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Reduced AMP kinase (AMPK) activity has been shown to play a key deleterious role in increased hepatic gluconeogenesis in diabetes, but the mechanism whereby this occurs remains unclear. In this article, we document that another AMP-dependent enzyme, AMP deaminase (AMPD) is activated in the liver of diabetic mice, which parallels with a significant reduction in AMPK activity and a significant increase in intracellular glucose accumulation in human HepG2 cells. AMPD activation is induced by a reduction in intracellular phosphate levels, which is characteristic of insulin resistance and diabetic states. Increased gluconeogenesis is mediated by reduced TORC2 phosphorylation at Ser171 by AMPK in these cells, as well as by the up-regulation of the rate-limiting enzymes PEPCK and G6Pc. The mechanism whereby AMPD controls AMPK activation depends on the production of a specific AMP downstream metabolite through AMPD, uric acid. In this regard, humans have higher uric acid levels than most mammals due to a mutation in uricase, the enzyme involved in uric acid degradation in most mammals, that developed during a period of famine in Europe 1.5 × 10(7) yr ago. Here, working with resurrected ancestral uricases obtained from early hominids, we show that their expression on HepG2 cells is enough to blunt gluconeogenesis in parallel with an up-regulation of AMPK activity. These studies identify a key role AMPD and uric acid in mediating hepatic gluconeogenesis in the diabetic state, via a mechanism involving AMPK down-regulation and overexpression of PEPCK and G6Pc. The uricase mutation in the Miocene likely provided a survival advantage to help maintain glucose levels under conditions of near starvation, but today likely has a role in the pathogenesis of diabetes., (© FASEB.)

We investigated the mechanism of action of uricase, which oxidizes uric acid to allantoin, in the rat. Allantoin may decompose chemically to urea and hydantoin, containing the carbons in positions 2 and 8 of the purine ring, respectively. These carbons are derived by formylation, catalyzed by formyltransferase, in two reactions of de novo synthesis. Since uric acid and allantoin are represented in equivalent amounts in the liver, we expected to find identical incorporation of radioactivity in C(2) and C(8) of both compounds after administration of (14)C-formate. In the case of (14)C-allantoin, this was true, but not for (14)C-uric acid extracted from rat liver. We interpret these results through a series of experiments and considerations.

Background: A diversity of endogenous and exogenous compounds have been demonstrated to cause proliferation of peroxisomes and variety of associated effects in rodents. The aim of our study was to see the effect of clofibrate on the enzymes associated with oxidative stress., Methods: Male Wistar rats weighting 250-350 g were treated with clofibrate in a dose of 250 mg/1000 g/ 24 h for 12 days. Whole liver homogenates and subsequent subcellular fractions wrere proceeded for enzyme measurements by spectrophotometric methods., Results: The activity of hydrogen producing enzymes D-aminoacid oxidase, Urate oxidase and Palmitoyl CoA oxidase was statistically significantly increased in the treated group in comparison with the control one. On the contrary to the massive increase of the Palmitoyl CoA oxidase--a marker enzyme for peroxisomal proliferation, there was only a limited increase of catalase (which inactivates hydrogen peroxide) activity. On the other hand superoxide dismutase and glutathione peroxidase activities in experimental group were down regulated in the treated group., Conclusion: Our results support the hypothesis that clofibrate (peroxisomal proliferators) application might produce oxidative stress in rat livers (Tab. 2, Fig. 8, Ref. 56) Full Text (Free, PDF) www.bmj.sk.

Dehydroepiandrosterone (DHEA) is an intermediate product in the synthesis of male and female sex hormones in the adrenal cortex of man. In livers of rats and mice DHEA increases the levels of cytochrome P450 IVA and peroxisomal beta-oxidation enzymes associated with peroxisome proliferation. Prolonged treatment of rats with DHEA induces liver tumors that are more frequent in females arising mainly in the periportal regions of the liver lobule (Metzger et al., Toxicol. Pathol. 23, 591-605, 1995). Because of paucity of information on hepatic zonation of peroxisomal response to DHEA and controversial reports on gender-specific differences of its effects the present study was undertaken using qualitative immunohistochemical and quantitative immunoelectron microscopical techniques in addition to Western blotting. Rats were treated for 24 weeks with 0.6% DHEA supplied with diet. Immunoblot analysis revealed marked induction of peroxisomal beta-oxidation enzymes, which by quantitative analysis was equally strong in male and female animals, whilst catalase and urate-oxidase were not increased. Cytochrome P450 IVA, in contrast, was induced significantly stronger in male than in female rats. Immunohistochemistry confirmed the induction of cytochrome P450 IVA showing a marked lobular gradient in female animals with strong induction in pericentral and almost no induction in periportal regions of the liver lobule. In male animals cytochrome P450 IVA was expressed more uniformly across the liver lobule. A similar sex specific zone-dependent response was observed for peroxisomes. DHEA induced in females a significant zonal gradient with marked peroxisome proliferation and a strong induction of peroxisomal hydratase/dehydrogenase in pericentral hepatocytes and a much smaller response in periportal regions. Livers of male animals, in contrast, showed a uniform peroxisomal proliferation to DHEA with only slight zonal differences. The striking homologies of the induction patterns of cytochrome P450 IVA and the peroxisome proliferation in both sexes support the notion of a functional relationship. In view of the almost exclusive periportal localization of DHEA-induced tumors in female rats in contrast to the pericentral localization of the peroxisomal proliferation shown by this study, it seems likely that other factors in addition to peroxisome proliferation may contribute to the hepatocarcinogenic effect of DHEA.

