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.

Interrelationship between aminopyrine oxidation and gluconeogenesis in hepatocytes prepared from fructose-pretreated mice.

Aminopyrine oxidation was studied in isolated hepatocytes prepared from 24-h-starved mice (i) after induction of the NADPH-generating malic enzyme and glucose-6-phosphate dehydrogenase, but not the mixed function oxygenases by fructose, (ii) after induction of both mixed function oxygenases and NADPH-generating malic enzyme and glucose-6-phosphate dehydrogenase by phenobarbital and (iii) without any pretreatment. Phenobarbital pretreatment, as expected, increased the rate of aminopyrine oxidation of isolated hepatocytes. However, fructose pretreatment also enhanced the rate of N-demethylation of aminopyrine by more than 100% supporting the view that the availability of NADPH is rate limiting in drug oxidation under certain conditions. The role of malic enzyme and glucose-6-phosphate dehydrogenase in the NADPH supply for aminopyrine oxidation was investigated by the addition of two groups of gluconeogenic precursors: lactate or alanine and glycerol or fructose with the simultaneous measurement of glucose synthesis and aminopyrine N-demethylation. There was a clear correlation between the increased rate of aminopyrine oxidation and the decreases of glucose production caused by aminopyrine. Gluconeogenesis in the presence of 1 mM aminopyrine was decreased by 70-80% when alanine or lactate were used as precursors, it was decreased by only 35-40% when glucose production was started from glycerol or fructose; in an accordance with the facts that NADPH generation and gluconeogenesis starting from alanine or lactate share two common intermediates--malate and glucose-6 phosphate--, while there is only one common intermediate--glucose-6 phosphate--if fructose or glycerol are used. Similar results were obtained with the addition of the structurally dissimilar hexobarbital. It is concluded that besides malic enzyme, glucose-6-phosphate dehydrogenase also takes part in NADPH supply for drug oxidation in glycogen-depleted hepatocytes.

Epinephrine and glucagon counteract inhibition of protein synthesis induced by D-galactosamine in isolated mouse hepatocytes.

10 mM D-galactosamine enhibited protein synthesis (1 h incubation time) by 67% in isolated mouse liver cells. Counteracting uridylate deficiency induced by D-galactosamine by preventive administration of 20 mM uridine did not decrease the extent of protein synthesis inhibition. 20 mM D-galactose reverted the inhibition of protein synthesis by D-galactosamine. 10(-5) M epinephrine and 10(-7) M glucagon decreased the incorporation of D-galactosamine into glycogen to 38% and 26% of the control value, respectively, after a 35 min incubation and reduced the inhibition of protein synthesis by D-galactosamine effectively. Experimental evidence supports the view that aminoglycogen formed after D-galactosamine treatment is responsible for the inhibition of protein synthesis.

Accumulation of S-D-lactoylglutathione and transient decrease of glutathione level caused by methylglyoxal load in isolated hepatocytes.

Methylglyoxal is converted to D-lactic acid through a conjugation with glutathione and S-D-lactoylglutathione is an intermediate of this pathway. In isolated hepatocytes prepared from fed mice incubated without nutrients (glucose, pyruvate and amino acids) the formation and release of S-D-lactoylglutathione and also a continuous lowering of cellular glutathione were demonstrated upon addition of methylglyoxal (20 mM). Under these incubation conditions, the glutathione content of the cells decreased in the controls. On the other hand, in hepatocytes incubated in a medium supplemented with the above-mentioned compounds an accumulation of S-D-lactoylglutathione and a transient decrease of glutathione were shown after addition of methylglyoxal. Under these experimental circumstances the glutathione content of the cells was preserved. Buthionine sulfoximine--an inhibitor of glutathione synthesis--prevented the restoration of glutathione level in hepatocytes observed in the presence of methylglyoxal; emetine--an inhibitor of protein synthesis--was ineffective. It is suggested that increased methylglyoxal formation may have a role in alterations of glutathione metabolism under conditions when serum acetone is increased and methylglyoxal production from acetone is elevated.

Effect of methylglyoxal on glucose formation, drug oxidation and glutathione content in isolated murine hepatocytes.

