Dependence of ATP citrate lyase activity on cell density of isolated rat hepatocytes

The activities of two enzymes involved in the lipogenic process, ATP citrate lyase and NADP-linked malic enzyme were evaluated as a function of cell density in isolated rat hepatocytes. The activity of ATP citrate lyase was profoundly affected by cell density with the activity/cell being higher at low cell densities than at high cell densities. Malic enzyme was not similarly affected, nor was cellular ATP content. The effect was observed regardless of dietary state but was most dramatic with hepatocytes from fasted-refed rats. Both an activator and an inhibitor of ATP citrate lyase have been isolated from conditioned medium from cells at low density and at high density, respectively. The activator fraction was heat stable while the inhibitor fraction was heat labile, and both factors had molecular weights in excess of 10,000 daltons.     

Effect of selenium deficiency and vitamin E deficiency on glutathione metabolism in isolated rat hepatocytes

Selenium deficiency and vitamin E deficiency both affect xenobiotic metabolism and toxicity. In addition, selenium deficiency causes changes in the activity of some glutathione-requiring enzymes. We have studied glutathione metabolism in isolated hepatocytes from selenium-deficient, vitamin E-deficient, and control rats. Cell viability, as measured by trypan blue exclusion, was comparable for all groups during the 5-h incubation. Freshly isolated hepatocytes had the same glutathione concentration regardless of diet group. During the incubation, however, the glutathione concentration in selenium-deficient hepatocytes rose to 1.4 times that in control hepatocytes. The selenium-deficient cells also released twice as much glutathione into the incubation medium as did the control cells. Total glutathione (intracellular plus extracellular) in the incubation flask increased from 47.7 +/- 8.9 to 152 +/- 16.5 nmol/10(6) selenium-deficient cells over 5 h compared with an increase from 46.7 +/- 7.1 to 92.0 +/- 17.4 nmol/10(6) control cells and from 47.7 +/- 11.7 to 79.5 +/- 24.9 nmol/10(6) vitamin E-deficient cells. This overall increase in glutathione concentration suggested that glutathione synthesis was accelerated by selenium deficiency. The activity of gamma-glutamylcysteine synthetase was twice as great in selenium-deficient liver supernatant (105,000 X g) as in vitamin E-deficient or control liver supernatant (105,000 X g). Hemoglobin-free perfused livers were used to determine the form of glutathione released and its route. Selenium-deficient livers released 4 times as much GSH into the caval perfusate as did control livers. Plasma glutathione concentration in selenium-deficient rats was found to be 2-fold that in control rats, suggesting that increased GSH synthesis and release is an in vivo phenomenon associated with selenium deficiency.     

Effect of insulin on glycogen metabolism in isolated catfish hepatocytes

Insulin effect on carbohydrate metabolism in catfish hepatocytes consisted of a significant decrease of cell glycogen concentration both in the absence and in the presence of glucose in the medium. The hormone did not influence either the output of glucose from the cell or the intracellular glucose level. Experiments with radioactive glucose showed a very low uptake of the sugar by the hepatocytes; correspondingly the incorporation of radioactivity into glycogen was very low and not influenced by insulin. The glycogen content in catfish liver cells was influenced by the hormone in the opposite way to rat liver cells.     

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.     

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.     

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.     

Acute effects of insulin on fatty acid metabolism in isolated rat hepatocytes

Isolated rat hepatocytes, previously shown to display enhanced rates of fatty acid biosynthesis upon a brief exposure to insulin, were used to study acute effects of this hormone on other aspects of hepatic fatty acid metabolism. Insulin activates the incorporation of exogenously added fatty acids into glycerolipids and depresses their utilization in the formation of ketone bodies. Insulin increases both the activity of acetyl-CoA carboxylase and the cellular content of malonyl-CoA. Evidence is presented that malonyl-CoA plays an important role in the insulin-mediated control of both ketogenesis and de novo fatty acid synthesis. All metabolic parameters studied are affected by glucagon in a manner opposite to that of insulin.     

