Characterization of the altered oligosaccharide composition of the insulin receptor on neural-derived cells

doi:10.1016/0006-8993(88)91592-2

Brain Research

Volume 443, Issues 1–2, 8 March 1988, Pages 1-11

https://doi.org/10.1016/0006-8993(88)91592-2 Get rights and content

Abstract

Typical insulin receptors are present on neuroblastoma cell lines. High affinity binding for insulin was present in membrane preparations from NG108 (a hybrid mouse neuroblastoma-rat glioma) as well as in membranes from SK-N-MC and SK-N-SH, two human neuroblastoma cell lines. Specific [¹²⁵I]insulin binding was 24.4% for NG108, 16.9% for SK-N-MC and 5.2% for SK-N-SH at membrane protein concentrations of 0.4 mg/ml. IC₅₀ for [¹²⁵I]insulin binding was 3.4 nM in NG108 membrane preparations and 0.9 nM for SK-N-SH and 1.8 nM in SK-N-MC membranes. Apparent mol. wt. for the α subunits (identified by specific immunoprecipitation using the anti-insulin receptor antiserum B₁₀) on SDS PAGE was 134 kDa for NG108; 124 kDa for SK-N-MC and 120 kDa for SK-N-SH. Neuraminidase digestion increased the mobility of the α subunit from both NG108 and SK-N-MC receptors to 120 kDa, whereas that from SK-N-SH were unaffected. Endoglycosidase H and endoglycosidase F digestions increased the mobility of the α subunits of all 3 cell lines to varying degrees, suggesting the presence of N-linked glycosy;ation. Insulin induced autophosphorylation of the insulin receptor β subunit in WGA-purified membranes from all 3 cell lines. In addition, phosphorylation of a protein with an apparent mol. wt. 105 kDa was stimulated by insulin in WGA purified membranes from NG108. Tyrosine-specific kinase activity was present in the membranes from each cell line and was stimulated by insulin in a dose-dependent manner from 10⁻⁹ to 10⁻⁶M. Proinsulin was about 100 times less potent in stimulating phosphorylation of the artificial substrate poly (Glu, Tyr)4:1 when compared to insulin in accordance with its lower binding affinity to the insulin receptor. Hexose transport was stimulated by insulin in all 3 cell lines. These results indicate that neuroblastoma cells contain specific insulin receptors and that they may be useful as models for studying the role of insulin in nervous tissue.

