Alterations in energy status by menadione metabolism in hepatocytes isolated from fasted and fed rats

The biochemical mechanism of cytotoxicity, induced by the quinoid compound 2-methyl 1,4-naphthoquinone (menadione), was investigated in hepatocytes freshly isolated from fasted and fed rats. Hepatocytes from fasted rats were significantly more vulnerable to the toxicity of menadione than hepatocytes from fed rats. Menadione (150 microM) induced a 50% loss of viability of cells (LT50) from fasted rats after 55 min of incubation, whereas a LT50 of 80 min was observed after exposure of hepatocytes from fed rats to menadione. Glutathione and NADPH levels were rapidly depleted by menadione metabolism. This depletion was sustained during the incubation period. No significant differences were found in the time course and extent of the menadione-induced glutathione and NADPH depletion in hepatocytes of both nutritional states. Menadione also affected the energy status of the hepatocytes. The ATP content of cells from fasted rats decreased to 50% (AT50) within 18 min of exposure to menadione, whereas a 50% loss of ATP content of hepatocytes from fed rats was reached at 65 min. In contrast to depletion of glutathione and NADPH, the time course and extent of menadione-induced ATP depletion correlated well with the time of onset and rate of cell killing. Our results suggest that menadione metabolism may interfere with both mitochondrial and glycolytic ATP production. Depletion of ATP might be a critical step in menadione-induced cytotoxicity.     

Effect of metal ion catalyzed oxidation of rifamycin SV on cell viability and metabolic performance of isolated rat hepatocytes

The effect of rifamycin SV on metabolic performance and cell viability was studied using isolated hepatocytes from fed, starved and glutathione (GSH) depleted rats. The relationships between GSH depletion, nutritional status of the cells, glucose metabolism, lactate dehydrogenase (LDH) leakage and malondialdehyde (MDA) production in the presence of rifamycin SV and transition metal ions was investigated. Glucose metabolism was impaired in isolated hepatocytes from both fed and starved animals, the effect is dependent on the rifamycin SV concentration and is enhanced by copper (II). Oxygen consumption by isolated hepatocytes from starved rats was also increased by copper (II) and a partial inhibition due to catalase was observed. Cellular GSH levels which decrease with increasing the rifamycin SV concentration were almost depleted in the presence of copper (II). A correlation between GSH depletion and LDH leakage was observed in fed and starved cells. Catalase induced a slight inhibition of the impairment of gluconeogenesis, GSH depletion and LDH leakage in starved hepatocytes incubated with rifamycin SV, iron (II) and copper (II) salts. Lipid peroxidation measured as MDA production by isolated hepatocytes was also augmented by rifamycin SV and copper (II), especially in hepatic cells isolated from starved and GSH depleted rats. Higher cytotoxicity was observed in isolated hepatocytes from fasted animals when compared with fed or GSH depleted animals. It seems likely that in addition to GSH level, there are other factors which may have an influence on the susceptibility of hepatic cells towards xenobiotic induced cytotoxicity.     

Insulin stimulates triacylglycerol secretion by perfused livers from fed rats but inhibits it in livers from fasted or insulin-deficient rats implications for the relationship between hyperinsulinaemia and hypertriglyceridaemia.

We determined whether the direction of the acute effect of insulin on hepatic triacylglycerol secretion is dependent on the prior physiological state or on the in vitro experimental system used. The effect of insulin on triacylglycerol secretion was studied using perfused livers isolated from rats under three metabolic conditions: fed normo-insulinaemic, 24-h fasted and fed, streptozotocin-diabetic (insulin-deficient). Insulin acutely activated triacylglycerol secretion (by 43%) in organs from fed, normo-insulinaemic animals, whereas it inhibited triacylglycerol secretion in livers isolated from fasted or insulin-deficient rats (by 30 and 33%, respectively). By contrast, in 24-h-cultured hepatocytes insulin invariably acutely inhibited triacylglycerol secretion irrespective of the metabolic state of the donor animals. It is concluded that the use of perfused livers enables the observation of a switch in the direction of insulin action on hepatic triacylglycerol secretion from stimulatory, in the normo-insulinaemic state, to inhibitory in the fasting or insulin-deficient state. The possible implications of this switch for the relationship between hyperinsulinaemia, increased hepatic very-low-density lipoprotein-triacylglycerol secretion and hypertriglyceridaemia observed in vivo are discussed.     

