Studies on the assay, activity and sedimentation behaviour of acetyl-CoA carboxylase from isolated hepatocytes incubated with insulin or glucagon.

The activity of acetyl-CoA carboxylase, measured in various ways, was studied in 15000g extracts of rat liver hepatocytes and compared with the rate of fatty acid synthesis in intact hepatocytes incubated with insulin or glucagon. Hepatocyte extracts were prepared by disruption of cells with a Dounce homogenizer or by solubilization with 1.5% (v/v) Triton X-100. Sucrose-density-gradient centrifugation demonstrated that the sedimentation coefficient of acetyl-CoA carboxylase from cell extracts was 30-35S, regardless of the conditions of incubation or disruption of hepatocytes. Solubilization of cells with 1.5% Triton X-100 yielded twice as much enzyme activity (measured by [14C]bicarbonate fixation) in the sucrose-gradient fractions as did cell disruption by the Dounce homogenizer. Analysis by high-performance liquid chromatography of acetyl-CoA carboxylase reaction mixtures showed that [14C]malonyl-CoA accounted for 10-60% of the total acid-stable radioactivity, depending on the method for disrupting hepatocytes and on the preincubation of the 15000g extract, with or without citrate, before assay. Under conditions in which incubation of cells with insulin or glucagon caused an activation or inhibition, respectively, of acetyl-CoA carboxylase, only 25% of the acid-stable radioactivity was [14C]malonyl-CoA and enzyme activity was only 13% (control), 16% (insulin), and 57% (glucagon) of the rate of fatty acid synthesis. Under conditions when up to 60% of the acid-stable radioactivity was [14C]malonyl-CoA and acetyl-CoA carboxylase activity was comparable with the rate of fatty acid synthesis, there was no effect of insulin or glucagon on enzyme activity.     

Lack of insulin stimulation on Percoll-prepared or high-speed-centrifuged rat liver hepatocytes

Rat liver hepatocytes isolated from a 30-31% percoll density gradient at 10,000g are refractory toward insulin stimulation of 14CO2 formation and 14C-incorporation into protein from [2,3-14C]succinate. Basal hepatocyte oxidation of succinate was not impaired by the presence of 5% percoll in the incubation medium nor was it impaired when percoll-free hepatocytes were used that had been isolated after centrifugation at 9000g; however, in both instances the stimulatory effect of insulin was lost. Hepatocyte damage may have occurred in these processes. This is in contrast to previous work which shows that insulin (10 mU/ml) will stimulate [2,3-14C]succinate oxidation and [2,3-14C]succinate carbon incorporation into protein in non-percoll-treated hepatocytes (isolated by centrifugation at 10g) by about 29%. We conclude that the latter procedure although more time consuming is the more gentle method of choice and leaves the hepatocyte in a form more closely related to an in vivo state than does treatment with a percoll density gradient at 10,000g.     

Although it is rapidly metabolized in cultured rat hepatocytes, lauric acid is used for protein acylation

This study was designed to examine the metabolic fate of exogenous lauric acid in cultured rat hepatocytes, in terms of both lipid metabolism and acylation of proteins. Radiolabeled [14C]-lauric acid at 0.1 mM in the culture medium was rapidly taken up by the cells (94.8 +/- 2.2% of the initial radioactivity was cleared from the medium after a 4 h incubation) but its incorporation into cellular lipids was low (24.6 +/- 4.2% of initial radioactivity after 4 h), due to the high beta-oxidation of lauric acid in hepatocytes (38.7 +/- 4.4% after the same time). Among cellular lipids, lauric acid was preferentially incorporated into triglycerides (10.6 +/- 4.6% of initial radioactivity after 4 h). Lauric acid was also rapidly converted to palmitic acid by two successive elongations. Protein acylation was detected after metabolic labeling of the cells with [11,12-3H]-lauric acid. Two-dimensional electrophoresis separation of the cellular proteins and autoradiography evidenced the incorporation of radioactivity into 35 well-resolved proteins. Radiolabeling of several proteins resulted from covalent linkage to the precursor [11,12-3H]-lauric acid or to its elongation product, myristic acid. The covalent linkages between these proteins and lauric acid were broken by base hydrolysis, indicating that the linkage was of the thioester or ester-type. Endogenous myristic acid produced by lauric acid elongation was used for both protein N-myristoylation and protein S-acylation. Therefore, these results show for the first time that, although it is rapidly metabolized in hepatocytes, exogenous lauric acid is a substrate for the acylation of liver proteins.     