Fenofibrate, the hypolipidemic drug and peroxisome proliferator, was given to mice (0.23% w/w in the diet) during 1-3 weeks and enzyme activities, H2O2 concentration, and H2O2 production rate were determined. A maximal increase of 150% in liver/body weight ratio was observed after 3 weeks of treatment. Acyl-CoA oxidase, catalase and uricase activities were increased by 712%, 506% and 41% respectively by treatment with fenofibrate. Se- and non Se-glutathione peroxidase and Mn-superoxide dismutase activities were increased by 331%, 188% and 130% respectively in the liver of 2 weeks-treated mice. Cu-Zn superoxide dismutase activity was not affected by fenofibrate treatment. H2O2 steady-state concentration showed an increase of 89% after 2 weeks of treatment. H2O2 production rates, and the steady-state concentrations of the intermediates HO, R and ROO, calculated using experimental data, were higher in the liver of fenofibrate-treated mice than in control animals. According to our findings, the imbalance between H2O2 production and its degradation by its metabolizing enzymes during peroxisome proliferation, would result in an increased level of H2O2 steady-state concentration, with the resulting oxidative stress which may lead to the generation of oxidative damage and to the induction of liver carcinogenesis.

Rat renal proximal tubule cell membranes have been reported to contain uricase-like proteins that function as electrogenic urate transporters. Although uricase, per se, has only been detected within peroxisomes in rat liver (where it functions as an oxidative enzyme) this protein has been shown to function as a urate transport protein when inserted into liposomes. Since both the uricase-like renal protein and hepatic uricase can transport urate, reconstitution studies were performed to further characterize the mechanism by which uricase may function as a transport protein. Ion channel activity was evaluated in planar lipid bilayers before and after fusion of uricase-containing proteoliposomes. In the presence of symmetrical solutions of urate and KCl, but absence of uricase, no current was generated when the voltage was ramped between +/- 100 mV. Following fusion of uricase with the bilayer, single channel activity was evident: the reconstituted channel rectified with a mean slope conductance of 8 pS, displayed voltage sensitivity, and demonstrated a marked selectivity for urate relative to K+ and Cl-. The channel was more selective to oxonate, an inhibitor of both enzymatic uricase activity and urate transport, than urate and it was equally selective to urate and pyrazinoate, an inhibitor of urate transport. With time, pyrazinoate blocked both its own movement and the movement of urate through the channel. Channel activity was also blocked by the IgG fraction of a polyclonal antibody to affinity purified pig liver uricase. These studies demonstrate that a highly selective, voltage dependent organic anion channel is formed when a purified preparation of uricase is reconstituted in lipid bilayers.

Rat liver urate oxidase synthesized from cDNA through coupled transcription and translation was incubated at 26 degrees C for 60 min with purified peroxisomes from rat liver. Urate oxidase was efficiently imported into the peroxisomes, as determined by resistance to externally added proteinase K. The amount of imported urate oxidase increased with time and the import was temperature dependent. A synthetic peptide composed of the C-terminal 10 amino acid residues of acyl-CoA oxidase (the C-terminal tripeptide is Ser-Lys-Leu) inhibited the import of urate oxidase, whereas other peptides, in which the C-terminal Ser-Lys-Leu (SKL) sequence was deleted or mutated, were not effective. Two mutant urate oxidase proteins in which the C-terminal Ser-Arg-Leu (SRL) sequence was deleted or mutated to Ser-Glu-Leu (SEL) were not imported into peroxisomes. With substitution of a lysine residue for arginine in the SRL tripeptide at the C-terminus the import activity was retained. These results show that urate oxidase is important into peroxisomes via a common pathway with acyl-CoA oxidase, and that the C-terminal SRL sequence functions as a peroxisomal-targeting signal.

Because of controversial data in the literature, we studied the localization of uric acid oxidase (UAOX) activity in rat liver by light microscopy (LM) and electron microscopy (EM). UAOX is partially inactivated by aldehyde fixation and therefore we developed a technique that permits the use of unfixed cryostat sections for both LM and EM studies. Sections of rat liver were mounted on a semipermeable membrane stretched over a gelled incubation medium containing urate as specific substrate for UAOX and cerium ions to capture H2O2 produced by oxidase activity. The specificity of the reaction was checked by comparing incubations in the presence of substrate with incubations either in the absence of substrate or in the presence of substrate and 2,6,8-trichloropurine, a competitive inhibitor of UAOX. After incubation the sections were either fixed immediately for EM or visualized for LM with a second-step incubation. At the LM level, final reaction product was found in a granular form, homogeneously distributed throughout the hepatocytes. EM revealed excellent subcellular morphology and electron-dense reaction product in both the core and the matrix of peroxisomes, but not in other organelles or the cytoplasmic matrix. After incubations without substrate or with substrate and inhibitor, hardly any reaction product was found. We conclude that, because of the use of unfixed tissue, UAOX is not inactivated, which results in localization of UAOX activity not only in the core of peroxisomes but also in the peroxisomal matrix.

In order to investigate the permeability properties of rat-liver peroxisomes in situ, we selectively permeabilized hepatocytes with digitonin in a medium mimicking the cytosol. This system permitted us to study the latency of peroxisomal oxidases by means of measurement of their activities in permeabilized compared to disrupted hepatocytes. The activity of peroxisomal oxidases was studied using three different methods: (1) measurement of the oxidase-mediated production of H2O2 in a system containing homovanillic acid, horseradish peroxidase and azide; (2) measurement of the rate of substrate utilization or product formation; (3) measurement of the production of H2O2 via the peroxidative action of catalase in the presence of an excess of methanol. The results obtained depended on which system was used to measure the activity of the different oxidases. Our observations lead us to conclude that method 1 cannot be used for latency studies, whereas methods 2 and 3 are suitable under defined circumstances. Based on the results of methods 2 and 3, we conclude that urate oxidase, L-alpha-hydroxyacid oxidase A and D-amino acid oxidase show no structure-linked latency in digitonin-permeabilized hepatocytes, suggesting that the substrates for these enzymes permeate freely through the peroxisomal membrane.

