Binding of [125I]insulin to specific receptors and stimulation of nucleotide incorporation in cells cultured from rat brain

The occurrence of insuling receptors and biological responses to insulin has been investigated in trypsin-dissociated fetal rat brain cells maintained in culture for 8 days. Binding of ¹²⁵]insulin to brain cells in culture was time- and pH-dependent and 85–90% specific. Porcine insulin competed for ¹²⁵]insulin binding in a dose-dependent manner. Unrelated polypeptides, including angiotensin II, glucagon, bovine growth hormone, and bovine prolactin did not compete for ¹²⁵]insulin binding. The half-life of ¹²⁵]insulin dissociation from receptors at 24°C was 15 min and a plot of lnB/Bo] vs time suggested two dissociation rate constants of2.7 × 10⁻⁴ sec⁻¹ and5.0 × 10⁻⁵ sec⁻¹. Scatchard analysis of the binding data gave a curvelinear plot which may indicate negative cooperativity or the occurrence of both high affinity(K_a = 2 × 10¹¹M⁻¹) and low affinity(K_a = 4 × 10¹⁰M⁻¹) sites. Of the estimated total of 4.9 × 10⁴ binding sites per cell, 28–30% appear to be high affinity sites.

Incubation of cultures with insuling caused a time- and dose-dependent stimulation of ³H]thymidine and ³H]uridine incorporation into TCA-precipitable material. Maximum stimulation of thymidine incorporation (2–5-fold) occured 11 h after incubation with 167 nM insulin. The same concentration of insulin caused a 2.2-fold increase in ³H]uridine incorporation in 2 h. These results indicate that cells cultured from rat brain contain specific insulin receptors capable of mediating effects of insulin on macromolecular synthesis in the central nervous system.