RESUMEN

Acetol and methylglyoxal are intermediates of the intrahepatic metabolism of acetone leading to pyruvate formation. In hepatocytes prepared from fasted streptozotocin-induced diabetic mice, net glucose production could be measured from methylglyoxal but not from acetone or acetol. Insulin increased glucose formation from methylglyoxal in a concentration-dependent manner, whereas it was ineffective when pyruvate was used as substrate. Drug oxidation, as evidenced by p-aminophenol formation from aniline, was enhanced by methylglyoxal, and insulin proved to be stimulatory in this case as well. It is concluded that insulin might be involved in the regulation of glucose formation from methylglyoxal, but its mode of action is not yet clear.

Asunto(s)

RESUMEN

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.

Asunto(s)

RESUMEN

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.

Asunto(s)

RESUMEN

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.

Asunto(s)

RESUMEN

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.

Asunto(s)

RESUMEN

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.

Asunto(s)

RESUMEN

1. To evaluate the condition under which net glucose production from acetone, added as sole substrate, occurs different pretreatments of mice, in combination with starvation, were used; (i) acetone pretreatment (acetone is a known inducer of cytochrome P-450 isozymes involved in this pathway), (ii) fructose pretreatment (to induce NADPH+H+ generating enzymes) or (iii) their combination. 2. There was net glucose formation from acetone only in that case, when the cells were prepared from 48 hr fasted animals pretreated with both acetone and fructose. However, using 2-14C-acetone, incorporation of 14C-carbon into glucose could be detected in all the cases and, at the same time, acetone was without any effect on protein synthesis. 3. The addition of acetone increased gluconeogenesis from alanine in almost all the cases. The only exception from this general rule was that the case, when hepatocytes were prepared from acetone pretreated 48 hr starved mice where, instead of the elevation of glucose formation, a decrease of that was caused by acetone. 4. Acetone decreased 14C-carbon incorporation into glucose from 14C-(U)-alanine added at saturating concentration in hepatocytes prepared from starved mice. 5. Similarly to acetone there was no net glucose formation from acetone either when added alone, however, it enhanced gluconeogenesis from alanine at non-saturating concentrations of the amino acid. 6. Methylglyoxal proved gluconeogenic in all the cases. 7. It is concluded that net glucose formation from acetone as sole substrate occurs only under those conditions which are far from a physiological situation, however, when gluconeogenesis from another substrate takes place, acetone can contribute to net glucose formation in hepatocytes prepared from fasted mice.

Asunto(s)

RESUMEN

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.

Asunto(s)

RESUMEN

Methylglyoxal by depleting glutathione stores increased Trypan-blue uptake by the cells incubated in glucose, pyruvate and amino acids free medium. Only a transient fall of glutathione concentration without any effect on cell viability was caused by methylglyoxal when the medium was supplemented with above-mentioned compounds. The role of gamma-glutamyl-transpeptidase is discussed.

Asunto(s)

RESUMEN

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.

Asunto(s)

RESUMEN

1. Gluconeogenesis from alanine can be prevented by the addition of monoiodo acetic acid (an inhibitor of glyceraldehyde 3-phosphate dehydrogenase), while glucose production from methylglyoxal is only partially inhibited by this compound. 2. It is supposed that methylglyoxal can enter gluconeogenic sequence not only at pyruvate.

Asunto(s)

RESUMEN

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.

Asunto(s)

RESUMEN

1. The effect of a redox cycler and arylator (menadione) and a pure arylator quinone (benzoquinone) was studied on different NADPH generating and consuming processes in isolated mouse hepatocytes. 2. Menadione inhibited gluconeogenesis from alanine but not from fructose or glycerol. 3. Drug oxidation measured as aniline hydroxylation and aminopyrine N-demethylation could be inhibited by menadione in microsomal membrane and in isolated hepatocytes both from fed or fasted animals. 4. Ureogenesis in isolated hepatocytes from fed mice could not be inhibited even by high concentration of menadione, while in cells from fasted animals menadione was inhibitory at high concentration in the presence of gluconeogenic precursor and at lower concentration in the absence of it. 5. Benzoquinone did not inhibit the above mentioned processes.

Asunto(s)

RESUMEN

1. A23187 increased the glucose production from methylglyoxal in isolated hepatocytes, and maximal stimulation was obtained at 10(-6) M. The effect of A23187 was dependent on the presence of Ca2+. 2. Glucose production from pyruvate (less than 1 mM) in isolated hepatocytes was stimulated by A23187 in the presence of 2.5 mM Ca2+ and was depressed at pyruvate concentrations above 1 mM. Both the virtual Km and the virtual Vmax of glucose production from pyruvate were decreased by A23187.

Asunto(s)

RESUMEN

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.

Asunto(s)

Asunto(s)

RESUMEN

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.

Asunto(s)

RESUMEN

Several reactions of the Ist and IInd phase of biotransformation have been reported to be under a negative cyclic-AMP dependent control. Conjugation of bilirubin and glucuronidation of morphine were investigated in isolated mouse hepatocytes and in mouse hepatocyte microsomes, respectively. N6,O2-dibutyryl cyclic-AMP did not inhibit conjugation of bilirubin in isolated hepatocytes. ATP and the dissociated catalytic subunit of the cyclic-AMP dependent protein kinase did not influence the glucuronidation of morphine, while inhibited the formation of p-nitrophenol glucuronide in microsomes prepared from isolated mouse hepatocytes.

Asunto(s)

RESUMEN

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.

Asunto(s)

RESUMEN

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.

Asunto(s)