Lactate-induced inhibition of glucose catabolism in guinea pig cortical brain slices.

Extracellular lactate concentration rises following ischaemic stroke in both the infarcted area and in the surrounding ischaemic penumbra. We investigated the effect of lactate accumulation on glucose metabolism in cortical slices from guinea pigs initially by varying superfusion medium to tissue volumes. Stable intracellular K+ concentrations indicated that a decrease in media/ tissue volume did not impair viability of the tissue, but 13C NMR demonstrated that lactate accumulation in the superfusion medium reduced glucose oxidation with inhibition of glial metabolism via pyruvate carboxylase. The concentration of lactate which had accumulated when significant inhibition was observed was approximately 0.85 mM. In independent experiments we found that superfusion of brain slices with lactate at this concentration (even using a 'high-volume' of superfusion fluid) decreased oxygen consumption by 40 +/- 3%. K(-)-induced depolarisation partially reversed this effect. These results suggest that even low extracellular lactate concentrations may depress metabolic rates in inactive and poorly perfused brain tissue in vivo through inhibition of glial metabolism of glucose.     

Choline and acetylcholine metabolism in rat neostriatal slices

Choline (Ch) uptake and release and acetylcholine (ACh) synthesis and release have been studied by gas chromatography mass spectrometry (GCMS) in slices of rat neostriatum in vitro to assess the effects of depolarization by 25 mM K+ and the influence of elevated concentrations of Ch in the incubation medium. During the first 60 min after preparation, 25 mM K+ increased ACh release by 182% and reduced ACh levels by 40%. The rate of ACh synthesis was unchanged. After a 1-h equilibration period, the rate of ACh synthesis was considerably less (2.41 nmol mg-1 h-1, compared to 9.78 nmol mg-1 h-1). Exposure to 25 mM K+ during the second hour increased the rate to 6.47 nmol mg-1 h-1. During the first 10 min of exposure to 25 mM K+, ACh synthesis was reduced, regardless of incubation. Increasing concentrations of external [2H4]Ch apparently favored initial rates of net ACh synthesis, since the rank order of initial net ACh synthesis rates is the same as the rank order of external [2H4] Ch concentration under both normal and depolarized conditions. However, the only significant effect of external [2H4]Ch on ACh metabolism was that it increased ACh release during the initial 10 min, when the preparation was depolarized with K+. The efflux of endogenous [2H0]Ch was increased initially (10 min) and slowed over a 60-min period by 25 mM K+, and increased when [2H4]Ch in the medium was increased. Changes in ACh synthesis and release were dependent upon the time exposure of slices to high K+, and the results suggest that Ch favors initial rates of ACh synthesis, but that Ch influences ACh release primarily under conditions of stress (i.e., depolarization).     

Production of DNA single-strand breaks in unstimulated splenocytes by dimethylnitrosamine

In an attempt to elucidate the mechanism whereby primary hepatocytes, but not liver S9 homogenates, generate immunosupprssive metabolites of dimethylnitrosamine (DMN), the production of DNA single-strand breaks (SSB) in unstimulated splenocytes was investigated with alkaline-elution analysis. Both hepatocytes and S9 homogenates induced SSB in cultured splenocytes by DMN - minimum detectable doses with the two metabolic activation systems (MAS) were 1 microM and 5 mM, respectively. DNA elution profiles were linear in splenocytes co-cultured with DMN and hepatocytes and convex in splenocytes incubated with DMN and S9 homogenates. Aminoacetonitrile (AAN; 10 mM), a DMN demethylase inhibitor, reversed SSB in splenocytes when incubated with either MAS. Addition of exogenous calf-thymus DNA to the hepatocyte co-culture medium did not affect the production of SSB. Rocking the hepatocyte-splenocyte cultures changed the elution profile from linear to convex. All of these treatments have been previously shown to block the immunosuppression by DMN in the hepatocyte co-culture system. These results indicate that the immunosuppression by DMN is not related to DNA damage, as measured by the production of SSB, and suggest that the metabolism of DMN to intermediates capable of producing genotoxicity and immunotoxicity may be qualitatively and/or quantitatively different.     

Studies on lung tumours. III. Oxidative metabolism of dimethylnitrosamine by rodent and human lung tissue.