Glycogenolysis - and not gluconeogenesis - is the source of UDP-glucuronic acid for glucuronidation

Differences in cofactor (NADPH and UDP-glucuronic acid) supply for various processes of biotransformation were studied by investigating the interrelations between glucose production (gluconeogenesis and glycogenolysis) and drug (p-nitrophenol, aminopyrine, phenolphthalein) biotransformation (hydroxylation and conjugation) in isolated murine hepatocytes. In glycogen-depleted hepatocytes prepared from animals fasted for 48 h (i) p-nitrophenol conjugation was decreased by 80% compared to the fed control, while aminopyrine oxidation was unaltered, (ii) addition of glucose or gluconeogenic substrates failed to increase the rate of p-nitrophenol conjugation, while the rate of p-nitrophenol and also aminopyrine oxidation was increased and (iii) gluconeogenesis was inhibited by 80% by aminopyrine oxidation: it was moderately decreased by p-nitrophenol oxidation and conjugation and remained unchanged by phenolphthalein conjugation. In hepatocytes prepared from fed mice (i) p-nitrophenol conjugation was independent of the extracellular glucose concentration, (ii) it was linked to the consumption of glycogen - addition of fructose inhibited p-nitrophenol glucuronidation only, while sulfation was unaltered and (iii) p-nitrophenol oxidation was not detectable: aminopyrine oxidation was not affected by fructose addition. It is suggested that UDP-glucuronic acid for glucuronidation derives predominantly from glycogen, while the NADPH generation for mixed function oxidation is linked to glucose uptake and / or gluconeogenesis in the liver.     

Fasting modulates metabolic responses to cortisol, GH and IGF‐I in Arctic charr hepatocytes

Hepatocytes in primary culture from fed and 2 month fasted Arctic charr Salvelinus alpinus were exposed to physiological doses of either cortisol, salmon growth hormone (GH), salmon insulin‐like growth factor‐I (IGF‐I) or a combination of salmon GH and salmon IGF‐I. Fasting significantly lowered medium glucose levels compared to the fed fish, but had no significant effects on hepatocyte glycogen content or on the activities of enzymes involved in the intermediary metabolism. Cortisol treatment had no effect on hepatocyte glycogen content or on the enzyme activities investigated, but resulted in a significant increase in medium glucose concentration in hepatocytes isolated from fasted, but not fed fish. GH and IGF‐I treatments, both singly and in combination, significantly increased the glycogen content of hepatocytes isolated from fed fish, with less pronounced effects on hepatocytes isolated from fasted fish. The combination of GH and IGF‐I significantly increased lactate dehydrogenase activity regardless of the feeding state and significantly reduced the phosphenolpyruvate carboxykinase activity and medium glucose concentration in hepatocytes isolated from fed fish. Further, GH and IGF‐I significantly increased the activities of alanine aminotransferase and aspartate aminotransferase in hepatocytes isolated from fasted fish, but not fed fish. There were no effects of GH, IGF‐I, or their combination, on glucose 6‐phosphate dehydrogenase or 3‐hydroxyacyl‐CoA dehydrogenase activities. The results demonstrated that nutritional status of the animal modulates hepatocyte responsiveness to metabolic hormones, and suggested a role for GH and IGF‐I in hepatic glycogen conservation.     

Effect of ethanol on hepatic phosphatidate phosphohydrolase: dose-dependent enzyme induction and its abolition by adrenalectomy and pyrazole treatment

Protein synthesis, measured as the incorporation of [¹⁴C]valine into cell proteins and into proteins secreted into the medium, and albumin production were studied in isolated rat liver hepatocytes. Protein synthesis was substantially higher in cells from fed rats than in cells from fasted rats. Addition of carbohydrates or amino acids increased protein synthesis in cells from fasted rats, whereas no effect was seen in cells from fed rats. Addition of oleate had no effect on protein synthesis. Ethanol inhibited protein synthesis in cells from fasted rats, whereas no or only small effect was seen in cells from fed rats. Simultaneous addition of carbohydrates diminished the inhibitory effect of ethanol, whereas addition of oleate increased the inhibitory effect of ethanol. It is suggested that the rate of protein synthesis in cells from fasted rats could be restricted by lack of precursors for synthesis of nonessential amino acids. The effect of ethanol is explained by an inhibition of gluconeogenesis.     