The first stage in the formation of glucose from acetone involves two oxidation steps catalyzed by isozymes of the cytochrome P-450 II E1 gene subfamily; methylglyoxal formed this way is further converted to pyruvate by a reversible conjugation with reduced glutathione. The effect of methylglyoxal on glucose formation, oxidation of aminopyrine, aniline and on reduced glutathione content was investigated in isolated hepatocytes prepared from (i) fasted or (ii) fasted and acetone (known to induce isozymes of P-450 II E1 gene subfamily) pretreated mice. Glucose formation and drug oxidation were increased by methylglyoxal at concentrations below 1 mM, but were severely decreased above 1 mM. Methylglyoxal also decreased protein synthesis at concentrations above 1 mM. If the addition of methylglyoxal was combined with that of other gluconeogenic precursors and glucose the initial increasing effect on drug oxidation was moderated or diminished and the decreasing effect (at high concentrations) was enhanced. The glutathione content of the cells was decreased by methylglyoxal in a concentration dependent manner. Acetone pretreatment of mice also resulted in a decreased glutathione content of the liver. Based on these observations it is assumed that methylglyoxal has contrasting effects in hepatocytes, and can contribute to the disturbed metabolism under circumstances when the acetone production is elevated.

Association of thrombin, plasmin, thrombin-antithrombin III complex and plasmin-antithrombin III complex with isolated hepatocytes.

The interaction of thrombin, plasmin or their antithrombin III complexes with isolated mouse hepatocytes was studied. Plasmin bound to hepatocytes in a concentration-dependent manner with an apparent Kd of 6.4.10(-8) M, attaining equilibrium within 10 min, and the interaction was inhibited by 6-amino-n-hexanoic acid. Plasmin treated with diisopropylfluorophosphate (DFP) bound to the cells in similar way as the untreated form of the enzyme. Thrombin bound also to hepatocytes, in a concentration-dependent manner, with a Kd of 5.4.10(-8) M reaching a steady state after 180 min. Thrombin inactivated with DFP, however, was inhibited in its binding to these cells. These data suggest that, whereas the kringle domains of plasmin are responsible for the enzyme-cell interaction, the active center of thrombin may be involved in the binding of this enzyme to hepatocytes. Plasmin-antithrombin III and thrombin-antithrombin III complexes were also associated with hepatocytes in a time-dependent manner, reaching a plateau after 180 min, and the two complexes competed in the interaction. While the interaction of active proteinases plasmin or thrombin with hepatocytes did not result in their internalization, the antithrombin III complexes were taken up by the cells, and thrombin-antithrombin III complex was degraded. These results indicate that hepatocytes may participate in the elimination of proteinase-antithrombin III complexes from the plasma, while the association of plasmin and thrombin with hepatocytes could imply distinct biological importance.

Association of alpha 2-macroglobulin-thrombin and alpha 2-macroglobulin-plasmin complexes with isolated hepatocytes.

125I-labelled alpha 2-macroglobulin complexed with thrombin or plasmin bound to hepatocytes in a concentration- and time-dependent manner. The apparent Kd values calculated from displacement experiments were 7.9 X 10(-8) M for alpha 2-macroglobulin-thrombin and 8.5 X 10(-8) M for alpha 2-macroglobulin-plasmin. Association of these complexes was only partially reversible; after a 180 min incubation period, 50-60% of the bound radioactivity was internalized by the cells. alpha 2-Macroglobulin itself bound also to hepatocytes, but the affinity of the alpha 2-macroglobulin complexes was higher than that of the inhibitor alone, and alpha 2-macroglobulin was not internalized, either. 125I-labelled thrombin or plasmin bound to hepatocytes as well. These bindings were also concentration-dependent and could be decreased with an excess of unlabelled ligands. Binding rates and amounts of the bound proteinases were higher than those of their alpha 2-macroglobulin complexes. The alpha 2-macroglobulin-thrombin complex competed with the alpha 2-macroglobulin-plasmin complex in binding to hepatocytes, whereas there was no competition between these complexes and the antithrombin III-thrombin complex. These results suggest that the binding sites of hepatocytes for alpha 2-macroglobulin-proteinase and antithrombin III-proteinase complexes are different.