Anabolic regulation of gluconeogenesis by insulin in isolated rat hepatocytes

The role of substrate availability in the regulation of gluconeogenesis in isolated rat hepatocytes was studied using [U-14C]alanine as a tracer in the presence of different concentrations of L-alanine in the incubation medium. At low alanine concentrations (0.5 mM) insulin decreased the 14C incorporation into the glucose pool and increased the incorporation of tracer carbons into the protein and lipid pools and into CO2. The net radioactivity lost from the glucose pool was only a small percentage of the total increase in the activity of the protein, lipid, CO2, or glycogen pools, supporting the notion that the effect of insulin in diminishing gluconeogenesis is secondary to its effects on pathways using pyruvate. At higher concentrations of alanine (2.5, 5.0, and 10.0 mM) in the incubation medium insulin increased the movement of alanine carbons into protein and glucose. This suggests that at higher substrate concentrations the ability of the liver to synthesize proteins is overwhelmed and the pyruvate carbons are forced into the gluconeogenesis pathway. These results were further confirmed by using [U-14C]lactate. The increases in observed specific activity of glucose following insulin administration would not be possible if insulin acted by affecting the activity of any enzyme directly involved in the formation or utilization of pyruvate, most of which have been proposed as sites of insulin action. Data presented show that insulin "inhibits" gluconeogenesis by affecting a change in substrate availability.     

The metabolism of cholesterol in the presence of liver mitochondria from normal and thyroxine-treated rats

1. [26-(14)C]- and [4-(14)C]-Cholesterol were incubated with liver mitochondria from normal and thyroxine-treated rats, and the radioactivity was measured in the carbon dioxide evolved during the incubation, in a butanol extract of the incubation mixture and in a volatile fraction containing substances of low molecular weight derived from the side chain of cholesterol. The butanol extract was separated by paper chromatography into three radioactive fractions, one of which contained the steroids more polar than cholesterol. 2. The butanol extract from incubations with [4-(14)C]cholesterol contained a radioactive substance moving with the same R(F) as cholic acid on thin-layer chromatography. 3. After incubation with [26-(14)C]-cholesterol, 60-80% of the radioactivity extracted by steam-distillation of the incubation mixture at acid pH was recovered as [(14)C]propionic acid. 4. In the presence of [26-(14)C]cholesterol, mitochondria from thyroxine-treated rats produced more radioactivity in carbon dioxide and in the volatile fraction, and less radioactivity in the fraction containing the polar steroids, than did mitochondria from normal rats. In the presence of [4-(14)C]cholesterol, mitochondria from thyroxine-treated rats produced the same amount of radioactivity in the polar steroids as did normal mitochondria. 5. Thyroxine treatment had no effect on the capacity of the mitochondria to oxidize propionate to carbon dioxide. 6. These results are best explained by supposing that thyroxine stimulates a rate-limiting reaction leading to the cleavage of the side chain of cholesterol, but has little or no influence on the hydroxylations of the ring system or on the oxidation of the C(3) fragment removed from the side chain.     

Pantethine inhibits cholesterol and fatty acid syntheses and stimulates carbon dioxide formation in isolated rat hepatocytes

The effects of pantethine on cholesterol and fatty acid metabolism were investigated in isolated rat hepatocytes. Preincubation of the cells with pantethine induced a concentration-dependent decrease of the radioactivity incorporated into carbon dioxide and lipids in incubations with [2-14C]acetate. When pantethine and the labeled substrate were simultaneously added to the cell suspension, there was an enhancement of carbon dioxide radioactivity at short incubation time (5 min) whereas, at longer incubation time, values were comparable to those of controls; lipid radioactivity, instead, was dramatically reduced by pantethine even at short incubation time and decreased further during the incubation, being 23% of that of controls at 60 min. Analysis of the incubation medium showed that pantethine induced a concentration- and time-dependent release of acetate into the medium. Results of the effect of the acetate concentration on the incorporation of [2-14C]acetate radioactivity into CO2 and lipids in control hepatocytes allowed the conclusion that the above-described modifications induced by pantethine are only partially attributable to the dilution of the labeled substrate, and that catabolism of acetate to carbon dioxide is stimulated by the disulphide pantethine, whereas cholesterol and fatty acid syntheses are inhibited.     

