The Effect of Bivalent Cation Chelating Agents on Some Actions of Adrenalin and Insulin in Rat Isolated Fat Cells

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Abstract
The stimulation of lipolysis by adrenalin in rat isolated fat cells was decreased when cells were incubated in media containing bivalent cation chelating agents (EDTA and EGTA) for up to 5 h. The loss of lipolytic response was closely paralleled by a decrease in cell ATP content and by decreased glucose uptake, and therefore appeared to reflect a general impairment of cell viability rather than a specific involvement of Ca2+ in the stimulation of lipolysis. The chelating agent EDTA was shown to penetrate approx. 64 % of the intracellular water space of fat cells within 30 min. The effect of chelating agents on cell viability was minimized by omitting Ca2+ but not Mg2+ in the presence of EGTA, and continuing incubations for only 30 min. In normal medium containing 1.3 mM Ca2+ the effect of insulin on lipolysis and cyclic AMP content in the presence of adrenalin was biphasic. Low concentrations of insulin (0.007-0.7 nM) inhibited lipolysis and lowered cyclic AMP content but high concentrations (7-700 nM) did not. Glucose uptake was stimulated by low concentrations of insulin and this effect was maintained at high concentrations. In medium containing 5 mM EGTA and no Ca2+, insulin at low concentrations (0.007-0.7 nM), inhibited lipolysis, lowered cyclic AMP content and stimulated glucose uptake as in normal medium. Under these conditions however there was only a partial loss of the antilipolytic effect at high insulin concentrations. In the absence of both Ca2+ and Mg2+ the antilipolytic effect of insulin was fully maintained, with no reversal of the effect even at the highest concentration (700 nM). It is concluded that the physiological effects of insulin in adipose tissue do not require changes in the flux of Ca2+ across the cell membrane, and there is no evidence that intracellular Ca2+ participates as a mediator of insulin action. Anomalous effects of high insulin concentrations are dependent on extracellular Ca2+ and Mg2+ however, consistent with the involvement of a separate class of receptors, or different intracellular mediator.