Ascorbic acid synthesis is stimulated by enhanced glycogenolysis in murine liver.

Ascorbic acid synthesis was stimulated by glucagon, dibutyryl cyclic AMP, as well as phenylephrine vasopressin or okadaic acid, in hepatocytes prepared from fed mice. However, no such effect was observed in glycogen-depleted cells from starved animals, either in the presence or absence of glucose. The rate of ascorbate synthesis showed close correlation with the glucose release by hepatocytes. In mice the injection of glucagon increased plasma ascorbate concentration fifteenfold, and caused a sixfold elevation of the ascorbate content of the liver. These results show that hepatic ascorbate synthesis is dependent on glycogenolysis, and indicate a regulatory role of ascorbate released by the liver.

Ascorbate synthesis-dependent glutathione consumption in mouse liver.

Ascorbate synthesis causes glutathione consumption in the liver. Addition of gulonolactone resulted in an increase of ascorbate production in isolated murine hepatocytes. At the same time, a decrease in reduced glutathione (GSH) level was observed. In hepatic microsomal membranes, ascorbate synthesis stimulated by gulonolactone caused an almost equimolar consumption of GSH. This effect could be counteracted by the addition of catalase or mercaptosuccinate, indicating the role of hydrogen peroxide formed during ascorbate synthesis in the depletion of GSH. The observed phenomenon may be one of the reasons why the evolutionary loss of ascorbate synthesis could be advantageous.

Latency is the major determinant of UDP-glucuronosyltransferase activity in isolated hepatocytes.

The glucuronidation of p-nitrophenol was measured in intact, saponin- and alamethicin-treated isolated mouse hepatocytes. In saponin-permeabilized cells the elevation of extrareticular UDP-glucuronic acid concentration enhanced the rate of glucuronidation threefold. When intracellular membranes were also permeabilized by alamethicin, a further tenfold increase in the glucuronidation of p-nitrophenol was present. Parallel measurements of the ER mannose 6-phosphatase activity revealed that saponin selectively permeabilized the plasma membrane, whereas alamethicin permeabilized both plasma membrane and ER membranes. The inhibition of p-nitrophenol glucuronidation by dbcAMP in intact hepatocytes was still present in saponin-treated cells and disappeared in alamethicin-permeabilized hepatocytes. It is suggested that the permeability of the endoplasmic reticulum membrane is a major determinant of glucuronidation not only in microsomes but in isolated hepatocytes as well.

Gluconeogenesis from ascorbic acid: ascorbate recycling in isolated murine hepatocytes.

Ascorbic acid synthesis and breakdown were investigated in isolated hepatocytes prepared from fasted mice. Stimulation of gluconeogenesis by alanine or xylitol led to ascorbate synthesis. On the other hand, ascorbate or dehydroascorbate addition resulted in concentration-dependent glucose production and elevation of the pentose phosphate pathway intermediate xylulose 5-phosphate. Stimulation of ascorbate oxidation and/or the inhibition of dehydroascorbate reduction increased glucose formation. Inhibition of the pentose phosphate pathway decreased glucose production from dehydroascorbate with increased accumulation of xylulose 5-phosphate. These results suggest that ascorbate can be recycled by a novel way involving intermediates of the pentose phosphate pathway, gluconeogenesis and hexuronic acid pathway.

Glutathione depletion induces glycogenolysis dependent ascorbate synthesis in isolated murine hepatocytes.

The relationship between glutathione deficiency, glycogen metabolism and ascorbate synthesis was investigated in isolated murine hepatocytes. Glutathione deficiency caused by various agents increased ascorbate synthesis with a stimulation of glycogen breakdown. Increased ascorbate synthesis from UDP-glucose or gulonolactone could not be further affected by glutathione depletion. Fructose prevented the stimulated glycogenolysis and ascorbate synthesis caused by glutathione consumption. Reduction of oxidised glutathione by dithiothreitol decreased the elevated glycogenolysis and ascorbate synthesis in diamide or menadione treated hepatocytes. Our results suggest that a change in GSH/GSSG ratio seems to be a sufficient precondition of altering glycogenolysis and a consequent ascorbate synthesis.

