Immunostimulatory principles from Chlorella pyrenoidosa--part 1: isolation and biological assessment in vitro.

Our proprietary preparation obtained by extraction of Chlorella pyrenoidosa cells, ONC-107 (Respondin), was recently found to selectively boost antibody response to the influenza vaccine in a human clinical trial. Respondin is a potent stimulator of mouse B cell proliferation and an activator of macrophages. Bioactivity-guided resolution concluded that Respondin is composed of a mixture of immunostimulatory principles of different chemical nature. A combination of size exclusion, anion exchange and hydrophobic interaction chromatography revealed that the bulk of the immunostimulatory activity resides in polysaccharide/protein complexes with molecular masses larger than 100 kDa that are composed primarily of galactose, rhamnose and arabinose.

An aqueous Chlorella extract inhibits IL-5 production by mast cells in vitro and reduces ovalbumin-induced eosinophil infiltration in the airway in mice in vivo.

An aqueous extract of the edible microalga (CP) (1), has recently been tested for its immunomodulatory effects in a human clinical trial. Here, the CP extract was dialyzed and fractionated using Sephadex G 100 chromatography. The effects of a dialyzed aqueous CP extract, fraction 2 , on mast cell mediator release in vitro and ovalbumin-induced allergic airway inflammation in vivo were examined. In vitro, treatment of mouse bone marrow-derived mast cells with 2 for 18 h significantly inhibited antigen (trinitrophenyl-BSA)-induced IL-5 production. In vivo, treatment of mice with 2 during ovalbumin sensitization and stimulation process significantly reduced eosinophil and neutrophil infiltration in the airways. Moreover, fractions obtained by size exclusion chromatography of 2 inhibited IgE-dependent cytokine GM-CSF production from human cord blood-derived mast cells. Taken together, these results suggest that 2 is composed of biopolymers with anti-allergic potential.

Insulin resistance and the modulation of rat cardiac K(+) currents.

K(+) currents were measured using a whole cell voltage-clamp method in enzymatically isolated rat ventricular myocytes obtained from two hyperinsulinemic, insulin-resistant models. Fructose-fed rats as well as genetically obese rats, both of which are resistant to the metabolic effects of insulin, were used. The normal augmentation of a calcium-independent sustained K(+) current was reduced or abolished in insulin-resistant states. This resistance can be reversed by the insulin-sensitizing drug metformin. Vanadyl sulfate (3-4 wk treatment or after 5-6 h in vitro) enhanced the sustained K(+) current. The in vitro effect of vanadyl was blocked by cycloheximide. Insulin resistance of the K(+) current was not reversed by vanadyl sulfate. The results show that insulin resistance is expressed in terms of insulin actions on ion channels, in addition to its actions on metabolism. This resistance can be reversed by the insulin-sensitizing drug metformin. Vanadate compounds, which mimic the effects of insulin on metabolism, also mimic the augmenting effects of insulin on a cardiac K(+) current in a manner suggesting synthesis of new channels.

Lipoprotein lipase activity is stimulated by insulin and dexamethasone in cardiomyocytes from diabetic rats.

Type 1 diabetes mellitus reduces lipoprotein lipase (LPL) activity in the heart. The diabetic phenotype of decreased LPL activity in freshly isolated cardiomyocytes persisted after overnight culture (16 h). Total cellular LPL activity was 311+/-56 nmol oleate released x h(-1) x mg(-1) cell protein in diabetic cultured cardiomyocytes compared with 661+/-81 nmol oleate released x h(-1) x mg(-1) cell protein for control cultured cells. Diabetes also resulted in lower heparin-releasable (HR) LPL activity compared with control cells (111+/-25 vs. 432+/-63 nmol x h(-1) x mg(-1) cell protein). In kinetic experiments, the reduction in total cellular LPL and HR-LPL activities in cultured cells from diabetic hearts was due to a decrease in maximal velocity, with no change in apparent Km for substrate (triolein). LPL activity in primary cultures of cardiomyocytes from control rats is stimulated by the combination of insulin (Ins) and dexamethasone (Dex). Overnight treatment of cultured cardiomyocytes from diabetic rats with Ins+Dex elicited an 84% increase in cellular LPL activity (to 572+/-65 nmol x h(-1) x mg(-1) cell protein) and a 194% increase in HR-LPL activity (to 326+/-46 nmol x h(-1) x mg(-1) cell protein). This stimulation occurred at subnanomolar concentrations of the hormones, but neither hormone was effective alone. The amount of immunoreactive LPL protein mass in cultured cardiomyocytes from diabetic hearts was unchanged by Ins+Dex treatment. Addition of oleic acid (60 microM) to the overnight culture medium inhibited the already reduced HR-LPL activity in diabetic cultured cells by 73% (to 30+/-4 nmol x h(-1) x mg(-1) cell protein). The presence of oleic acid also reduced hormone-stimulated HR-LPL activity. Increasing the glucose concentration in the culture medium to 26 mM had no effect on total cellular LPL or HR-LPL activities.

