title: Low pH accelerates dissociation of receptor-bound insulin.
abstract: We have examined the effect of pH of the kinetics of [125I]insulin binding in isolated rat adipocytes and found that a lowering of extracellular pH from 7.8 to 7.0 reduced tracer insulin binding (0.2 ng/ml) from 3.3% to 1.0% due to a decrease in receptor affinity. Further studies, performed at 16 C to eliminate the confounding affects of insulin internalization on binding kinetics, revealed that the t1/2 of insulin dissociation was shortened from 44 min at pH 7.8 to 8 min at pH 7.0, while the association rate constant was slightly increased from 1.61 X 10(9) to 2.09 X 10(9) M-1 min-1 at pH 7.8 and 7.0, respectively. Thus, the overall decrease in binding affinity at low pH is due predominantly to an accelerated rate of insulin dissociation. Kinetic studies performed at 37 C also revealed an effect of pH to accelerate insulin dissociation; however, the magnitude of the effect was greatly reduced. This was explained by the observation that when [125I]insulin was prebound to adipocytes at 37 C approximately half of the cell-associated radioactivity resided within the cell at the start of the dissociation experiment. An additional amount of insulin was internalized during the dissociation time course due to endocytotic uptake of cell surface ligand. Consequently, only a small portion of cell-associated [125I]insulin was on the cell surface and susceptible to extracellular pH changes. To assess the effect of low pH (7.0) on receptor-mediated insulin degradation, we prebound [125I]insulin to adipocytes at 16 C, then warmed the cells (37 C) to initiate insulin endocytosis and ligand degradation. Under these conditions, insulin degradation was markedly reduced at the lower pH. These results indicate that 1) low pH reduced insulin binding affinity by accelerating the rate of insulin dissociation, and 2) this is accompanied by a reduction in the rate of receptor-mediated insulin degradation.
mesh_terms: D000273:Adipose Tissue / Q000378:metabolism; D000818:Animals; D002738:Chloroquine / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D007328:Insulin / Q000378:metabolism*; D007700:Kinetics; D008297:Male; D051381:Rats; D011919:Rats, Inbred Strains; D011972:Receptor, Insulin / Q000187:drug effects / Q000378:metabolism*; D013696:Temperature
chemical_list: D007328:Insulin; D002738:Chloroquine; D011972:Receptor, Insulin