Ethanol metabolism in guinea pig: Ethanol oxidation and its effect on NADNADH ratios,oxygen consumption,and ketogenesis in isolated hepatocytes of fed and fasted animals

Ethanol metabolism was studied in isolated hepatocytes of fed and fasted guinea pigs. Alcohol dehydrogenase (EC 1.1.1.1) activities of fed or fasted liver cells were 2.04 and 1.88 μmol/g cells/min, respectively. Under a variety of in vitro conditions, alcohol dehydrogenase operates in fed hepatocytes at 34–74% and in fasted liver cells at 23–61% of its maximum velocity, respectively. Hepatocytes of fed animals, incubated in Krebs-Ringer bicarbonate buffer, oxidized ethanol at an average rate of 0.69 μmol/g wet weight cells/min, whereas cells of 48-h fasted animals consumed only 0.44 μmol/g/min under identical conditions. Various substrates and metabolites of intermediary metabolism significantly enhanced ethanol oxidation in fed liver cells. Maximum stimulatory effects were achieved with alanine (+138%) and pyruvate (+102%), followed in decreasing order by propionate, lactate, fructose, dihydroxyacetone, and galactose. In contrast to substrate couples such as lactate/pyruvate and glycerol/dihydroxyacetone, sorbitol with or without fructose significantly inhibited ethanol oxidation. The addition of hydrogen shuttle components such as malate, aspartate, or glutamate to fasted hepatocytes resulted in significantly higher stimulation of ethanol uptake than in fed hepatocytes. Also, the degree of inhibition of shuttle activity by n-butylmalonate was more pronounced in fasted liver cells (77% inhibition) than in fed cells (59% inhibition). These data as well as oxygen kinetic studies in intact guinea pig hepatocytes utilizing uncouplers (carbonyl cyanide-p-trifluoromethoxyphenylhydrazone, dinitrophenol), electron-transport inhibitors (rotenone, antimycin), and malate-aspartate shuttle inhibitors (aminooxyacetate, n-butylmalonate) strongly suggested that the malate-aspartate shuttle is the predominant hydrogen transport system during ethanol oxidation in guinea pig liver.Comparison of the alcohol dehydrogenase-inhibitors 4-methylpyrazole and pyrazole on ethanol oxidation demonstrated that the alcohol dehydrogenase system is quantitatively the most important alcohol-metabolizing pathway in guinea pig liver. Supporting this conclusion, it was found that the H₂O₂-forming substrate glycolate slightly increased ethanol oxidation in liver cells of control animals (+26%), but prior inhibition of catalase by 3-amino-1,2,4-triazole resulted in a significant increase (+25%) instead of a decrease in alcohol oxidation. This finding does not support a quantitatively important role of peroxidatic oxidation of ethanol by catalase in liver.Cytosolic $\text{NAD}\text{NADH}$ ratios were greatly shifted toward reduction during ethanol oxidation. These reductive shifts were even more pronounced when cells were incubated in the presence of fatty acids (octanoate, oleate) plus ethanol. Inhibitor studies with 4-methylpyrazole demonstrated that the decrease of the cytosolic $\text{NAD}\text{NADH}$ ratio during fatty acid oxidation was due to an inhibition of hydrogen transport from cytosol to mitochondria and not the result of transfer of hydrogen, generated by fatty acid oxidation, from mitochondria to cytosol. Lactate plus pyruvate formation was slightly inhibited by ethanol in fed hepatocytes but greatly accelerated in fasted cells; this latter effect was mostly the result of increased lactate formation. Such regulation may represent a hepatic mechanism of alcoholic lactic acidosis as observed in human alcoholics. The ethanol-induced decrease of the mitochondrial $\text{NAD}\text{NADH}$ ratio was prevented by addition of 4-methylpyrazole. Endogenous ketogenesis was greatly increased (+80%) by ethanol in fed liver cells. This effect of ethanol was blunted in the presence of glucose. Propionate, by competing with fatty acid oxidation, was strongly antiketogenic. This effect was alleviated by ethanol. In 48-h fasted hepatocytes, endogenous ketogenesis was enhanced by 84%. Although ethanol did not further stimulate endogenous ketogenesis under these conditions, alcohol significantly increased ketogenesis in the presence of octanoate or oleate. This stimulatory effect of ethanol was almost completely prevented by 4-methylpyrazole. These findings demonstrate that the syndrome of alcoholic ketoacidosis may be due, at least partially, to the additional stimulation of ketogenesis by or from ethanol during fatty acid oxidation in the fasting state.     

