Keratinocyte growth factor and beta-cell differentiation in human fetal pancreatic endocrine precursor cells

AIMS AND HYPOTHESIS: Keratinocyte growth factor (KGF) is a member of the heparin-binding fibroblast growth factor family with a high degree of specificity for epithelial cells in vitro and in vivo. Our aim was to study the effect of KGF on beta-cell growth and differentiation on islet-like cell clusters derived from human fetal pancreas. METHODS: We investigated the effects of KGF, in vitro, on beta-cell differentiation from undifferentiated pancreatic precursor cells and in vivo after transplantating human fetal pancreatic cells into athymic rats treated with KGF. RESULTS: Treatment of islet-like cell clusters with KGF in vitro did not change the number of insulin producing cells, as measured by the measurement of insulin content or DNA. The in vivo treatment of recipient rats with KGF increased the number of beta cells within the grafts 8 weeks after transplantation. At this time, glucose-stimulated insulin secretion was evaluated by glucose stimulation tests in rats bearing the transplants. Measurements of human C-peptide concentrations after glucose challenge showed that the newly differentiated beta cells in the KGF-treated group were functionally competent as opposed to the control group, where the graft failed to release insulin appropriately. CONCLUSION/INTERPRETATION: These findings suggest that in vivo, KGF is capable of inducing human fetal beta-cell expansion. The growth promoting effect of KGF on beta cells occurred mainly through the activation of ductal cell proliferation and their subsequent differentiation into beta cells.     

Stimulation of pancreatic islet beta-cell replication by oncogenes.

Although the growth potential of the pancreatic islet beta cells is limited, glucose, cAMP, and certain polypeptide growth factors have been reported by other workers to exert modest stimulatory effects on beta-cell replication. To further assess means through which beta-cell growth can be stimulated, selected oncogene constructs linked to a rat insulin promoter were introduced by means of electroporation into free islet cells prepared from fetal rats and adult hyperglycemic obese (ob/ob) mice. The uptake and expression of the added oncogenes were sufficiently efficient to exert effects on beta-cell physiology in short-term experiments (less than or equal to 4 days). Stimulation of islet cell [3H]thymidine incorporation was observed after transfection with src alone or the combination of myc and ras. The effect observed in the fetal islet cells with src was more pronounced than any effect previously reported. Transfection with the src oncogene resulted in phosphorylation of lipocortin I and was paralleled by an increased immunofluorescence against src-like immunoreactivity in a majority of the electroporated cells. It is concluded that electroporation can induce sufficiently efficient expression of added oncogene constructs to study their effects on cells that are not readily transformable into continuously growing cell lines. Furthermore, the results suggest that beta-cell replication might be manipulated extrinsically by inserting appropriate growth-promoting genes into these cells.     

妊娠期母体胰岛的适应性变化

妊娠时随着胎儿对母体物质需求的增加,孕妇出现了糖代谢的改变以及生理性胰岛素抵抗。为了适应胰岛素需求的增加,妊娠期母体胰岛在结构和功能上都会发生相应的改变,包括葡萄糖刺激下胰岛素分泌能力的增强,以及伴随的葡萄糖刺激阈值的下降。此外,还出现母体胰岛素合成增多,胰岛细胞增殖以及胰岛容积的增加。这些变化与妊娠期激素特别是胎盘生乳素(HPL)、催乳素(PRL)及生长激素(GH)等密切相关。不仅如此,脂肪细胞因子、炎症因子、细胞因子信号传递抑制体(SOCS)、胰岛血管内皮细胞等因素对妊娠期母体胰岛的适应性变化也会产生影响。     

胰岛细胞抗体与胰岛β细胞功能关系的探讨

对251例1型糖尿病（T1DM）患者进行胰岛细胞抗体（ICA）检测,发现ICA阳性组中谷氨酸脱羧酶抗体（GADA）和蛋白酪氨酸磷酸酶抗体（IA-2A）阳性率均显著高于ICA阴性组（P〈0.01）,空腹和餐后胰岛素水平明显低于ICA阴性组（P〈0.05,P〈0.01）,胰岛素抵抗指数亦明显下降（P〈0.05）,提示T1DM患者中ICA阳性更易于合并有GADA、1A-2A阳性,且对胰岛β细胞功能的破坏更大。     

