Influence of ambient glucose and insulin concentrations on adipocyte insulin binding

To elucidate factors of importance for insulin binding, fat cells from humans and rats were incubated under various experimental conditions for different periods of time. Human adipocytes incubated for 24 hours in the absence of insulin showed no significant difference in insulin binding compared with cells from freshly excised tissue. After 48 hours, however, an increased rate of binding (average 54%; P less than 0.05) was obtained. The addition of insulin (2000 microU/ml) to the culture medium resulted in a decrease in insulin binding (average 33%; P less than 0.05) compared with cells maintained in the absence of insulin. There was no apparent difference in receptor affinity, indicating that the altered binding was due to a change in receptor number. In the absence of insulin, elevating the glucose concentration of the medium from 0.8 mM to 22.4 mM did not significantly influence insulin binding. Rat adipocytes showed similar but more rapid changes. Thus, incubation for 24 hours without insulin caused an increase in insulin binding (average 37%; P less than 0.05). This up-regulation was seen even in a high glucose concentration (28 mM) but was completely prevented by the presence of insulin in the medium. Furthermore, when rat adipocytes were incubated with insulin in the presence of a high glucose concentration (28 mM) there was a significant further decrease in insulin binding compared with that of parallel incubations performed in 5.6 mM glucose. Thus, even in the absence of TRIS buffer, insulin-dependent regulation of the number of binding sites is shown for both human and rat adipocyte tissue in vitro. Although this perturbation could be directly due to hormone-receptor interaction at the membrane level, the finding of rat adipocytes that the ambient glucose concentration can modulate this effect suggests the importance of post-receptor events.