Calyculin A and okadaic acid: inhibitors of protein phosphatase activity

Calyculin A and okadaic acid induce contraction in smooth muscle fibers. Okadaic acid is an inhibitor of phosphatase activity and the aims of this study were to determine if calyculin A also inhibits phosphatase and to screen effects of both compounds on various phosphatases. Neither compound inhibited acid or alkaline phosphatases, nor the phosphotyrosine protein phosphatase. Both compounds were potent inhibitors of the catalytic subunit of type-2A phosphatase, with IC50 values of 0.5 to 1 nM. With the catalytic subunit of protein phosphatase type-1, calyculin A was a more effective inhibitor than okadaic acid, IC50 values for calyculin A were about 2 nM and for okadaic acid between 60 and 500 nM. The endogenous phosphatase of smooth muscle myosin B was inhibited by both compounds with IC50 values of 0.3 to 0.7 nM and 15 to 70 nM, for calyculin A and okadaic acid, respectively. The partially purified catalytic subunit from myosin B had IC50 values of 0.7 and 200 nM for calyculin A and okadaic acid, respectively. The pattern of inhibition for the phosphatase in myosin B therefore is similar to that of the type-1 enzyme.     

Interactions of okadaic acid with insulin action in hepatocytes: role of protein phosphatases in insulin action.

The regulation of carbohydrate metabolism involves changes in the phosphorylation state of enzymes. We used okadaic acid, a potent inhibitor of protein phosphatases type 2A (IC50 0.05-2 nM) and type 1 (IC50 10-20 nM) to determine the role of these phosphatases in the control of carbohydrate metabolism by insulin in rat hepatocytes. In the absence of insulin, okadaic acid caused total inhibition of glycogen synthesis at 100 nM and half-maximal inhibition at 8-9 nM. In the presence of insulin, lower concentrations of okadaic acid (to which type 2A phosphatases are sensitive) were effective at inhibiting glycogen synthesis. 2.5 nM okadaic acid caused total inhibition of the 2-fold stimulation of glycogen synthesis by insulin but had no effect on the basal unstimulated rate of glycogen synthesis. This suggests the involvement of type 2A protein phosphatases in the stimulation of glycogen synthesis by insulin. Okadaic acid (5 nM), partially suppressed but did not abolish the increase in glucokinase mRNA levels caused by insulin, indicating that dephosphorylation mechanisms may be involved in the control of glucokinase mRNA levels by insulin. It is concluded that activation of protein phosphatases type 1 and/or type 2A by insulin may have a widespread role in the control of glucose metabolism at various sites.     

Osmotic Shock Inhibits Insulin Signaling by Maintaining Akt/Protein Kinase B in an Inactive Dephosphorylated State

We have previously reported that insulin and osmotic shock stimulate an increase in glucose transport activity and translocation of the insulin-responsive glucose transporter isoform GLUT4 to the plasma membrane through distinct pathways in 3T3L1 adipocytes (D. Chen, J. S. Elmendorf, A. L. Olson, X. Li, H. S. Earp, and J. E. Pessin, J. Biol. Chem. 272:27401-27410, 1997). In investigations of the relationships between these two signaling pathways, we have now observed that these two stimuli are not additive, and, in fact, osmotic shock pretreatment was found to completely prevent any further insulin stimulation of glucose transport activity and GLUT4 protein translocation. In addition, osmotic shock inhibited the insulin stimulation of lipogenesis and glycogen synthesis. This inhibition of insulin-stimulated downstream signaling occurred without any significant effect on insulin receptor autophosphorylation or tyrosine phosphorylation of insulin receptor substrate 1 (IRS1). Furthermore, there was no effect on either the insulin-stimulated association of the p85 type I phosphatidylinositol (PI) 3-kinase regulatory subunit with IRS1 or phosphotyrosine antibody-immunoprecipitated PI 3-kinase activity. In contrast, osmotic shock pretreatment markedly inhibited the insulin stimulation of protein kinase B (PKB) and p70S6 kinase activities. In addition, the dephosphorylation of PKB was prevented by pretreatment with the phosphatase inhibitors okadaic acid and calyculin A. These data support a model in which osmotic shock-induced insulin resistance of downstream biological responses results from an inhibition of insulin-stimulated PKB activation.     

Okadaic acid inhibits a protein phosphatase activity involved in formation of the mitotic spindle of GH4 rat pituitary cells.

