins1 and Ins2 (Duvillié B, et al., Proc. Natl. Acad. Sci. USA 94:5137-5140, 1997). The characterization of single homozygous null mutants for Ins1 or Ins2 is described here. Neither kind of mutant mice was diabetic. Immunocytochemical analysis of the islets showed normal distribution of the endocrine cells producing insulin, glucagon, somatostatin, or pancreatic polypeptide. Analysis of the expression of the functional insulin gene in Ins1-/- or Ins2-/- mice revealed a dramatic increase of Ins1 transcripts in Ins2-/- mutants. This compensatory response was quantitatively reflected by total pancreatic insulin content similar for both types of mutants and wild-type mice. Moreover, both mutants had normal plasma insulin levels and normal glucose tolerance tests. The determination of beta-cell mass by morphometry indicated beta-cell hyperplasia in the mutant mice. The beta-cell mass in Ins2-/- mice was increased almost threefold, which accounts for the increase of Ins1 transcripts in Ins2-/-mutants. This study thus contributes to evaluate the potential of increasing the beta-cell mass to compensate for low insulin production.

Pancreatic development and adult diabetes.

Low birth weight is an important risk factor for type 2 diabetes in later life. Maturity-onset diabetes of the young has been linked to genetic sequence abnormalities in transcription factors known to be involved in endocrine pancreatic development. These observations suggest that both the maternal environment and the fetal genome can influence the number and/or function of pancreatic beta cells in early life, and that this has life-long implications for postnatal diabetes. This article reviews the evidence that suggests that beta cells derive from a neogenic process within the pancreatic ductal epithelium, controlled by specific transcription factors and locally acting peptide growth factors. In rodents, many of the fetal phenotypes of beta cells are destroyed during neonatal life in a developmental apoptosis and are replaced by a second wave of neogenesis. This results in islets with insulin release characteristics suited to postnatal life. The timing and amplitude of these ontological events are altered by nutritional sufficiency, and this may be mediated by changes in pancreatic growth factor expression, particularly of the IGF axis. Because beta-cell plasticity after the perinatal period is limited, a dysfunctional programming of beta-cell ontogeny may present a long-term risk factor for glucose intolerance and type 2 diabetes. This critical window of pancreatic development is likely to occur in third trimester of human development.

Genetic manipulation of insulin action and beta-cell function in mice.

Transgenic and gene targeting approaches have now been applied to a number of genes in order to investigate the metabolic disorders that would result by manipulating insulin action or pancreatic beta-cell function in the mouse. The availability of such mutant mice will allow in the future to develop animal models in which the pathophysiologies resulting from polygenic defects might be reconstituted and studied in detail. Such animal models hopefully will lead to better understanding of complex polygenic diseases such as non-insulin-dependent diabetes mellitus (NIDDM).

Imprinting at the mouse Ins2 locus: evidence for cis- and trans-allelic interactions.

The mouse gene encoding preproinsulin 2 (Ins2) is located on the distal end of chromosome 7 in a region of several hundred kilobases that contains several imprinted genes. The exclusive expression of the Ins2 paternal allele in the visceral yolk sac during the last part of gestation indicates that Ins2 also is imprinted. However, in other tissues in which Ins2 is expressed, both alleles are active at all developmental stages. Taking advantage of two mouse strains carrying different null mutations introduced at the Ins2 locus via homologous recombination in ES cells, we examined whether genes inserted at the Ins2 locus become imprinted and have the same restricted pattern of monoallelic expression. In the first null allele, Ins2 was replaced by LacZ, under the control of the endogenous Ins2 promoter, and a Neo cassette with its own promoter was inserted 3' to LacZ (Zneo allele). In the second null allele, Ins2 and its promoter were replaced by the same Neo cassette (Neo allele). Expression of the maternally and paternally inherited genes was monitored by RT-PCR performed on various reciprocal crosses involving the two mutants and the wildtype alleles. In (Zneo x wildtype) F1 embryos, the pattern of LacZ expression was similar to that of Ins2; i.e., LacZ is expressed in the yolk sac only when paternally inherited, while its expression in the embryo proper is independent of its paternal or maternal origin. For both of the mutant alleles, Neo was transcribed only when paternally inherited, in the yolk sac as well as in the embryo. Unexpectedly, we found that LacZ transcription on the maternal chromosome varied depending on the nature of the allele on the paternal chromosome. While fully expressed in the embryo when the paternal chromosome carries the wildtype allele, the maternally inherited LacZ is extinguished when the paternal allele is the Neo allele. The major conclusion from our results is that individual genes introduced into an imprinted chromosomal domain can become imprinted, indicating the influence of long-range cis-acting effects. In addition, our data suggest that the two parental alleles may "communicate" with each other and influence the transcription at the locus.

Phenotypic alterations in insulin-deficient mutant mice.

Two mouse insulin genes, Ins1 and Ins2, were disrupted and lacZ was inserted at the Ins2 locus by gene targeting. Double nullizygous insulin-deficient pups were growth-retarded. They did not show any glycosuria at birth but soon after suckling developed diabetes mellitus with ketoacidosis and liver steatosis and died within 48 h. Interestingly, insulin deficiency did not preclude pancreas organogenesis and the appearance of the various cell types of the endocrine pancreas. The presence of lacZ expressing beta cells and glucagon-positive alpha cells was demonstrated by cytochemistry and immunocytochemistry. Reverse transcription-coupled PCR analysis showed that somatostatin and pancreatic polypeptide mRNAs were present, although at reduced levels, accounting for the presence also of delta and pancreatic polypeptide cells, respectively. Morphometric analysis revealed enlarged islets of Langherans in the pancreas from insulin-deficient pups, suggesting that insulin might function as a negative regulator of islet cell growth. Whether insulin controls the growth of specific islet cell types and the molecular basis for this action remain to be elucidated.