Insulin and dexamethasone stimulation of cardiac lipoprotein lipase activity involves the actin-based cytoskeleton.

Lipoprotein lipase (LPL) activity in cultured ventricular cardiomyocytes from adult rat hearts was stimulated by the combination of insulin (100 nM) and dexamethasone (100 nM) during an overnight (16 h) incubation. Wortmannin (100 nM), rapamycin (30 ng/ml) or PD98059 (50 microM) did not prevent this stimulation, suggesting that phosphatidylinositol 3-kinase, p70 S6 kinase and the mitogen-activated protein kinase cascade are not involved in transducing the hormonal signal. In contrast, cytochalasin D (2 microM) completely abolished the stimulatory effect of insulin and dexamethasone on both heparin-releasable LPL and total cellular LPL activities. The potential role of the actin cytoskeleton in the stimulation of LPL activity by insulin and dexamethasone appears to be distal to the initial signalling events since cytochalasin D is still effective in preventing the stimulation when added 2 h after the hormones.

Insulin stimulation of rat ventricular K+ currents depends on the integrity of the cytoskeleton.

1. The effect of insulin on K+ currents was studied with enzymatically dispersed ventricular myocytes from insulin-deficient (type I) diabetic rats. Diabetic conditions were induced by a single intravenous injection of streptozotocin (100 mg kg-1) given 8-13 days before the experiments. Measurements of plasma glucose and insulin levels confirmed the diabetic status of the animals. 2. A Ca2+-independent transient outward K+ current, It, and a slowly inactivating, quasi-steady-state current, Iss, which are depressed in diabetic myocytes, could be restored by exposure to 1, 10 or 100 nM insulin. This was only observed after a delay of 5-6 h, although an insulin exposure of only 1 h was sufficient to initiate its stimulatory action on It and Iss. The stimulatory effect of insulin on these K+ currents was prevented by 2 microM cycloheximide, which in itself had no direct effect on these currents. 3. Disruption of the actin microfilament network with 1 microM cytochalasin D (CD) also prevented the stimulatory effect of 100 nM insulin on both It and Iss. Since CD was added 1 h after insulin, inhibitory effects on insulin signalling were ruled out. Adding CD (1 microM) 5-9 h after insulin, when currents were already augmented, had no effect (up to 50 min exposure). Incubating control cells for 6-10 h with 1 microM CD had no effect on any of the currents measured. 4. Stabilization of the actin network by pre-exposure to 2.5 microM phalloidin restored the stimulatory effect of insulin, in the continued presence of CD, ruling out any effects of CD on components other than the cytoskeleton. 5. The stimulatory effect of insulin was also prevented by incubating cells with insulin in the presence of the microtubule-disrupting agent colchicine (5 microM). 6. These results suggest that the insulin-mediated augmentation of K+ currents in diabetic myocytes requires protein synthesis, possibly of K+ channels, as well as an intact cytoskeleton. The possibility that newly formed channels translocate to the plasma membrane in a process dependent on different elements of the cytoskeleton is discussed.

Dexamethasone stimulates the expression of GLUT1 and GLUT4 proteins via different signalling pathways in L6 skeletal muscle cells.