Repartition of ATP, ADP and PO4 in isolated hepatocytes from fed and fasted rats

1. The digitonin fractionation procedure [Zuurendonk, P. F. and Tager, J. M. (1974) Biochim. Biophys. Acta, 333, 393-399] was used to determine the repartition of adenine nucleotides and inorganic phosphate in isolated hepatocytes from fed and fasted rats. 2. This repartition is not significantly modified in the presence of pyruvate or alanine or lactate + pyruvate for isolated hepatocytes from fasted rats. 3. In isolated hepatocytes from fasted rats, the mitochondrial ATP/ADP X PO4 ratio is two-fold lower than in isolated hepatocytes from fed rats. 4. The cytosolic ATP/ADP X PO4 ratio depends on the nutritional state and (or) on the added substrate for neoglucogenesis.     

Fasting enhances glycogen synthase activation in hepatocytes from insulin-resistant genetically obese (fa/fa) rats.

Glycogen synthase activation and phosphorylase inactivation by glucose were studied in hepatocytes isolated from fed or overnight-fasted lean or genetically obese (fa/fa) rats. In cells from fed animals, both the time course and dose-response to glucose of synthase activation were the same in both groups, despite higher levels of phosphorylase a in hepatocytes from obese animals. In contrast, in cells from fasted obese animals synthase activation with or without glucose was enhanced severalfold over that of lean controls, despite similar levels of phosphorylase a and of total (a + b) synthase activities. In both nutritional conditions glucose 6-phosphate concentrations were 2-3-fold higher in obese-rat hepatocytes than in lean-rat cells. In addition, synthase activation was transient in the fasted lean group, but was sustained in obese-rat hepatocytes. The rate of synthase activation was, however, comparable in lean- and obese-rat liver Sephadex G-25 filtrates, irrespective of the nutritional state of the donor rats. It is concluded that enhanced synthase activation in hepatocytes from starved obese rats might be due to an unbalanced synthase interconversion brought about by elevated glucose 6-phosphate concentrations and impaired kinase [van de Werve & Massillon (1990) Biochem. J. 269, 795-799], rather than to an intrinsic change in synthase phosphatase.     

Effect of D-galactosamine in vitro on [U-14C]palmitate oxidation, triacylglycerol synthesis and secretion in isolated hepatocytes

Isolated rat hepatocytes were used to study in vitro effects of 10 mM D-galactosamine (GalN) on hepatic fatty acids metabolism. At this concentration, membrane integrity and biochemical competence (i.e., gluconeogenesis and ureogenesis) remained unaffected. Protein synthesis and secretion, as measured by the incorporation of [U-14C]leucine into total and medium protein, was significantly inhibited when incubated for more than 2 h. GalN activated the incorporation of [U-14C]palmitate into triacylglycerols and depressed its utilization in the formation of labelled ketone bodies and 14CO2. Hepatocytes isolated from fasted rats exposed to GalN in vitro did not show any variation in prelabelled triacylglycerol secretion. GalN induced a rapid inhibition of prelabelled triacylglycerol secretion by hepatocytes isolated from fed rats in which this secretion occurred to a larger extent than in hepatocytes isolated from fasted rats. The data reported here suggest that GalN induces a rise of triacylglycerol synthesis by inhibiting the palmitate oxidation pathway and a decrease of triacylglycerol secretion through an early derangement of the secretory pathway.     

Dependence with nutritional status of the ethanol effects on fatty acid metabolism in rat hepatocytes

1. The effects of ethanol on fatty acid synthesis, esterification and oxidation were studied in hepatocytes isolated from fed and 24 hr fasted rats. 2. [3H]H2O was preferentially incorporated into the glycerol backbone of triglycerides and phospholipids. Addition of ethanol markedly increased the incorporation of this label in both classes of glycerolipids; the increase was higher in fasted rat hepatocytes, both in the glycerol backbone and acyl groups of glycerolipids. 3. Ethanol increased [U-14C]palmitate incorporation into triglycerides only in hepatocytes from fasted rats. 4. [14C]CO2 and total acid soluble product formation from [1-14C]palmitate resulted inhibited by ethanol both in the fed and the fasted state.     