Secretion of the newly synthesized cholesterol by rat hepatocytes in primary culture

We used monolayer cultured rat hepatocytes as an experimental model to study the secretion of the newly synthesized cholesterol by the liver. Cellular cholesterol was labeled by exposing cultured hepatocytes to [14C]acetate prior to the study of secretion. Secretion of the newly synthesized cholesterol was measured by extracting cholesterol in the culture medium and assaying for the radioactivity of [14C]cholesterol. We found that: (a) cultured hepatocytes could secrete newly synthesized cholesterol in serum-free medium; (b) secreted [14C]cholesterol was bound to macromolecule(s) and the secretion rate was not affected by cycloheximide for up to 5 h; (c) serum added to the culture medium greatly enhanced hepatic cholesterol secretion; (d) serum high-density lipoproteins were most effective, lipoprotein-deficient serum (d greater than 1.21) less effective in stimulating cholesterol secretion, whereas low-density and very-low-density lipoproteins had little effect; (e) when the serum-free culture medium was fractionated by ultracentrifugation, a major portion of the secreted [14C]cholesterol was found in the high-density lipoprotein fraction; (f) part of the medium [14C]cholesterol also turned up in the high-density lipoprotein fraction when lipoprotein-deficient serum was added as the acceptor; (g) secreted [14C]cholesterol was found only in free form, although some of the cellular [14C]cholesterol was found as esters.     

Fatty acid metabolism in Atlantic salmon (Salmo salar L.) hepatocytes and influence of dietary vegetable oil

Isolated hepatocytes from Atlantic salmon (Salmo salar), fed diets containing either 100% fish oil or a vegetable oil blend replacing 75% of the fish oil, were incubated with a range of seven (14)C-labelled fatty acids. The fatty acids were [1-(14)C]16:0, [1-(14)C]18:1n-9, 91-(14)C]18:2n-6, [1-(14)C]18:3n-3, [1-(14)C]20:4n-6, [1-(14)C]20:5n-3, and [1-(14)C]22:6n-3. After 2 h of incubation, the hepatocytes and medium were analysed for acid soluble products, incorporation into lipid classes, and hepatocytes for desaturation and elongation. Uptake into hepatocytes was highest with [1-(14)C]18:2n-6 and [1-(14)C]20:5n-3 and lowest with [1-(14)C]16:0. The highest recovery of radioactivity in the cells was found in triacylglycerols. Of the phospholipids, the highest recovery was found in phosphatidylcholine, with [1-(14)C]16:0 and [1-(14)C]22:6n-3 being the most prominent fatty acids. The rates of beta-oxidation were as follows: 20:4n-6>18:2n-6=16:0>18:1n-9>22:6n-3=18:3n-3=20:5n-3. Of the fatty acids taken up by the hepatocytes, [1-(14)C]16:0 and [1-(14)C]18:1n-9 were subsequently exported the most, with the majority of radioactivity recovered in phospholipids and triacylglycerols, respectively. The major products from desaturation and elongation were generally one cycle of elongation of the fatty acids. Diet had a clear effect on the overall lipid metabolism, with replacing 75% of the fish oil with vegetable oil resulting in decreased uptake of all fatty acids and reduced incorporation of fatty acids into cellular lipids, but increased beta-oxidation activity and higher recovery in products of desaturation and elongation of [1-(14)C]18:2n-6 and [1-(14)C]18:3n-3.     