The alterations of hepatic peroxisomes and their enzymes during aging were investigated in male rats. Peroxisomes in the livers of young (2 months) and old (39 months) male Wistar rats were analyzed by morphometry and quantitative immunocytochemistry, as well as by immunoblotting of highly purified peroxisomal fractions. Immunoblots showed that catalase and acyl-CoA oxidase were decreased in peroxisomes of old animals but the trifunctional enzyme, thiolase, and urate oxidase were increased. The morphometrical analysis revealed a heterogeneous distribution of peroxisomes in the liver lobule of the old animals, with a significant elevation of peroxisomal volume density in pericentral over periportal hepatocytes, in contrast to the uniform pattern in the young rats. Furthermore, age-related lobular gradients were also observed by quantitative immunocytochemistry in the peroxisomal concentrations of trifunctional enzyme (central > portal) and, inversely, for catalase (portal > central). Whereas acyl-CoA oxidase was diminished across the liver lobule, the enzyme 3-ketoacyl-CoA thiolase was elevated. These observations show that peroxisomes are significantly altered in aged animals and suggest that these alterations may contribute to the disturbance of lipid metabolism in aged animals. Moreover, the diminution in catalase and the elevation of urate oxidase could contribute to the oxidative stress which is considered to be of fundamental importance in the aging process.

The uricosuric drug benzbromarone (3,5-dibromo-4-hydroxyphenyl)-1-(2-ethyl-3-benzofuranyl)methanone, a benzofurane derivative, was studied for its effects on parameters related to hepatic peroxisome proliferation. Groups of male F-344 rats were fed either basal diet, the peroxisome proliferator clofibrate at 5000 ppm as a comparison compound, or benzbromarone at two doses, 1000 and 2000 ppm. Benzbromarone and clofibrate produced hepatomegaly and increases in the activities of catalase, acyl CoA oxidase, malate dehydrogenase, and glycerol-3-phosphate dehydrogenase. Benzbromarone and clofibrate also both induced similar histologic and ultrastructural changes in hepatocytes, including induction of peroxisomes. Therefore, benzbromarone acted as a peroxisome-proliferating agent in rats under these conditions. Benzbromarone differs from other peroxisome proliferators in its chemical structure, uricosuric action, and the morphology of liver peroxisomes that were induced by exposure.

Tissue distribution of uricase (urate oxidase, EC 1.7.3.3) was studied by immunoblotting and RNA slot blot analysis. For immunoblotting, highly specific monoclonal antibodies against rat liver uricase were obtained, and for mRNA detection, a cloned uricase cDNA was used. Among seven tissues studied, uricase was immunologically detected only in the liver. The contents of uricase in other tissues, i.e., brain, thymus, heart, spleen, kidney and lactating mammary gland, were estimated to be less than 2% of that in the liver. Uricase mRNA was also detected only in the liver. The steady-state level of the mRNA in the isolated hepatocytes was relatively constant during the 8-day culture period when compared with those of other mRNAs expressed in the liver, suggesting a unique control mechanism of its expression.

A circannual study of tyrosine aminotransferase and other metabolic enzymes in frog liver is reported. The subcellular distribution of all enzymatic activities under investigation was also studied. Results show significant oscillations of all enzymatic activities throughout the year; in particular tyrosine aminotransferase has a marked summer maximum. The subcellular distribution of tyrosine aminotransferase shows significant variations: the soluble activity of the enzyme presents a bimodal circannual distribution, which has its counterpart in an increased activity of heavier fractions.

The compartmentalization of catalase, fatty acyl-CoA oxidase and urate oxidase was examined in the livers of mice, rats and guinea pigs, using the technique of digitonin extraction in order to avoid the trauma associated with centrifugation procedures. The results are interpreted as indicating that an appreciable proportion of catalase activity occurs in the cytoplasmic compartment of these cells. Following treatment of the animals with clofibrate, the specific activity in both peroxisomal and cytoplasmic compartments was increased, with a higher proportion of cytoplasmic catalase being evident in mice. The results for catalase were compared with those for fatty acyl-CoA oxidase and urate oxidase both of which were indicated as showing a closer association with the peroxisomal compartment than was the case for catalase. These data have been discussed in relation to their significance on present understanding of peroxisomal structure and function.

The uptake rate of uric acid into liver slices of the Dalmatian dog was found to be significantly lower compared with that in the beagle. Since there was no difference in the hepatic uricase activity between the two breeds, the abnormality of uric acid metabolism in the Dalmatian dog is considered to result from a defective hepatic transport system for uric acid.

The processes associated with the biogenesis of peroxisomes in mouse liver have been studied by following the incorporation of radiolabelled leucine into major enzymic components of this organelle. Maximal incorporation of label into peroxisomal catalase and urate oxidase occurred within 2 hr, with the urate oxidase being labelled before catalase, but subsequent to the incorporation of phospholipid into this organelle. Subsequently, immunoprecipitation of catalase from the large granular fraction of mouse liver was shown to result in the isolation of a catalase molecule which had lost a peptide of approx. 2000 dalton from each subunit by comparison with the newly-synthesized enzyme. It was observed that the modification of catalase was obviated by the presence of leupeptin and iodoacetamide and this information has enabled the purification of both modified and unmodified forms of the enzyme. The possible significance of these data has been discussed and the major features incorporated into a working model of peroxisomal biogenesis.