It was recently demonstrated that dexamethasone treatment of L6 skeletal muscle cells resulted in an elevation of GLUT1 protein. However, the level of GLUT4 protein under these conditions was not examined. In addition, the signalling mechanism(s) leading to dexamethasone-induced expression of GLUT1 protein was not investigated. In the present study we investigated the effect of dexamethasone on the expression of GLUT1 and GLUT4 proteins in differentiated L6 muscle cells and the signalling mechanism(s) via which dexamethasone may act. Dexamethasone (300 nM) treatment for 24 h elevated GLUT1 and GLUT4 proteins by 68% and 94%, respectively, above control levels. These increases were due to de novo synthesis as shown by metabolic labelling with [35S]methionine. Incubation of cells with 100 nM wortmannin or 30 ng/ml rapamycin prevented the dexamethasone-stimulated elevation of GLUT1 protein. In contrast, neither of these inhibitors affected the elevation of GLUT4 protein by dexamethasone. Furthermore, dexamethasone down-regulated insulin receptor substrate-1 protein content by 42% and insulin-induced tyrosine phosphorylation of insulin receptor substrate-1 by 28%. The p70 ribosomal S6 kinase was not activated by dexamethasone and instead, dexamethasone attenuated the stimulation of this enzyme activity by insulin. These results suggest that dexamethasone induces the expression of GLUT1 and GLUT4 protein by independent signalling mechanisms with a concomitant depression of intracellular signalling by insulin.

Type I and II models of diabetes produce different modifications of K+ currents in rat heart: role of insulin.

1. Several K+ currents were measured and compared in enzymatically dispersed ventricular myocytes from control and diabetic rats. 2. Diabetic conditions were established either with a single intravenous injection of streptozotocin (STZ, 100 mg kg-1; 6-14 days duration) or by feeding with a fructose-enriched diet for 4-10 weeks. Both groups became hyperglycaemic, with the former having decreased and the latter having elevated levels of plasma insulin. These conditions therefore mimic type I (insulin-dependent) and type II (non-insulin-dependent) diabetes mellitus, respectively. 3. As reported previously, a Ca(2+)-independent transient outward K+ current, I(t), was attenuated in the type I model. This was not observed in the type II model. The two models differed greatly in the changes observed in a quasi-steady-state K+ current denoted Iss. In the STZ model Iss was substantially attenuated, whereas in the fructose-fed model it was augmented. In both models, the background inwardly rectifying current, IK1, was unchanged. Concomitantly, there was a substantial prolongation of the action potential in the STZ model but not in the fructose-fed model. 4. Incubation of control myocytes with insulin (100 nM) for 5-9 h caused a significant augmentation of Iss, with no effect on I(t) or on IK1. Incubation of myocytes from STZ-diabetic rats with insulin reversed the attenuation of I(t), but not of Iss. 5. The effect of insulin was not blocked by wortmannin, an inhibitor of phosphatidylinositol 3-kinase. However, inhibition of the mitogen-activated protein kinase pathway with PD98059 prevented restoration of I(t). Insulin action on I(t) may therefore involve changes in transcription or expression of channel proteins, rather than changes in cellular metabolism.

Dexamethasone stimulates the expression of GLUT1 and GLUT4 proteins via different signalling pathways in L6 skeletal muscle cells.

It was recently demonstrated that dexamethasone treatment of L6 skeletal muscle cells resulted in an elevation of GLUT1 protein. However, the level of GLUT4 protein under these conditions was not examined. In addition, the signalling mechanism(s) leading to dexamethasone-induced expression of GLUT1 protein was not investigated. In the present study we investigated the effect of dexamethasone on the expression of GLUT1 and GLUT4 proteins in differentiated L6 muscle cells and the signalling mechanism(s) via which dexamethasone may act. Dexamethasone (300 nM) treatment for 24 h elevated GLUT1 and GLUT4 proteins by 68% and 94%, respectively, above control levels. These increases were due to de novo synthesis as shown by metabolic labelling with [35S]methionine. Incubation of cells with 100 nM wortmannin or 30 ng/ml rapamycin prevented the dexamethasone-stimulated elevation of GLUT1 protein. In contrast, neither of these inhibitors affected the elevation of GLUT4 protein by dexamethasone. Furthermore, dexamethasone down-regulated insulin receptor substrate-1 protein content by 42% and insulin-induced tyrosine phosphorylation of insulin receptor substrate-1 by 28%. The p70 ribosomal S6 kinase was not activated by dexamethasone and instead, dexamethasone attenuated the stimulation of this enzyme activity by insulin. These results suggest that dexamethasone induces the expression of GLUT1 and GLUT4 protein by independent signalling mechanisms with a concomitant depression of intracellular signalling by insulin.

Regulation of hepatic glutaminase in the streptozotocin-induced diabetic rat.

