Differentiation and function of pancreatic ␤ 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 ␤ 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. NGFstimulated changes in morphology and function are mediated by high-affinity Trk A receptors in other mammalian cells. Trk A receptors are present in ␤ 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 ␤-cell NGF, which, in turn, could be related to the pathogenesis of diabetes mellitus where serum NGF levels are diminished.Nerve growth factor (NGF) has been implicated in the survival and differentiation of neuronal and non-neuronal systems (1). In particular, it induces morphological and physiological changes in pancreatic ␤ cells, including an increase in voltagedependent sodium current density (2) and the extension of neurite-like processes. This latter effect is potentiated by dibutyryl cAMP (dbcAMP) (3). Furthermore, pancreatic ␤ cells treated with NGF in culture (5 days) secrete more insulin in response to stimulation with 20.6 mM glucose than with 5.6 mM glucose. In contrast, with increasing time in culture, insulin secretion of control cells tends to be similar in both glucose concentrations (3). The effects of NGF on ␤ cells are mediated through the high-affinity receptor Trk A (4), which has been shown to exist in pancreatic ␤ cells (5, 6).Together, these data suggest that NGF is an important factor in the maintenance of the endocrine function of ␤ cells in vitro. However, pancreatic NGF has not been detected. A possible source is the pancreatic ␤ cell itself, since it has been observed that endogenous NGF levels in diabetic animals (7) and NGF serum levels in type II diabetic patients (8) are decreased. Nevertheless, despite efforts to detect NGF expression in fetal ␤ cells or in ␤ cell lines by Northern blot analysis (9), to date, neither synthesis nor secretion of NGF by ␤ cells has been demonstrated.In the present study, we demonstrate NGF synthesis and secretion by single adult rat pancreatic ␤ cells. Furthermore, since regulation of the levels of Trk A receptors can modulate responsiveness to NGF (10, 11), we also examined whether NGF and/or dbcAMP modulate ␤ cell Trk A mRNA levels.

Function and survival of cells depend in part on the presence of growth factors. We explored the autocrine regulation of insulin and nerve growth factor (NGF) on single adult rat pancreatic ␤-cell survival and hormone secretion. When NGF or insulin signaling were blocked in culture media, cell survival decreased compared with control cells, with apoptosis being the main mechanism of cell death. To further explore the role of glucose in ␤-cell survival, we cultured the cells for 16 h in 2.6 mmol/l glucose and observed that nearly 17% of the cells developed apoptosis; this effect was partially prevented by NGF and almost completely inhibited by insulin treatment. A high K ؉ concentration had the same effect, suggesting that insulin and NGF secretion by the cells was responsible for the survival effects and not glucose per se. Blocking NGF signaling with an NGF antibody or with K252a reduced insulin biosynthesis and secretion in the cells that survived the treatment. Moreover, the functional ␤-cell subpopulation with a higher insulin secretion rate is more susceptible to K252a. These results further indicate that NGF and insulin play important autoregulatory roles in pancreatic ␤-cell survival and function and strongly suggest the need to explore new focuses in diabetes treatment.

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 ␤-cells. After a 1-h exposure to NGF (50 ng/ml), single ␤-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 ␤-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 ␤-cell function. Two targets of short-term NGF-modulation are insulin secretion and calcium-channel activity.

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We studied the effects of nerve growth factor (NGF), fibroblast growth factor (FGF), and dibutyryl-cAMP (dbcAMP) on rat pancreatic β-cell morphology and of NGF and dbcAMP on insulin secretion. After 2 wk in culture, nearly 3% of β-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 β-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 β-cells increased to 50%. β-cells cultured under these conditions maintained their immunoreactivity to insulin and nearly all β-cells and their neurite-like processes were also positive to GABA, tubulin, tau protein, and N-CAM. FGF increased the percentage of adult β-cells bearing neurite-like processes to 13%, and FGF and dbcAMP applied together to 40%. β-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 β-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.