László Braun

Ascorbate metabolism and its regulation in animals

This article provides a comprehensive review on ascorbate metabolism in animal cells, especially in hepatocytes. The authors deal with the synthesis and the breakdown of ascorbate as a part of the antioxidant and carbohydrate metabolism. Hepatocellular and interorgan cycles with the participation of ascorbate are proposed, based on experiments with murine and human cells; reactions of hexuronic acid pathway, non-oxidative branch of the pentose phosphate cycle, glycolysis and gluconeogenesis are involved. Besides the well-known redox coupling between the two major water-soluble antioxidants (glutathione and ascorbate), their metabolic links have been also outlined. Glycogenolysis as a major source of UDP-glucuronic acid determines the rate of hexuronic acid pathway leading to ascorbate synthesis. Glycogenolysis is regulated by oxidized and reduced glutathione; therefore, glycogen, ascorbate and glutathione metabolism are related to each other. Hydrogen peroxide formation, due to the activity of gulonolactone oxidase catalyzing the last step of ascorbate synthesis, also affects the antioxidant status in hepatocytes. Based on new observations a complex metabolic regulation is supposed. Its element might be present also in humans who lost gulonolactone oxidase but they need and metabolize ascorbate. Finally, the obvious disadvantages and the possible advantages of the lost ascorbate synthesizing ability in humans are considered.

GULONOLACTONE OXIDASE ACTIVITY-DEPENDENT INTRAVESICULAR GLUTATHIONE OXIDATION IN RAT LIVER MICROSOMES

The orientation of gulonolactone oxidase activity was investigated in rat liver microsomes. Ascorbate formation upon gulonolactone addition resulted in higher intravesicular than extravesicular ascorbate concentrations in native microsomal vesicles. The intraluminal ascorbate accumulation could be prevented or the accumulated ascorbate could be released by permeabilising the vesicles with the pore‐forming alamethicin. The formation of the other product of the enzyme, hydrogen peroxide caused the preferential oxidation of intraluminal glutathione in glutathione‐loaded microsomes. In conclusion, these results suggest that the orientation of the active site of gulonolactone oxidase is intraluminal and/or the enzyme releases its products towards the lumen of the endoplasmic reticulum.

Ascorbate‐mediated electron transfer in protein thiol oxidation in the endoplasmic reticulum

Addition of, or gulonolactone oxidase‐dependent in situ generation of, ascorbate provoked the oxidation of protein thiols, which was accompanied by ascorbate consumption in liver microsomal vesicles. The maximal rate of protein thiol oxidation was similar upon gulonolactone, ascorbate or dehydroascorbate addition. Cytochrome P450 inhibitors (econazole, proadifen, quercetin) decreased ascorbate consumption and the gulonolactone or ascorbate‐stimulated thiol oxidation. The results demonstrate that the ascorbate/dehydroascorbate redox couple plays an important role in electron transfer from protein thiols to oxygen in the hepatic endoplasmic reticulum, even in gulonolactone oxidase deficient species.

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.

The regulation of the induction of vascular endothelial growth factor at the onset of diabetes in spontaneously diabetic rats

Early induction of VEGF was studied in liver, kidney and lung of spontaneously diabetic rats. Western blot analysis, northern hybridization were applied to show the expression of VEGF in different organs. Radiolabelled hypoxia responsive element (HRE) and cAMP responsive element (CRE) oligonucleotides were assayed by electrophoretic mobility shift (EMSA) or supershift using anti ARNT and anti CREB-1 monoclonal antibodies. An increase in VEGF expression at the level of protein and mRNA was demonstrated at the beginning of the disease. EMSAs showed: a.) a binding of HIF-1 to HRE and/or CRE, b.) in the same time the binding of CREB- I was detected to both HRE and/or CRE sequences in the liver, kidney and lung of diabetic animals. Based on these in vivo observations it is supposed that HRE and CRE through the interaction between HIF-1 and CREB-1 are equally involved in the regulation of VEGF expression at the onset of diabetes.

Transcriptional induction of bilirubin UDP-glucuronosyltransrase by ethanol in rat liver

Several drug-metabolizing enzymes including bilirubin UDP-glucuronosyltransferase (UGT1A1) are influenced by long-term ethanol consumption. In the present study, the activity and expression of UGT1A1 were investigated in livers of ethanol-treated rats. Animals were treated daily for 15 days with ethanol or isocaloric amount of glucose solution by gastric intubation. Microsomes and total RNA were prepared from the liver of rats and analyzed by Western blot and Northern hybridization using UGT1A1 specific antibody and cDNA probe. Microsomal bilirubin UGT activity was also measured. The elevation of UGT1A1 mRNA was observed in the liver of ethanol consumer animals with the simultaneous increase in microsomal UGT1A1 protein leading to stimulated bilirubin glucuronidation both in vivo and in microsomal vesicles. These results arise the possibility of the transcriptional induction and/or the mRNA stabilization by ethanol consumption, which can be caused by ethanol itself or the metabolic changes due to the treatment.

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.

Induction and peroxisomal appearance of gulonolactone oxidase upon clofibrate treatment in mouse liver.

Various antihyperlipemic peroxisome proliferators are known to be carcinogenic in rodents but not in human, other primates and guinea pig, which species lost their ability to synthesize ascorbate due to mutations in the gulonolactone oxidase gene. Ascorbate synthesis is accompanied by H2O2 production, consequently its induction can be potentially harmful; therefore, the in vivo effect of the peroxisome proliferator clofibrate was investigated on gulonolactone oxidase expression in mouse liver. Liver weights and peroxisomal protein contents were increased upon clofibrate treatment. Elevated plasma ascorbate concentrations were found in clofibrate‐treated mice due to the higher microsomal gulonolactone oxidase activities. Remarkable gulonolactone oxidase activity appeared in the peroxisomal fraction upon the treatment. Increased activity of the enzyme was associated with an elevation of its mRNA level. According to the present results the evolutionary loss of gulonolactone oxidase may contribute to the explanation of the missing carcinogenic effect of peroxisome proliferators in humans.

Methylglyoxal and cell viability.

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.

Different induction of gulonolactone oxidase in aromatic hydrocarbon- responsive or -unresponsive mouse strains

The role of aromatic hydrocarbon receptor (AhR)‐mediated signal transduction pathways was investigated in the regulation of ascorbate synthesis by using Ah‐responsive and Ah‐unresponsive mouse strains. In vivo 3‐methylcholanthrene treatment increased hepatic and plasma ascorbate concentrations only in the Ah‐responsive strain. The mRNA level of gulonolactone oxidase and the microsomal ascorbate production from p‐nitrophenyl glucuronide, D‐glucuronic acid or gulonolactone in the liver of Ah‐responsive and Ah‐unresponsive mice were compared. In Ah‐responsive mice, these parameters were higher originally, and they further increased upon in vivo addition of 3‐methylcholanthrene, while in Ah‐unresponsive mice the treatment was not effective. These results suggest that the transcription of gulonolactone oxidase gene is regulated by an Ah receptor‐dependent signal transduction pathway.