A protocol has been developed for maintaining isolated rat adipose cells in primary tissue culture. Using this protocol, cells remain fully viable and responsive to insulin for at least 24 h, as assessed by measuring 3-0-methylglucose transport, lipogenesis from [U-14C]-glucose, and the incorporation of [35S] methionine into total membrane protein. The acute insulin-induced internalization of its own receptor was then examined by biosynthetically labeling cells in culture with either [35S] methionine or [3H[ glucosamine, maximally inducing receptor internalization with a 30-min incubation in the presence of saturating insulin, and preparing plasma and low-density microsomal membrane fractions by differential ultracentrifugation. Receptors were immunoprecipitated with anti-receptor antiserum, and the receptor subunits separated by NaDodSO4-PAGE under reducing conditions and analyzed by autoradtography. When cells not acutely treated with insulin are examined, both the 135K α- and 95K β-receptor subunits are prominently labeled in the plasma membrane fraction, but only faintly labeled in the low-density microsomal membrane fraction. Following the induction of maximal acute receptor internalization, both subunits are decreased by 20–30% in the plasma membrane fraction and concomitantly increased in the low-density microsomal membrane fraction. However, the relative molecular weights and labeling intensities of the two subunits remain constant and correspond to those observed in the biosynthetically labeled human lymphocyte receptor. A minor band of Mr ≃ 190K is also labeled, but its labeling intensity is similar in the two membrane fractions from basal cells and does not change in response to insulin. This band has not been previously detected in the rat adipose cell, but is thought to represent either a minor receptor component or a precursor of either or both of the major receptor subunits in human lymphocytes. Thus, an experimental system is now available for studying the chronic regulation of both the insulin receptor and the response to insulin in a primary target cell for insulin action. In addition, this system has been used to demonstrate that the acute insulin-induced internalization of its own receptor in the isolated rat adipose cell appears to comprise the subcellular translocation of intact receptors.

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