Ultrastructural changes in pancreatic beta cells treated with NGF and dbcAMP.

Nerve growth factor (NGF) induces morphological and physiological changes in cultured pancreatic beta-cells, including the extension of neurite-like processes. This latter effect is potentiated by dibutyryl cAMP (dbcAMP). beta-cells cultured under these conditions maintain their immunoreactivity to insulin and gamma-amino-butyric acid (GABA). NGF, dbcAMP, and high glucose concentrations also increase the expression of the GABA-synthesizing enzyme glutamic acid decarboxylase-65 in cultured beta-cells. The aim of this work was to study the effect of NGF alone or in combination with dbcAMP on pancreatic beta-cell ultrastructural morphology, after 10 days in culture. We used light microscopy, scanning electron microscopy, and transmission electron microscopy to analyze the modifications in cell surface and neurite-like projections. Morphometric analysis showed that NGF and/or dbcAMP treatment substantially increased the insulin and GABA content in granules and rough endoplasmic reticulum. Given that pancreatic beta-cells express NGF receptors and that NGF is synthesized and secreted by beta-cells, these results further suggest that NGF could have trophic actions on pancreatic hormone synthesis and/or storage.

Nerve growth factor increases insulin secretion and barium current in pancreatic beta-cells.

We analyzed the effect of a brief exposure to nerve growth factor (NGF) on insulin secretion and macroscopic barium currents of single adult rat pancreatic beta-cells. After a 1-h exposure to NGF (50 ng/ml), single beta-cells show a 2.5-fold increase in the insulin secretion index in 5.6 mmol/l glucose and a nearly twofold increase in 15.6 mmol/l glucose compared with control cells. We have recently demonstrated that pancreatic beta-cells synthesize and secrete NGF. We analyzed the effect of endogenous NGF on insulin secretion by incubating islet cells in the presence of an anti-NGF monoclonal antibody for 1 h in different glucose concentrations. Although the basal insulin secretion index (5.6 mmol/l glucose) is not affected, glucose-stimulated insulin secretion (15.6 mmol/l glucose) is decreased by 41% in the presence of the antibody. This effect is mediated by the activation of the NGF receptor TrkA because the specific inhibitor of Trk phosphorylation K252a also blocks NGF-induced increase in insulin secretion, both in the presence and absence of exogenous NGF. Using the whole-cell variation of the patch-clamp technique, we found that cells exposed to NGF for 5 min exhibit a 32% increase in the average barium current density. These results suggest that the effects of NGF on insulin secretion are partially mediated by an increase in calcium current through Ca channels. These results further suggest that NGF plays an important autoregulatory role in pancreatic beta-cell function. Two targets of short-term NGF-modulation are insulin secretion and calcium-channel activity.

Nerve and fibroblast growth factors as modulators of pancreatic beta cell plasticity and insulin secretion.

Trophic factors such as nerve and fibroblast growth factors are important modulators of beta cell physiology. These two factors induce the extension of neurite-like processes in primary cultures of adult rat beta cells. Moreover, both NGF and FGF enhance glucose-induced insulin secretion. Since beta cells synthesize NGF and pancreatic islet cells produce FGFs, it is possible that autocrine/paracrine interactions may be major regulators of insulin secretion, and impairment of these interactions could lead to pathological states such as diabetes mellitus.

Heterogeneity in glutamic acid decarboxylase expression among single rat pancreatic beta cells.

AIMS/HYPOTHESIS: An isoform of glutamic acid decarboxylase, (GAD)65 has been identified as a pancreatic beta-cell autoantigen in Type I (insulin dependent) diabetes mellitus. We investigated the expression of GAD isoforms among single rat beta cells in culture, under different conditions and the correlation between GAD65 expression and insulin secretion-rate. RESULTS: Independent of culture conditions, 100 % of fresh and cultured beta cells express GAD67. In contrast, considerable heterogeneity in GAD65 expression among single beta cells was observed. After 2 days in culture in 2.6 mmol/l glucose, only 24 % of the beta cells express GAD65. This percentage increases to 39 % in 5.6 mmol/l glucose and to 54 % and 56 % in 11.6 mmol/l and 20.6 mmol/l glucose, respectively. Moreover, reducing glucose concentration from 11.6 to 2.5 mmol/l for 2 days, reduces GAD65 expression by nearly 30 %. After 11 days in culture with 11.6 mmol/l glucose, 50 % of beta cells continue expressing GAD65, this percentage is further increased to nearly 75 % by including either nerve growth factor or dibutyryl cyclic AMP or both in the culture medium. When beta cells are challenged for 1 h with 20.6 mmol/l glucose, 67 % respond forming insulin-immunoplaques. More than two-thirds of insulin-secretors are GAD65-positive, in contrast to only 11 % of the non-secreting cells. Moreover, 87 % of beta cells that have a high insulin secretory rate express GAD65. CONCLUSION/INTERPRETATION: These results show that the most active beta cells, which secrete more insulin, also express GAD65 and that manipulating extracellular glucose may modify the expression of the enzyme and possibly the autoimmune attack in Type I diabetes.