Role of a nonmitochondrial Ca2+ pool in the synergistic stimulation by cyclic AMP and vasopressin of Ca2+ uptake in isolated rat hepatocytes.

The subcellular distribution of 45Ca2+ accumulated by isolated rat hepatocytes exposed to dibutyryl cyclic AMP (dbcAMP) followed by vasopressin (Vp) was studied by means of a nondisruptive technique. When treated with dbcAMP followed by vasopressin, hepatocytes obtained from fed rats accumulated an amount of Ca2+ approximately fivefold higher than that attained under control conditions. Ca2+ released from the mitochondrial compartment by the uncoupler carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP) accounted for only a minor portion of the accumulated Ca2+. The largest portion was released by the Ca2+ ionophore A23187 and was attributable to a nonmitochondrial compartment. DbcAMP + Vp-treatment also caused a maximal stimulation of glucose production and a twofold increase in cellular glucose 6-phosphate levels. In hepatocytes obtained from fasted rats, dbcAMP + Vp-stimulated Ca2+ accumulation was lower, although with the same subcellular distribution, and was associated with a minimal glucose production. In the presence of gluconeogenetic substrates (lactate plus pyruvate) hepatocytes from fasted rats were comparable to cells isolated from fed animals. However, Ca2+ accumulation and glucose 6-phosphate production could be dissociated in the absence of dbcAMP, in the presence of lactate/pyruvate alone. Under this condition in fact Vp induced only a minimal accumulation of Ca2+ in hepatocytes isolated from fasted rats, although glucose production was markedly increased. Moreover, treatment of fed rat hepatocytes with 1 mM ATP caused a maximal activation of glycogenolysis, but only a moderate stimulation of cellular Ca2+ accumulation. In this case, sequestration of Ca2+ occurred mainly in the mitochondrial compartment. By contrast, the addition of ATP to dbcAMP-pretreated hepatocytes induced a large accumulation of Ca2+ in a nonmitochondrial pool. Additional experiments using the fluorescent Ca2+ indicator Fura-2 showed that dbcAMP pretreatment can enlarge and prolong the elevation of cytosolic free Ca2+ caused by Vp. A nonmitochondrial Ca2+ pool thus appears mainly responsible for the Ca2+ accumulation stimulated by dbcAMP and Vp in isolated hepatocytes, and cyclic AMP seems able to activate Ca2+ uptake in such a nonmitochondrial pool.     

Critical evaluation of methods used for the isolation of rat liver hepatocytes for metabolic studies.

Hepatocytes were isolated from normal fed, fasted and alloxan diabetic animals. The best cell preparations were obtained by using low concentrations of collagenase (10–20 mg) and exposing the liver for a very short period of time (10–15 min). Addition of hyaluronidase significantly decreased the glycogen content of the isolated hepatocytes. Glucagon (10⁻¹²M) stimulated glycogenesis in hepatocytes containing high glycogen whereas, in cells containing low glycogen much higher concentration of glucagon was needed (10⁻⁹M). Addition of insulin (100 μunits) stimulated both glycogen and protein synthesis in isolated hepatocytes containing high glycogen. Under these conditions glycogen synthase activity was stimulated by 40%. Incorporation of ¹⁴C phenylalanine into protein was linear for only 3–4 hr in cells containing low glycogen whereas, in cells containing high glycogen incorporating was linear for 8–10 hr. These studies suggest that intracellular glycogen plays an important role in the hormonal regulation of metabolism in hepatocytes.     

Alterations in inositol phosphate production during oxidative stress in isolated hepatocytes

The level of inositol phosphates was measured in rat hepatocytes treated with 2-methyl-1,4-naphthoquinone (menadione) or tert-butyl hydroperoxide, which cause Ca2+ mobilization from intracellular stores and an increase in cytosolic free Ca2+ concentration. Although neither agent produced any apparent changes in the resting level of inositol phosphates, pretreatment of hepatocytes with either menadione or tert-butyl hydroperoxide, as well as with several sulfhydryl reagents, markedly inhibited the increase in inositol phosphates induced by both hormonal and nonhormonal stimuli. Addition of dithiothreitol to menadione- or tert-butyl hydroperoxide-treated hepatocytes reversed this inhibition and reestablished responsiveness to extracellular stimuli. Our findings suggest that the inhibition of the inositol phosphate response by menadione and tert-butyl hydroperoxide occurs through the modification of critical sulfhydryl group(s) and that the alterations in intracellular Ca2+ homeostasis occurring during the metabolism of menadione and tert-butyl hydroperoxide in hepatocytes are not mediated by inositol phosphates.     