The oxidative metabolism of the carcinogen dimethylnitrosamine (DMN) was studied in mouse, rat, hamster and human respiratory tissue. [14C]DMN was purified by Dowex-1-bisulfite column chromatography to remove a contaminant (probably [14C]formaldehyde) interfering with the enzyme assay. Since formaldehyde and methyl carbonium ions - yielding methanol with water - are considered to be the primary products of DMN metabolism, tissue slices were assayed for the production of [14C]CO2 from 14C-labelled methanol, formaldehyde, formate, and DMN. Oxidation of formaldehyde to formate was not, but oxidation of formate to CO2 was very much rate-limiting. This rate-limiting step was circumvented by introducing quantitative chemical oxidation of formate to CO2 by mercury(II)chloride following the enzymic reaction. Since oxidation of methanol to CO2 proved to be insignificant, production of CO2 from DMN by lung tissue enzymes and HgCl2 may serve as a parameter for N-demethylating activity and the production of the suspected carcinogenically active methyl carbonium ions. The DMN-N-demethylating activities of lung tissue slices of two mouse strains with widely different susceptibilities to formation of lung adenomas by DMN differed significantly, but the difference seemed too small to explain the divergence in tumourigenic response. The enzymatic activities decreased in hamster bronchus, hamster trachea, hamster lung, GRS/A mouse lung, C3Hf/A mouse lung, human lung, Sprague-Dawley rat lung, in that order. The reported resistance of the hamster respiratory system to tumour induction by DMN may therefore not be due to poor DMN-N-demethylating capacity.     

Effects of dimethylnitrosamine on metabolism of N,N-dimethylaniline by rat liver microsomes. A comparative study of treatment in vivo,in isolated liver perfusion and in the microsomal system

The effect of dimethylnitrosamine (DMN) on rat liver microsomal detoxication was studied, using the non-carcinogenic aromatic amine N,N-dimethylaniline (dimethylaniline) as substrate. Prior to the preparation of microsomes, the rat liver was exposed to DMN either in vivo (by i.p. injection) or in the isolated liver perfusion system (by addition to the perfusion medium). DMN treatment in vivo (20 mg/kg body wt.) caused a 40% increase in dimethylaniline N-oxygenation and a 30% decrease in dimethylaniline C-oxygenation. When DMN was added to the perfusion medium to a final concentration of 5 or 25 mM, a similar effect was observed. With the 5 mM dose, C-oxygenation was decreased by 20% with a non-significant increase in N-oxygenation. The higher dose caused a 50% increase in N-oxygenation, whereas the decrease in C-oxygenation remained at 20%.When microsomes were incubated with both DMN (5 mM) and dimethylaniline (5 mM) in the system, a small but significant decrease in both N- and C-oxygenation of dimethylaniline was observed. The effect of DMN on the amino acid incorporation into liver and plasma proteins was also studied in the liver perfusion system. The synthesis of both liver and plasma proteins was reduced by DMN.     

Localization and role of pyruvate kinase isoenzymes in the regulation of carbohydrate metabolism and pyruvate recycling in rat kidney cortex

This work was performed to gain more information on the role of pyruvate kinase isoenzymes in the regulation of renal carbohydrate metabolism. Immunohistochemically, pyruvate kinase type L is shown to be localized in the proximal tubule of the nephron and pyruvate kinase type M2 in the distal tubule and the collecting duct. a tight relationship between gluconeogenesis and pyruvate recycling was found. The rate of gluconeogenesis (8 mumol/g wet wt. per 30 min) was of the same order of magnitude as the rate of pyruvate recycling (10.92 mumol/g wet wt. per 30 min). Stimulation of gluconeogenesis from 20 mM lactate in kidney cortex slices of 24-h-starved rats by dibutyryl-cAMP, alanine and parathyroid hormone was connected with a decrease in pyruvate recycling; inhibition of gluconeogenesis due to a lack of Ca2+ in the incubation medium was linked with an increase in pyruvate recycling. The degradation of [6-14C]glucose to lactate, pyruvate, ketone bodies and CO2 and of [2-14C]lactate was unaffected by dibutyryl-cAMP, alanine, epinephrine, vasopressin or the omission of Ca2+ from the incubation medium. 1 mM dibutyryl-cAMP or 5 mM alanine did not alter the activities of oxaloacetate decarboxylase, 'malic' enzyme and malate dehydrogenase from rat kidney cortex. Since aerobic glycolysis in the distal tubules and the collecting ducts is not influenced by hormones, dibutyryl-cAMP and Ca2+, pyruvate kinase type M2 residing in this tissue is unlikely to be a control point of glycolysis. Since this tissue degrades only one-seventh of the glucose formed via gluconeogenesis, it does not contribute significantly to pyruvate recycling. Therefore, the decrease of pyruvate recycling in the presence of dibutyryl-cAMP and alanine in rat kidney cortex slices, leading to increased renal gluconeogenesis, has to be ascribed to the regulation of pyruvate kinase type L.     