The liver of diabetic animals removes increased quantities of glutamine. We therefore examined factors that affect hepatic glutaminase activity in hepatocytes and mitochondria. Glutamine use, through glutaminase, was measured in isolated rat hepatocytes by monitoring the production of 14CO2 from [1-(14)C]glutamine. Hepatocytes from streptozotocin-induced diabetic rats use glutamine more rapidly than do hepatocytes from normal or insulin-maintained diabetic rats. Glutamine use in all of these hepatocytes was stimulated by glucagon and epinephrine. Glutaminase activity, assayed in broken mitochondrial membranes, was increased approximately 2.5-fold in diabetic rats. The sensitivity of glutaminase, measured in intact liver mitochondria, to phosphate was markedly left-shifted in mitochondria from diabetic rats compared with those from controls. In fact, glutaminase was increased 10-fold at 2.5 mmol/l phosphate compared with controls. This increased sensitivity of glutaminase to physiological concentrations of phosphate is characteristic of its hormonal activation. Therefore, activation of glutaminase plays a major role in diabetes and is as important as increases in its total enzyme amount in determining the increased glutamine uptake in diabetes.

Lipoprotein lipase activity in rat cardiomyocytes is stimulated by insulin and dexamethasone.

Lipoprotein lipase (LPL) activity was studied in rat cardiomyocytes after overnight culture (16 h) in the presence of insulin (100 nM) and/or dexamethasone (100 nM). Insulin in combination with dexamethasone (INS/DEX) increased heparin-releasable LPL activity by 71% over the control level (566+/-85 versus 331+/-48 nmol/h.mg cell protein). This was accompanied by a 61% increase in total cellular LPL activity (914+/-89 versus 567+/-64 nmol/h.mg cell protein). The increase in LPL activity occurred at sub-nanomolar concentrations of the hormones, but neither hormone was effective alone. LPL protein mass, quantified by ELISA, was the same in both control and INS/DEX-treated cells (27.7 versus 28.6 ng/mg cell protein, respectively), thus LPL specific activity in cardiomyocytes was increased by INS/DEX treatment (0.113 versus 0.069 mU/ng LPL protein). These findings emphasize the importance of hormonal interactions in the regulation of LPL in heart tissue.

Fatty acids reduce heparin-releasable LPL activity in cultured cardiomyocytes from rat heart.

Varying glucose and fatty acid (FA) concentrations in the medium of cultured cardiomyocytes from adult rat hearts were tested for effects on lipoprotein lipase (LPL) activity. Glucose (5.5, 11, and 25 mM in the culture medium for 18-22 h) had no effect on either heparin-releasable LPL (HR-LPL) or on cellular LPL (C-LPL) activities. When cardiomyocytes were cultured overnight with 60 microM oleate, HR-LPL activity was reduced to 20% of control, with no change in C-LPL activity or total C-LPL mass. Similar results (HR-LPL and C-LPL activities) were obtained with 60 microM concentrations of palmitate and myristate; linoleate and eicosapentaenoate did reduce C-LPL activity, but the decrease in HR-LPL activity was much greater. Oxfenicine, an FA oxidation inhibitor, did not alter the inhibitory effect of 60 microM oleate on HR-LPL. Short-term incubations (1 and 3 h) of cultured cardiomyocytes with 60 microM oleate did not displace LPL into the medium. Immunodetectable LPL on the cell surface of oleate-treated cultured cardiomyocytes was increased compared with control cells, but heparin treatment released the same amount of LPL mass that had reduced catalytic activity.

Stimulation of glucose uptake by the natural coenzyme alpha-lipoic acid/thioctic acid: participation of elements of the insulin signaling pathway.

Thioctic acid (alpha-lipoic acid), a natural cofactor in dehydrogenase complexes, is used in Germany in the treatment of symptoms of diabetic neuropathy. Thioctic acid improves insulin-responsive glucose utilization in rat muscle preparations and during insulin clamp studies performed in diabetic individuals. The aim of this study was to determine the direct effect of thioctic acid on glucose uptake and glucose transporters. In L6 muscle cells and 3T3-L1 adipocytes in culture, glucose uptake was rapidly increased by (R)-thioctic acid. The increment was higher than that elicited by the (S)-isomer or the racemic mixture and was comparable with that caused by insulin. In parallel to insulin action, the stimulation of glucose uptake by thioctic acid was abolished by wortmannin, an inhibitor of phosphatidylinositol 3-kinase, in both cell lines. Thioctic acid provoked an upward shift of the glucose-uptake insulin dose-response curve. The molar content of GLUT1 and GLUT4 transporters was measured in both cell lines. 3T3-L1 adipocytes were shown to have >10 times more glucose transporters but similar ratios of GLUT4:GLUT1 than L6 myotubes. The effect of (R)-thioctic acid on glucose transporters was studied in the L6 myotubes. Its stimulatory effect on glucose uptake was associated with an intracellular redistribution of GLUT1 and GLUT4 glucose transporters, similar to that caused by insulin, with minimal effects on GLUT3 transporters. In conclusion, thioctic acid stimulates basal glucose transport and has a positive effect on insulin-stimulated glucose uptake. The stimulatory effect is dependent on phosphatidylinositol 3-kinase activity and may be explained by a redistribution of glucose transporters. This is evidence that a physiologically relevant compound can stimulate glucose transport via the insulin signaling pathway.

