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Observations that insulin-induced stimulation of glycogen synthetase occurs without detectable reduction in cyclic AMP suggests an alternative controlling mechanism. The hypothesis that this stimulation might be mediated through calcium was tested using procaine HC1, a drug whose insulin-like action is not associated with reduction in basal or adrenaline-stimulated cyclic AMP levels. Pretreatment of adipose tissue with insulin or porcaine for 30 minutes increased homogenate glucose-6-phosphate independent ("I") form activity to 58% and 48% respectively with no significant change in total activity. Insulin and procaine also had similar effects on calcium efflux from isolated rat adipocytes. Following an initial displacement of calcium from the adipocytes by insulin (but not by procaine) both agents decreased efflux of calcium, measured following 10, 20 and 30 minutes incubation. In adipose tissue homogenates increasing the free calcium concentration from 10(-8) to 10(-3) M increased "I" form glycogen synthetase activity without significantly altering total activity. A complex interrelationship with ATP and calcium was also observed. In the presence of ATP 0.5 mM maximal activation occurred at 10(-6) M calcium concentration. These findings suggest that insulin-induced activation of glycogen synthetase may be effected by alteration of intracellular free calcium with consequent effects on glycogen synthetase-related enzymes in a mechanism independent of or complementary to effects on cyclic AMP levels.

A unifying hypothesis is proposed for the mechanism of insulin action in adipose tissue. Insulin both induces displacement of Ca++ from a membrane-bound pool and inhibits efflux of the ion, thereby facilitating a rise in intracellular free Ca++ concentration. The former effect could enhance the transport of substrates and ions into the cell, while the latter modulates the activity of some intracellular enzymes to stimulate glycogenesis, lipogenesis, and decrease lipolysis and glycogenolysis. The calcium ion might act as the missing second messenger for insulin action.

The in-vitro antilipolytic response to insulin and procaine hydrochloride by adipose tissue from young rats (150 - 180 g) and lean mice has been compared with that from aged Wistar rats (600 g) and obese (ob/ob) hyperglycaemic mice. 1. The adipose tissue from the obese mice showed diminished responsiveness to insulin and to procaine hydrochloride. Response to these agents, however, was restored by prewashing the tissue, suggesting that the apparent resistance in this tissue reflected saturation of the insulin receptors to endogenous insulin. 2. In adipose tissue of old Wistar rats the antilipolytic effect of insulin was also impaired, but this was not restored after extensive washing. Unlike adipose tissue ghosts prepared from young rats, insulin did not decrease the binding of calcium to ghost membrane preparations from old rats. Neither did insulin inhibit the adrenaline stimulated 45 calcium efflux from perifused isolated fat cells prepared from old rats. These results suggest that the insulin response of fat cells from old rats is impaired because of a defect either in their insulin receptors or in their post-receptor responses. 3. Procaine-hydrochloride, however, when added to the medium perifusing fat cells of these old rats inhibited the adrenaline stimulated lipolysis, reduced the Ca efflux and decreased the binding of Ca to fat cell ghosts; as it did with similar preparations of young rats. Thus the cells from old rats still show full post-receptor responsiveness to an insulin-like stimulus, provided the stimulus for such a response is given at a point beyond the insulin receptor itself. The results suggest that the insulin resistance observed in old rat fat cells may be related to some by deficiency in the insulin receptors, possibly due to their lower replacement with age.

The reaction steps involved in the activation and inactivation of the hormone sensitive lipase have been studied in human adipose tissue. Homogenates prepared from adipose tissue shreds or isolated fat cells contained a cyclic-AMP dependant protein kinase. This enzyme was partially purified from the 100,000 g supernatant using DEAE Cellulose Column Chromatography. The activity of the purified preparation was stimulated 3-4 fold with cyclic-AMP and inhibited with progressive increases in free calcium concentration in the reaction mixture. Calcium seems to inhibit the binding of cyclic-AMP to the protein kinase regulatory units and thus abolishes cyclic-AMP stimulation. In the presence of cyclic-AMP and ATP the protein kinase phosphorylates a lipase preparation partially purified from the same tissue with synchronous increase in the lipolytic activity. Inactivation of the lipase is preceeded by its dephosphorylation by a phosphatase(s) which is present in 105,000 g supernatant. Increasing free calcium concentration between 10-8 and 10-3M produced progressive inhibition of the lipase activity. This inhibition can be attributed to a dual mechanism. First, calcium ions inhibited the protein kinase activation of the lipase. Second, calcium ions stimulated the adipose tissue phosphatase(s) and this increased the rate of inactivation of the hormone sensitive lipase. A role for calcium ions as well as cyclic-AMP in the control of lipolysis is postulated.

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. -l 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 × 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. The 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.