Okadaic acid, a selective inhibitor of serine/threonine protein phosphatases, was utilized to investigate the requirement for phosphatases in cell cycle progression of GH4 rat pituitary cells. Okadaic acid inhibited GH4 cell proliferation in a concentration-dependent manner with a half-maximal inhibition (IC50) of approximately 5 nM. Treatment of GH4 cells with 10 nM okadaic acid resulted in a 40-60% decrease in phosphatase activity and an increase in the proportion of phosphorylated retinoblastoma (RB) protein. Cell cycle analysis indicated that okadaic acid increased the percentage of cells in G2-M, decreased proportionally the percentage of cells in G1 phase, and had little effect on the percentage of cells in S-phase. The absence of a change in the proportion of S-phase cells indicates that G1-specific phosphatases responsible for dephosphorylation of RB protein were not inhibited by 10 mM okadaic acid. Mitotic index revealed that 10 nM okadaic acid decreased proliferation of GH4 cells specifically by slowing the progression through mitosis. Immunostaining with anti-tubulin demonstrated that 10 nM okadaic acid-treated mitotic cells contained mitotic spindles; however, the spindle apparatus in these cells frequently contained multiple poles. These results suggest that the organization of spindle microtubules during prometaphase requires a protein phosphatase that is sensitive to nanomolar concentrations of okadaic acid. Chromosomes in 10 nM okadaic acid-treated cells appear to be attached to spindle microtubules and the nuclear envelope is absent. The effects of okadaic acid on the spindle differ from those elicited by the calcium channel blocker, nimodipine, indicating that this okadaic acid sensitive phosphatase is not part of the calcium signalling events which participate in mitotic progression.     

Identification of high levels of type 1 and type 2A protein phosphatases in higher plants.

Extracts of Brassica napus (oilseed rape) seeds contain type 1 and type 2A protein phosphatases whose properties are indistinguishable from the corresponding enzymes in mammalian tissues. The type 1 activity dephosphorylated the beta-subunit of phosphorylase kinase selectively and was inhibited by the same concentrations of okadaic acid [IC50 (concentration causing 50% inhibition) approximately 10 nM], mammalian inhibitor 1 (IC50 = 0.6 nM) and mammalian inhibitor 2 (IC50 = 2.0 nM) as the rabbit muscle type 1 phosphatase. The plant type 2A activity dephosphorylated the alpha-subunit of phosphorylase kinase preferentially, was exquisitely sensitive to okadaic acid (IC50 approximately 0.1 nM), and was unaffected by inhibitors 1 and 2. As in mammalian tissues, a substantial proportion of plant type 1 phosphatase activity (40%) was particulate, whereas plant type 2A phosphatase was cytosolic. The specific activities of the plant type 1 and type 2A phosphatases were as high as in mammalian tissue extracts, but no type 2B or type 2C phosphatase activity was detected. The results demonstrate that the improved procedure for identifying and quantifying protein phosphatases in animal cells is applicable to higher plants, and suggests that okadaic acid may provide a new method for identifying plant enzymes that are regulated by reversible phosphorylation.     

Protein kinase C(19-31) pseudosubstrate inhibition of insulin action in rat adipocytes

Treatment of intact adipocytes with the autoregulatory PKC pseudosubstrate PKC(19-31) inhibited insulin-stimulated hexose uptake and lipogenesis, with no effect on basal values. The effect was dose-dependent with respective IC50 values of 30 microM and 600 microM for insulin-stimulated hexose uptake in electroporated and intact adipocytes. These studies indicate that PKC may play a role in the mediation of insulin action in adipocytes.     

Effects of prostaglandin E2, indomethacin and adenosine deaminase on basal and insulin-stimulated glucose metabolism in human adipocytes