Effects of epidermal growth factor (urogastrone) on gluconeogenesis, glucose oxidation, and glycogen synthesis in isolated rat hepatocytes

Using isolated rat hepatocytes, we studied the effect of epidermal growth factor (urogastrone) (EGF-URO) on the incorporation of [3-14C]pyruvate into glucose and glycogen, on the incorporation of [U-14C]glucose into glycogen, and on the oxidation of [U-14C]glucose to 14CO2. The effects of EGF-URO were compared with those of glucagon and insulin. EGF-URO, with an EC50 of 0.2 nM, enhanced by 34% (maximal stimulation) the conversion of [3-14C]pyruvate into glucose; no effect was observed on the oxidation of glucose to CO2 and on the incorporation of either pyruvate or glucose into glycogen. The effect of EGF-URO on pyruvate conversion to glucose was observed only when hepatocytes were preincubated with EGF-URO for 40 min prior to the addition of substrate. Glucagon (10 nM) increased the incorporation of [3-14C]pyruvate into glucose (44% above control); however, unlike EGF-URO, glucagon stimulated gluconeogenesis better without than with a preincubation period. Neither insulin nor EGF-URO (both 10 nM) affected the incorporation of [U-14C]glucose into glycogen during a 20-min incubation period. However, at longer time periods of incubation with the substrate (60 instead 20 min), insulin (but not EGF-URO) increased the incorporation of [14C]glucose into glycogen; EGF-URO counteracted this stimulatory effect of insulin. In contrast with previous data, our work indicates that EGF-URO can, under certain conditions, counteract the effects of insulin and, like glucagon, promote gluconeogenesis in isolated rat hepatocytes.     

Effects of dexamethasone and insulin on the synthesis of triacylglycerols and phosphatidylcholine and the secretion of very-low-density lipoproteins and lysophosphatidylcholine by monolayer cultures of rat hepatocytes.

Rat hepatocytes in monolayer culture were preincubated for 19 h with 1 microM-dexamethasone, and the incubation was continued for a further 23 h with [14C]oleate, [3H]glycerol and 1 microM-dexamethasone. Dexamethasone increased the secretion of triacylglycerol into the medium in particles that had the properties of very-low-density lipoproteins. The increased secretion was matched by a decrease in the triacylglycerol and phosphatidylcholine that remained in the hepatocytes. Preincubating the hepatocytes for the total 42 h period with 36 nM-insulin decreased the amount of triacylglycerol in the medium and in the cells after the final incubation for 23 h with radioactive substrates. However, insulin had no significant effect on the triacylglycerol content of the cell and medium when it was present only in the final 23 h incubation. Insulin antagonized the effects of dexamethasone in stimulating the secretion of triacylglycerol from the hepatocytes, especially when it was present throughout the total 42 h period. The labelling of lysophosphatidylcholine in the medium when hepatocytes were incubated with [14C]oleate and [3H]glycerol was greater than that of phosphatidylcholine. The appearance of this lipid in the medium, unlike that of triacylglycerol and phosphatidylcholine, was not stimulated by dexamethasone, or inhibited by colchicine. However, the presence of lysophosphatidylcholine in the medium was decreased when the hepatocytes were incubated with both dexamethasone and insulin. These findings are discussed in relation to the control of the synthesis of glycerolipids and the secretion of very-low-density lipoproteins and lysophosphatidylcholine by the liver, particularly in relation to the interactions of glucocorticoids and insulin.     

Lipogenesis and cholesterogenesis in the liver of rats after cholesterol loading

Intensity of fatty acids and separate classes of lipids synthesis was studied in vitro in the liver of white rats at loading by cholesterol in the dose of 300 mg/kg once a day during 30 days by incubation of organ homogenate with [6-(14)C] glucose, [2-(14)C] lysine, [1-(14)C] palmitic acid with following determination of radioactivity of fatty acids, phospholipids, cholesterol, acylglycerols radioactivity was investigated. The inhibition of fatty acids and separate classes of lipids synthesis in vitro in the liver of white rats at loading by cholesterol at the use of [6-(14)C] of glucose and [2-(14)C] lysine, as predecessors of fatty acids and lipids and stimulation of lipids synthesis at the use of [1-(14)C] palmitic acid as the predecessor was established. The loading of white rats by cholesterol results in its synthesis inhibition in the liver during incubation of its homogenates with [6-(14)C] glucose and does not influence the cholesterol synthesis during incubation of homogenates with [2-(14)C] lysine and [1-(14)C] palmitic acid. Thus synthesis of fatty acids and their use in the phospholipids and acylglycerols synthesis in the liver of white rats with hypercholesterolemia sharply decreases during incubation of their homogenates with [6-(14)C] glucose and [2-(14)C] lysine, and the synthesis of cholesterol, phospholipids and acylglycerols - increases during incubation with [1-(14)C] palmitic acid.     