In the rat brown fat peroxisomes - thermogenetic organules - an peroxisomal enzyme activities undergo remarkable changes during the adaptation to cold of the animals (see 3). In this paper was show that changes of peroxisomal enzyme activities occur also in liver and kidney during cold-adaptation. Catalase, L-hydroxyacid oxidase, uricase and D-aminoacid oxidase (DAO) were assayed as in (6). During cold-adaptation, the activity of the former three enzymes (Table 2) increases with the weight of the organs (Table 1) whereas that of DAO exhibits a much larger increase (Table 3). Results are discussed with regard to the contribution of the liver to non-shivering thermogenesis.

Carp liver was fractionated by differential and density gradient centrifugation and assayed for enzymes of purine catabolism. While urate oxidase is an exclusively peroxisomal enzyme, only a very small percentage of the enzymes xanthine oxidase, allantoinase and allantoicase is associated with subcellular or ganelle fractions. There is no general purine catabolizing subcellular compartment. There is some but not yet conclusive evidence for the assumption that urate oxidase is a membrane bound enzyme.

We examined the effects of chronic dietary iron overload on hepatic mitochondrial oxidative metabolism. Experimental iron overload was produced by feeding rats a chow diet supplemented with carbonyl iron over a 7-week period. Biochemical and histologic evaluations of liver tissue confirmed moderate degrees of hepatic parenchymal iron overload. Electron microscopy showed no abnormalities in hepatic mitochondrial ultrastructure in blocks of tissue or in mitochondrial fractions from iron-loaded liver. Studies of mitochondrial oxidative metabolism revealed a consistent and progressive decrease in state 3 (ADP-stimulated) respiration and in respiratory control ratios at hepatic iron concentrations above 1,000 micrograms per gm for all three substrates studied, glutamate, beta-hydroxybutyrate and succinate. Changes in state 4 (ADP-limited) respiration and ADP/O ratios were not progressive with increasing hepatic iron concentrations. At hepatic iron concentrations at which there were decreases in state 3 respiration and respiratory control ratios, there was also evidence of lipid-conjugated diene formation, indicative of mitochondrial lipid peroxidation. There were no changes in mitochondrial function when iron as either ferritin or hemosiderin or as a combination of ferritin, hemosiderin and ferric nitrilotriacetate was added in vitro to normal liver homogenates. Use of density gradient centrifugation to reduce iron and lysosomal contamination of mitochondrial fractions failed to prevent the reduction in mitochondrial function. We conclude that moderate degrees of chronic hepatic iron overload in vivo result in an inhibitory defect in the mitochondrial electron transport chain as evidenced by a decrease in state 3 respiration and respiratory control ratios.

1. Liver catalase, D-amino acid oxidase, urate oxidase of Alytes obstetricans and Xenopus laevis (anuran amphibians) and fatty acyl-CoA oxidase of Alytes were present at all post-embryonic stages. 2. Catalase and D-amino acid oxidase activities increased during spontaneous metamorphosis of the two species. 3. During triiodothyronine-induced metamorphosis of Alytes larvae, catalase and D-amino acid oxidase activities increased after a latent period. 4. Our results suggest that expression of some hepatic peroxisomal enzymes is modulated by thyroid hormones.

Poly(ethylene glycol) of 5 000 daltons has been attached covalently to preparations of urate oxidase (urate: oxygen oxidoreductase, EC 1.7.3.3) from hog liver and Candida utilis. Attachment of sufficient poly(ethylene glycol) to either urate oxidase renders the enzyme incapable of eliciting antibody production in mice, or of reacting with antibodies to the unmodified enzyme. The poly(ethylene glycol) : urate oxidase conjugates exhibit higher Km and lower V values than the unmodified urate oxidases. Optimal pH values are increased for the poly(ethylene glycol) : urate oxidases, and optimal temperatures are decreased. The blood circulating lives of the modified urate oxidases following intravenous injection are much longer than those of the unmodified urate oxidases: repetitive injections over a period of 90 days dd not alter the blood circulating lives of the poly(ethylene glycol) : urate oxidases. The unmodified enzymes, on the other hand, were cleared from the blood with extreme rapidity after a few intravenous injections.

In the livers of fasted rats, the activity of peroxisomal palmitocyl-CoA oxidation (NADH production) was increased more rapidly and markedly than that of mitochondrial carnitine palmitoyltransferase, which is the rate limiting enzyme of mitochondrial beta-oxidation. The peroxisomal oxidizing activity was about twice that of the control throughout the period of fasting (1-7 days). carnitine acetyltransferase activity was increased to a similar extent in both peroxisomes and mitochondria. A possible physiological role of liver peroxisomes may thus be as an effective supply of NADH2, acetyl residues and short and medium-length fatty acyl-CoA in the cells on the enhancement of peroxisomal beta-oxidation of the animals under starvation; these substances thus produced may be transported into the mitochondria as energy sources.

