The Role of Calcium in Insulin Action

Abstract

Abstract. The effects of adrenaline, insulin and procaine‐HCl on Ca distribution in intact fat cells and on Ca binding to fat cell ghost membranes have been investigated. 1. Fat cells incubated in 45Ca containing media till isotopic equilibrium indicated that the exchangeable Ca in these cells averages 25.7 ± 3.2 nmol/mg protein, which represents approximately 9.8% of their total Ca content. 2. Perifusion of 45Ca prelabelled fat cells gave washout curves whose analysis conformed with three kinetically distinct Ca pools (Fig. 1). The fast exchangeable pool (Compartment A) had an efflux rate constant of 0.193 ± 0.013 min.−1. The release of Ca from the second and third pools (Compartments B and C) was much slower with efflux rate constants of 0.032 ± 0.0018 min.−l and 0.0042 ± 0.0006 min.−1 respectively. Changing the Ca concentration in the perifusing medium modified the initial fast phase and its rate constant, while added dinitrophenol (DNP) inhibited the efflux rate from the latter compartments. The results suggest that the initial fast component of the 45Ca washout represents Ca exchange from an extracellular pool probably present on the outer surface of cell membranes, while the release of Ca during the second and third slow phases represent Ca derived from two distinct intracellular compartments. The rate constant for 45Ca efflux from compartment (B) is of the same order of magnitude as the transmembrane fluxes measured in other cells and presumably represents Ca efflux from the cytoplasmic pool. The efflux of Ca from compartment (C), on the other hand, occurs at a much slower rate and probably reflects the release of Ca from a storage pool bound to the plasma membrane and/or endoplasmic reticulum. 3. Analysis of Ca binding to fat cell ghosts suggests that the membrane structures in these preparations contain two classes to Ca binding sites, a high affinity site with K (assoc) of 1.4 times 10−6 M and a low affinity site with K (assoc) of 1.3 times 10−5 M. The maximum amount of Ca that could be stored in these membranes would be approximately 60 nmol/mg protein. 4. In perifused 45Ca prelabelled fat cells adrenaline increased the 45Ca efflux from intracellular compartments prior to its stimulation of lipolysis. Previous perifusion with insulin or procaine‐HCl prevented the effects of adrenaline on 45Ca efflux and lipolysis. Moreover, both insulin and procaine‐HCl decreased the binding of Ca by ghost membranes. The dose response of these effects compared with that for their effects on lipolysis in isolated fat cells. Pretreatment of fat cells with trypsin to delete the insulin receptor abolished the effects of insulin both on Ca binding and on lipolysis while the insulin like response for procain‐HCl remained. These experiments suggest that the effects of insulin and procaine‐HCl on fat cells could be mediated by increasing mobilisation of Ca from membrane bound stores and inhibition of Ca efflux thereby facilitating an increase in intracellular Ca concentration. These effects of insulin require the presence of an intact insulin receptor while procaine‐HCl operates beyond the insulin receptor site.