Inhibition by insulin of glucagon-dependent phospholipid methyltransferase phosphorylation in rat hepatocytes

Addition of insulin to isolated rat hepatocytes prelabeled with [32P]phosphate inhibited glucagon-dependent phospholipid methyltransferase phosphorylation and activation. Insulin alone had no effect on either the phosphorylation of the enzyme or on its activity. The effect of insulin on glucagon-dependent phospholipid methyltransferase phosphorylation was dose-dependent and occurred at physiological doses of the hormone (10(-11)-10(-10) M). Analysis of 32P-labeled peptides after digestion with trypsin revealed only one site of phosphorylation regulated by glucagon (10(-8) M) in isolated rat hepatocytes. This site, as analyzed by HPLC and thin-layer chromatography, coincided with that phosphorylated by the cAMP-dependent protein kinase using purified rat liver phospholipid methyltransferase.     

Epidermal growth factor (urogastrone)-stimulated gluconeogenesis in isolated mouse hepatocytes

In freshly isolated mouse hepatocytes obtained from fasted animals, we have studied the receptors for epidermal growth factor urogastrone (EGF-URO) in terms of the electrophoretic profile, ligand affinity, and numbers of EGF-URO receptors present on the cells, and also in terms of the ability of EGF-URO to stimulate gluconeogenesis, as reflected by the increased incorporation of [3-14C]pyruvate into glucose. The effects of EGF-URO were compared with those of glucagon. Ligand-binding studies revealed that the mouse hepatocytes possess an unusually high number of EGF-URO receptors (about 3 X 10(6) binding sites/cell), with a ligand dissociation constant of 4.4 nM. The binding of EGF-URO by mouse hepatocytes was more than 10-fold higher than the previously measured binding of EGF-URO by rat hepatocytes. Crosslink-labeling studies, coupled with gel electrophoretic analysis, demonstrated the presence of intact EGF-URO receptors, although some receptor processing had occurred during the isolation procedure. EGF-URO was able to stimulate the incorporation of 3-14C-labeled pyruvate into glucose; glucagon was unable to do so. In contrast, in rat hepatocytes isolated and assayed under identical conditions, glucagon (10 nM) caused a marked (250%) stimulation of the incorporation of pyruvate into glucose. Maximally, EGF-URO caused a 34% increase in the incorporation of [3-14C]pyruvate into glucose; a half-maximal effect was observed at a concentration of 2.5 nM EGF-URO. The stimulatory effect of EGF-URO was not dependent on the concentration of pyruvate, lactate, glucose, or calcium in the incubation medium. Although raising the concentration of pyruvate in the incubation medium increased the incorporation of [3-14C]pyruvate into glycogen, EGF-URO did not cause any change in the incorporation of radioactivity into glycogen. Overall, our data point to marked differences between rat and mouse liver preparations, in terms of the hormonal regulation of glucose metabolism, and our work documents a potential role for the remarkably high number of mouse hepatocyte EGF-URO receptors in terms of the modulation of gluconeogenesis in the mouse.     

Binding properties of high-density lipoprotein subfractions and low-density lipoproteins to rabbit hepatocytes

