Effects of Preincubation on the Ability of Rat Adipocytes to Bind and Respond to Growth Hormone*

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Abstract
To examine whether the binding of human GH (hGH) to target cells changes when metabolic responsiveness to the hormone changes, the stimulation by hGH of glucose oxidation and incorporation into lipids and the binding of [125I]iodohGH were measured simultaneously in isolated adipocytes of normal rats. When studied at 37 C, freshly prepared adipocytes failed to respond metabolically to hGH added in vitro but did bind hGH rapidly and reversibly. In contrast, adipocytes that were preincubated for 3 h at 37 C were metabolically responsive to hGH at concentrations as low as 100 ng/ml. The specific binding of hGH to preincubated adipocytes was also increased over a wide range of hGH concentrations tested. Metabolic responses to hGH and increased hGH binding were also observed in cells that were preincubated at 37 C for only 90 min. Preincubation did not alter the rate of dissociation of hGH from the cells. Although some degradation of [125I]iodo-hGH occurred when the hormone was incubated with the cells at 37 C, the amount of degradation was not different in the presence of fresh or preincubated cells. Preincubation also increased the ability of adipocytes of hypophysectomized rats to bind hGH, indicating that the increased binding of hGH to preincubated normal rat adipocytes is not due exclusively to removal of endogenous GH. Inhibitors of protein synthesis added to the preincubation medium prevented both the induction of metabolic responsiveness and the increase in hGH binding to the cells. Moreover, when the cells were preincubated at 24 C rather than at 37 C, the appearance of metabolic responsiveness and increased hGH binding did not occur. Finally, a second exposure of preincubated adipocytes to collagenase also prevented the induction of responsiveness and the increase in binding, suggesting that the digestion procedure used to isolate the adipocytes damages the GH receptor-effector mechanism and that this damage is reversed to some extent during the preincubation period at 37 C, a condition compatible with metabolism and protein synthesis. Thus, these studies indicate that preincubation renders adipocytes of normal rats responsive to hGH and increases the ability of the cells to bind the hormone. Under all conditions tested, the binding of hGH and the metabolic responses of the cells to the hormone changed in parallel.