The effects of prostaglandin E2 were studied on glucose metabolism (3-O-methylglucose transport, CO2 production and lipogenesis) in human adipocytes. Initially, the effects of endogenously produced adenosine and prostaglandins were indirectly demonstrated by using adenosine deaminase and indomethacin in the incubations. From these studies it was found that adenosine deaminase (5 micrograms/ml) had a pronounced effect on adipocyte glucose metabolism in vitro. In the basal (nonhormonal-stimulated) state, glucose transport, CO2 production and lipogenesis were inhibited by about 30% (P less than 0.05). Furthermore, adenosine deaminase significantly inhibited the isoproterenol- and insulin-stimulated CO2 production and lipogenesis (P less than 0.01). Indomethacin (50 microM) had a consistently inhibitory effect on the insulin-stimulated CO2 production (P less than 0.05), whereas indomethacin had no significant effects on basal or isoproterenol-stimulated glucose metabolism. In contrast to the relatively minor effect of endogenous prostaglandins, the addition of exogenous prostaglandin E2 significantly stimulated the glucose transport, glucose oxidation and lipogenesis in human adipocytes, especially in the presence of adenosine deaminase. Half-maximal stimulation was obtained at prostaglandin E2 concentrations of 2.2, 0.8 and 0.8 nM, respectively. The effect of prostaglandin E2 was specific, since the structurally related prostaglandin, prostaglandin F2 alpha, had practically no effect on glucose metabolism. The maximal effect of prostaglandin E2 (1 microM) on glucose metabolism was 30-35% of the maximal insulin (1 nM) effect. When insulin and prostaglandin E2 were added together, the effect of prostaglandin E2 on glucose metabolism was additive at all insulin concentrations tested.     

Dexamethasone inhibits receptor-activated phosphoinositide breakdown in rat basophilic leukemia (RBL-2H3) cells

The activation of rat basophilic leukemia cells for histamine release is accompanied by Ca2+ influx and arachidonic acid release. IgE receptor but not A23187 ionophore stimulation of these cells also resulted in phosphoinositide breakdown. In these experiments, the culture of these cells with dexamethasone inhibited IgE- and ionophore-mediated histamine release. The concentration for 50% of maximal inhibition was 12 nM, and prolonged exposure to the drug was required, with maximal effect observed in 8 to 15 hr. The inhibitory effect of dexamethasone was reversible (t1/2 for recovery was 16 hr). Dexamethasone blocked the IgE-mediated 45Ca2+ influx and the release of [14C]-arachidonic acid (IC50 of 1 nM and 10 nM respectively). Dexamethasone inhibited the IgE receptor-mediated phosphoinositide breakdown (IC50 of 5 nM). It also decreased arachidonic acid release after A23187 stimulation demonstrating an effect on phospholipase A2. Therefore, exposure of the cells to dexamethasone results in the inhibition of both phospholipase A2 and phospholipase C pathways of arachidonic acid generation.     

Postreceptor regulation of insulin action in primary cultures of rat hepatocytes by oral hypoglycemic agents: effects of linogliride and chlorpropamide

We have previously demonstrated the ability of the sulfonylurea tolazamide to potentiate insulin action in primary cultures of hepatocytes prepared from normal and streptozotocin-diabetic rats. To determine whether the pirogliride derivative linogliride, a non-sulfonylurea orally effective hypoglycemic agent, can potentiate insulin action, we evaluated the ability of linogliride to affect insulin-stimulated lipogenesis in primary cultures of hepatocytes prepared from normal rats. In addition, we also evaluated the ability of the sulfonylurea chlorpropamide to affect insulin-stimulated lipogenesis in the same in vitro system. The exposure of hepatocytes for 18 h to either linogliride (100 ug/ml) or chlorpropamide (175 ug/ml) resulted in dose-dependent (0.1 to 100 nM insulin) increases in insulin-stimulated lipogenesis, although the effects of chlorpropamide are approximately two times those of linogliride. This increase in insulin responsiveness was not associated with any change in insulin sensitivity (ED50) or insulin binding. The results provide evidence for an extra-pancreatic effect of linogliride and chlorpropamide in the liver and indicate that these structurally unrelated oral hypoglycemic agents enhance insulin responsiveness through postbinding mechanisms.     

Intracellular signaling by a mutant human insulin receptor lacking the carboxyl-terminal tyrosine autophosphorylation sites.