Primary cultures of rabbit hepatocytes which were preincubated for 20 h in a medium containing lipoprotein-deficient serum subsequently bound, internalized and degraded 125I-labeled high-density lipoproteins2 (HDL2). The rate of degradation of HDL2 was constant in incubations from 3 to 25 h. As the concentration of HDL2 in the incubation medium was increased, binding reached saturation. At 37 degrees C, half-maximal binding (Km) was achieved at a concentration of 7.3 micrograms of HDL2 protein/ml (4.06 X 10(-8)M) and the maximum amount bound was 476 ng of HDL2 protein/mg of cell protein. At 4 degrees C, HDL2 had a Km of 18.6 micrograms protein/ml (1.03 X 10(-7)M). Unlabeled low-density lipoproteins (LDL) inhibited only at low concentrations of 125I-labeled HDL2. Quantification of 125I-labeled HDL2 binding to a specific receptor (based on incubation of cells at 4 degrees C with and without a 50-fold excess of unlabeled HDL) yielded a dissociation constant of 1.45 X 10(-7)M. Excess HDL2 inhibited the binding of both 125I-labeled HDL2 and 125I-labeled HDL3, but excess HDL3 did not affect the binding of 125I-labeled HDL3. Preincubation of hepatocytes in the presence of HDL resulted in only a 40% reduction in specific HDL2 receptors, whereas preincubation with LDL largely suppressed LDL receptors. HDL2 and LDL from control and hypercholesterolemic rabbits inhibited the degradation of 125I-labeled HDL2, but HDL3 did not. Treatment of HDL2 and LDL with cyclohexanedione eliminated their capacity to inhibit 125I-labeled HDL2 degradation, suggesting that apolipoprotein E plays a critical role in triggering the degradative process. The effect of incubation with HDL on subsequent 125I-labeled LDL binding was time-dependent: a 20 h preincubation with HDL reduced the amount of 125I-labeled LDL binding by 40%; there was a similar effect on LDL bound in 6 h but not on LDL bound in 3 h. The binding of 125I-labeled LDL to isolated liver cellular membranes demonstrated saturation kinetics at 4 degrees C and was inhibited by EDTA or excess LDL. The binding of 125I-labeled HDL2 was much lower than that of 125I-labeled LDL and was less inhibited by unlabeled lipoproteins. The binding of 125I-labeled HDL3 was not inhibited by any unlabeled lipoproteins. EDTA did not affect the binding of either HDL2 or HDL3 to isolated liver membranes. Hepatocytes incubated with [2-14C]acetate in the absence of lipoproteins incorporated more label into cellular cholesterol, nonsaponifiable lipids and total cellular lipid than hepatocytes incubated with [2-14C]acetate in the presence of any lipoprotein fraction. However, the level of 14C-labeled lipids released into the medium was higher in the presence of medium lipoproteins, indicating that the effect of those lipoproteins was on the rate of release of cellular lipids rather than on the rate of synthesis.     

Insulin release and metabolism of calcium, adenine and adenosine 3',5'-cyclic monophosphate.

In order to assess further the mechanisms involved in insulin release, we prelabeled rat pancreatic islets of Langerhans by incubating either 45Ca or [2-3H]adenine. When prelabeled islets were perfused with a glucose-free medium (the experiment with 45Ca) and a medium containing 2.8 mM glucose (the experiment with [2-3H]adenine) respectively, a constant rate of efflux of the radioactivity was established by 30 min in each case. D-Glucose at 16.7 mM concentration elicited a rapid efflux of 45Ca and [2-3H]adenine derivatives ([3H]Ad) within 4 to 6 min after commencing the step-wise stimulation by glucose, concomitantly with insulin release. However, L-glucose and D-galactose littel stimulated both 45Ca and [3H]Ad release. Lanthanum chloride caused a burst peak of 45Ca release in the absence of glucose. A rapid efflux of 45Ca was caused by beta-D-glucose and D-glyceraldehyde to much lesser extent than by alpha-D-glucose. The slowly rising concentration of glucose at 0.1 mM/min of gradient level failed to elicit any rapid efflux of 45Ca or [3H]Ad, although insulin release occurred in accordance with an increase in glucose concentration. Even when the gradient of glucose concentration was raised to 0.7 mM/min, glucose failed to stimulate an efflux of [3H]Ad but the subsequent stimulation by 16.7 mM glucose caused a rapid efflux of [3H]Ad concomitantly with the release of insulin. No rapid efflux of 45Ca was observed under a slow-rise glucose stimulation until the gradient level of the glucose concentration was raised to 6.7 mM. Analysis of distribution of the radioactive adenine derivatives after incubation showed that the adenosine fraction had the highest radioactivity in the medium followed by the ATP, adenine and cAMP fraction in that order, and the ATP fraction had the highest radioactivity in the islet. The ratio of radioactivity in the cAMP fraction in the medium to the total count was the highest among all. On the basis of these results, it was suggested that the discharge of [3H]Ad and 45Ca might occur with the alteration of the membrane permeability induced by a rapid change of the glucose concentration, and that their discharge might perhaps link to the glucoreceptor mechanism directly controlling insulin release.     

