Alpha-adrenergic stimulation of glutamine metabolism in isolated rat hepatocytes.

The mechanisms by means of which phenylephrine stimulates glutamine metabolism were studied in isolated rat hepatocytes. In the first 2 min after phenylephrine addition there was a rapid fall in the concentrations of intracellular 2-oxoglutarate and glutamate, presumably owing to activation of 2-oxoglutarate dehydrogenase. This was followed 2-3 min later by activation of glutaminase and by increases in glutamate and 2-oxoglutarate. Activation of glutaminase by phenylephrine was due to direct stimulation of the enzyme rather than to reversal of inhibition by the decrease in 2-oxoglutarate and glutamate. The stimulation of glutaminase by phenylephrine is partly due to an increase in the affinity of the enzyme for ammonia, its essential activator. It is concluded that stimulation of steady-state flux through the pathway from glutamine to glucose and urea can only be achieved by stimulation of glutaminase, the first enzyme in the pathway.     

alpha-adrenergic stimulation of respiration in isolated rat hepatocytes.

1. The alpha-adrenergic agonists noradrenaline (in the presence of beta-blocker) and phenylephrine cause a transient stimulation of the respiration in isolated rat hepatocytes. After a lag period of 12s, this activation first attains its maximal value (+24%) for about 1 min and then falls to a sustained value (+15%). The effect is blocked by the alpha-antagonists phenoxybenzamine and phentolamine. It is dose-dependent, with an half-maximal stimulation by 16 nM-noradrenaline, which is similar to that found for other cell responses to the hormone. 2. Vasopressin and ATP, which in common with alpha-agonists are believed to increase intracellular [Ca2+], induce similar activation in the respiration rate. 3. The alpha-adrenergic-mediated respiration depends on extracellular Ca2+. The activation is decreased or abolished when extracellular [Ca2+] is decreased by adding EGTA, or when the Ca2+ antagonists Mn2+ and La3+ are present in the incubation medium. 4. It is suggested that the activation of the mitochondrial respiration rate results from the increase in cytosolic Ca2+ concentration, presumably via Ca2+ influx or Ca2+ release from the plasma membrane or endoplasmic reticulum.     

Uptake and metabolism of S-adenosyl-L-methionine by isolated rat hepatocytes

In the present study, direct evidence is given to SAMe capability of crossing the membrane of isolated rat hepatocytes. The kinetics of SAMe uptake is biphasic: a fast phase being completed in less than 15 sec and a slower one with an apparent K_m of 8.33 μM and a V_max of 10.6 pmol/min/mg protein. Both processes are pH and temperature dependent. Analysis of the fast phase by a Scatchard plot discloses two sets of binding sites of high and low affinity, respectively. Experiments carried out incubating isolated hepatocytes with double-labelled SAMe (methyl-³H, carboxyl-¹⁴C) have shown that about 70% of SAMe uptake by the cell is rapidly decarboxylated.     

Transport and metabolism of thiamin in isolated rat hepatocytes.

This study examines thiamin transport in isolated rat hepatocytes and its relationship to thiamin phosphorylation. In an Na+ medium, [35S]thiamin, 3 microM, was accumulated rapidly by the cells, and a near study state intra-/extracellular distribution ratio of 3 was attained in 1 min. However, the uptake of radioactivity continued to increase with time owing principally to the accumulation of [35S]thiamin pyrophosphate (TPP). In a choline, Li+ or K+ medium, the steady state intra-/extracellular distribution ratio of [35S]thiamin was decreased to less than or equal to 1.1. Accordingly, the rate of formation of [35S]TPP also decreased. Ouabain and uncouplers of oxidative phosphorylation significantly lowered the distribution ratio of intra-/extracellular [35S]thiamin. These data indicate that thiamin transport in liver is concentrative, Na+-dependent, and dependent on biological energy. Additionally, they suggest that thiamin transport plays a significant role in governing the rate of synthesis of TPP. Neither pyrithiamin, an inhibitor of thiamin pyrophosphokinase nor o-benzoylthiamin disulfide, a permeable thiamin analog, affected the distribution ratio of intra-/extracellular [35S]thiamin, but preferentially inhibited the phosphorylation of [35S]thiamin. By contrast, amprolium primarily inhibited uptake. These data suggest that thiamin transport and phosphorylation can be differentiated by the action of appropriate inhibitors.     

