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.

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.

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.

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.

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.

Ascorbate as a substrate for glycolysis or gluconeogenesis: evidence for an interorgan ascorbate cycle.

Ascorbate catabolism was investigated in murine and human cells unable to synthesize ascorbate due to the missing gulonolactone oxidase activity. In HepG2 cells the addition of ascorbate or dehydroascorbate resulted in high glucose production, while human erythrocytes, MCF7 cells and the cellular elements of the murine blood were able to metabolize ascorbate or dehydroascorbate to lactate. The oxidative agent menadione stimulated, while the transketolase inhibitor oxythiamine inhibited, the metabolism of dehydroascorbate in each of these three cell types. Our results suggest that ascorbate breakdown through the pentose phosphate pathway can reach the glycolytic/gluconeogenic route in different cells. In ascorbate synthesizing species the ascorbate-lactate route in peripheral cells may form a catabolic branch of an interorgan ascorbate cycle, where hepatocytes are responsible for ascorbate synthesis. The catabolic part of this cycle using exogenous ascorbate could be demonstrated even in humans cells.

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.

Inhibition of glucuronidation by an acyl-CoA-mediated indirect mechanism.

The mechanism of the inhibition of glucuronidation by long-chain fatty acyl-CoAs was studied in rat liver microsomal membranes and in isolated hepatocytes. Palmitoyl- and oleoyl-CoA did not affect p-nitrophenol UDP-glucuronosyltransferase activity in native microsomes but were inhibitory in permeabilised vesicles. The extent of inhibition was dependent on the effectiveness of permeabilisation and was constant in time in fully permeabilised microsomes. Fatty acyl-CoAs mobilised calcium from calcium-loaded microsomes. Elevation of the intracellular acyl-CoA level by the addition of palmitate or oleate inhibited the glucuronidation of p-nitrophenol in isolated hepatocytes. This effect could be abolished by emptying the intracellular calcium stores. Therefore, it is concluded that fatty acyl-CoAs inhibit glucuronidation indirectly, presumably via calcium mobilisation.

Beta-glucuronidase latency in isolated murine hepatocytes.

The physiological function of microsomal beta-glucuronidase is unclear. Substrates may be either glucuronides produced in the lumen of endoplasmic reticulum (ER) or those taken up by hepatocytes. In the latter case, efficient inward transport of glucuronides at the plasma membrane and the ER membrane would be required. Therefore, the potential role of beta-glucuronidase in ER was investigated. Isolated mouse hepatocytes and mouse and rat liver microsomal vesicles were used in the experiments. Selective permeabilization of the plasma membrane of isolated hepatocytes with saponin or digitonin resulted in an almost 4-fold elevation in the rate of beta-nitrophenol glucuronide hydrolysis, while the permeabilization of plasma membrane plus ER membrane by Triton X-100 caused a further 2-fold elevation. In microsomal vesicles, the p-nitrophenol glucuronide or phenolphthalein glucuronide beta-glucuronidase activity showed about 50% latency as revealed by alamethicin or Triton X-100 treatment. A light-scattering study indicated that the microsomes are relatively impermeable to both glucuronides and to glucuronate. On the basis of our results, the role of liver microsomal beta-glucuronidase in the deconjugation of glucuronides taken up by the liver seems unlikely. Hydrolysis of the glucuronides produced in the ER lumen may play a role in substrate supply for ascorbate synthesis or in "proofreading" of glucuronidation.

Endoplasmic reticulum stress underlying the pro-apoptotic effect of epigallocatechin gallate in mouse hepatoma cells.