Subcellular organellles from livers of rats three days prenatal to 50 weeks postnatal were separated on sucrose gradients. The peroxisomes had a constant density of 1.243 g/ml throughout the life of the animal. The density of the mitochondria changed from about 1.236 g/ml at birth to a constant value of 1.200 g/ml after two weeks. The peroxisomal and mitochondrial fatty acid beta-oxidation and the peroxisomal and supernatant activities of catalase and glycerol-3-phosphate dehydrogenase were measured at each age, as well as the peroxisomal core enzyme, urate oxidase, and the mitochondrial matrix enzyme, glutamate dehydrogenase. All of these activities were very low or undetectable before birth. Mitochondrial glutamate dehydrogenase and peroxisomal urate oxidase reached maximal activities per g of liver at two and five weeks of age, respectively. Fatty acid beta-oxidation in both peroxisomes and mitochondria and peroxisomal glycerol-3-phosphate dehydrogenase exhibited maximum activities per g of liver between one and two weeks of age before weaning and then decreased to steady state levels in the adult. Peroxisomal beta-oxidation accounted for at least 10% of the total beta-oxidation activity in the young rat liver, but became 30% of the total in the liver of the adult female and 20% in the adult male due to a decrease in mitochondrial beta-oxidation after two weeks of age. The greatest change in beta-oxidation was in the mitochondrial fraction rather than in the peroxisomes. At two weeks of age, four times as much beta-oxidation activity was in the mitochondria as in the peroxisomal fraction. Peroxisomal glycerol-3-phosphate dehydrogenase activity accounted for 5% to 7% of the total activity in animals younger than one week, but only 1% to 2% in animals older than one week. Up to three weeks of age, 85% to 90% of the liver catalase was recovered in the peroxisomes. The activity of peroxisomal catalase per g of rat liver remained constant after three weeks of age, but the total activity of catalase further increased 2.5- to 3-fold, and all of the increased activity was in the supernatant fraction.

The effect of the plasticizer di(2-ethylhexyl)phthalate on the intracellular membranes of hepatocytes was investigated. Supplementation of the diet with 2% plasticizer resulted in the appearance of a large number of peroxisomes, and the number of mitochondria was also greatly increased. No significant change in the amount or appearance of the endoplasmic reticulum was detected. The oxidation of palmitoyl-CoA in peroxisomes and the activities of carnitine-acyltransferases are increased to a great extent in both mitochondria and peroxisomes. Intact respiratory control and oxidative phosphorylation indicated that mitochondrial integrity was maintained during the induction. In microsomes, cytochrome P-450 and NADPH-cytochrome c reductase are elevated. The increased incorporation of glycerol into phospholipids indicated an increased rate of synthesis. The induction of peroxisomal and mitochondrial membranes and enzymes, but not of the membranes of the endoplasmic reticulum, by phthalate esters is an unusual and valuable induction pattern not seen with other inducers.

The properties of H2O2 production in the "haemoglobin-free", "non-circulatory" perfused liver of rats were examined. The H2O2 production with 1 mM-lactate and 0.15 mM-pyruvate was 82nmol/min per g of liver or 333nmol/min per 100g body wt. in the liver of fed rats at 30 degrees C. This rate decreased to almost half in the livers of starved and phenobarbital-pretreated rats. When H2O2 production was stimulated by urate infusion, almost all of the H2O2 produced by the uricase reaction was decomposed by the catalase reaction. During the demethylation reaction of aminopyrine, no change in H2O2 production was detected by the present method; thus microsomal H2O2 production observed in isolated subcellular fractions appeared not to contribute significantly to the H2O2 production in the whole organ. Whereas the rate of the glycolate-dependent H2O2 production was halved at an intracellular O2 concentration that caused a 10 percent increase in the reduction state of cytochrome c, the half-maximal rate of H2O2 production with lactate and pyruvate was observed at an O2 concentration that caused a 40 percent increase in the reduction state of cytochrome c in the liver. No further increase in the rates of H2O2 production was obtained by increasing O2 pressure up to 5 times 10(5) Pa. The rate of ethanol oxidation through the catalase "peroxidatic" reaction varied, depending on the substrate availability. The maximal capability of this pathway in ethanol oxidation reached approx. 1.5 mumol/min per g of liver, when a mixture of urate, glycollate and octanoate was infused to enhance H2O2 production.

Effects of vitamin E deficiency and its restoration on biochemical characteristics of hepatic peroxisomes were studied. Rats were maintained on the vitamin E-deficient diet for 25 weeks and then on a diet supplemented with vitamin E for 5 weeks. Blood hemolysis by hydrogen peroxide and lipid peroxidation in the liver increased markedly in vitamin E-deficient rats. The former returned to the control level after the resupplying of vitamin E, but the latter did not. Of liver peroxisomal enzymes, the activities of catalase, D-amino-acid oxidase and urate oxidase decreased in vitamin E-deficient rats. On the other hand, activities of fatty acyl-CoA oxidase and carnitine acetyltransferase increased significantly in vitamin E-deficient rats. All activities of these peroxisomal enzymes were restored to the control levels in vitamin E-supplemented rats. The activities of the mitochondrial, lysosomal and microsomal enzymes tested showed no apparent change except that the change of mitochondrial palmitoyltransferase was shown to be similar to that of peroxisomal fatty acid oxidation. These results were also supported by cell fractionation techniques. Following the methods of aqueous polymer two-phase systems, the characteristics of peroxisomal surface membranes altered in respect of their hydrophobicity, but not in respect of the surface charge of peroxisomal membranes. These results indicate that peroxisomal functions, especially those of the fatty acid oxidation system, change their activities more sensitively than other intracellular organelles in response to the condition of vitamin E deficiency.