Muscle subcellular localization and recruitment by insulin of glucose transporters and Na+-K+-ATPase subunits in transgenic mice overexpressing the GLUT4 glucose transporter.

Insulin-stimulated glucose uptake in skeletal muscle is mediated through the GLUT4 glucose transporter. Transgenic (TG) mice overexpressing human GLUT4 in skeletal muscle show an increased ability to handle a glucose load. Here, the participation of the overexpressed GLUT4 in the response to insulin was examined. In TG mouse muscle, the GLUT4 protein content was 10-fold higher in crude membrane (CM), sevenfold higher in internal membrane (IM), and 15-fold higher in a plasma membrane (PM)-rich fraction, relative to non-TG littermates. This suggested partial saturation of the normal sorting mechanisms. The distribution and abundance of the GLUT1 glucose transporter was not affected. Insulin injection (4.3 U/kg body wt) increased GLUT4 in the PM-rich fraction; the increase was threefold higher in TG than in non-TG mice. Insulin decreased the GLUT4 content of the IM in both animal groups and of a second, heavier intracellular membrane fraction only in TG mice. The net content of Na+-K+-pump subunits was 40-65% lower in CM from TG compared with non-TG littermates. In spite of this, insulin caused a three- to sixfold higher translocation of the alpha2 and beta1 subunits of the Na+-K+-pump in TG compared with non-TG animals. The results suggest that overexpression of GLUT4 confers to the muscle increased ability to translocate subunits of the Na+-K+-pump either as a direct consequence of the recruitment of glucose transporters or as an adaptation to the more demanding metabolic state.

Syntaxin 4 in 3T3-L1 adipocytes: regulation by insulin and participation in insulin-dependent glucose transport.

Syntaxins are thought to be membrane receptors that bind proteins of the synaptobrevin/vesicle-associated membrane protein (VAMP) family found on transport vesicles. Recently, we detected synaptobrevin II and cellubrevin on immunopurified vesicles containing the glucose transporter 4 (GLUT4) in insulin-responsive cells. In an effort to identify the plasma membrane receptors for these vesicles, we now examine the expression of syntaxins in the 3T3-L1 adipocyte cell line. Neither syntaxin 1A nor 1B was found, in keeping with the neuronal restriction of these isoforms. In contrast, syntaxins 2 and 4 were readily detectable. By subcellular fractionation and estimation of protein yields, 67% of syntaxin 4 was localized to the plasma membrane, 24% to the low-density microsomes, and 9% to the high-density microsomes. Interestingly, acute insulin treatment decreased the content of syntaxin 4 in low-density microsomes and caused a corresponding gain in the plasma membrane fraction, reminiscent of the recruitment of GLUT4 glucose transporters. In contrast, there was no change in the distribution of syntaxin 2, which was mostly associated in the plasma membrane. A fraction of the intracellular syntaxin 4 was recovered with immunopurified GLUT4-containing vesicles. Moreover, anti-syntaxin 4 antibodies introduced in permeabilized 3T3-L1 adipocytes significantly reduced the insulin-dependent stimulation of glucose transport, in contrast to the introduction of irrelevant immunoglobulin G, which was without consequence. We propose that either the plasma membrane and/or the vesicular syntaxin 4 are involved in docking and/or fusion of GLUT4 vesicles at the cell surface of 3T3-L1 adipocytes.

Hormonal regulation of the Na(+)-K(+)-ATPase: mechanisms underlying rapid and sustained changes in pump activity.