Accumulation of phenols and catechols in isolated mouse hepatocytes in starvation or after pretreatment with acetone.

Conditions leading to the accumulation of unconjugated phenols and catechols were investigated in mouse livers. The formation of unconjugated hydroxylated products of added p-nitrophenol and aniline was investigated in isolated hepatocytes prepared from 48 hr fasted or fed mice or from fed mice after acetone pretreatment. 4-Nitrocatechol and p-aminophenol--the hydroxylated products of p-nitrophenol and aniline--were accumulated in cells prepared from fasting animals, while in cells prepared from fed mice these unconjugated derivatives were not detectable. The accumulation of 4-nitrocatechol and p-aminophenol was also shown in isolated hepatocytes prepared from acetone pretreated fed mice. Inhibition of glucuronidation by N6,O2-dibutyryl cAMP or by D-galactosamine increased the accumulation of 4-nitrocatechol upon addition of p-nitrophenol in cells prepared from fasted mice. Both 48 hr starvation and acetone pretreatment enhanced the activity of microsomal p-nitrophenol and aniline hydroxylase by 300% and 600%, respectively, whereas p-nitrophenol conjugation in isolated hepatocytes as well as in hepatocyte homogenates was decreased by about 80% after 48 hr starvation. Acetone pretreatment did not alter the rate of p-nitrophenol conjugation measured in liver homogenates. It is suggested that a shift from conjugation toward hydroxylation in starvation gives rise to the formation of hazardous metabolites.

Endotoxin inhibits glucuronidation in the liver. An effect mediated by intercellular communication.

Endotoxin [lipopolysaccharide (LPS) 50 micrograms/mL] added to the perfusion medium increased glucose production and inhibited the glucuronidation of p-nitrophenol in perfused mouse liver both in recirculating and non-recirculating systems, while sulfation of p-nitrophenol was unchanged. The effects of endotoxin could be prevented by the addition of cyclooxygenase inhibitors, while PGD2 and PGE2 also caused a decrease in p-nitrophenol glucuronidation in perfused liver. In isolated hepatocytes endotoxin failed to affect p-nitrophenol conjugation, while PGD2 and PGE2 decreased the rate of it. Our results suggest that endotoxin inhibits glucuronidation through an intercellular communication presumably mediated by eicosanoids.

Cyclic AMP-dependent phosphorylation in the control of biotransformation in the liver.

The possibility of a short-term cAMP-dependent regulation of mixed-function oxidation and of glucuronide formation was investigated in isolated mouse hepatocytes and in mouse liver microsomal membranes. N6, O2-dibutyryl cAMP (in accordance with its increasing effect on gluconeogenesis) decreased aminopyrine oxidation and p-nitrophenol conjugation in isolated hepatocytes, while the phenolphthalein conjugation remained unaltered. Similar to dibutyryl cAMP the Ca2+ ionophore A 23187 also decreased aminopyrine oxidation. In cell-free systems the phosphorylation of isolated microsomal membranes by the exogenous cAMP-dependent protein kinase was inhibitory on aminopyrine oxidation and p-nitrophenol glucuronide formation but aniline oxidation and phenolphthalein glucuronidation were not affected. The correlation between the negative cAMP-dependent control of certain processes of biotransformation and the positive cAMP-dependent regulation of gluconeogenesis is discussed.

Changes of prostacyclin and thromboxane synthesis in the course of mouse liver perfusion. Stimulated thromboxane A2 synthesis of freshly prepared isolated mouse hepatocytes.

The formation of prostacyclin and thromboxane A2 (measured as 6-keto PGF1 alpha and TXB2 by radioimmunoassay) was investigated during a 30 min perfusion of mouse liver in a recirculation system. After cannulation of the portal vein an immediate increase of de novo synthesis and secretion of PGI2 occurred followed by a sharp decrease. Increased PGI2 synthesis was also followed by a continuous increase of TXA2 synthesis and secretion reaching a maximum at the end of the 30 min perfusion. Elevated TXA2 synthesis was also shown in freshly isolated hepatocytes investigated in the course of a 20 min incubation period immediately after the perfusion. However, the elevated TXA2 formation was not observed when it was measured after a 120 min preincubation of the cells. Both PGI2 and TXA2 production could be provoked to a similar extent by the addition of arachidonate and A 23187 immediately after the perfusion or after a 120 min preincubation.