The stimulation of glycogenolysis in isolated hepatocytes by opioid peptides.

The opioid peptides [Leu]enkephalin and dynorphin-(1-13) were shown to enhance glycogen breakdown when added directly to hepatocytes. This was the result of a concerted effect on the enzymes of glycogen metabolism, with a stimulation of glycogen phosphorylase activity and a simultaneous decrease in glycogen synthase I activity. The latter only became significant when the enzyme was activated by incubating the cells in presence of 20 mM- or 40 mM-glucose. The effect of the opioid peptides was independent of an increase in cyclic AMP or any change in the activity ratio of the cyclic AMP-dependent protein kinase and was abolished by depleting the cells of Ca2+. Both [Leu]enkephalin and dynorphin-(1-13) produced a significant decrease in cyclic AMP formation, suggesting that in liver, as in neuronal tissue, they may act by inhibiting adenylate cyclase activity.     

Inability of glucagon to regulate glycogen metabolism in rat hepatocytes isolated after fasting and refeeding high-carbohydrate diets

Studies are described which demonstrate that the ability of glucagon, epinephrine, and dibutyryl-cAMP to stimulate glycogenolysis is impaired in rat hepatocytes isolated from animals starved for 24 h and then refed a sucrose-rich diet or refed standard rat chow. The impaired regulation of glycogenolysis by glucagon was observed within 24 h after refeeding and persisted for at least 3 days. The inability of glucagon to stimulate glycogen breakdown in the refed condition appeared to be due to a suppressed activation of glycogen phosphorylase and phosphorylase b kinase by the hormone. The capacity of glucagon to regulate pyruvate kinase and glycolysis was not altered by refeeding, suggesting that the defect lies beyond interaction of the hormone at its receptor. Prolonged incubation of hepatocytes from refed rats was accompanied by depletion of glycogen reserves and was accompanied by restoration of hormonal stimulation of glycogenolysis. Addition of glycogen to cell-free extracts was found to inhibit phosphorylase b kinase but not phosphorylase. The findings of this investigation are consistent with the interpretation that high levels of glycogen present of liver after refeeding may lead to a diminished activity of phosphorylase b kinase and its hormonal regulation.     

Studies of isoniazid metabolism in isolated rat hepatocytes by mass fragmentography

Isoniazid metabolism in isolated rat hepatocytes was studied by mass fragmentography using single ion monitoring. Isoniazid and its metabolites were determined as the trimethylsilylated derivatives of acetylisoniazid and diacetylhydrazine and of the benzaldehyde hydrazones of isoniazid and acetylhydrazine. Deuterated analogues served as internal standards. Hydrazine was quantitated as benzalazine using ¹⁵N-labeled hydrazine as an internal standard. The method is well suited for the microanalysis of isoniazid metabolites in specificity and reliability to demonstrate the overall pathway of isoniazid metabolism, from which it was clarified that the greater part of hydrazine, a hazardous metabolite of isoniazid, was formed through the direct hydrolysis of isoniazid itself as expected.     

Ethanol inhibition of vinyl chloride metabolism in isolated rat hepatocytes.

Isolated rat liver cells convert [14C]vinyl chloride into non-volatile metabolites. The metabolism is not increased by in vivo pretreatment with phenobarbital. It is sensitive to inhibition by ethanol, which at a concentration of 4 mM inhibits vinyl chloride metabolism to 50% in hepatocyte suspensions. The metabolic activity is NADPH-dependent and is localized in the microsomal fraction of the liver. The enzyme is also strongly inhibited by tetrahydrofuran, indicating that it could be identical to an ethanol-inducible cytochrome P-450 described in the literature [1].     