The sodium-potassium-activated adenosinetriphosphatase (Na(+)-K(+)-ATPase; Na(+)-K+ pump) is a ubiquitous plasma membrane enzyme that catalyzes the movement of K+ into cells in exchange for Na+. In addition, it provides the driving force for the transport of other solutes, notably amino acids, sugar, and phosphate. The regulation of Na(+)-K(+)-ATPase in various tissues is under the control of a number of circulating hormones that impart both short- and long-term control over its activity. The molecular mechanisms by which hormones alter Na(+)-K(+)-ATPase activity have only begun to be studied. In this review, we assess the acute and long-term actions of a number of hormones (aldosterone, thyroid hormone, catecholamines, insulin, carbachol) on the Na(+)-K+ pump. The long-term regulation exerted by thyroid hormone and aldosterone is mediated by changes in gene expression. The short-term regulation exerted by catecholamines is mediated by reversible phosphorylation of the pump catalytic subunit. Recent evidence supports regulation of the pump by phosphorylation in vitro and in intact cells. Finally, in some tissues the rapid action of insulin, aldosterone, and carbachol involves changes in the subcellular distribution of pump units.

Activation of hepatic glutaminase in the endotoxin-treated rat.

The basis for the accelerated hepatic utilization of glutamine that occurs during endotoxemia was investigated. In rats treated with Escherichia coli lipopolysaccharide, glutaminase activity, measured in membranes of freezed-thawed liver mitochondria, was unchanged compared with that of controls. However, flux through glutaminase in intact mitochondria was increased more than 3.5-fold by the endotoxin treatment. The effect was associated with an increase in the sensitivity of glutaminase flux to phosphate, an activator of the enzyme. These findings are similar to the activation of glutaminase by glucogenic hormones. We, therefore, propose that the increased hepatic consumption of glutamine during endotoxemia is due to an activation of glutaminase that is only evident in intact mitochondria.

Hormonal control of hepatic glutaminase.

(1) Glucagon activates hepatic glutaminase in vivo. Mitochondria from glucagon-injected rats retain an enhanced capacity to catabolize glutamine and this is more sensitive to activation by inorganic phosphate. The glucagon-elicited stimulation of glutaminase is not evident in broken mitochondria. A similar activation of glutaminase occurs in a number of situations which are associated with elevated glucagon levels in vivo, i.e., after a high-protein meal, after injection of bacterial endotoxin and in diabetes mellitus. (2) Studies in isolated hepatocytes revealed that glutaminase could be activated, not only by glucagon, but also by a cell-permeable protein kinase A activator (Sp-cAMPS) and by a cell-permeable protein phosphatase 1 and 2A inhibitor (okadaic acid). However, the activation of glutaminase by glucagon was not inhibited by a cell-permeable protein kinase A inhibitor (Rp-8-Br-cAMPS). We suggest that the signalling pathway, for glutaminase activation by glucagon, is complex and possibly contains redundant elements.

Rapid activation of hepatic glutaminase in rats fed on a single high-protein meal.

We report that hepatic glutaminase is rapidly activated in rats fed on a single high-protein (60% casein) meal. Rats previously fed on a normal-protein (15% casein) diet for 3-4 days were given a high-protein meal for 2 h. The high-protein meal increased the rate of flux through glutaminase in intact liver mitochondria nearly 3-fold (20.6 +/- 1.7 nmol/min per mg of protein versus 7.5 +/- 2.9 nmol/min per mg of protein) at a P(i) concentration of 10 mM. The activation of flux through glutaminase by a high-protein meal involved an increased sensitivity of glutaminase to P(i), an activator of the enzyme. The Ka for P(i) was 1.0 mM and 24.1 mM in mitochondria from rats fed on the high-protein and normal-protein meals respectively. We measured the concentration of P(i) in the mitochondrial matrix and found that it did not differ in mitochondria from rats fed on the high-protein and normal-protein meals, suggesting that the effect of the high-protein meal on the P(i)-sensitivity of glutaminase was not due to a change in the distribution of P(i) across the mitochondrial inner membrane. Glutaminase activity was measured by using mitochondrial membranes from frozen-thawed mitochondria. Glutaminase activity and its dependence on P(i) were similar for preparations from rats fed on high-protein and normal-protein meals. These findings show that hepatic glutaminase is stimulated rapidly by a high-protein meal. This is part of the physiological hepatic response to increased protein intake which permits the liver to cope with the influx of glutamine occurring at this time.