Insulin and insulin-like growth factor II permit nerve growth factor binding and the neurite formation response in cultured human neuroblastoma cells.

Abstract

In serum-free medium, SH-SY5Y human neuroblastoma cells specifically and reversibly lost the capacity to bind 1251-labeled nerve growth factor (NGF) to the highaffinity sites (slow sites) and to respond by neurite outgrowth, unless physiological concentrations of insulin or insulin-like growth factor II were present. In serum-containing medium, anti-insulin antiserum decreased the neurite formation response to NGF, and insulin supplementation increased the number of available NGF slow sites. The low-affinity NGF fast sites are absent from SH-SY5Y cells and did not emerge on treatment with insulin. Insulin potentiated the induction of neurites by NGF in rat pheochromocytoma PC12 cells also. These results implicate a wider role for insulin and its homologs in the nervous system. Nerve growth factor (NGF) is important to the development of the sympathetic and sensory neurons of vertebrates and can cause outgrowth of axons in vitro and in vivo (1-3). We have used the human neuroblastoma SH-SY5Y cell as a model in which to study the mechanism of neurite formation (4-6). Cloned SH-SY5Y cells (7) respond to NGF with increased neurite outgrowth (4, 8) and veratridine-dependent Na' uptake (8). The neurites show ultrastructural maturation in cells treated with NGF (8) and end in typical neuronal growth cones (9).High-affinity sites (slow sites) and low-affinity sites (fast sites) that bind 1251-labeled NGF are present in sensory (10, 11), sympathetic (12), and pheochromocytoma PC12 (13,14) cells. Both sites are also present in some human neuroblastoma cell lines, but only the slow type site is present in SH-SYSY cells (4), suggesting that binding to the fast sites is not required for neurite outgrowth. The hypothesis that the slow sites are the receptors for neurite outgrowth would be considerably more attractive if it were possible to activate and inactivate the capacity of these sites to bind NGF and to show corresponding activation and inactivation of the capacity to respond by neurite outgrowth. We show that SH-SY5Y cells reversibly lose virtually all capacity to bind NGF and respond by neurite outgrowth in serum-free media (SFM). Moreover, both capacities are retained in media containing insulin or insulin-like growth factor II (IGF-II).MATERIALS AND METHODS Materials. Pork pancreatic insulin (24 units/mg) and phorbol 12,13-dibutyrate (PBt2) were from Sigma. Insulin was dissolved in 0.01 M HCl and stored at -20°C. Rat liver cell multiplication-stimulating activity, which is the same as IGF-II (15), of about 85% purity, was from Collaborative Research. The ,3 subunit of NGF was prepared from male mouse saliva (16); purity was confirmed by the single band in polyacrylamide gels subjected to isoelectric focusing (pH 3.5-10), or NaDodSO4 gel electrophoresis and by bioassay (17). Anti-insulin antiserum was from Cappel Laboratories (Cochranville, PA). The cloned cell line SH-SY5Y (7) was a kind gift from June L. Biedler and Barbara A. Spengler. Cells between passage numbers 9 and 29 were studied. The cloned PC12 ce...

Insulin in the Brain: Sources, Localization and Functions

“…However, they observed that the anti-NGF antiserum is not able to completely inhibit the effects of insulin on neurite outgrowth, while the anti-insulin antiserum abolished the neurite outgrowth process. Therefore, they not only did not rule out the possible direct effect of insulin on neurite growth, but also proposed that insulin might be acting indirectly through the remaining non-neuronal cells in the cultures they used [242]. Two years later, these investigators [243] reported similar results in sympathetic and sensory neurons.…”

“…Recio-Pinto et al [242] reported that insulin and IGF-2 are necessary for NGF to bind its receptor and stimulate neurite formation and that, in the absence of insulin, the SH-SY5Y and PC12 cells fail to respond to NGF. So, insulin and its homologous IGF-2 may have a permissive role in NGF-directed neurite outgrowth.…”

Identification of a key regulatory element for the basal activity of the human insulin-like growth factor II gene promoter P3

“…Mice carrying IGF-II-null mutations exhibit a body weight decrease to 60 %, revealing the importance of IGF-II for fetal development [2]. IGF-II is also a potent stimulator of cell differentiation, as was shown for neuronal cells [3] and muscle cells [4]. In addition to its differentiation potential, IGF-II also stimulates the proliferation of many different cells in culture [5].…”

Cocaine differentially inhibits neuronal differentiation and proliferation in vitro.

“…Neuronal differentiation was estimated after 72 h exposure to NGF by morphological parameters, specifically by the striking appearance of axondendritic processes > 40 gm long (or about four cell diameters) detected by phase contrast microscopy (20,21,23). Differentiation was quantitated as the percentage of cells bearing axondendritic processes greater than four cell diameters in length (20,21 ).…”