The effect of feeding rats with partially hydrogenated marine oil or rapeseed oil on the chain shortening of erucic acid in perfused heart.

1. The metabolism of [14(-14)C]erucic acid and [U-14C]palmitic acid was studied in perfused hearts from rats fed diets containing hydrogenated marine oil, rapeseed oil or peanut oil for three weeks. 2. [14C]Erucic acid was shortened to [14C]eicosenoic acid (20 : 1, n -- 9) and [14C]oleic acid (18 : 1, n -- 9) in perfused rat hearts from all diet groups. The rapeseed oil diet caused a three-fold increase and the marine oil diet a four-fold increase in the amount of chain-shortened products recovered in heart lipids at the end of perfusion, compared to peanut oil diet. 3. The content of C16:1, C18:1 and C20:1 fatty acids was increased in heart lipids of rats fed hydrogenated marine oil or rapseed oil diet, compared to peanut oil diet. 4. Feeding hydrogenated marine oil or rapeseed oil to the rats induced a 85% increase in catalase activity, a 20% increase in the activity of cytochrome oxidase and a 30--40% increase in the content of total CoA in the heart compared to rats fed peanut oil diet. 5. It is suggested that [14(-14)C]erucic acid is shortened by the beta-oxidation system of peroxisomes in the heart. The increased chain shortening in the hearts from animals fed rapeseed oil or partially hydrogenated marine oil for three weeks may be an important part of an adaptation process.     

The stimulation of glycogenolysis and gluconeogenesis in isolated hepatocytes by opioid peptides.

The opioid agonists [leucine]enkephalin, [D-Ala2,D-Leu5]enkephalin and dynorphin-(1-13)-peptide, but not morphine, stimulated the conversion of [2-14C]pyruvate into glucose and glycogenolysis when added directly to isolated hepatocytes. Naloxone produced a small but significant inhibition of both the basal and stimulated rate of incorporation of label into glucose but had no effect on the total glucose output by the cells. The effects of the opioid peptides were mediated by a cyclic AMP-independent mechanism.     

The effect of an NADPH-regenerating system on biphenyl metabolism in isolated rat hepatocytes.

Biphenyl 4-hydroxylation was studied in isolated rat hepatocytes. It was found that there was in inter-relationship between 4-hydroxylase activity and glucuronidase activity, removal of 4-hydroxybiphenyl by conjugation being necessary to stimulate a second phase of hydroxylation. Addition of an NADPH-regenerating system resulted in an initial depression of both processes, but later their activities were enhanced. This action could not be explained by the presence of non-viable cells.     

Inhibition of oxidative metabolism by propionic acid and its reversal by carnitine in isolated rat hepatocytes.

The present study was designed to study the interaction of propionic acid and carnitine on oxidative metabolism by isolated rat hepatocytes. Propionic acid (10 mM) inhibited hepatocyte oxidation of [1-14C]-pyruvate (10 mM) by 60%. This inhibition was not the result of substrate competition, as butyric acid had minimal effects on pyruvate oxidation. Carnitine had a small inhibitory effect on pyruvate oxidation in the hepatocyte system (210 +/- 19 and 184 +/- 18 nmol of pyruvate/60 min per mg of protein in the absence and presence of 10 mM-carnitine respectively; means +/- S.E.M., n = 10). However, in the presence of propionic acid (10 mM), carnitine (10 mM) increased the rate of pyruvate oxidation by 19%. Under conditions where carnitine partially reversed the inhibitory effect of propionic acid on pyruvate oxidation, formation of propionylcarnitine was documented by using fast-atom-bombardment mass spectroscopy. Propionic acid also inhibited oxidation of [1-14C]palmitic acid (0.8 mM) by hepatocytes isolated from fed rats. The degree of inhibition caused by propionic acid was decreased in the presence of 10 mM-carnitine (41% inhibition in the absence of carnitine, 22% inhibition in the presence of carnitine). Propionic acid did not inhibit [1-14C]palmitic acid oxidation by hepatocytes isolated from 48 h-starved rats. These results demonstrate that propionic acid interferes with oxidative metabolism in intact hepatocytes. Carnitine partially reverses the inhibition of pyruvate and palmitic acid oxidation by propionic acid, and this reversal is associated with increased propionylcarnitine formation. The present study provides a metabolic basis for the efficacy of carnitine in patients with abnormal organic acid accumulation, and the observation that such patients appear to have increased carnitine requirements ('carnitine insufficiency').     