It has been recently reported that tea flavanols, including epigallocatechin gallate (EGCG), efficiently inhibit glucosidase II in liver microsomes. Since glucosidase II plays a central role in glycoprotein processing and quality control in the endoplasmic reticulum we investigated the possible contribution of endoplasmic reticulum stress and unfolded protein response (UPR) to the pro-apoptotic activity of EGCG in mouse hepatoma cells. The enzyme activity measurements using 4-methylumbelliferyl-alpha-d-glucopyranoside substrate confirmed the inhibition of glucosidase II in intact and alamethicin-permeabilized cells. EGCG treatment caused a progressive elevation of apoptotic activity as assessed by annexin staining. The induction of CHOP/GADD153, the cleavage of procaspase-12 and the increasing phosphorylation of eIF2alpha were revealed in these cells by Western blot analysis while the induction of endoplasmic reticulum chaperones and foldases was not observed. Time- and concentration-dependent depletion of the endoplasmic reticulum calcium stores was also demonstrated in the EGCG-treated cells by single-cell fluorescent detection. The massive alterations in the endoplasmic reticulum morphology revealed by fluorescent microscopy further supported the development of UPR. Collectively, our results indicate that EGCG interferes with protein processing in the endoplasmic reticulum presumably due to inhibition of glucosidase II and that the stress induces an incomplete unfolded protein response with dominantly pro-apoptotic components.

Enhancement of interleukin-6 production by fibrinogen degradation product D in human peripheral monocytes and perfused murine liver.

The effect of fibrinogen degradation products D and E (FDP-D, FDP-E) on IL-6 production in perfused mouse livers and peripheral monocytes is studied. Similarly to bacterial endotoxin FDP-D is highly potent to augment the IL-6 production measured in perfused mouse livers, while FDP-E is not stimulatory. FDP-D but not FDP-E is able to stimulate the in vitro IL-6 production of human peripheral monocytes, as well. Plasmin alone is almost ineffective on IL-6 production both in perfused livers and monocytes. Our findings suggest a direct positive feedback circuit, among fibrinogen, FDP and IL-6.

Ryanodine receptor channel-dependent glutathione transport in the sarcoplasmic reticulum of skeletal muscle.

We found that glutathione transport across endo/sarcoplasmic reticulum membranes correlates with the abundance of ryanodine receptor type 1 (RyR1). The transport was the fastest in muscle terminal cisternae, fast in muscle microsomes and slow in liver, heart, and brain microsomes. Glutathione influx could be inhibited by RyR1 blockers and the inhibitory effect was counteracted by RyR1 agonists. The effect of blockers was specific to glutathione, as the transport of other small molecules was not hindered. Therefore, the glutathione transport activity seems to be associated with RyR1 in sarcoplasmic reticulum.

Ascorbate oxidation is a prerequisite for its transport into rat liver microsomal vesicles.

Oxidation and uptake of ascorbate show similar time courses in rat liver microsomal vesicles: a rapid burst phase is followed by a slower process. Inhibitors of ascorbate oxidation (proadifen, econazole or quercetin) also effectively decreased the uptake of ascorbate. The results show that dehydroascorbate is the transport form of ascorbate at the membrane of the endoplasmic reticulum.

Regulation of glucuronidation by glutathione redox state through the alteration of UDP-glucose supply originating from glycogen metabolism.

The effect of altered redox state of glutathione was investigated on p-nitrophenol glucuronidation in isolated mouse hepatocytes. Decrease of GSH/GSSG ratio provoked by various agents caused increased glucuronidation which was accompanied by stimulated glycogenolysis and elevated UDP-glucose content. The stimulation of glycogenolysis and glucuronidation by glutathione consumption could be prevented by the reduction of oxidized glutathione with dithiothreitol and by the glycogenolysis inhibitor fructose. In permeabilized hepatocytes glycogen metabolism, bypassed by the addition of UDP-glucose, stimulated glucuronidation which was insensitive to glutathione depletion. In liver microsomes either UDP-glucuronosyltransferase activity or UDP-glucuronic acid transport was not influenced by GSH/GSSG ratio. These results suggest that alteration of the GSH/GSSG ratio regulates glucuronidation by affecting enzymes of the glycogen metabolism via the modification of UDP-glucuronate supply.

Evidence for an UDP-glucuronic acid/phenol glucuronide antiport in rat liver microsomal vesicles.