Betaine aids in the osmoregulation of duodenal epithelium of broiler chicks, and affects the movement of water across the small intestinal epithelium in vitro.

In Experiment 1, the water holding capacity of broiler chick intestinal tissue was studied in vitro. The chicks were fed with corn-based diets with or without a 0.2% betaine supplementation in the drinking water. Slices from duodenum and jejunum were incubated in iso-osmotic (300 mM) or hyperosmotic saline (600 mM) with or without 10 mM betaine. The water volume of tissue slices was studied by adding tritiated water in the incubation medium while [14C]inulin was used to correct for the adherent water. After 30 min of incubation, by which time the steady-state of tritium influx had been achieved, the 3H and 14C-activities of the tissue slices were measured. The ileal and duodenal tissues incubated in the hyperosmotic saline accumulated less tritium than those incubated in iso-osmotic saline. Duodenal slices incubated in hyperosmotic saline with the presence of betaine showed a tritium content similar to slices incubated in iso-osmotic saline. The data suggest that the presence of betaine helped the duodenal, but not jejunal, epithelium to maintain water balance in hyperosmotic conditions. The dietary betaine supplementation diminished the differences between the incubation treatments in duodenal, but not in ileal tissue. In Experiment 2, the same double labeling method, but with shorter incubation times, was used to assess the rate of water flux from the incubation medium to duodenal or jejunal slices. The dietary treatments (as in Experiment 1) had little effect on the results. Betaine in the hyperosmotic saline significantly decreased the rate of tritium accumulation into the tissue slices, indicating that betaine slowed down the influx of water to the epithelium. We suggest that betaine affects the movement of water across the intestinal epithelium and has a role in the osmoregulation of small intestine of broiler chicks.     

Effect of 1,10-phenanthroline on acute liver injury induced by dimethylnitrosamine in the rat

Acute liver damage was induced in rats by intragastric doses of dimethylnitrosamine (DMN, 3 mg/100 g body weight) and measured 24 hours later by morphological and biochemical methods. 1, 10-Phenanthroline (1, 10-P, 2 mg/100 g) administered simultaneously with DMN prevented the development of the characteristic morphological picture of liver injury. At the same time, the amount and synthesis of total liver proteins, the activity and distribution of liver β-glucuronidase, and the level of seromucoid and isocitric dehydrogenase (ICDH) activity in the serum, significantly changed by DMN, was within the range of control values when 1, 10-P was simultaneously administered. The protective effect of 1, 10-P against acute DMN hepatotoxicity paralleled the inhibition of some liver microsomal drug-metabolizing enzymes (aniline hydroxylase, morphine demethylase, content of cytochrome P-450). At higher doses of DMN (6 mg and 10 mg/100 g), the administration of 1, 10-P was no longer protective, although the content of cytochrome P-450 was only 20% of the value for normal liver microsomes. Therefore, in acute administration within a certain range of concentration of DMN, 1, 10-P might inhibit the microsomal drug-oxidizing enzymes, thus inhibiting the metabolism of the drug to a more toxic product.     

Efficient analysis of hepatic glucose output and insulin action using a liver slice culture system.