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Insulin action regulates the metabolic functions of the classically insulin-responsive tissues: liver, adipose, and skeletal muscle. Evidence also suggests that insulin acts on neural tissue and can modulate neural metabolism, synapse activity, and feeding behaviors. Insulin receptors are expressed on both the vasculature and neurons of the retina, but their functions are not completely defined. Insulin action stimulates neuronal development, differentiation, growth, and survival, rather than stimulating nutrient metabolism, e.g., glucose uptake as in skeletal muscle. Insulin receptors from retinal neurons and blood vessels share many similar properties with insulin receptors from other peripheral tissues, and retinal neurons express numerous proteins that are attributed to the insulin signaling cascade as in other tissues. However, undefined neuron-specific signals downstream of the insulin receptor are likely to also exist. This review compares retinal insulin action to that of peripheral tissues, and demonstrates that the retina is an insulin-sensitive tissue. The review also addresses the hypothesis that dysfunctional insulin receptor signaling in the retina contributes to cell dysfunction and death in retinal diseases.
Binding of enterostatin to the human neuroepithelioma cell line SK-N-MC
1998, Peptides
SK-N-MC cells were found to possess binding sites for enterostatin, a peptide with central effects on appetite and sympathetic activation of brown adipose tissue during high-fat feeding. Scatchard analyses of the binding indicated one high-affinity binding (K_d = 0.5–1.5 nM) and one low-affinity binding (K_d = 15–30 nM) for ³H-enterostatin (APGPR). ¹²⁵I-YGGAPGPR showed similar binding parameters as for the low affinity binding of ³H-APGPR. Met-enkephalin and β-casomorphin_1–5 were found to displace the binding of ³H-APGPR to the SK-N-MC cells. Affinity purification of solubilized cells revealed an APGPR-binding protein estimated to 53 kDa which may represent a distinct enterostatin receptor. Cross-linking of ¹²⁵I-YGGAPGPR to intact cells labeled one major protein with the same molecular size. There was no binding of enterostatin to four other human neuroblastoma/neuroepithelioma cell lines, named IMR-92, LAN#5, NB-1 #14 and SH5-SY.
Insulin binding and fluid-phase endocytosis stimulation in the mouse neuroblastoma cell line 41A3
1996, International Journal of Developmental Neuroscience
As well as many other hormones and growth factors, insulin is known to influence several processes in the CNS; its specific effects, however, are still poorly understood. Neuroblastoma cell lines represent a useful experimental system for the analysis of the insulin-specific effect on neurons, in the absence of possible regulatory mechanisms elicited by other neuronal/glial cells and/or soluble factors. The expression and the binding properties of insulin receptors, as well as the insulin effects on both membrane fluidity and cell surface architecture, have been investigated in 41A3 mouse neuroblastoma cells, respectively by radioligand-binding fluorescence spectroscopy and scanning electron microscopy. In the same cells, insulin-induced modifications on cytoskeletal organisation also have been studied.
Binding studies were performed using ¹²⁵I-insulin, while the cationic fluorescent probe trimethylammonium 1,6-diphenyl-1,3,5-hexatriene was used for biophysical investigations.
The results presented in this paper provide evidence that insulin interacts with 41A3 neuroblastoma cells through a receptor-mediated mechanism and that, in these cells, insulin binding modifies the cell surface morphology and stimulates endocytosis.
Insulin receptor in mouse neuroblastoma cell line N18TG2: Binding properties and visualization with colloidal gold
1992, International Journal of Developmental Neuroscience
Insulin function in the nervous system is still poorly understood. Possible roles as a neuromodulator and as a growth factor have been proposed (Baskin et al., 1987, Ann. Rev. Physiol. 49, 335–347). Stable cell lines may provide an appropriate experimental system for the analysis of insulin action on the various cellular components of the central nervous system.
We report here a study to investigate the presence and the properties of insulin specific binding sites in the murine neuroblastoma line, N18TG2, together with insulin action on cell growth and metabolism. Also, receptor internalization has been studied. Binding experiments, carried out in standard conditions at 20°C, enabled us to demonstrate that these cells bind insulin in a specific manner, thus confirming previous findings on other cell lines. Saturation curves showed the presence of two binding sites with Kd 0.3 and 9.7 nM. Competition experiments with porcine and bovine insulin showed an IC50 of 1 and 10 nM, respectively. Competition did not occur in the presence of the unrelated hormones ACTH and FSH. Dissociation experiments indicated the existence of an internalization process of the ligandreceptor complex; this was confirmed by an ultrastructural study using gold conjugated insulin. As far as the insulin action in N18TG2 cells is concerned, physiological concentrations stimulate cell proliferation, whereas no stimulation of glucose uptake was observed, indicating that insulin action in these cells is not mediated by general metabolic effects.
On the basis of these data, N18TG2 line appears to be a very suitable model for further studies of the neuronal type insulin receptors, and possibly insulin specific action on the nervous system.
Insulin and IGF-I receptors in neuroblastoma cells: Increases in mRNA and binding produced by glyburide
1989, Neuropeptides
Insulin and IGF-I binding to neuroblastoma cells (SK-N-MC) was increased by 13% and 7% respectively following a 24 hr. incubation with the sulphonylurea glyburide. This increase in binding was associated with increased steady-state levels of insulin receptor and IGF-I receptor mRNA levels. Though insulin and IGF-I both stimulate glucose uptake into these cells, the increased binding following glyburide treatment was not associated with any change in glucose uptake.
TPA-induced neurite formation in a neuroblastoma cell line (SH-SY5Y) is associated with increased IGF-I receptor mRNA and binding
1989, Molecular Brain Research

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Research reportCharacterization of the altered oligosaccharide composition of the insulin receptor on neural-derived cells

Abstract

Cell

FEBS Lett.

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

Neuroscience

J. Biol. Chem.

Biochem. Biophys. Res. Commun.

Biochem. Biophys. Acta

Biochem. Biophys. Res. Commun.

Quantitative autoradiographic evidence for insulin receptors in the choroid plexus of the rat brain

Diabetes

Insulin receptors and insulin modulation of norepinephrine uptake in neuronal cultures from rat brain

J. Biol. Chem.

Insulin receptors on cultured hypothalamic cells: functional and structural differences from receptors on peripheral target cells

Endocrinology

Insulin binds to specific receptors and stimulates 2-deoxy-D-glucose uptake in cultured glial cells from rat brain

J. Biol. Chem.

A direct in vitro demonstration of insulin binding to isolated brain microvessels

Diabetes

Insulin receptor of human cerebral gliomas: structure and function

J. Clin. Invest.

Identification of the insulin receptor of cerebral microvessels

Am. J. Physiol.

Insulin receptors are widely distributed in the central nervous system of the rat

Nature (London)

Research report
Characterization of the altered oligosaccharide composition of the insulin receptor on neural-derived cells