The transport of glucuronides synthesized in the luminal compartment of the endoplasmic reticulum by UDP-glucuronosyltransferase isoenzymes was studied in rat liver microsomal vesicles. Microsomal vesicles were loaded with p-nitrophenol glucuronide (5 mM), phenolphthalein glucuronide or UDP-glucuronic acid, by a freeze-thawing method. In was shown that: (i) the loading procedure resulted in millimolar intravesicular concentrations of the different loading compounds; (ii) addition of UDP-glucuronic acid (5 mM) to the vesicles released both intravesicular glucuronides within 1 min; (iii) glucuronides stimulated the release of UDP-glucuronic acid from UDP acid-loaded microsomal vesicles; (iv) trans-stimulation of UDP-glucuronic acid entry by loading of microsomal vesicles with p-nitrophenol glucuronide, phenolphthalein glucuronide, UDP-glucuronic acid and UDP-N-acetyl-glucosamine almost completely abolished the latency of UDP-glucuronosyltransferase, although mannose 6-phosphatase latency remained unaltered; (v) the loading compounds by themselves did not stimulate UDP-glucuronosyltransferase activity. This study indicates that glucuronides synthesized in the lumen of endoplasmic reticulum can leave by an antiport, which concurrently transports USP-glucuronic acid into the lumen of the endoplasmic reticulum.

Protein-disulfide isomerase- and protein thiol-dependent dehydroascorbate reduction and ascorbate accumulation in the lumen of the endoplasmic reticulum.

The transport and intraluminal reduction of dehydroascorbate was investigated in microsomal vesicles from various tissues. The highest rates of transport and intraluminal isotope accumulation (using radiolabeled compound and a rapid filtration technique) were found in hepatic microsomes. These microsomes contain the highest amount of protein-disulfide isomerase, which is known to have a dehydroascorbate reductase activity. The steady-state level of intraluminal isotope accumulation was more than 2-fold higher in hepatic microsomes prepared from spontaneously diabetic BioBreeding/Worcester rats and was very low in fetal hepatic microsomes although the initial rate of transport was not changed. In these microsomes, the amount of protein-disulfide isomerase was similar, but the availability of protein thiols was different and correlated with dehydroascorbate uptake. The increased isotope accumulation was accompanied by a higher rate of dehydroascorbate reduction and increased protein thiol oxidation in microsomes from diabetic animals. The results suggest that both the activity of protein-disulfide isomerase and the availability of protein thiols as reducing equivalents can play a crucial role in the accumulation of ascorbate in the lumen of the endoplasmic reticulum. These findings also support the fact that dehydroascorbate can act as an oxidant in the protein-disulfide isomerase-catalyzed protein disulfide formation.

Role of vitamin E in ascorbate-dependent protein thiol oxidation in rat liver endoplasmic reticulum.

Addition of ascorbate or its generation from gulonolactone causes the oxidation of protein thiols and a simultaneous dehydroascorbate formation in rat liver microsomes. The participation of vitamin E in the phenomenon was studied. We measured ascorbate and protein thiol oxidation and lipid peroxidation in vitamin E deficient liver microsomes. Vitamin E deficiency partly uncoupled the two processes: ascorbate oxidation increased, while protein thiol oxidation decreased. These changes were accompanied with an accelerated lipid peroxidation in the vitamin E-deficient microsomes, which indicates the accumulation of reactive oxygen species. All these effects were reduced by the in vitro addition of vitamin E to the deficient microsomes, supporting its direct role in the process. The results demonstrate that vitamin E is a component of the protein thiol oxidizing machinery in the hepatic endoplasmic reticulum transferring electrons from the thiol groups towards oxygen.

Prostaglandin-independent stimulation of interleukin-6 production by fibrinogen degradation product D in perfused murine liver.

Bacterial endotoxin (LPS) and fibrinogen degradation product D (FDP-D) are both potent stimulators of interleukin-6 (IL-6) production in liver, however, there are differences in their metabolic effects. The aim of the present study was to compare the role of prostaglandins in the enhancement of IL-6 production by LPS or FDP-D in perfused mouse livers. Indomethacin inhibited the effect of LPS significantly but was ineffective in the case of FDP-D. Accordingly, production of prostaglandins D2 and E2 was not elevated following the addition of FDP-D, while their formation was increased several fold by LPS. At the same time interleukin-1 (IL-1) production in perfused liver rose markedly upon the addition of FDP-D. It is suggested that prostaglandins are not involved in the effects of FDP-D on the liver. The stimulatory effect of FDP-P on IL-6 production might be the consequence of elevated IL-1 levels.