Tamás Kardon

On the role of 4-hydroxynonenal in health and disease

Polyunsaturated fatty acids are susceptible to peroxidation and they yield various degradation products, including the main α,β-unsaturated hydroxyalkenal, 4-hydroxy-2,3-trans-nonenal (HNE) in oxidative stress. Due to its high reactivity, HNE interacts with various macromolecules of the cell, and this general toxicity clearly contributes to a wide variety of pathological conditions. In addition, growing evidence suggests a more specific function of HNE in electrophilic signaling as a second messenger of oxidative/electrophilic stress. It can induce antioxidant defense mechanisms to restrain its own production and to enhance the cellular protection against oxidative stress. Moreover, HNE-mediated signaling can largely influence the fate of the cell through modulating major cellular processes, such as autophagy, proliferation and apoptosis. This review focuses on the molecular mechanisms underlying the signaling and regulatory functions of HNE. The role of HNE in the pathophysiology of cancer, cardiovascular and neurodegenerative diseases is also discussed.

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.

Tissue transglutaminase (TG2) acting as G protein protects hepatocytes against Fas‐mediated cell death in mice

Tissue transglutaminase (TG2) is a protein cross‐linking enzyme known to be expressed by hepatocytes and to be induced during the in vivo hepatic apoptosis program. TG2 is also a G protein that mediates intracellular signaling by the alpha‐1b‐adrenergic receptor (AR) in liver cells. Fas/Fas ligand interaction plays a crucial role in various liver diseases, and administration of agonistic anti‐Fas antibodies to mice causes both disseminated endothelial cell apoptosis and fulminant hepatic failure. Here we report that an intraperitoneal dose of anti‐Fas antibodies, which is sublethal for wild‐type mice, kills all the TG2 knock‐out mice within 20 hours. Although TG2−/− thymocytes exposed to anti‐Fas antibodies die at the same rate as wild‐type mice, TG2−/− hepatocytes show increased sensitivity toward anti‐Fas treatment both in vivo and in vitro, with no change in their cell surface expression of Fas, levels of FLIPL (FLICE‐inhibitory protein), or the rate of I‐κBα degradation, but a decrease in the Bcl‐xL expression. We provide evidence that this is the consequence of the impaired AR signaling that normally regulates the levels of Bcl‐xL in the liver. In conclusion, our data suggest the involvement of adrenergic signaling pathways in the hepatic regeneration program, in which Fas ligand‐induced hepatocyte proliferation with a simultaneous inhibition of the Fas‐death pathway plays a determinant role. (HEPATOLOGY 2005.)

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.

Maintenance of luminal NADPH in the endoplasmic reticulum promotes the survival of human neutrophil granulocytes

The present study demonstrates the expression of hexose‐6‐phosphate dehydrogenase and 11β‐hydroxysteroid dehydrogenase type 1 in human neutrophils, and the presence and activity of these enzymes in the microsomal fraction of the cells. Their concerted action together with the previously described glucose‐6‐phosphate transporter is responsible for cortisone–cortisol interconversion detected in human neutrophils. Furthermore, the results suggest that luminal NADPH generation by the cortisol dehydrogenase activity of 11β‐hydroxysteroid dehydrogenase type 1 prevents neutrophil apoptosis provoked by the inhibition of the glucose‐6‐phosphate transporter. In conclusion, the maintenance of the luminal NADPH pool is an important antiapoptotic factor in neutrophil granulocytes.

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.

Immunodetection of the expression of microsomal proteins encoded by the glucose 6-phosphate transporter gene.

Glucose 6-phosphate transport has been well characterized in liver microsomes. The transport is required for the functioning of the glucose-6-phosphatase enzyme that is situated in the lumen of the hepatic endoplasmic reticulum. The genetic deficiency of the glucose 6-phosphate transport activity causes a severe metabolic disease termed type 1b glycogen storage disease. The cDNA encoding a liver transporter for glucose 6-phosphate was cloned and was found to be mutated in patients suffering from glycogen storage disease 1b. While related mRNAs have been described in liver and other tissues, the encoded protein(s) has not been immunologically characterized yet. In the present study, we report (using antibodies against three different peptides of the predicted amino acid sequence) that a major protein encoded by the glucose 6-phosphate transporter gene is expressed in the endoplasmic reticulum membranes of rat and human liver. The protein has an apparent molecular mass of approx. 33 kDa using SDS/PAGE, but several lines of evidence indicate that its real molecular mass is 46 kDa, as expected. The glucose 6-phosphate transporter protein was also immunodetected in kidney microsomes, but not in microsomes derived from human fibrocytes, rat spleen and lung, and a variety of cell lines. Moreover, little or no expression of the glucose 6-phosphate transporter protein was found in liver microsomes obtained from three glycogen storage disease 1b patients, even bearing mutations that do not directly interfere with protein translation, which can be explained by a (proteasome-mediated) degradation of the mutated transporter.

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.

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.