Modulation of basal glucose transporter K(m) in the adipocyte by insulin and other factors

R. R. Whitesell, N. A. Abumrad

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Abstract

We have previously described experimental conditions where basal methylglucose transport in adipocytes exhibited an apparent K(m) of approximately 35 mM. Under those conditions insulin stimulated transport predominantly by decreasing the transport K(m) (Whitesell, R.R., and Abumrad, N.A. (1985) J. Biol. Chem. 260, 2894-2899). Our findings were in contrast with earlier reports that the K(m) of basal glucose transport was low (3-5 mM) and similar to that of transport in insulin-treated cells. In this study we have investigated the effect of different experimental conditions on the kinetics of basal glucose transport in adipocytes. When transport was assayed at 37°C, cell agitation for 10 min prior to the transport assay decreased the basal K(m) from 35 to 12 mM. Deprivation of metabolic substrate produced a further reduction down to 2 mM. Refeeding starved cells with 1 mM glucose returned the K(m) back up to 12 mM in agitated cells and to 40 mM in stabilized cells. The effects of agitation to lower and of glucose to raise the basal K(m) were prevented by preincubating cells with dinitrophenol. Cell agitation or substrate lack did not alter the V(max) of basal transport and were without effect on both K(m) and V(max) in insulin-treated cells. The temperature dependencies of the kinetics of basal and stimulated transport were studied. A decrease in the assay temperature from 37 to 23°C caused both basal K(m) and V(max) to drop proportionately from 25 to 5 mM, and 13 to 3.6 nmol (μl·min), respectively. In insulin-stimulated cells, only the V(max) was decreased (K(m) went from 3.5 to 3 mM, V(max) from 45 to 17 nmol/(μl·min)). The results support the concept that experimental conditions can produce large changes in the K(m) of basal glucose transporters. Furthermore they explain why, under certain assay conditions (with temperatures around 23°C or with deprivation of metabolic substrate), the effect of insulin on transport K(m) is not observed. Our data also suggest that basal transport characteristics do not persist in insulin-treated cells. We would propose that one of the actions of insulin (in addition to raising V(max)) is to change the characteristics of basal transporters by overriding metabolic factors which keep the K(m) high. Alternatively, insulin could cause the disappearance of basal transporters as new and different ones are recruited from intracellular stores.

Original languageEnglish
Pages (from-to)15090-15096
Number of pages7
JournalJournal of Biological Chemistry
Volume261
Issue number32
StatePublished - 1986

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