We have recently characterized a mutant insulin receptor (Y/F2) in which the two tyrosines in the carboxyl terminus (Tyr1316, Tyr1322) were mutated to phenylalanine. Compared with wild type receptors, the Y/F2 receptor exhibited markedly enhanced sensitivity to insulin-stimulated DNA synthesis with normal insulin-stimulated glucose uptake (Takata, Y., Webster, N. J. G., and Olefsky, J. M. (1991) J. Biol. Chem. 266, 9135-9139). In this paper, we present further evidence for the divergence of the metabolic and mitogenic signaling pathways utilized by the insulin receptor. The mutant receptor showed normal sensitivity and responsiveness for insulin-stimulated glucose incorporation into glycogen. The insulin sensitivity for phosphorylation of two substrates (pp180 and pp220) was the same in both Y/F2 cells and HIRc cells. Phosphotyrosine content, however, was greater in Y/F2 cells than in HIRc cells, especially in the basal state. Insulin stimulated S6 kinase activity 2-6-fold, with an ED50 of -10 nM in Rat 1 cells and 0.5 nM in HIRc cells. The sensitivity to insulin was enhanced in Y/F2 cells with an ED50 of 0.1 nM. These effects were insulin-specific, since insulin-like growth factor (IGF)-I-stimulated mitogenesis was normal. In summary: 1) Y/F2 receptors exhibit normal metabolic and enhanced mitogenic signaling; 2) the enhanced mitogenic signaling is specific for the insulin receptor in the Y/F2 cells, since IGF-I-stimulated mitogenesis is normal; 3) Y/F2 cells display increased endogenous substrate phosphorylation and augmented insulin-stimulated S6 kinase activity placing these responses among insulin's mitogenic effects; and 4) these results are consistent with the concept that the COOH-terminal tyrosine residues of the insulin receptor are normally inhibitory to mitogenic signaling.     

Inhibition of Clathrin-Mediated Endocytosis Selectively Attenuates Specific Insulin Receptor Signal Transduction Pathways

To examine the role of clathrin-dependent insulin receptor internalization in insulin-stimulated signal transduction events, we expressed a dominant-interfering mutant of dynamin (K44A/dynamin) by using a recombinant adenovirus in the H4IIE hepatoma and 3T3L1 adipocyte cell lines. Expression of K44A/dynamin inhibited endocytosis of the insulin receptor as determined by both cell surface radioligand binding and trypsin protection analysis. The inhibition of the insulin receptor endocytosis had no effect on either the extent of insulin receptor autophosphorylation or insulin receptor substrate 1 (IRS1) tyrosine phosphorylation. In contrast, expression of K44A/dynamin partially inhibited insulin-stimulated Shc tyrosine phosphorylation and activation of the mitogen-activated protein kinases ERK1 and -2. Although there was an approximately 50% decrease in the insulin-stimulated activation of the phosphatidylinositol 3-kinase associated with IRS1, insulin-stimulated Akt kinase phosphorylation and activation were unaffected. The expression of K44A/dynamin increased the basal rate of amino acid transport, which was additive with the effect of insulin but had no effect on the basal or insulin-stimulated DNA synthesis. In 3T3L1 adipocytes, expression of K44A/dynamin increased the basal rate of glucose uptake, glycogen synthesis, and lipogenesis without any significant effect on insulin stimulation. Together, these data demonstrate that the acute actions of insulin are largely independent of insulin receptor endocytosis and are initiated by activation of the plasma membrane-localized insulin receptor.     

Insulin stimulates ecdysteroid production through a conserved signaling cascade in the mosquito Aedes aegypti.

Selective activators and inhibitors of insulin signaling cascades in mammalian cells were tested for their effects on insulin stimulated steroidogenesis by ovaries of Aedes aegypti. Bovine insulin in the concentration range of 1.7 microM to 85 microM stimulated ecdysteroidogenesis in vitro. Pervanadate, an inhibitor of tyrosine kinase phosphatase, stimulated ecdysteroid production at concentrations of 250 microM to 1 microM. Okidaic acid, a serine/threonine phosphatase inhibitor, stimulated steroidogenesis with an ED50 of 77.39 nM. A selective inhibitor of tyrosine kinase activity, HNMPA-(AM3), inhibited ecdysteroid production with an IC50 of 14.2 microM. Two selective inhibitors of phosphatidylinositol 3-kinase, wortmannin and LY294002, inhibited ecdysteroid production at low concentrations (IC50 = 1.6 nM and 30 nM, respectively). These concentrations are similar to those inhibiting insulin action in mammalian cells. A selective inhibitor of mitogen-activated protein kinase, PD098059, had no effect on ecdysteroid production even up to 100 microM. Thus, insulin stimulation of ecdysteroid production by ovaries in vitro appears to be controlled by the tyrosine kinase activity of the mosquito insulin receptor and the signaling cascade involving phosphatidylinositol 3-kinase and protein kinase B.     

