The action of phenylarsine oxide on insulin stimulation of glucose transport in rat adipocytes

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It is widely accepted that the insulin receptor contains two distinct glycoprotein subunits, a and /?, held together by interchain disulphide bonds and strong non-covalent interactions to yield a (/?-S-S-a)-S-S-(a-S-S-/?) receptor complex (Massague et al., 1980). D-Glucose transporters exist in intracellular storage sites. Stimulation of cells with insulin results in the transporters being sequestered to the plasma membrane (Kono et al., 1981; Karnieli et al., 1981; Wardzala & Jeanrenaud, 1981). These transporters contain exoand endo-facial sulphydryl groups, which appear to be necessary for the proper functioning of the carrier. The exofacial groups, in particular, are required for maximal activity of the transporter and may even be involved in the regulation of transport rates by insulin (May, 1985). It has previously been reported that phenylarsine oxide (PhAsO) inhibits the internalization of the insulinreceptor complex in rat hepatocytes (Draznin et al., 1984) and 3T3-C2 fibroblasts (Knutson et al., 1983). This inhibitory effect is attributed to the functional presence of two vicinal or paired thiol groups which form a stable cyclic thioarsinite complex with PhAsO (Wallace & Ho, 1972; Walker-Smith & Payne, 1983). Our investigations revealed that PhAsO inhibited glucose uptake, implying that PhAsO interacts with sulphydryl groups in the receptors and transporters, resulting in transport inhibition. By treating stimulated and unstimulated cells with varying concentrations of PhAsO we were able to separate the effect of PhAsO on transporters and on receptors. Adipocytes were prepared by collagenase digestion of epididymal fat pads as previously described (More & Jones, 1983). Aliquots of (1 or 2ml) adipocytes were treated with insulin, PhAsO or both, added simultaneously or separately. D-Glucose uptake measurements have been previously described (More & Jones, 1983). In this work D-glucose uptake was routinely measured after 60 s time intervals. Fig. l(a) shows the inhibitory effect of PhAsO ( 1 0 ~ ~ ) on glucose uptake in insulin-stimulated cells. This inhibition was significantly, though only partially reversed by dithiothreitol (200 PM). In unstimulated cells the plasma membrane contains approx. 85% of the cellular insulin receptors (Knutson et al., 1983) but relatively few transporters; 10% of cell’s transporters are on the surface in basal state (Simpson et al., 1983). Simultaneous treatment of these cells with insulin and PhAsO (1 PM) results in partial inhibition of glucose uptake due to the inhibition of a fraction of the receptors. After 20 min of insulin stimulation, receptors and transporters would be redistributed between the plasma membrane and the intracellular pools. Approx. 65% of receptors are internalized in 15min (Knutson et al., 1983), while the transporters on the membrane have increased 65-fold (Simpson et al., 1983). Addition of PhAsO (1 PM) at this stage causes a significant inhibition of transport within 1 min (Fig. 16). However, the glucose uptake begins to recover and after 20min returns to approx. 93% of the initial transport activity. This dramatic recovery is probably due to internalized receptors,