Dietary fat type alters glucose metabolism in isolated rat hepatocytes

Dietary fat type can influence the regulation of carbohydrate metabolism in multiple tissue types. The influence of feeding high-fat (40% of kilocalories) diets containing either menhaden oil (MO) or coconut oil (CO) on hepatic glycogenolytic and gluconeogenic capacities was studied in isolated rat hepatocytes. Estimates of both glycogenolytic and gluconeogenic capacities were performed on hepatocytes isolated from fed and fasted animals, respectively. In MO-fed animals, both basal and hormone-stimulated rates of glucose production were significantly greater than those in CO-fed animals. However, both groups displayed a similar maximal increase in glucose production above basal for glucagon and epinephrine (2.3- and 1.9-fold, respectively). Basal rates of adenosine 3′,5′-cyclic phosphate (cAMP) production were not different between groups whereas glucagon-stimulated cAMP production was increased twofold in the MO-fed group. In both MO and CO groups, the addition of 10 nM insulin reduced glucose production in fed animals to similar absolute rates. In animals fasted for 24 hours, gluconeogenic capacity was estimated using 10 mM pyruvate, lactate, or glycerol. Glucose production from all substrates was significantly greater in CO-fed animals. In addition to increased gluconeogenic rates, maximal phosphoenolpyruvate carboxykinase (PEPCK) activity was increased in the CO-fed group. Insulin reduced glucose production in both dietary groups, but the absolute rate of glucose production was 28% greater in the CO-fed group relative to the MO-fed group. In summary, dietary fat type can markedly influence the regulation of hepatic glucose metabolism in multiple metabolic pathways. MO feeding promoted glycogenolysis and sensitivity to insulin whereas CO feeding favored gluconeogenesis and reduced insulin sensitivity.     

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].     

Action of insulin on the subcellular metabolism of polyphosphoinositides in isolated rat hepatocytes

The effect of insulin on 32Pi incorporation into phospholipids in various subcellular sites of isolated rat hepatocytes was investigated. After labeling the phospholipids of hepatocytes from rats previously starved for 24 h with 32Pi (10 mu Ci/10(6) cells) for 90 min, either saline or insulin (32 nM) was added. Following incubations of 1, 5, and 30 min, chilled cells were rapidly washed, homogenized in the presence of inhibitors of phospholipid degradation, and fractionated into the major subcellular organelles. Phospholipids were extracted from plasma membranes, microsomes, lysosomes, mitochondria, and nuclei with acidic chloroform:methanol. The aqueous deacylation products were separated by anion exchange high performance liquid chromatography, and the 32Pi incorporated into all the major diacylglycerophospholipids was determined. In parallel experiments, the specific radioactivity of 32Pi and [gamma-32P]ATP was determined. The results revealed that insulin had no effect on the turnover of the major phospholipids, including the polyphosphoinositides, of all subcellular compartments analyzed relative to the control. In addition, there were no significant differences in the amount and 32P labeling of cellular orthophosphate between saline- and insulin-treated cells. The specific radioactivity of [gamma-32P]ATP was increased by 20% after 30-min treatment with insulin, requiring appropriate correction of 32P-labeled phosphatidic acid, phosphatidylinositol 4-phosphate, and phosphatidylinositol 4,5-bisphosphate for estimation of mass changes at near steady-state labeling of cellular ATP.     

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.     

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 an 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.     

Methionine metabolism and ultrastructural changes with D-galactosamine in isolated rat hepatocytes

The biochemical and morphological effects of 2, 10 and 100 mM of D-galactosamine (GalN) were studied in isolated rat hepatocytes during 2 h of incubation. Lactate dehydrogenase (LDH), alanine aminotransferase (ALAT) and cell viability did not change, whatever the concentration used. The variations observed, which were dose dependent, included a large drop in ATP levels and inhibition of RNA and protein synthesis. A very high concentration of GalN was necessary, however, to induce a significant decline in methionine adenosyltransferase activity compared to control cells.The use of L-[methyl-¹⁴C]methionine during cell incubation with GalN demonstrated a decrease of S-adenosyl-L-methionine (SAMe) and an accumulation of L-methionine content related to the GalN concentration. These results suggested that an hepatotoxic agent such as GalN was able to induce disturbances of methionine metabolism.Some of the ultrastructural changes observed were different from those previously found in vivo, in rats given GalN intraperitoneally, underlining the marked difference between in vivo and in vitro intoxication.     

Effect of berenil on polyamine metabolism in primary cultured rat hepatocytes

• 1.1. Putrescine and spermidine content increased in hepatocytes during culture. In the presence of 10 μM Berenil, putrescine content was further increased, while the increase of spermidine was prevented.
• 2.2. Ornithine decarboxylase activity was markedly reduced, and to a lesser extent also S-adenosyl-methionine decarboxylase activity.
• 3.3. Berenil appears to promote an increase in the transformation of spermidine into putrescine, and to inhibit the polyamine efflux.