Uridine catabolism in Kupffer cells, endothelial cells, and hepatocytes

Kupffer cells, endothelial cells, and hepatocytes were separated by centrifugal elutriation. The rate of uracil formation from [2-14C]uridine, the first step in uridine catabolism, was monitored in suspensions of the three different liver cell types. Kupffer cells demonstrated the highest rate of uridine phosphorolysis. 15 min after the addition of the nucleoside the label in uracil amounted to 51%, 13%, and 19% of total radioactivity in the medium of Kupffer cells, endothelial cells, and hepatocytes, respectively. If corrected for Kupffer cell contamination, hepatocyte suspensions demonstrated similar activities as endothelial cells. In contrast to non-parenchymal cells, hepatocytes continuously cleared uracil from the incubation medium. The lack of uracil consumption by Kupffer cells and endothelial cells points to uracil as the end-product of uridine catabolism in these cells. Kupffer cells and endothelial cells did not produce radioactive CO2 upon incubation in the presence of [2-14C]uridine. Hepatocytes, however, were able to degrade uridine into CO2, beta-alanine, and ammonia as demonstrated by active formation of volatile radioactivity from the labeled nucleoside. There was almost no detectable formation of thymine from thymidine or of cytosine, uracil, or uridine from cytidine by any of the different cell types tested. These results are in line with low thymidine phosphorolysis and cytidine deamination in rat liver. Our studies suggest a co-operation of Kupffer cells, endothelial cells, and hepatocytes in the breakdown of uridine from portal vein blood with uridine phosphorolysis predominantly occurring in Kupffer cells and with uracil catabolism restricted to parenchymal liver cells.     

Myristic acid, unlike palmitic acid, is rapidly metabolized in cultured rat hepatocytes

This study was designed to examine and compare the metabolism of myristic and palmitic acids in cultured rat hepatocytes. [1-(14)C]-Labeled fatty acids were solubilized with albumin at 0.1 mmol/L in culture medium. Incubation with 24-hr cultured hepatocytes was carried out for 12 hr. Myristic acid was more rapidly (P < 0.05) taken up by the cells than was palmitic acid (86.9 +/- 0.9% and 68.3 +/- 5.7%, respectively, of the initial radioactivity was cleared from the medium after 4 hr incubation). Incorporation into cellular lipids, however, was similar after the same time (33.4 +/- 2.8% and 34.9 +/- 9.3%, respectively, of initial radioactivity). In the early phase of the incubation (30 min), myristic acid was more rapidly incorporated into cellular triglycerides than was palmitic acid (7.4 +/- 0.9% and 3.6 +/- 1.9%, respectively, of initial radioactivity). However, after 12 hr incubation, the radioactivity of cellular triglycerides, cellular phospholipids, and secreted triglycerides was significantly higher with palmitic acid as precursor. Myristic acid oxidation was significantly higher than that of palmitic acid (14.9 +/- 2.2% and 2.3 +/- 0.6%, respectively, of the initial radioactivity was incorporated into the beta-oxidation products after 4 hr). Myristic acid was also more strongly elongated to radiolabeled palmitic acid (12.2 +/- 0.8% of initial radioactivity after 12 hr) than palmitic acid was to stearic acid (5.1 +/- 1.3% of initial radioactivity after 12 hr). The combination of elongation and beta-oxidation results in the rapid disappearance of C14:0 in hepatocytes whereas C16:0 is esterified to form glycerolipids. This study provides evidence that myristic acid is more rapidly metabolized in cultured hepatocytes than is palmitic acid.     

Studies on the effects of insulin and acetylcholine on activation of glycogen synthase and on glycogenesis in hepatocytes

The addition of insulin (4.0 × 10^?11 M) or acetylcholine (10^?6 M) to isolated hepatocytes stimulated glycogen accumulation and this stimulation was more pronounced when the medium glucose was raised from 50 to 300 mg percent. Studies with [¹⁴C]-glucose showed a two-fold stimulation in glycogen synthesis by the addition of insulin (4.0 × 10^?11 M) or acetylcholine (10^?6 M). A sixteen percent increase in the activity of glycogen synthase was observed in cells incubated for 10 minutes with insulin (4.0 × 10^?11 M) or acetylcholine (10^?6 M), whereas at one hour incubation a 40 percent increase in activity was observed with the same concentration of insulin or acetylcholine. The effects of insulin and acetylcholine were not additive.     

