Adenosine triphosphate-dependent inhibition of insulin-stimulated glucose transport in fat cells. Possible role of membrane phosphorylation.

Abstract Addition of low concentrations of ATP, but not other nucleoside triphosphates, to fat cells inhibits insulin-stimulated glucose oxidation under conditions that measure glucose transport. Basal rates of glucose transport are not inhibited. The inhibition cannot be overcome by increasing the concentration of insulin, and the dose-response relationships indicate no gross alteration in the apparent affinity of the cells for the hormone. The binding of 125I-labeled insulin is not altered by ATP. Under the conditions used, ATP does not alter oxidation by affecting cytoplasmic metabolic processes. The effects of exogenous ATP on glucose transport were confirmed by direct measurements of 3H-labeled 3-O-methyl-d-glucose uptake in isolated fat cells. ATP inhibits the insulin-stimulated but not the basal rate of 3-O-methylglucose transport. The inhibition by ATP is relatively selective since it represents a fall in the apparent affinity for glucose of the insulin-activated component of the glucose transport system. The inhibition can be overcome by high glucose concentrations. The antilipolytic activity of insulin in ATP-treated cells is unaffected while insulin-stimulated transport is markedly depressed; these activities of insulin can thus be dissociated. ATP also inhibits the enhanced rates of glucose transport observed with concanavalin A, wheat germ agglutinin, spermine, spermidine, cysteine, and glutathione, substances that activate the same glucose transport system as insulin. The ouabain-stimulated glucose oxidation, which occurs by mechanisms different from those of insulin, is not affected by ATP. The ATP analog, α,β-methylene-adenosine 5'-triphosphate (Ap(CH2)pp), can mimic the effect of ATP. Another analog, β,γ-methylene-adenosine 5'-triphosphate (App(CH2)p), does not suppress insulin-stimulated transport but it can effectively inhibit the ATP effect; this phosphonic acid analog cannot donate its terminal phosphoryl moiety in phosphorylation reactions. Phloretin, a strong competitive inhibitor of d-glucose transport, can protect the cells from the ATP effect. Phlorizin, another competitive inhibitor, potentiates the effect of ATP. Fat cells exposed to low concentrations (5 to 50 µm) of ATP for a short time (5 min) at 24° continue to exhibit a diminished insulin response (glucose transport) after thorough washing to remove the ATP in the medium. The recovery of insulin sensitivity during subsequent incubation at 37° is slow, pointing to the protracted and relatively irreversible effect of such mild treatment with ATP. Brief treatment of cells with [γ-32P]ATP results in the selective phosphorylation of two plasma membrane components which are estimated to be of molecular weight 16,000 and 22,000 by sodium dodecyl sulfate gel electrophoresis. Phosphorylation of the latter is inhibited by phloretin, and phosphorylation of both is inhibited by App(CH2)p. The possibility is entertained that the procedures utilized may selectively label membrane components involved in insulin-mediated glucose transport (glucose carriers). Consideration is given to the possible physiological significance of such selective membrane phosphorylation in the control of insulin sensitivity in normal as well as insulin-resistant states.