Assay of insulin receptors in mouse brain

We previously demonstrated that mouse adrenergic system undergoes age-related impairments which can be reversed by grafting thymus into old animals. Recently, our attention is devoted to brain insulin receptors (InsRs), because of their possible involvement in neuromodulation of monoaminergic systems. The paucity of information on brain InsRs in general, and on mouse in particular, prompted us to look for methods by which brain InsR characteristics can be determined accurately, before beginning a study on possible age-dependent modifications of this receptor system and their eventual recovery by thymus graft. Brain insulin receptor characteristics were studied in a group of young Balb/c-nu mice by binding competition experiments, set up incubating fresh brain membranes with a constant amount of 125I-insulin in presence of increasing concentrations of cold insulin. Experimental data were analysed using both one-site and two-site models. Comparison of results demonstrates that curvilinear Scatchard plot of brain InsRs is indicative of the presence of two binding sites with high and low affinity, respectively. Data also shows that density and affinity of the high affinity receptor subset can be determined accurately, while the low affinity receptor subpopulation presents a high degree of interindividual variability for both density and affinity. It can be concluded that this method of determination of InsR characteristics can be safely used to deepen the study of thymus graft-induced recovery of age-related modifications of brain InsR system.