BACKGROUND: Liver slices have been reported to retain histological integrity and metabolic capacity for over 24 hours in flask culture systems, and they have been used for pharmacological and toxicological studies before. However, whether this method is suitable to measure hepatic glucose output is unknown. METHODS: Precision-cut liver slices were prepared from fresh male rat liver. After high-glucose pre-incubation (11.2 mmol/l), medium was changed to low-glucose conditions (0.5 mmol/l). Glucose and lactate levels as well as aspartate aminotransferase activity were monitored for 50 minutes with or without addition of insulin (600 pmol/l) and/or epinephrine (0.5 micromol/l). Slice potassium content and histology were examined to prove liver viability. RESULTS: We observed a stable glucose production from the liver slices of 0.3-0.4 micromol/g liver/min. Epinephrine increased (by 82+/-30%) and insulin decreased (by 80+/-8%) liver slice glucose output. Significant signs of ischemia were not detected. CONCLUSIONS: Hepatic glucose release can be reliably measured in a liver slice culture system, and it is regulated by major hormone systems. This method may be helpful for further characterization of direct insulin action and resistance in a complex tissue as the liver; however, pharmacological applications such as the analysis of drug effects on hepatic glucose metabolism can also be envisioned.     

Lipid peroxidation measured as thiobarbituric acid-reactive substances in tissue slices: characterization and comparison with homogenates and microsomes

Liver slices were used to measure lipid peroxidation induced by bromotrichloromethane, tert-butyl hydroperoxide (t-BOOH), or ferrous iron. The responses of liver homogenates and microsomes to oxidative conditions were compared with the response of tissue slices. Lipid peroxidation was evaluated by the production of thiobarbituric acid-reactive substances (TBARS). As was observed in homogenates and microsomes, TBARS production by liver slices depended upon the amount of tissue, the incubation time, inducer, the amount of inducer, and the presence of antioxidant. Control liver slices incubated at 37 degrees C for 2 h produced 19 nmol of TBARS per g of liver. When slices were incubated in the presence of 1 mM BrCCl3, 1 mM t-BOOH, or 50 microM ferrous iron, TBARS production increased 4.6-, 8.2-, or 6.7-fold over the control value, respectively. Comparable induction of TBARS by liver homogenates and microsomes was observed when these preparations were incubated with the same inducers. Addition of 5 microM butylated hydroxytoluene (BHT) prevented the induction of TBARS by 50 microM ferrous iron by liver slices. The results indicate the usefulness of tissue slices to measure lipid peroxidation. The usefulness of tissue slices is emphasized when a number of compounds or tissues are studied and tissue integrity is desired as in toxicological, pharmacological, and nutritional studies where reduced numbers of experimental animals is a relevant issue.     

Benzoate stimulates glutamate release from perfused rat liver.

In isolated perfused rat liver, benzoate addition to the influent perfusate led to a dose-dependent, rapid and reversible stimulation of glutamate output from the liver. This was accompanied by a decrease in glutamate and 2-oxoglutarate tissue levels and a net K+ release from the liver; withdrawal of benzoate was followed by re-uptake of K+. Benzoate-induced glutamate efflux from the liver was not dependent on the concentration (0-1 mM) of ammonia (NH3 + NH4+) in the influent perfusate, but was significantly increased after inhibition of glutamine synthetase by methionine sulphoximine or during the metabolism of added glutamine (5 mM). Maximal rates of benzoate-stimulated glutamate efflux were 0.8-0.9 mumol/min per g, and the effect of benzoate was half-maximal (K0.5) at 0.8 mM. Similar Vmax. values of glutamate efflux were obtained with 4-methyl-2-oxopentanoate, ketomethionine (4-methylthio-2-oxobutyrate) and phenylpyruvate; their respective K0.5 values were 1.2 mM, 3.0 mM and 3.8 mM. Benzoate decreased hepatic net ammonia uptake and synthesis of both urea and glutamine from added NH4Cl. Accordingly, the benzoate-induced shift of detoxication from urea and glutamine synthesis to glutamate formation and release was accompanied by a decreased hepatic ammonia uptake. The data show that benzoate exerts profound effects on hepatic glutamate and ammonia metabolism, providing a new insight into benzoate action in the treatment of hyperammonaemic syndromes.     

