In vitro recovery of insulin binding after downregulation in cultured normal human monocytes

Abstract

When normal human peripheral blood monocytes were treated with insulin in vitro, surface insulin receptors disappeared rapidly, but total insulin receptors (surface and internalized receptors), measured in detergent-solubilized extracts of total cellular membranes, decreased slowly. Surface receptors decreased to 51 +/- 4, 36 +/- 12, and 34 +/- 12%, of control levels after 2, 6, and 18 h of insulin pretreatment, respectively. Total receptors decreased to 86 +/- 12, 69 +/- 17, and 34 +/- 12% of control levels in the same periods. Chloroquine, a lysosomotropic agent, inhibited the removal of surface receptors, indicating that lysosomal proteases play a role in this process. Unlike monocytes, IM-9 lymphocytes lost surface receptors and total receptors at the same rate when incubated with insulin. Monocytes treated with insulin for 18 h, washed free of unbound insulin and recultured for 48 h regained 94 +/- 7% of control insulin binding, indicating that cultured monocytes are competent to regenerate their insulin receptors. Monocytes treated with insulin for 6 h also required 48 h to recover their insulin binding, despite the fact that substantial numbers of insulin receptors remained intact within these cells. Two-hour pretreated monocytes recovered somewhat faster, attaining control levels of receptors after 24 h of reculture. This suggests that internalized insulin receptors pass from a recyclable pool to a nonrecyclable one.