Notch signalling suppresses apoptosis in adult human and mouse pancreatic islet cells

Aims/hypothesis The pathogenesis of diabetes and the success of islet transplantation depend on the control of pancreatic beta cell fate. The Notch signalling pathway is essential for normal prenatal pancreatic development, but the presence and function of this gene network in adult islets has received much less attention. Methods The presence of Notch signalling components was assessed in vitro using RT-PCR, western blotting and immunofluorescence. The functional consequences of altering Notch signalling on insulin secretion and programmed cell death were examined. Results Adult mouse islets, human islets and mouse insulinoma MIN6 cells possess key components of the Notch pathway. RT-PCR, western blotting and immunofluorescence indicated that the Notch target gene, neurogenin3 (Ngn3, also known as Neurog3), is also present in adult islet cells. Inhibiting Notch signalling with N-[N-(3,5-difluorophenacetyl-l-alanyl)]-S-phenylglycine t-butyl ester (DAPT) increased Ngn3 mRNA expression and protein levels in adult islets. The activated notch homologue 1 (NOTCH1) protein level was decreased upon serum withdrawal, as well as after treatment with a phosphatidylinositol 3-kinase inhibitor, or hydroxy-2-naphthalenylmethylphosphonic acid, an insulin receptor inhibitor. While islets cultured in DAPT did not exhibit defects in insulin secretion, indicating that differentiation is unaltered, inhibiting gamma-secretase-dependent Notch activation led to a dose-dependent increase in caspase-3-dependent apoptosis in both MIN6 cells and human islets. Conversely, gamma-secretase overactivity resulted in an accumulation of cleaved NOTCH1 and protection from apoptosis. Conclusions/interpretation Together these results show that the Notch/Ngn3 signalling network is intact and functional in adult islets. This pathway represents an attractive target for modulating beta cell fate in diabetes, islet transplantation and efforts to derive beta cell surrogates in vitro.     

Imaging of pancreatic islet cells

At present, the onset and progress of diabetes, and the efficacy of potential treatments, can only be assessed through indirect means, i.e. blood glucose, insulin, or C-peptide measurements. The development of non-invasive and reliable methods for (1) quantification of pancreatic beta islet cell mass in vivo, (2) determining endogenous islet function and survival, and (3) visualizing the biodistribution, survival, and function of transplanted exogenous islets are critical to further advance both basic science research and islet cell therapy in diabetes. Islet cell imaging using magnetic resonance, bioluminescence, positron emission tomography, or single photon emission computed tomography may provide us with a direct means to interrogate islet cell distribution, survival, and function. Current state-of-the-art strategies for beta-cell imaging are discussed and reviewed here in context of their clinical relevance.     

Monolayer cultures of pancreatic islet cells from adult rats: effect of 2-deoxyglucose

Techniques for the monolayer culture of pancreatic islet cells from adult rats and the responsiveness of B cells are described. Whole pancreatic tissue was enzymatically dispersed and then cultured for 30 days in tissue culture medium 199 containing 5.5 mmol glucose/l, with or without 1 mmol 2-deoxyglucose/l. In the absence of 2-deoxyglucose, the responsiveness of B cells diminished to almost zero by day 15 and islets degenerated. In contrast, addition of 2-deoxyglucose to the medium resulted in a selective degeneration of fibroblasts, yielding monolayers that consisted mostly of islet cells. In this stationary system in which monolayers of islet cells were maintained in medium with 2-deoxyglucose, insulin secretion from B cells on days 15 and 30 increased in a dose-dependent fashion in response to increasing concentrations of glucose, leucine and 2-ketoisocaproate. Similarly, when exposed to 16.7 mmol glucose/l, perifused B cells showed a biphasic pattern of insulin secretion on day 15. Addition of 10 mumol forskolin/l and 200 nmol 12-O-tetradecanoyl phorbol-13-acetate/l remarkably enhanced this response. Likewise, the response to 10 mmol leucine/l or 10 mmol 2-ketoisocaproate/l was biphasic. These results suggest that these monolayer cultures retain the functional properties of the adult rat pancreas, and may be useful not only as a model for the in-vitro study of B cell function, but also for implantation.     