Glucose Metabolism Assessed with 2-Deoxyglucose and the Effect of Glutamate in Subdivisions of Rat Hippocampal Slices

A new approach to the study of glucose phosphorylation in brain slices is described. It is based on timed incubation with nonradioactive 2-deoxyglucose (DG), after which the tissue levels of DG and 2-deoxyglucose-6-phosphate (DG6P) are measured separately with sensitive enzymatic methods applied to specific small subregions. The smallest samples had dry weights of approximately 0.5 microgram. Direct measurements in different regions of hippocampal slices showed that within 6 min after exposure to DG, the ratios of DG to glucose in the tissue were almost the same as in the incubation medium, which simplifies the calculation of glucose phosphorylation rates and increases their reliability. Data are given for ATP, phosphocreatine, sucrose space, and K+ in specific subregions of the slices. DG6P accumulation proceeded at a constant rate for at least 10 min, even when stimulated by 10 mM glutamate in the medium. The calculated control rate of glucose phosphorylation was 2 mmol/kg (dry weight)/min. In the presence of 10 mM glutamate it was twice as great. The response to 10 mM glutamate of different regions of the slice was not uniform, ranging from 164% of control values in the molecular layer of CA1 to 256% in the stratum radiatum of CA1. There was a profound fall in phosphocreatine levels (75%) in response to 10 mM glutamate despite a 2.4-fold increase in glucose phosphorylation. Even in the presence of 1 mM glutamate, the increase in glucose phosphorylation (50%) was not great enough to prevent a significant drop in phosphocreatine content.     

Factors that modify the metabolism of ethanol in rat liver and adaptive changes produced by its chronic administration

1. 2,4-Dinitrophenol (0.1mm) increases by 100-160% the rate of ethanol metabolism by rat liver slices incubated in a medium saturated with a gas mixture containing O(2)+CO(2)+N(2) (18:5:77). Similar effects are produced by relatively low concentrations of arsenate (10mm). At higher concentrations (37.5 and 50mm) arsenate inhibits the rate of ethanol metabolism. 2. When liver slices are incubated under an atmosphere containing O(2)+CO(2) (95:5) the metabolism of ethanol increases by about 100% over that obtained with O(2)+CO(2)+N(2) (18:5:77). However, under these conditions the activating effect of dinitrophenol is no longer observed. 3. Chronic administration of ethanol to rats for 3-4 weeks, in doses from 3 to 8g/kg per day, increases by 70-90% the ability of the liver to metabolize ethanol. In the liver slices of these rats, although an O(2)+CO(2)+N(2) (18:5:77) mixture was used, dinitrophenol does not further increase the metabolism of ethanol. If the chronic administration of ethanol is discontinued for two weeks, the rate of ethanol metabolism is lowered to control values and the activating effect of dinitrophenol is recovered. 4. No change in alcohol dehydrogenase activity was found in the liver of the rats in which the metabolism of ethanol had been increased as a result of the chronic ethanol treatment; a 40% increase in the activity of succinate dehydrogenase was observed.     

The effect of actinomycin D on the synthesis of ribonucleic acid and protein in rat liver parenchymal cells in suspension and liver slices

1. Rat liver parenchymal cells in suspension are shown to require a higher concentration of actinomycin D than liver slices for equivalent inhibition of the incorporation of [(14)C]adenine, [(14)C]uracil and [(32)P]phosphate into RNA, and of (14)C-labelled amino acids into protein; protein synthesis is much less susceptible to actinomycin D inhibition than RNA synthesis in both the tissue preparations. Possible causes for these differences are discussed. 2. The uptake of [(3)H]actinomycin D in the first few minutes was much greater in the cell suspensions than in the tissue slices; that in the next 1-4hr. was about the same in both the cases. The uptake by both the tissue preparations was at all times proportional to the concentration of the drug within the range 0.5-2.0mug./ml. 3. In the slices actinomycin D taken up initially was concentrated almost exclusively in the nuclei; with time the concentration of the drug in the mitochondria and the supernatant increased more rapidly than in the nuclei though at no stage did it exceed that in the nuclei. In the cell suspension the largest concentration of the drug taken up initially was found in the supernatant; most of the drug taken up subsequently also stayed in the supernatant. 4. When the drug concentration in the incubation medium was 1mug./ml., its concentration within the parenchymal cells in suspension and the parenchymal cells in the slices reached 2.2 and 1.6mug./cm.(3) of cellular volume respectively. On average, 7% of the drug was removed from the medium by the cells in suspension and 23% by the cells in the slices; the average ratio of intracellular to extracellular concentration was 2.4 in the former and 2.1 in the latter case.     