     

Effect of growth hormone on protein phosphorylation in isolated rat hepatocytes

Hepatocytes from male rats were incubated with [32P]Pi for 40 min at 37 degrees C, thereby equilibrating the cellular ATP pool with 32P. Subsequent exposure to bovine growth hormone for 10 additional min did not change the specific activity of cellular [gamma-32P]ATP. Two-dimensional gel electrophoresis or chromatofocusing followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis was used to fractionate phosphoproteins solubilized from control or hormone-stimulated cells. Stimulation of hepatocytes with 5 nM growth hormone for 10 min at 37 degrees C affected the phosphorylation of a number of proteins including an Mr 46,000 species of pI 4.7 whose phosphorylation was augmented (2.65 +/- 0.50)-fold. A significant fraction of the maximal effect of growth hormone on phosphorylation of the Mr 46,000 species was elicited by 1-5% receptor occupancy. Bovine growth hormone, which binds to somatogenic receptors with great specificity, or recombinant human growth hormone, which is not contaminated with other hormones, affected phosphorylation of hepatic proteins similarly. The Mr 46,000 phosphoprotein was isolated in a fraction enriched in cytosol after centrifugation of cellular homogenates. Phosphorylation of the Mr 46,000 phosphoprotein was also increased (1.75 +/- 0.35)-fold and (2.15 +/- 0.50)-fold by insulin and glucagon, respectively. These observations are consistent with the possibility that selective changes in the phosphorylation state of cellular proteins may mediate growth hormone actions in cells.     

Effect of hypoxic cell radiosensitizers on glutathione level and related enzyme activities in isolated rat hepatocytes

A comparative study of the effect of misonidazole and novel radiosensitizers on glutathione (GSH) levels and related enzyme activities in isolated rat hepatocytes was performed. Incubation of hepatocytes with 5 mM radiosensitizers led to a decrease in the intracellular GSH level. The most pronounced decrease in cellular GSH was evoked by 2,4-dinitroimidazole-1-ethanol (DNIE); after incubation for only 15 min, GSH was hardly detected. DNIE-mediated GSH loss was dependent upon its concentration. DNIE reacted with GSH nonenzymatically as well as with diethylmaleate, while misonidazole and 1-methyl-2-methyl-sulfinyl-5-methoxycarbonylimidazole (KIH-3) did not. Addition of partially purified glutathione S-transferase (GST) did not enhance DNIE-mediated GSH loss in a cell-free system. DNIE inhibited glutathione peroxidase (GSH-Px), GST, and glutathione reductase (GSSG-R) activities in hepatocytes, while misonidazole and KIH-3 did not. GSH-Px activity assayed with H2O2 as substrate was the most inhibited. Inhibition of GSH-Px activity assayed with cumene hydroperoxide as substrate and GST was less than that of GSH-Px assayed with H2O2 as substrate. GSSG-R activity was decreased by DNIE, but not significantly. Incubation of purified GSH-Px with DNIE resulted in a little change in the activity when assayed with H2O2 as substrate.     

Effect of D-Penicillamine on iron uptake by isolated rat hepatocytes

d-penicillamine (β,β-dimethylcysteine) promotes the incorporation of iron into isolated rat hepatocytes. The mechanism for doing this remains unknown. No differences in iron distribution between control and treated cells has been observed. Ferritin appears as the main destination of internalized iron in both cases. Therefore, increasing iron storage may appear as a side effect of the use ofd-penicillamine as a therapeutic agent for several diseases.     

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.     

Interactions of dietary fat and 2,5-anhydro-D-mannitol on energy metabolism in isolated rat hepatocytes

The fructose analog 2,5-anhydro-D-mannitol (2,5-AM) stimulates feeding in rats by reducing ATP content in the liver. These behavioral and metabolic effects occur with rats fed a high-carbohydrate/low-fat (HC/LF) diet, but they are prevented or attenuated when the animals eat high-fat/low-carbohydrate (HF/LC) food. To examine the metabolic bases for this effect of diet, we assessed the actions of 2,5-AM on ATP content, oxygen consumption, and substrate oxidation in isolated hepatocytes from rats fed one of the two diets. Compared with cells from rats fed the HC/LF diet ("HC/LF" cells), cells from rats fed the HF/LC diet ("HF/LC" cells) had similar ATP contents but lower oxygen consumption, decreased fructose, and increased palmitate oxidation. 2,5-AM did not decrease ATP content or oxygen consumption in HF/LC cells as much as it did in HC/LF hepatocytes, and it only affected fructose and palmitate oxidation in HC/LF cells. 31P-NMR spectroscopy indicated that differences in phosphate trapping accounted for differences in depletion of ATP by 2,5-AM. These results suggest that intake of the HF/LC diet prevents the eating response and attenuates the decline in liver ATP by shifting hepatocyte metabolism to favor fat over carbohydrate as an energy-yielding substrate.     

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.