Delayed intracellular dissociation of the insulin-receptor complex impairs receptor recycling and insulin processing in cultured Epstein-Barr virus-transformed lymphocytes from insulin-resistant subjects.

Insulin-receptor internalization and processing are defective in insulin-resistant subjects. To assess the reversibility of these defects, we cultured Epstein-Barr virus-transformed-lymphoblasts from six normal, six obese, and six non-insulin-dependent diabetic (NIDDM) subjects in media containing low (5 mmol/l) or high (25 mmol/l) glucose concentrations, and studied the insulin-receptor internalization and processing in vitro. In cells from normal, obese, and NIDDM subjects cultured in low glucose concentrations, exposure to 100 nmol/l insulin for 30 min at 37 degrees C reduced cell-surface 125I-insulin binding to a similar extent (82 +/- 2, 77 +/- 5, and 82 +/- 5% of initial values, respectively). The same results were obtained with cells cultured in high glucose concentrations. In cells cultured under both glucose conditions, and exposed to 100 nmol/l insulin for 30 min at 37 degrees C, a complete recovery of the initial 125I-insulin binding was observed in normal but not in obese and NIDDM subjects. Release of intracellular insulin and its degradation in vitro was determined by incubating cells with 600 pmol/l of 125I-insulin for 60 min at 37 degrees C, acid washing cells, and re-incubating in insulin-free buffer at 37 degrees C. The radioactivity released by cells was characterized by trichloroacetic acid precipitability, Sephadex G-50 column chromatography, and re-binding to fresh cells. Rates of release of internalized radioactivity were reduced in obese and NIDDM subjects (t1/2 = 61 +/- 9 min, p < 0.02; 58 +/- 10 min, p < 0.05; and 38 +/- 4 min in obese, NIDDM, and normal subjects, respectively). The percentage of intact insulin released from cells was significantly higher in obese and NIDDM subjects than in the normal subjects. The t1/2 of intracellular dissociation of insulin-receptor complexes measured by a polyethylene glycol assay was lower in normal (6 +/- 1 min) than in obese (12 +/- 2 min, p < 0.03) and NIDDM subjects (14 +/- 3 min, p < 0.02). The results suggest that in insulin-resistant subjects a primary defect in intracellular dissociation of insulin is responsible for alterations of receptor recycling and insulin processing.