1. The present study has confirmed that the hypolipidemic and carcinogenic agent clofibrate induces a marked increase in the specific activity of some peroxisomal marker enzymes in rat liver homogenates, notably of the palmitoyl-CoA dependent dehydrogenase and catalase activities. 2. Clofibrate was found to induce a marked polydispersity of the peroxisomes as determined by analytical differential centrifugation of homogenates and morphometric analysis of hepatocytes. 3. Two major populations of peroxisomes were detected by analytical differential centrifugation under conditions which reduce the hydrostatic pressure effects on the organelle to a minimum. Using urate oxidase as the marker enzyme, the S4,B-values of the two populations were estimated to 1 1 860 S and 4240 S, both different from that of the homogenous population of peroxisomes in the control animals (S4,B approximately equal to 6680 S). The 4240 S-population induced by clofibrate revealed a high specific activity relative to that of of urate oxidase and particularly relative to that of catalase, which was very low. In addition, a less distinct population of particles (870 S lees than S lees than 4240 S) contained more than 50% of the total particle-bound palmitoyl-CoA dependent dehydrogenase activity sedimented at a centrifugal effect of t integral of 0 rmp(2)dt = 1.5 x 10(10) min(-1), but not urate oxidase and catalase activities. This fraction was not observed in the homogenates of normal rats. As in the normal controls, the palmitoyl-CoA dependent dehydrogenase activity was found to be particle-bound (S greater than 870 S). 4. Morphometric analyses of randomly selected hepatocytes revealed that after clofibrate treatment the relative volume fraction of the peroxisomes increased by a factor of 5.5 and thier average diameter and volume by a factor of 1.3 and 2.1, respectively. Furthermore, the frequency of electron-dense matrix cores decreased on clofibrate treatment. In contrast, no change was observed in the average size of the mitochondria, and their relative volume fraction increased only by a factor of 1.2. 5. The clofibrate induced changes in eh morphological and biochemical properties of rat liver peroxisomes appears to be a very useful model system in which to study the biogenesis as well as the biochemical and physiological role(s) of this organelle in mammalian cells.

Urate oxidase and catalase were purified from rat liver peroxisomes, and respective antibodies were prepared from rabbits by the administration of these enzymes. Although urate oxidase generally precipitates in immunoprecipitation-possible pH ranges (pH 4.5--9.5), the enzyme remained soluble in 50 mM glycine buffer (pH 9.5) containing 50% glycerol up to concentration of 0.3 mg/ml. Anti-urate oxidase reacted with purified urate oxidase as well as with the crude preparation. After [3H]leucine was injected to rats, urate oxidase and catalase were purified from rat liver at certain intervals, and further precipitated by respective antibodies. The half-life of the catalase was 39 h and that of urate oxidase, 20 h. When the sonicated light mitochondrial fraction was incubated at 37 degrees C and at pH 7.0 or 5.6, inactivation of catalase did not seem to differ between these pH values, and approximately 80% of the catalase activity remained even after 8 h. Urate oxidase was inactivated very rapidly at pH 5.6; only 30% of its activity survived incubation for 6 h. This inactivation was found to occur by some proteolytic process. From these findings, the turnover rate of urate oxidase was found to be different from that of catalase, and this distinction seemed to be due to different sensitivity to some degradative enzymes.

Administration of ethionine resulted in a dose- and time-dependent enhancement of the activities of peroxisomal beta-oxidation, carnitine palmitoyltransferase and omega-oxidation, especially the 12-hydroxylation of lauric acid. The mitochondrial and, especially, the microsomal palmitoyl-CoA hydrolase activities were increased, whereas the peroxisomal and cytosolic activities were decreased. Ethionine administration decreased the catalase and urate oxidase activities in both a dose- and time-related manner. The liver cells and the volume fraction of cytoplasma decreased 40% in ethionine-exposed animals, whereas the average nuclei volume fraction increased approximately 50%. The volume fraction and the total number of mitochondria increased 1.5-fold after ethionine exposure and an accumulation of lipid in large droplets of the hepatocytes was observed. No proliferation of peroxisomes was observed after treatment; the volume fraction and the number of peroxisomes decreased. However, the size of peroxisomes in livers of ethionine-exposed rats tended to be greater than controls; a 1.5-fold increase in average size was observed. As there was no induction of the protein content of the bifunctional enoyl-CoA hydratase, an enzyme involved in peroxisomal beta-oxidation, it is considered that ethionine selectively stimulates the peroxisomal beta-oxidation due to increased peroxisome surface area rather than evoked a peroxisome proliferation capacity. Increased peroxisomal beta-oxidation was also observed in the kidney of ethionine-exposed rats at a dose of 750 mg/day/kg body weight. At that dose the amount of reduced glutathione (GSH) was significantly increased in kidney. The amount of GSH and the level of peroxisomal beta-oxidation were significantly increased in liver at an ethionine dose of 100 mg/day/kg body weight. These responses in liver were evident within 2 days of ethionine exposure and then leveled off whereas a significant increase in GSH and peroxisomal beta-oxidation in kidney was observed within 12 days. Whether the acute H2O2-generating peroxisomal oxidation of long-chain fatty acids in the liver may also make this organ susceptible to the long-term effects of low-dose ethionine and be an important step in the chain of events which eventually results in tumour development should be considered.

After Wistar male rats had been fed on a diet containing 0.25% of ethyl p-chlorophenoxyisobutyrate (CPIB) for 28 days, changes in the enzyme activities and centrifugal behavior of rat liver peroxisomes were investigated. (1) Compared with control rats fed on the basal diet, the catalase [EC 1.11.1.6] activity of rat livers after the administration of CPIB increased about 2.5-fold, while urate oxidase [EC 1.7.3.3] activity did not change significantly. Though D-amino acid oxidase [EC 1.4.3.3] activity markedly decreased to approximately one-sixth of the control, the activity of L-alpha-hydroxy acid oxidase [EC 1.1.3.15], a flavin enzyme like D-amino acid oxidase, was not affected significnatly after the administration of CPIB. (2) When the hepatic cells of CPIB-treated rats were fractionated by differential centrifugation, most of the increase of catalase activity appeared in the supernatant fraction. A decrease in the hepatic D-amino acid oxidase activity of CPIB-treated rats was observed in all the fractions. As for the subcellular distribution of the particle-bound enzymes, the specific activities of both catalase and urate oxidase of CPIB-treated rat livers were higher in the light mitochondrial fraction than in other fractions. (3) Sedimentation patterns in a sucrose density gradient did not show any difference between normal peroxisomers, and CPIB-treated ones. (4) In the case of CPIB-treated rats, studies of their sedimentation patterns by Ficoll density gradient centrifugation showed two main particulate peaks containing both catalase and urate oxidase, although only a single peak was observed in the case of control rats.