Role of peroxisomal fatty acid beta-oxidation in ethanol metabolism

The contribution of peroxisomal fatty acid beta-oxidation to ethanol metabolism was examined in deermice hepatocytes. Addition of 1 mM oleate to hepatocytes isolated from fasted alcohol dehydrogenase (ADH)-positive deermice in the presence of 4-methylpyrazole or to hepatocytes from fasted or fed ADH-negative deermice produced only a slight and statistically not significant increase in ethanol oxidation. Lactate (10 mM), which is not a peroxisomal substrate, showed a greater effect on ethanol oxidation. There was also a lack of oleate effect on the oxidation of ethanol by hepatocytes of ADH-positive deermice. Furthermore, in ADH-negative deermice, the catalase inhibitor azide (0.1 mM) did not inhibit the increase in ethanol oxidation by oleate and lactate. The rate of oleate oxidation by hepatocytes from fasted ADH-negative deermice was much lower than that of ethanol. These results indicate that in deermice hepatocytes, peroxisomal fatty acid oxidation does not play major role in ethanol metabolism.     

The role of oxidative processes in the cytotoxicity of substituted 1,4-naphthoquinones in isolated hepatocytes

In order to clarify the role of oxidative processes in cytotoxicity we have studied the metabolism and toxicity of 2-methyl-1,4-naphthoquinone (menadione) and its 2,3 dimethyl (DMNQ) and 2,3 diethyl (DENQ) analogs in isolated rat hepatocytes. The two analogs, unlike menadione, cannot alkylate nucleophiles directly and were considerably less toxic than menadione. This decreased toxicity was consistent with the inability of DMNQ and DENQ to alkylate but we also found them to undergo lower rates of redox cycling in hepatocytes and a higher ratio of two electron as opposed to one electron reduction relative to menadione. Thus, facile analysis of the respective roles of alkylation and oxidation in cytotoxicity was not possible using these compounds. In hepatocytes pretreated with bischloroethyl-nitrosourea (BCNU) to inhibit glutathione reductase, all three naphthoquinones caused a potentiation of reduced glutathione (GSH) removal/oxidized glutathione (GSSG) generation and cytotoxicity relative to that observed in control cells. These data show that inhibition of hepatocyte glutathione reductase by BCNU results in enhanced naphthoquinone-induced oxidative challenge and subsequent cellular toxicity. That DMNQ and DENQ are cytotoxic, albeit at high concentrations, and that this cytotoxicity is potentiated by BCNU pretreatment suggest that oxidative processes alone can be a determinant of cytotoxicity.     

Oxygen dependence and subcellular partitioning of hepatic menadione-mediated oxygen uptake. Studies with isolated hepatocytes, mitochondria, and microsomes from rat liver in an oxystat system

Using an oxystat system, menadione (2-methyl-1,4-naphthoquinone)-mediated oxygen uptake was investigated in isolated rat hepatocytes, in malate/glutamate-supplemented mitochondria, and in NADPH-reduced microsomes at steady-state oxygen partial pressures (pO2) between 0.1 to 100 mm Hg (0.2-150 microM O2). Menadione-mediated stimulation of oxygen uptake was half-maximal at pO2 of 0.5, 0.2, and 0.9 mm Hg, respectively. In hepatocytes and mitochondria half-maximal concentrations of menadione were 15 and 4 microM. However, in microsomes saturation with menadione was not reached at concentrations up to 300 microM. Antimycin A inhibited menadione-mediated oxygen uptake in hepatocytes and mitochondria by about three-fourths, while rotenone was without inhibitory effect; KCN inhibited practically completely. In mitochondria menadione-stimulated oxygen uptake was significantly inhibited by dicoumarol but further enhanced by the addition of ADP, even in the presence of rotenone. The results suggest that menadione-mediated hepatocellular oxygen uptake proceeds almost independently of pO2 in most regions of the liver lobule but that in areas of low pO2 such as the centrolobular regions limitation by oxygen may occur. They also demonstrate that in the intact hepatocyte menadione-mediated oxygen uptake predominantly (greater than 90%) results from electron transfer in the mitochondrial respiratory chain by menadione.     