Stimulation of arachidonic acid metabolism by CCl4 in isolated rat hepatocytes

Arachidonic acid metabolism was evaluated in isolated rat hepatocytes after CCl4 exposure. CCl4 induced dose-dependently the synthesis and release of prostacyclin (PGI2) and thromboxane (TXB2). Treatment with prostaglandin E2 (PGE2) 30 min after exposure to CCl4, significantly reduced the cell damage as well as the release of TXB2 from the cells.     

Rapid stimulation of free glucuronate formation by non-glucuronidable xenobiotics in isolated rat hepatocytes

Vitamin C synthesis in rat liver is enhanced by several xenobiotics, including aminopyrine and chloretone. The effect of these agents has been linked to induction of enzymes potentially involved in the formation of glucuronate, a precursor of vitamin C. Using isolated rat hepatocytes as a model, we show that a series of agents (aminopyrine, antipyrine, chloretone, clotrimazole, metyrapone, proadifen, and barbital) induced in a few minutes an up to 15-fold increase in the formation of glucuronate, which was best observed in the presence of sorbinil, an inhibitor of glucuronate reductase. They also caused an approximately 2-fold decrease in the concentration of UDP-glucuronate but little if any change in the concentration of UDP-glucose. Depletion of UDP-glucuronate with resorcinol or d-galactosamine markedly decreased the formation of glucuronate both in the presence and in the absence of aminopyrine, confirming the precursor-product relationship between UDP-glucuronate and free glucuronate. Most of the agents did not induce the formation of detectable amounts of glucuronides, indicating that the formation of glucuronate is not due to a glucuronidation-deglucuronidation cycle. With the exception of barbital (which inhibits glucuronate reductase), all of the above mentioned agents also caused an increase in the concentration of ascorbic acid. They had little effect on glutathione concentration, and their effect on glucuronate and vitamin C formation was not mimicked by glutathione-depleting agents such as diamide and buthionine sulfoximine. It is concluded that the stimulation of vitamin C synthesis exerted by some xenobiotics is mediated through a rapid increase in the conversion of UDP-glucuronate to glucuronate, which does not apparently involve a glucuronidation-deglucuronidation cycle.     

The assessment of viability in isolated rat hepatocytes.

Isolated rat hepatocytes are used in many metabolic studies, but the viability of these cell preparations is often not adequately established. The present study shows that ATP content is a more reliable index of metabolic viability than trypan blue exclusion. At some of the low trypan blue exclusion levels quoted in the literature, a high percentage of cell preparations is likely to be nonviable by the criterion of ATP content. We suggest that ATP content measured on initial cell preparations and at the end of all incubation procedures is essential for establishing cell viability for metabolic studies on isolated hepatocytes.     

Effect of cell density on metabolism in isolated rat hepatocytes

Freshly isolated rat hepatocytes show many changes in metabolic activities as a function of cell density in the incubation flask. Fatty acid synthesis, cholesterol synthesis, general protein synthesis, and rates of accumulation of pyruvate, lactate, citrate, acetyl-CoA, acetoacetate and beta-hydroxybutyrate decrease as the cell density increases between 1 mg/ml and 60 mg/ml. Glucose release only decreases between 1-5 mg/ml and the concentration of ATP does not vary at any density. There is a small increase in the lactate/pyruvate ratio and a dramatic decrease in the beta-hydroxybutyrate/acetoacetate ratio with increasing cell concentration. When cells at 8 or 28 mg/ml were incubated with added lactate and pyruvate, or alanine, a two fold increase in fatty acid synthesis and 50% decrease in cholesterol synthesis were observed as compared to rates with endogenous substrate. With added glucose the synthetic rates were similar to those obtained with endogenous substrate. However, regardless of the type of substrate used, the less dense cells gave rates up to three times greater than that of the more dense cells. When conditioned medium isolated after incubation of cells at high density was added to the less dense cells, a decrease in the rates of fatty acid synthesis and cholesterol synthesis was observed in the less dense cells; however, when medium from the less dense cells after incubation for the same period was added to the more dense cells, there was no significant change in fatty acid or cholesterol synthesis. These results suggest that a factor may be released into the medium of incubating hepatocytes that progressively inhibits certain metabolic processes as the cell density increases.     