Islet endothelial cells and pancreatic beta-cell proliferation: studies in vitro and during pregnancy in adult rats

The growth of both tumors and nonneoplastic tissues may be influenced by signals from the vascular endothelium. In the present investigation we show that purified proliferating endothelial cells from pancreatic islets can stimulate beta-cell proliferation through secretion of hepatocyte growth factor (HGF). This secretion could be induced by soluble signals from the islets, such as vascular endothelial growth factor-A (VEGF-A) and insulin. During pregnancy, the pancreatic beta-cells display a highly reproducible physiological proliferation. We show that islet endothelial cell proliferation precedes beta-cell proliferation in pregnant animals. Vascular growth was closely associated with endocrine cell proliferation, and prominent expression of HGF was observed in islet endothelium on d 15 of pregnancy, i.e. coinciding with the peak of beta-cell proliferation. In summary, our results suggest the existence of an endothelial-endocrine axis within adult pancreatic islets, which is of importance for adult beta-cell proliferation.     

GLP-1 receptor agonists are growth and differentiation factors for pancreatic islet beta cells

Glucagon-like peptide-1 (GLP-1) is an incretin hormone that, when given exogenously, is capable of normalizing blood glucose in individuals with type 2 diabetes. Until recently most of the research on this compound had been related to its insulinotropic properties. However, GLP-1 also regulates insulin synthesis and proinsulin gene expression, as well as the components of the glucose-sensing machinery. In addition to regulating insulin release, it is involved in regulating the secretion of at least two other islet hormones--glucagon and somatostatin. Extraislet effects of GLP-1 include a role in the central nervous system stress response, hypothalamic-pituitary function, and the suppression of gastric emptying. Recent studies from our own and other laboratories show that GLP-1 can regulate islet growth and is a differentiation factor of the endocrine pancreas. This leads us to propose that GLP-1 and GLP-1 receptor agonists, in the context of long-term treatment of type 2 diabetes, will have broader biological action on the endocrine pancreas than was earlier anticipated. We propose that GLP-1 is a growth factor for pancreatic endocrine cells and can increase islet cell mass. Here we review those reports that have highlighted its role as a factor for islet cell growth and differentiation.     

Proendocrine genes coordinate the pancreatic islet differentiation program in vitro

In the developing pancreas, the basic helix-loop-helix (bHLH) protein Neurogenin3 (Ngn3) specifies which precursor cells ultimately will become endocrine cells and initiates the islet differentiation program. NeuroD1, a closely related bHLH protein and a downstream target of Ngn3, maintains the differentiation program initiated by Ngn3. We have developed an in vitro model of Ngn3-dependent differentiation by infecting pancreatic duct cell lines with an Ngn3-expressing adenovirus. We found that both Ngn3 and its downstream target NeuroD1 activated the islet differentiation program in these cells by inducing the expression of genes with early roles in the differentiation cascade, as well as genes characteristic of fully differentiated islet cells. Induction of these genes, as exemplified by the insulin1 gene, involved alteration of the local chromatin structure. Interestingly, the subsets of genes activated by Ngn3 and NeuroD1 were not completely overlapping, indicating that these two bHLH proteins serve specific functions in the development of the endocrine pancreas. In addition, microarray gene expression analysis identified a previously uncharacterized group of Ngn3-induced genes with potentially important roles in islet development and function. These studies demonstrate how Ngn3 initiates islet differentiation and provide us with a model for testing methods for producing islet cells for people with diabetes.     