Uptake of arachidonic acid, arachidic acid, oleic acid and their incorporation into phospholipids and triacylglycerols of isolated murine hepatocytes. Effect of thrombin-antithrombin III complex.

Uptake and metabolism of arachidonic acid, arachidic acid and oleic acid were investigated in isolated hepatocytes prepared from mouse liver with the collagenase perfusion method. The rate of uptake of arachidonic acid was time- and concentration- dependent. 94-98% of the arachidonic acid was incorporated into the phospholipid and triacylglycerol fractions following a 60 min incubation period at 37 degrees C. In the presence of thrombin-anti-thrombin III complex a change in the distribution of arachidonic acid incorporated into lipid fractions was found, i.e. increased incorporation into phosphatidyl-serine and phosphatidylethanolamine, whereas the uptake was not altered. There was no change in the uptake and incorporation of arachidic acid and oleic acid.

Specific binding of thrombin-antithrombin III complex to hepatocytes.

Thrombin-antithrombin III complex binds selectively to isolated hepatocytes, whereas antithrombin III alone does not. The binding is time and concentration dependent at 37 degrees C: the apparent Km value is 0.8/microM. The rate of binding is approximately 1.6 X 10(5) molecules h-1 cell-1 at this concentration. At 4 degrees C there is no measurable interaction between the complex and the hepatocytes. The binding is also prevented by pretreatment of cells with trypsin. On the other hand, about 80% of the thrombin-antithrombin III complex bound to hepatocytes is releasable by trypsin digestion. NaF or carboxyatractyloside does not inhibit the process. The interaction of thrombin-antithrombin III complex with hepatocytes seems to be specific, since the complexes of antithrombin III with other proteinases, like trypsin or plasmin, are not bound at the concentrations used. Based on these data, a mechanism for the binding of the inactive complexed form of thrombin to hepatocytes is suggested.

Increased oxidation and decreased conjugation of drugs in the liver caused by starvation. Altered metabolism of certain aromatic compounds and acetone.

Starvation causes several changes in the various processes of biotransformation. The focus of this review is on biotransformation of various aromatic and other compounds whose metabolism is catalyzed in phase I by isozymes belonging to the CYP2E1 gene subfamily, while in phase II phenol-UDPGT or conjugation with GSH play a dominant role. The other ways of conjugation are beyond the scope of this review. The reason why this aspect has been chosen is that the capacity of these reactions is profoundly altered by nutritional conditions. There is a balance between the two phases of biotransformation. Therefore, under standard circumstances in a well-fed state the intermediate formed in the course of phase I is converted to a conjugated compound rapidly, as a result of phase II. However, in starvation the pattern of drug metabolism is altered and the balance between the two phases is changed. This alteration of drug metabolism upon starvation is partly connected to the changes of cofactor supplies due to the metabolic state.

Accumulation of phenols in isolated hepatocytes after pretreatment with methylglyoxal.

The effects of a single intraperitoneal injection of methylglyoxal (50-800 mg/kg body wt.) in mice were investigated in the liver after 24 h. The administration of methylglyoxal (400 mg/kg body wt.) resulted in an increase in aniline hydroxylase activity in liver microsomes. At the same time an accumulation of p-amino-phenol, the hydroxylated product of aniline, was observed in isolated hepatocytes upon addition of aniline similarly to conditions (starvation, diabetes mellitus, pyrazole pretreatment) when aniline hydroxylase was induced. Methylglyoxal also decreased the reduced glutathione content in the liver, while the activity of serum glutamate pyruvate transaminase was increased, suggesting the onset of liver injuries. It is assumed that the increased oxidation of aniline hydroxylase combined with decreased glutathione levels after methylglyoxal treatment favours the formation of potentially hazardous phenol derivatives in the liver.