Pancreatic beta cells synthesize and secrete nerve growth factor.

Differentiation and function of pancreatic beta cells are regulated by a variety of hormones and growth factors, including nerve growth factor (NGF). Whether this is an endocrine or autocrine/paracrine role for NGF is not known. We demonstrate that NGF is produced and secreted by adult rat pancreatic beta cells. NGF secretion is increased in response to elevated glucose or potassium, but decreased in response to dibutyryl cAMP. Moreover, steady-state levels of NGF mRNA are down-regulated by dibutyryl cAMP, which is opposite to the effect of cAMP on insulin release. NGF-stimulated changes in morphology and function are mediated by high-affinity Trk A receptors in other mammalian cells. Trk A receptors are present in beta cells and steady-state levels of Trk A mRNA are modulated by NGF and dibutyryl cAMP. Taken together, these findings suggest endocrine and autocrine roles for pancreatic beta-cell NGF, which, in turn, could be related to the pathogenesis of diabetes mellitus where serum NGF levels are diminished.

Neuron-like phenotypic changes in pancreatic ß-cells induced by NGF, FGF, and dbcAMP.

We studied the effects of nerve growth factor (NGF), fibroblast growth factor (FGF), and dibutyryl-cAMP (dbcAMP) on rat pancreatic ß-cell morphology and of NGF and dbcAMP on insulin secretion. After 2 wk in culture, nearly 3% of ß-cells extended neurite-like processes spontaneously; when cells were treated with NGF, almost 30% of them extended processes. In the presence of dbcAMP, almost all ß-cells flattened, and the extension of neurite-like processes was more pronounced in fetal than in adult cells. The most prominent effect, regardless of age, was observed in cells treated with NGF and dbcAMP together, since the percentage of neurite-like bearing ß-cells increased to 50%. ß-cells cultured under these conditions maintained their immunoreactivity to insulin and nearly all ß-cells and their neurite-like processes were also positive to GABA, tubulin, tau protein, and N-CAM. FGF increased the percentage of adult ß-cells bearing neurite-like processes to 13%, and FGF and dbcAMP applied together to 40%. ß-cells treated with NGF and dbcAMP for 5 to 7 d preserved their capability to secrete the hormone in response to different extracellular glucose concentrations. Insulin secretion of dbcAMP-treated ß-cells was 2.5-fold higher than in control cells. NGF-treated cells were able to discriminate between different glucose concentrations, a property lost in control cells with time in culture.

Functional heterogeneity of single pancreatic beta-cells stimulated by L-leucine and the methyl ester of succinic or glutamic acid.

Single pancreatic beta-cells exposed to D-glucose in the absence or presence of L-leucine and to the amino acid in the absence or presence of either the monomethyl ester of succinic acid (SME) or the dimethyl ester of glutamic acid (GME) were examined in a reverse hemolytic plaque assay for insulin release. At a D-glucose concentration of 11.1 mM, plaque-forming cells amounted to 84.8 +/- 1.0% including 11.4 +/- 4.1% of cells forming large plaques. These percentages were not increased by the incorporation of L-leucine (5 mM) into the incubation medium despite the fact that the amino acid, when tested in the absence of D-glucose, caused sizeable secretory activity. When the secretory response evoked by L-leucine was increased by either SME (10 mM) or GME (3 mM), marked heterogeneity of individual beta-cells in terms of both the occurrence and magnitude of hemolytic plaques was again observed. These findings argue against the view that the heterogeneity in secretory behavior of isolated purified beta-cells can be accounted for solely by differences in hexose metabolism as conceivably attributable to individual variations in glucokinase activity.