The activities of five peroxisomal enzymes (fatty acyl-CoA oxidase, FAO; uricase, U; L-hydroxyacid oxidase, LHAO; D-aminoacid oxidase, DAO; and catalase, C and the triglyceride content were determined in the regenerating liver of rats after partial hepatectomy. The FAO activity was significantly increased on day 3. Thus, its rise followed the increase of the triglyceride content and preceded the disappearance of steatosis. For U and LHAO a similar progressive increase in activities was obtained on days 3, 5, and 7 of regeneration. DAO exhibited a highly significant increase on day 1. C increased on day 1 and then again after day 3. It is concluded that peroxisomal functions are not restored at parallel rates during liver regeneration.

The effect of ionic strength and pH on the release of some enzymes of the matrix of peroxisomes in rat's liver was studied. Catalase, L ALpha-hydroxy acid oxidase, isocitrate dehydrogenase, glycerophosphate dehydrogenase and lactate dehydrogenase were easily released from the particles during their lysis and treatment with 0.16 M KCl, whereas urate oxidase, NADH cytochrome c reductase and D-amino acid oxidase were not solubilized. After the solubilization of peroxisomal membrane by 0.2% Triton X-100, the remaining core contained about 50% amino acid oxidase activity, and had 1.28--1.30 g/cm3 density. These results suggest that D-amino acid oxidase associates with urate oxidase in the peroxisomal core.

The biogenesis of peroxisomes has been investigated in the model of regenerating rat liver after partial hepatectomy using ultrastructural cytochemical staining methods: catalase as a marker of the peroxisomal matrix and uricase for the cores. The peroxisomes in regenerating rat liver showed several distinctive features: a) marked variation in shape and size, e.g., peroxisomes with tail-like extensions and tortuously elongated rod-shaped ones, b) formation of peroxisomal clusters and, c) interconnections between adjacent peroxisomes suggesting cleavage or budding. Whereas the reaction product for catalase was present at all intervals after hepatectomy in the matrix of all peroxisomes, the pattern of localization of uricase case varied with the time. It was confined to the cores in controls and at 10 days after the operation, while at 24 and 48 h it showed, in addition, a diffuse reaction in the matrix of some peroxisomes. In interconnected apparently dividing peroxisomes, the core with positive uricase reaction was present only in one half, while the other half was devoid of the reaction product. Similarly, the diffuse uricase staining was confined to the half which contained the core with the other half remaining unstained. These observations are consistent with the concept that new peroxisomes are formed from preexisting ones by budding and segmentation. While catalase is transferred uniformly to all new segments, uricase is compartmentalized in certain portions, of the apparently growing "peroxisomal reticulum".

A report is made (during cold exposure) of: a) the morphometric-stereologic analysis both on the whole rat liver peroxisomal population, and on 2 peroxisomal subpopulations having a diameter greater than 0.5 microns, and less than 0.5 microns, called Pe (Peroxisomes) and sPe (small Peroxisomes), respectively; b) the uricase and palmitoyl-coenzyme A oxidase activity assay on 2 classes of rat liver peroxisomes, sedimenting at 10,000 g and 27,000 g and containing Pe and sPe, respectively. The peroxisomal volume and number densities increase during cold exposure, reaching a maximum (+67% and +130%, respectively, P less than 0.05) at 10 days. These modifications are accompanied by an appreciable reduction of the average peroxisome volume (from 0.27 +/- 0.05 microns 3 to 0.19 +/- 0.02 microns 3) due to a major percentage increase of sPe during cold exposure. At a qualitative level, the formation of "clusters" and a stricter association between mitochondria and peroxisomes is also observed. Cold exposure increases the oxidative capacity of the whole peroxisomal compartment; in the Pe fraction the palmitoyl-CoA oxidase and uricase specific activity ratio is constant (about 0.1), during cold exposure, but in the sPe fraction this ratio increases significantly (from 0.05 to 0.09). The results indicate that the peroxisomal population is influenced during cold exposure, with the formation of a new peroxisomal population which is on average smaller and more specialized for the beta-oxidative activity. The possible involvement of peroxisomes in thermoregulatory thermogenesis during cold exposure is also discussed.

Urate oxidase was mostly recovered in a 40 000 g for 20 min subcellular fraction from conventional homogenated. Different agents were able to solubilize the sedimentable enzyme when tested on this crude peroxisomal fraction. The agents increased also total enzyme activity which was recovered in supernatants after centfiguation. If the possible effect of absorption to pellets is to be discounted, the enzyme was practically 100% extracted by six alternated freezing and thawing high alkaline pH, the detergent Hyamine 2389 and high ionic strength of calcium chloride. Triton X-100 was very effective in extracting proteins from the fraction, while it left most of the enzyme activity insoluble. It is suggested that the forces responsible for the integrity of the crystalloid, which was observed by other authors at the electron microscope, and to which the enzyme is believed to be related, are the electrostatic nature.