Regulation of hepatic glycine catabolism by glucagon

Glucagon stimulates 14CO2 production from [1-14C] glycine by isolated rat hepatocytes. Maximal stimulation (70%) of decarboxylation of glycine by hepatocytes was achieved when the concentration of glucagon in the medium reached 10 nM; half-maximal stimulation occurred at a concentration of about 2 nM. A lag period of 10 min was observed before the stimulation could be measured. Inclusion of beta-hydroxybutyrate (10 mM) or acetoacetate (10 mM) did not affect the magnitude of stimulation suggesting that the effects of glucagon were independent of mitochondrial redox state. Glucagon did not affect either the concentration or specific activity of intracellular glycine, thus excluding the possibilities that altered concentration or specific activity of intracellular glycine contributes to the observed stimulation. The stimulation of decarboxylation of glycine by glucagon was further studied by monitoring 14CO2 production from [1-14C]glycine by mitochondria isolated from rats previously injected with glucagon. Glycine decarboxylation was significantly stimulated in the mitochondria isolated from the glucagon-injected rats. We suggest that glucagon is a major regulator of hepatic glycine metabolism through the glycine cleavage enzyme system and may be responsible for the increased hepatic glycine removal observed in animals fed high-protein diets.     

Pyridine Nucleotide Changes In Hepatocytes Exposed To Quinones

Quinones may be toxic by a number of mechanisms. including arylation and oxidative stress caused by redox cycling. Using isolated hepatocytes, we have studied the cytotoxicity of four quinones. with differing abilities to arylate cellular nucleophiles and redox cycle. in relation to their effects on cellular pyridine nucleotides. High concentrations of menadione (redox cycles and arylates). 2-hydroxy-1,4-naphthoquinone (neither arylates nor redox cycles via a one electron reduction) 2.3-dimethoxy-1.4-naphthoquinone (a pure redox cycler) and p-benzoquinone (a pure arylator) caused an initial decrease in NAD⁺ and loss of viability, which was not prevented by 3-aminobenzamide. an inhibitor of poly(ADP-ribose)polymerase. In contrast. 3-aminobenzamide inhibited the loss of NAD' and viability caused by dimethyl sulphate so implicating poly(ADP-ribose)polymerase in its toxicity but not that of the quinones. Non-toxic concentrations of menadione. 2.3-dimethoxy-1.4-naphthoquinone and 2-hydroxy-1.4-naphthoquinone all caused markedly similar changes in cellular pyridine nucleotides. An initial decrease in NAD⁺ was accompanied by a small. transient increase in NADP⁺ and followed by a larger. prolonged increase in NADPH and total NADP⁺ + NADPH. Nucleotide changes were not observed with non-toxic concentrations of p-benzoquinone. Our findings suggest that a primary event in the response of the cell to redox cycling quinones is to bring about an interconversion of pyridine nucleotides. in an attempt to combat the effects of oxidative stress     

Effect of sodium fluoride on cyclic AMP production in rat hepatocytes.

The effect of NaF on cAMP production was studied in hepatocytes isolated from fed and fasted rats. A four-six fold increase in hepatocyte cAMP production was observed in the presence of 10-20 mM NaF in cells isolated from either fed or fasted rats. The maximal stimulation of cAMP production was observed after a 10 min incubation in the presence of 1 mM theophylline. However, as little as 0.05-0.15 mM NaF induced a significant increase in cAMP production. It was also found that NaF would alter the production of glucose in isolated rat hepatocytes. When hepatocytes from fed rats were incubated with 0.05-5 mM NaF there was an increase in amount of glucose released from endogenous sources. Also NaF resulted in a decrease in lactate and pyruvate production. Similarly NaF stimulated glucose production in hepatocytes from fasted rats. The maximal stimulation was observed with about 0.15-0.25 mM NaF. At NaF concentrations greater than 1.5 mM a decrease in glucose production was observed. It is concluded that NaF increases the level of cAMP and alters glucose metabolism in intact hepatocytes.