Insulin Binding and Metabolism by Primary Cultures of Rat Hepatocytes: Evidence for Intracellular Location of the Low Affinity Binding Sites*

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Abstract
Insulin binding, dissociation, and degradation were studied in primary cultures of rat hepatocytes. These cells present an advantage over freshly isolated hepatocytes, since they have repaired any damage to the plasma membrane occasioned during isolation and do not leak proteolytic enzymes into the medium. Intact cells were compared to isolated hepatocyte plasma membrane. It was found that: 1) the cultured hepatocytes had high and low affinity binding sites, while membrane had only one type of site, with a Ka corresponding to that of the high affinity sites of intact cells; 2) after binding to equilibrium at 22 C, the rate of dissociation of insulin from intact cells was biphasic, while dissociation from membrane showed a single rate, very similar to the more rapid of the rates of dissociation from intact cells; 3) the amount of insulin associated with the plasma membrane of cells which had previously been allowed to bind to equilibrium (32% of the total) corresponded to that which dissociated rapidly (28–32%); 4) 85% or more of cell-associated insulin was intact after binding to equilibrium at both 37 and 4 C; 5) The relationship between the amount of insulin bound to cells and the rate of degradation was biphasic; degradation increased slowly as the amount of bound insulin increased to the calculated maximum binding of high affinity sites, while it increased rapidly as binding increased beyond that level; and 6) chloroquine caused a doubling of the low affinity binding without affecting the Ka. It was also found that after short periods of binding at 22 or 37 C, dissociation from plasma membrane was biphasic; subsequently, dissociation became monophasic, at a rate corresponding to the slower of the two initial rates. This change occurred after a shorter time at 37 than at 22 C and did not occur at 4 C. The following interpretations were made from this evidence. 1) The high affinity sites are the receptors on the plasma membrane, while insulin bound to low affinity sites is intracellular and largely intact. The latter dissociates more slowly from the cell and is the immediate substrate for degradation. 2) There is a temperature- and time-dependent conformational change in the membrane-bound receptor resulting from occupancy by insulin.

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