Internalization of 3H-glibenclamide in pancreatic islet cells

Summary As judged by autoradiographic criteria at the optical level, rat islet cells progressively accumulate ³H-glibenclamide over 1 to 30 min incubation at 24°C. The labeled material associates with all endocrine cells, but is preferentially seen within B cells. The total uptake of ³H-glibenclamide (1.0 mol/l) is little affected by the presence of unlabeled glibenclamide (0.2 mmol/l), but is significantly decreased at 4°C (p < 0.05). At the electron microscopic level, less than 15% of the autoradiographic grains are located at the B cell plasma membrane and 72–79% of the grains found over the cytoplasm are associated with insulin secretory granules. Such a pattern is observed both after short (1 min) or prolonged (30 min) incubation, and at 4 or 24 °C. It is proposed that the insulinotropic action of glibenclamide is not necessarily attributable to primary events located solely at the cell surface.     

Lysosomes and pancreatic islet function: adaptation of beta-cell lysosomes to various metabolic demands

The effect of various functional demands on the lysosomes of pancreatic islet beta cells was studied in vivo. To expose pancreatic islets to different metabolic situations, normal syngeneic mouse islets were transplanted to either lean mice, alloxan-diabetic mice, or obese hyperglycemic mice. Two weeks after transplantation, primary and secondary beta-cell lysosomes of the islet grafts were analyzed by morphometry. The beta-cell lysosomes and secretory granules of the endogenous islets of lean and obese hyperglycemic mice were also measured. The beta cells of the islets transplanted to lean normoglycemic mice showed only a moderately developed synthetic apparatus and a great number of secretory granules. They had mainly secondary lysosomes, frequently containing secretory granule material, indicating a high crinophagic activity. The islet beta cells exposed to the high serum glucose concentration of alloxan-diabetic and obese hyperglycemic mice had an extensive synthetic apparatus, but a very small content of secretory granules. In these beta cells, there was a predominance of small primary lysosomes, indicating a low crinophagic activity. It is concluded that crinophagy may provide a mechanism for the pancreatic beta cell to moderate its content of insulin. When its secretory granule stores are diminished due to increased demands on insulin secretion, the beta cell seems able to drastically decrease the crinophagic activity. The detailed morphometric analysis of the endogenous islets of the lean and obese hyperglycemic mice showed that the beta cells of the obese hyperglycemic mice had a smaller number and size of the secretory granules.(ABSTRACT TRUNCATED AT 250 WORDS)     

Basement membrane exposure defines a critical window of competence for pancreatic duct differentiation from undifferentiated pancreatic precursor cells

We previously showed that the undifferentiated pancreatic epithelium can differentiate into islets, ducts, or acini depending on its milieu and that laminin is necessary for pancreatic duct formation. Therefore we wanted to study the plasticity of laminin-induced duct differentiation the better to understand mechanisms of pancreatic duct lineage selection induced by basement membrane. Mouse embryonic pancreases were dissected at gestational day 11 (E11.5), and epithelium was isolated from its surrounding mesenchyme. Some epithelia were cultured in a collagen gel devoid of laminin. These epithelia were "rescued" at days 1-7 of culture by transferring them to a laminin-rich matrix (Matrigel) for 7 additional days. Other epithelia were instead first cultured in Matrigel, and then placed into collagen. Immunohistochemistry was performed for insulin, amylase, and carbonic anhydrase II. Pancreatic epithelia rescued from collagen into laminin during days 1-4 after harvest were still able to form ducts, whereas epithelia deprived of laminin for longer than this 4-day window were not. Pancreatic epithelia exposed to laminin for as little as 1 day, and then placed into collagen, still retained the ability to make ducts. Thus there is a clear cut-off in the development of the pancreatic epithelium at E11.5, after which laminin appears necessary to induce duct formation. We believe that such "windows of competence" in embryonic development imply that developmental programs in the embryo allow some flexibility.     

Human pancreatic islet tumor cells maintained in vitro

Summary Tumor cells from two human insulinomas were maintained in culture for more than 6 months. The cultured cells synthesized and secreted proinsulin and insulin; and responded to cyclic AMP (1 mM) and tolbutamide (1 mM) stimulation with increased insulin release. In culture, these cells did not respond to stimulation by high concentrations of glucose (300 mg%) or by glucagon (10 g/ml).