Attenuation of insulin-stimulated insulin receptor substrate-1 serine 307 phosphorylation in insulin resistance of type 2 diabetes

Insulin resistance is a primary characteristic of type 2 diabetes and likely causally related to the pathogenesis of the disease. It is a result of defects in signal transduction from the cell surface receptor of insulin to target effects. We found that insulin-stimulated phosphorylation of serine 307 (corresponding to serine 302 in the murine sequence) in the immediate downstream mediator protein of the insulin receptor, insulin receptor substrate-1 (IRS1), is required for efficient insulin signaling and that this phosphorylation is attenuated in adipocytes from patients with type 2 diabetes. Inhibition of serine 307 phosphorylation by rapamycin mimicked type 2 diabetes and reduced the sensitivity of IRS1 tyrosine phosphorylation in response to insulin, while stimulation of the phosphorylation by okadaic acid, in cells from patients with type 2 diabetes, rescued cells from insulin resistance. EC(50) for insulin-stimulated phosphorylation of serine 307 was about 0.2 nM with a t(1/2) of about 2 min. The amount of IRS1 was similar in cells from non-diabetic and diabetic subjects. These findings identify a molecular mechanism for insulin resistance in non-selected patients with type 2 diabetes.     

Diacylglycerols modulate insulin action in rat adipocytes

Effect of 1,2-diacylglycerols on the insulin receptor function and insulin action in rat adipocytes was studied. 1,2-dioctanoylglycerol (100 micrograms/ml) did not alter insulin binding but it did stimulate phosphorylation of the beta-subunit of the insulin receptor as well as its tyrosine kinase activity. However, dioctanoylglycerol inhibited insulin-stimulated receptor autophosphorylation. This concentration of dioctanoylglycerol inhibited insulin-stimulated CO2 metabolism, lipogenesis and 3-O-methyl-glucose transport in a dose-dependent manner but did not alter any of these bioeffects in absence of insulin. While there was no direct link between diacylglycerol effect on tyrosine kinase activity of the insulin receptor and insulin action in rat adipocytes, the parallel inhibition of insulin-stimulated receptor autophosphorylation and insulin bioeffects by dioctanoylglycerol suggests its direct or indirect role in insulin signalling in rat fat cells.     

Mechanisms of leptin secretion from white adipocytes

The mechanisms regulating leptin secretion were investigated in isolated rat white adipocytes. Insulin (1-100 nM) linearly stimulated leptin secretion from incubated adipocytes for at least 2 h. The adrenergic agonists norepinephrine, isoproterenol (two nonselective beta-agonists), or CL-316243 (potent beta3) all inhibited insulin (10 nM)-stimulated leptin release. The inhibitory effects of norepinephrine and isoproterenol could be reversed not only by the nonselective antagonist propranolol but also by the selective antagonists ICI-89406 (beta1) or ICI-118551 (beta2), the beta2-antagonist being less effective than the beta1. Insulin-stimulated leptin secretion could also be inhibited by a series of agents increasing intracellular cAMP levels, such as lipolytic hormones (ACTH and thyrotropin-stimulating hormone), various nonhydrolyzable cAMP analogs, pertussis toxin, forskolin, methylxanthines (caffeine, theophylline, IBMX), and specific inhibitors of phosphodiesterase III (imazodan, milrinone, and amrinone). Significantly, antilipolytic agents other than insulin (adenosine, nicotinic acid, acipimox, and orthovanadate) did not mimic the acute stimulatory effects of insulin on leptin secretion under these conditions. We conclude that norepinephrine specifically inhibits insulin-stimulated leptin secretion not only via the low-affinity beta3-adrenoceptors but also via the high-affinity beta1/beta2-adrenoceptors. Moreover, it is suggested that 1) activation of phosphodiesterase III by insulin represents an important metabolic step in stimulation of leptin secretion, and 2) lipolytic hormones competitively counterregulate the stimulatory effects of insulin by activating the adenylate cyclase system.     

Okadaic acid inhibition of KCl cotransport. Evidence that protein dephosphorylation is necessary for activation of transport by either cell swelling or N-ethylmaleimide