Alpha-adrenergic suppression of very-low-density-lipoprotein triacylglycerol secretion by isolated rat hepatocytes.

The effect of adrenaline on triacylglycerol synthesis and secretion was examined in isolated rat hepatocytes. Cells were incubated with 0.5 mM-[1-14C]oleate, and the accumulation of triacylglycerol and [14C]triacylglycerol was measured in the incubation medium. Triacylglycerol appearing in the medium was present in a form with properties similar to very-low-density lipoproteins. Triacylglycerol, [14C]triacylglycerol and [14C]phospholipid contents of hepatocytes were also determined. Addition of 10 microM-(-)adrenaline decreased accumulation of glycerolipid in the incubation medium and also decreased cellular [14C]phospholipid content. Prazosin abolished these effects, whereas propranolol did not. The hormone did not affect cellular triacylglycerol content or rates of incorporation of [1-14C]oleate into cell triacylglycerol. The effect of adrenaline on the removal of newly secreted triacylglycerol and the secretion of synthesized glycerolipid was also examined. The catecholamine did not affect rates of removal of newly secreted triacylglycerol. Adrenaline did inhibit the secretion of pre-synthesized lipid by the cells, as assessed by the appearance of radiolabelled triacylglycerol from hepatocytes that had been preincubated with [1,2,3-3H]-glycerol. Adrenaline did not affect rates of fatty acid uptake by hepatocytes, but did stimulate oxidation of [1-14C]oleate, principally to 14CO2.     

In vivo covalent binding of [14C]trinitrotoluene to proteins in the rat.

When a single dose of [14C]trinitrotoluene was administered intraperitoneally (i.p.) to rats at 1, 10 or 50 mg/kg of body weight, covalently bound radioactivity was detected in globin, plasma proteins and proteins in the liver and kidney. The extent of covalent binding was dose dependent and was highest in plasma and renal proteins at all times up to 4 h after dosing. Covalent adduct levels in globin, however, decline slower than others. At a dose of 50 mg/kg of body weight, globin covalent adduct levels peaked at 1 h after dosing at 182 pmol/mg protein and subsequently decreased to approximately 50 pmol/mg protein between days 1 and 8. Of the covalent adduct levels in liver and kidney, those in the 10,000 x g and microsomal fractions were found to be higher than that in the cytosolic fraction. Radioactivity covalently bound to globin and the hepatic proteins was susceptible to dilute acid hydrolysis from which 2-amino-4,6-dinitrotoluene (2A) and 4-amino 2,6-dinitrotoluene (4A) were the major products recovered by solvent extraction. Upon acetylation, the hydrolysate gave rise to derivatives identified as the acetates of 2A and 4A on the basis of mass spectrometry and HPLC cochromatography with authentic samples. Four hours after an i.p. dose of [14C]TNT at 50 mg/kg of body weight about 0.4% of the dose was found as bound adducts to hemoglobin, of which approximately 48% was recovered as solvent extractable radioactivity after acid hydrolysis. About 2% of the radioactive dose was in the liver, of which approximately 30% was covalently bound to hepatic proteins, and approximately 49% of that was convertible to solvent extractable radioactivity upon acid hydrolysis. In vitro incubation of [14C]TNT with blood showed that there was a linear increase of covalent adducts in globin during the first 2 h of incubation; the concentration of covalent adducts was slightly higher than that with plasma proteins. The major compounds recovered from the hydrolysate of the globin adducts were also 2A and 4A as obtained from globin in the in vivo studies. On the basis of the in vitro and in vivo study results, we have confirmed the formation of protein adducts following a single i.p. administration of [14C]TNT at 1, 10 or 50 mg/kg of body weight to the rat or by in vitro incubation with blood.(ABSTRACT TRUNCATED AT 400 WORDS)