Calcium metabolism in rat hepatocytes.

1. The total calcium concentration in rat hepatocytes was 7.9 microgram-atoms/g dry wt.; 77% of this was mitochondrial. Approx. 20% of cell calcium exchanged with 45Ca within 2 min. Thereafter incorporation proceeded at a low rate to reach 28% of total calcium after 60 min. Incorporation into mitochondria showed a similar time course and accounted for 20% of mitochondrial total calcium after 60 min. 2. The alpha-adrenergic agonists phenylephrine and adrenaline + propranolol stimulated incorporation of 45Ca into hepatocytes. Phenylephrine was shown to increase total calcium in hepatocytes. Phenylephrine inhibited efflux fo 45Ca from hepatocytes perifused with calcium-free medium. 3. Glucagon, dibutryl cyclic AMP and beta-adrenergic agonists adrenaline and 3-isobutyl-1-methyl-xanthine stimulated calcium efflux from hepatocytes perifused with calcium-free medium. The effect of glucagon was blocked by insulin. Insulin itself had no effect on calcium efflux and it did not affect the response to dibutyryl cyclic AMP. 4. Incorporation of 45Ca into mitochondria in hepatocytes was stimulated by phenylephrine and inhibited by glucagon and by carbonyl cyanide p-trifluoromethoxyphenylhydrazone. The effect of glucagon was blocked by insulin. 5. Ionophore A23187 stimulated hepatocyte uptake of 45Ca, uptake of 45Ca into mitochondria in hepatocytes and efflux of 45Ca into a calcium-free medium.     

Phosphatidylinositol metabolism in rat hepatocytes stimulated by vasopressin.

In isolated rat hepatocytes, vasopressin evoked a large increase in the incorporation of [32P]Pi into phosphatidylinositol, accompanied by smaller increases in the incorporation of [1-14C]oleate and [U-14C]glycerol. Incorporation of these precursors into the other major phospholipids was unchanged during vasopressin treatment. Vasopressin also promoted phosphatidylinositol breakdown in hepatocytes. Half-maximum effects on phosphatidylinositol breakdown and on phosphatidylinositol labelling occurred at about 5 nM-vasopressin, a concentration at which approximately half of the hepatic vasopressin receptors are occupied but which is much greater than is needed to produce half-maximal activation of glycogen phosphorylase. Insulin did not change the incorporation of [32P]Pi into the phospholipids of hepatocytes and it had no effect on the response to vasopressin. Although the incorporation of [32P]Pi into hepatocyte lipids was decreased when cells were incubated in a Ca2+-free medium, vasopressin still provoked a substantial stimulation of phosphatidylinositol labelling under these conditions. Studies with the antagonist [1-(beta-mercapto-beta, beta-cyclopentamethylenepropionic acid),8-arginine]vasopressin indicated that the hepatic vasopressin receptors that control phosphatidylinositol metabolism are similar to those that mediate the vasopressor response in vivo. When prelabelled hepatocytes were stimulated for 5 min and then subjected to subcellular fractionation. The decrease in [3H]phosphatidylinositol content in each cell fraction with approximately in proportion to its original phosphatidylinositol content. This may be a consequence of phosphatidylinositol breakdown at a single site, followed by rapid phosphatidylinositol exchange between membranes leading to re-establishment of an equilibrium distribution.