The mechanism of activation of KCl cotransport has been examined in rabbit red blood cells. Previous work has provided evidence that a net dephosphorylation is required for activation of transport by cell swelling. In the present study okadaic acid, an inhibitor of protein phosphatases, was used to test this idea in more detail. We find that okadaic acid strongly inhibits swelling-stimulated KCl cotransport. The IC50 for okadaic acid is approximately 40 nM, consistent with the involvement of type 1 protein phosphatase in transport activation. N-Ethylmaleimide (NEM) is well known to activate KCl cotransport in cells of normal volume. Okadaic acid, added before NEM, inhibits the activation of transport by NEM, indicating that a dephosphorylation is necessary for the NEM effect. Okadaic acid added after NEM inhibits transport only very slightly. After a brief exposure to NEM and rapid removal of unreacted NEM, KCl cotransport activates with a time delay that is similar to that for swelling activation. Okadaic acid causes a slight increase in the delay time. These findings are all consistent with the idea that NEM activates transport not by a direct action on the transport protein but by altering a phosphorylation-dephosphorylation cycle. The simplest hypothesis that is consistent with the data is that both cell swelling and NEM cause inhibition of a protein kinase. Kinase inhibition causes net dephosphorylation of some key substrate (not necessarily the transport protein); dephosphorylation of this substrate, probably by type 1 protein phosphatase, causes transport activation.     

Stimulation of glucose transport and glucose transporter phosphorylation by okadaic acid in rat adipocytes

Okadaic acid, an inhibitor of Type I and IIa protein phosphatases, was recently found to stimulate 2-deoxyglucose uptake in rat adipocytes (Haystead, T. A. J., Sim, A. T. R., Carling, D., Honnor, R. C., Tsukitani, Y., Cohen, P., and Hardie, D. G. (1989) Nature 337, 78-81). In the present experiments the effect of okadaic acid on the phosphorylation and subcellular distribution of the insulin-regulatable glucose transporter (IRGT) was investigated. At maximally effective concentrations, insulin and okadaic acid increased the amount of IRGT in the plasma membrane by 10- and 4-fold, respectively. Thus, the stimulation of glucose transport by okadaic acid was apparently due to an increase in the surface concentration of the IRGT. However, despite its stimulatory actions, okadaic acid partially inhibited the ability of insulin to enhance glucose transport and translocation of the transporter. When cells were incubated with okadaic acid alone or in combination with insulin, phosphorylation of the IRGT in the plasma membrane was increased by approximately 3-fold relative to the intracellular pool of transporters in control cells. Phosphorylation of the IRGT was confined to the presumed cytoplasmic domain at the COOH terminus of the protein. Glucose transporters were dephosphorylated in vitro by Type I or Type IIa protein phosphatases, indicating that inhibition of one or both of these phosphatases could account for the increased phosphorylation produced by okadaic acid. The observation that okadaic acid stimulated translocation of the IRGT implicated a serine/threonine phosphorylation event in triggering movement of the intracellular IRGT-containing vesicles (GTV) to the cell surface. Immunoadsorption of GTV from 32P-labeled adipocytes revealed that the IRGT was the major phosphoprotein in these vesicles. The phosphorylation of at least three other GTV proteins was increased by okadaic acid, and these species would appear to be candidates for regulators of GTV movement to the plasma membrane. It is unlikely that phosphorylation of the IRGT is the signal for translocation because insulin did not increase phosphorylation of the protein. Rather, the inhibitory effect of okadaic acid on insulin-stimulated translocation is consistent with the hypothesis that phosphorylation of the IRGT promotes its internalization.     

Effects of arachidonic acid on leptin secretion and expression in primary cultured rat adipocytes

Leptin, a hormone produced in adipocytes, is a key signal in the regulation of food intake and energy expenditure. Several studies have suggested that leptin can be regulated by macronutrients intake. Arachidonic acid is a dietary fatty acid known to affect cell metabolism. Controversial effects of this fatty acid on leptin have been reported. The aim of this experimental trial was to evaluate the effect of the arachidonic acid on basal and insulin-stimulated leptin secretion and expression in isolated rat adipocytes. Because insulin-stimulated glucose metabolism is an important regulator of leptin expression and secretion by the adipocytes, the effects of the arachidonic acid on indices of adipocyte metabolism were also examined. Isolated adipocytes were incubated with arachidonic acid (1-200 microM) in the absence and presence of insulin (1.6 nM). Leptin secretion and expression, glucose utilization and lactate production were determined at 96 h. The arachidonic acid (200 microM) inhibited both the basal and insulin stimulated leptin secretion and expression. Glucose utilization was not affected by the acid. Basal lactate production was increased by the fatty acid at the highest concentration used (200 microM), however lactate production in presence of insulin was not modified. Finally, the percentage of glucose carbon released as lactate was significantly increased (200 microM). These results suggest that the inhibitory effect of the arachidonic acid on leptin secretion and expression may be due, al least in part, to the increase in the anaerobic utilization of glucose.