The peroxisomal core from the liver of rats was purified 450-fold as a marker of urate oxidase [EC 1.7.3.3.] activity. This preparation has a high specific activity of urate oxidase but not of other peroxisomal enzymes: D-amino acid oxidase [EC 1.4.3.3.], L-alpha-hydroxy acid oxidase [EC 1.1.3.15], or catalase [EC 1.11.1.6]. No activity of marker enzymes for other subcellular particles; cytochrome c oxidase [EC1.9.3.1] (mitochondria), acid phosphatase [EC 3.1.3.2] (lysosomes), or glucose-6-phosphatase [EC 3.1.3.9] (microsomes), was detected in this preparation. The core obtained showed a single protein band in sodium dodecyl sulfate-polyacrylamide gel electrophoresis and the position of the band was found to correspond to a molecular weight 35,000. When the peroxisomal core was subjected to treatment at various pH's with 0.1 M carbonate buffer, urate oxidase was almost completely solubulized at pH 11.0, although approximately 35% of the core protein still remained in the pellet After solubilization of the core at pH 11.0, the specific activity of urate oxidase in the supernatant increased about 1.6 times; the density of the insoluble protein remaining in the pellet was identical with the that of the original core on sucrose density gradient centrifugation.

Peroxisome proliferation has been induced with 2-methyl-2-(p-[1,2,3,4-tetrahydro-1-naphthyl]-phenoxy)-propionic acid (Su-13437). DNA, protein, cytochrome oxidase, glucose-6-phosphatase, and acid phosphatase concentrations remain almost constant. Peroxisomal enzyme activities change to approximately 165%, 50%, 30%, and 0% of the controls for catalase, urate oxidase, L-alpha-hydroxy acid oxidase, and D-amino acid oxidase, respectively. For catalase the change results from a decrease in particle-bound activity and a fivefold increase in soluble activity. The average diameter of peroxisome sections is 0.58 +/- 0.15 mum in controls and 0.73 +/- 0.25 mum after treatment. Therefore, the measured peroxisomal enzymes are highly diluted in proliferated particles. After tissue fractionation, approximately one-half of the normal peroxisomes and all proliferated peroxisomes show matric extraction with ghost formation, but no change in size. In homogenates submitted to mechanical stress, proliferated peroxisomes do not reveal increased fragility; unexpectedly, Su-13437 stabilizes lysosomes. Our results suggest that matrix extraction and increased soluble enzyme activities result from transmembrane passage of peroxisomal proteins. The changes in concentration of peroxisomal oxidases and soluble catalase after Su-13437 allow the calculation of their half-lives. These are the same as those found for total catalase, in normal and treated rats, after allyl isopropyl acetamide: about 1.3 days, a result compatible with peroxisome degradation by autophagy. A sequential increase in liver RNA concentration, [14C]leucine incorporation into DOC-soluble proteins and into immunoprecipitable catalase, and an increase in liver size and peroxisomal volume per gram liver, characterize the trophic effect of the drug used. In males, Su-13437 is more active than CPIB, another peroxisome proliferation-inducing drug; in females, only Su-13437 is active.

1. The activity of uricase, the densities of peroxisomes and cores in liver samples of baby pigs up to 4 weeks of age were investigated. 2. From 1 to 4 weeks of age, uricase activity as well as counts of cores and peroxisomes increased 5.5-, 3.3- and 2-fold. 3. Uricase activity and counts of cores and peroxisomes were correlated (P less than 0.001) with age in linear relationships. 4. Calculated for time of birth uricase activity was very low and ratio of cores to peroxisomes was 1:7. 5. From 0 to 28 days of age the calculated increases of uricase activity and counts of cores and peroxisomes were 178-, 10- and 3-fold.

1. The metabolism of palmitate and especially of erucate was studied in hepatocytes isolated from rats fed for 3 weeks a diet containing peanut oil (diet, 1), rapeseed oil (diet 2) and partially hydrogenated marine oil (diet 3). 2. The metabolism of palmitate was not significantly influenced by the diet. The rapeseed oil diet caused 1.4 fold and 1.3 fold increase and marine oil diet 3 fold and 2.2 fold increase in the oxidation and chain-shortening respectively of [14-14C]erucic acid in isolated hepatocytes. 3. Cyanide and antimycin A did not inhibit the chain-shortening of erucate in liver cells of rats fed rapeseed oil and peanut oil. The high capacity of the chain-shortening system in hepatocytes of marine oil-fed rats was partially inhibited. 4. Inhibition of the transfer of fatty acids into the mitochondria by lowering the intracellular carnitine concentration and/or by addition of (+)-decanoyl-carnitine resulted in a very pronounced apparent stimulation of the chain-shortening of erucic acid. It is suggested that the chain-shortening system may be virtually independent of the mitochondria, unless the availability of the extramitochondria NAD+ and/or NADP+ is rate-limiting under conditions of extremely low redox potential of the mitochondria. 5. Feeding marine oil or rapeseed oil to the rats induced a 30% increase in catalase activity, a 25--30% increase in urate oxidase activity and a 50% increase in the total CoA in the liver compared to rats fed peanut oil. 6. It is suggested that the increased metabolism of erucate in hepatocytes of marine oil and rapeseed oil-fed rats may be due to the increase in ther peroxisomal beta-oxidation.

Biochemical and morphometric analysis reveal that the peroxisomal content of rat liver cells is markedly reduced during pneumococcal sepsis. It is suggested that during some bacterial infections, hepatic synthesis of acute-phase serum proteins occurs at the expense of peroxisomal protein synthesis and results in reduction of the peroxisomal protein pool and number of peroxisomes.