Glucose and amino acid metabolism in an established line of skeletal muscle cells

The suitability of an established myogenic line (L₆) for the study of skeletal muscle intermediary metabolism was investigated. Myoblasts were grown in tissue culture for ten days at which time they had differentiated into multinucleated myotubes. Myotube preparations were then incubated for up to 96 hours in 10 ml of Dulbecco's modified Eagle medium containing 10% fetal calf serum. Glucose was utilized at a nearly linear rate, 3.0 nmol/min/mg protein. Intracellular glucose was detectable throughout the incubation, even when medium glucose was as low as 16 mg%. During the initial 28 hours of incubation, when net lactate production was observed, only 35% of the glucose utilized was converted to lactate. Alanine was produced in parallel to lactate at an average rate of 0.6 nmol/min/mg protein. In concert with active glutamine utilization, high rates of ammoniagenesis were observed as medium glutamine decreased from 3.3 mM to 0.49 mM and medium ammonia increased from 2.3 mM to 6.2 mM, between zero time and 96 hours of incubation, respectively. The cells maintained stable ATP and citrate levels, and physiologic intracellular lactate/pyruvate ratios (10–24) throughout 96 hours of incubation. These results suggest (1) glucose utilization by skeletal muscle in tissue culture is limited by phosphorylation, not transport; (2) as much as 50% of glucose-derived pyruvate enters mitochondrial pathways; (3) glutamine carbon may be utilized simultaneously with glucose consumption and this process accounts for high rates of ammoniagenesis.     

Effect of Passive Avoidance Training on In Vitro Protein Synthesis in Forebrain Slices of Day-Old Chicks

Slices from the forebrains of day-old chicks represent a highly active in vitro protein-synthesising system. The in vitro incorporation of L-[14C]leucine into protein of slices was estimated to be 2.5 mmol/mg protein/h. Incorporation was linear over 90 min of incubation and was suppressed by 92% by 1 mM cycloheximide. The highest incorporation was into microsomal and cell-soluble fractions. Under the electron microscope, slices appeared vacuolated near the cut surfaces, but well preserved internally (greater than 40 micron from the edge). Autoradiography showed that radioactivity was incorporated evenly across the slice with no decrease in label in the central part of the tissue. The rate of incorporation was only weakly dependent on leucine concentration in the medium (0.04-1 mM). Addition of a mixture of unlabelled amino acids (1 mM) produced a 20-50% inhibition of incorporation of radioactive L-leucine depending on the amino acids involved. In slices prepared from chicks 1 h after training on a one-trial passive avoidance paradigm, L-[14C]leucine incorporation was 23% higher (p less than 0.01) in the forebrain roof than in slices from control chicks. This figure is comparable to the one previously reported in vivo. Subcellular fractionation of incubated slices from the forebrain roof of trained and control birds revealed that the increased protein synthesis was due mainly to an elevated leucine incorporation into the soluble fraction.     

Neutral red (NR) assay for cell viability and xenobiotic-induced cytotoxicity in primary cultures of human and rat hepatocytes

Neutral red (NR) in medium was absorbed and concentrated in lysosomes of cultured rat and human hepatocytes. NR uptake increased with the time of incubation and reached a plateau in 2 hr. Uptake was proportional to the concentration of the NR solution and the numbers of viable liver cells. Prolonged culture of hepatocytes increased the numbers of lysosomes, and thus, the dye accumulation. The NR can be extracted from lysosomes for quantitative measurement of hepatocyte viability and cytotoxicity of xenobiotics. With this assay, several serum-free media (e.g., Waymouth's, MEM, LHC-8, etc.) were compared for the maintenance of viable hepatocytes in vitro. Interestingly, LHC-8 medium, which is used to grow human bronchial epithelial cells, best preserved viable rat hepatocytes. The cytotoxic effects of dimethylnitrosamine (DMN) and aflatoxin B₁ (AFB₁) were examined by NR assay on rat and human hepatocyte cultures and were found to be dependent on dose and time of the exposures. NR₅₀ was 20 mM for DMN and 0.072 µM for AFB₁ in rat hepatocytes with 24 hr of exposures and reduced to 12.5 mM for DMN and 0.053 µ uM for AFB₁ with 48 fr exposures. Human hepatocytes were more resistant to the toxicity of both chemicals; NR₅₀ values were 100 mM DMN and 1.8 µM AFB₁ respectively, for 24 hr treatments. Compared with lactate dehydrogenase (LDH) leakage test, the NR assay was simpler and more sensitive in determining the viability and cytotoxicity of xenobiotics in primary cultures of hepatocytes.Abbreviations NR Neutral Red - MEM Eagle's Minimum Essential Medium - DMN dimethylnitrosamine - AFB₁ aflatoxin B₁ - LDH lactate dehydrogenase - HBSS Hanks balanced salt solution; - EDTA ethylene bis (oxyethylenenitrilo)-tetraacetic acid - L-15 Leibovitz's 15 - NADH B-nicotinamide adenine dinu - FBS fetal bovine serum - IA immediate autopsy Contribution No. 2816 from Laboratory of Genotoxicology.     

Glyceroneogenesis and the supply of glycerol-3-phosphate for glyceride-glycerol synthesis in liver slices of fasted and diabetic rats

The pathways of glycerol-3-phosphate (G3P) generation for glyceride synthesis were examined in precision-cut liver slices of fasted and diabetic rats. The incorporation of 5 mM [U-(14)C]glucose into glyceride-glycerol, used to evaluate G3P generation via glycolysis, was reduced by approximately 26-36% in liver slices of fasted and diabetic rats. The glycolytic flux was reduced by approximately 60% in both groups. The incorporation of 1.0 mM [2-(14)C]pyruvate into glyceride-glycerol (glyceroneogenesis) increased approximately 50% and approximately 36% in slices of fasted and diabetic rats, respectively, which also showed a two-fold increase in the activity phosphoenolpyruvate carboxykinase. The increased incorporation of 1.0 mM [2-(14)C]pyruvate into glyceride-glycerol by slices of fasted rats was not affected by the addition of 5 mM glucose to the incubation medium. The activity of glycerokinase and the incorporation of 1 mM [U-(14)C]glycerol into glyceride-glycerol, evaluators of G3P formation by direct glycerol phosphorylation, did not differ significantly from controls in slices of the two experimental groups. Rates of incorporation of 1 mM [2-(14)C]pyruvate and [U-(14)C]glycerol into glucose of incubation medium (gluconeogenesis) were approximately 140 and approximately 20% higher in fasted and diabetic slices than in control slices. It could be estimated that glyceroneogenesis by liver slices of fasted rats contributed with approximately 20% of G3P generated for glyceride-glycerol synthesis, the glycolytic pathway with approximately 5%, and direct phosphorylation of glycerol by glycerokinase with approximately 75%. Pyruvate contributed with 54% and glycerol with 46% of gluconeogenesis. The present data indicate that glyceroneogenesis has a significant participation in the generation of G3P needed for the increased glyceride-glycerol synthesis in liver during fasting and diabetes.     

Modulation of paracetamol metabolism by Kupffer cells: a study on rat liver slices

Recent studies support the hypothesis that non parenchymal cells (mainly macrophages) may play a role in the metabolism and cellular effects of paracetamol. In order to investigate this hypothesis, male Wistar rats were intravenously injected with either 7.5 mg/kg gadolinium chloride (Gd+) or NaCl 0.9% (Gd-). The treatment with GdCl3 decreased the number and the function of Kupffer cells in liver tissue, as assessed by the histological examination of the liver after colloidal carbon injection in the portal vein. Precision-cut liver slices (PCLS) were prepared from both groups of rats and cultured for 8h in Waymouth's medium in the presence and absence of 5 mM paracetamol. Interestingly, PCLS obtained from Gd+ rats exhibited a lower release of tumor necrosis factor (TNF-alpha) and a better viability than PCLS from control (Gd-) rats. Incubation with paracetamol led to a decreased glycogen level in liver slices from Gd+ or Gd-, without modifying neither liver morphology nor ATP level nor LDH release. A higher proportion of paracetamol glucuronide, was secreted from the slices obtained from Gd+ rats. These data suggest that Kupffer cells could affect the viability of PCLS in culture and are involved in the regulation of phase II metabolism in the adjacent hepatocytes. We propose that PCLS in culture is a suitable model to elucidate the biochemical mechanism underlying the modulation of metabolism occurring through hepatocytes-Kupffer cells interactions.