Effect of clofibrate application on morphology and enzyme content of liver peroxisomes.

Male albino rats (Sprague Dawley) were fed for 2-6 weeks on a diet containing 0.75% clofibrate. Liver cell fractions obtained from these animals were assayed for peroxisomal enzymes. In the cell homogenate the catalase activity was doubled, whereas the activity of urate oxidase was found to be only slightly depressed. The activity of carnitine acetyltransferase increased several times. In liver peroxisomes purified by isopycnic gradient centrifugation the specific activity of urate oxidase decreased appreciably showing that peroxisomes formed under the proliferative influence of clofibrate are not only modified with respect to their morphological characteristics but also to their enzymic equipment. This is also obvious from the changes in peroxisomal carnitine acetyltransferase activity which was enhanced by clofibrate to more than the fivefold amount. In purified mitochondria this enzyme was even more active: clofibrate advances both, the peroxisomal and the mitochondrial moiety of carnitine acetyltransferase. Morphological and cytochemical studies showed an increase in the number of microbodies and as compared to the controls microbodies were lying in groups more frequently. Small particles located closely adjacent to "normal" sized peroxisomes were found particularly after short feeding periods. While the number of coreless microbodies increased studies gave no clear evidence for an increase in marked shape irregularities of the peroxisomes.     

Effects of clofibrate (alpha-p-chlorophenoxyisobutyryl-ethyl-ester) on male rat liver

In male rats, fed 0.5% clofibrate in their diet for 8 days and 21 days, the ultrastructural morphometric alterations of the hepatocytes were evaluated and compared with the biochemical data. The morphologic alterations of the microbodies were particularly related to the changes of the catalase activity of the liver homogenates. The results showed a marked hypertrophy of the liver and an increase in the volume of the individual hepatocyte. The numerical density and, even more pronounced, the volume density of the microbodies increased excessively during the treatment. The numerical density of the mitochondria decreased markedly after 21 days of administration. The surface of the rough endoplasmic reticulum showed a significant decrease, whereas the surface of the smooth endoplasmic reticulum showed a hypertrophy. The catalase activity of the liver homogenates increased 2-fold after 8 days and remained at this new steady-state after 21 days of treatment. The results suggest that the enzyme content of the microbodies changed after treatment with clofibrate, and support the suggestion that clofibrate may induce the synthesis of a yet unidentified peroxisomal protein.     

Effect of citrate feeding on free radical induced changes in experimental urolithiasis.

Feeding calculi producing diet (CPD) to rats for 4 weeks produced calcium oxaltate stones. Supplementation of sodium citrate to CPD (c-CPD) prevented stone formation. Except oxalate, the excretion of calcium, phosphorus and magnesium was restored to normal in c-CPD fed rats. The CPD fed rats exhibited increase in glycolic acid oxidase (GAO) and lactate dehydrogenase (LDH) activities and only GAO activity was partially restored in c-CPD fed rats. Kidney sub-cellular fractions of calculi producing diet (CPD) fed rats showed increased susceptibility for lipid peroxidation in presence of promotors. Antioxidant enzyme activities of superoxide dismutase (SOD), catalase and glutathione peroxidase and antioxidant concentrations of reduced glutathione, total thiols, ascorbic acid and vitamin E were significantly decreased while the xanthine oxidase activity, and concentrations of hydroxyl radical, diene conjugates and hydroperoxides were significantly increased in CPD fed rats. The susceptibility to lipid peroxidation, activities of antioxidant enzymes, and the concentration of antioxidants were not normalized by feeding citrate.     

Effect of chronic ethanol, catalase inhibitor 3-amino-1,2,4-triazole and clofibrate treatment on lipid peroxidation in rat myocardium

1. The effect of chronic alcohol consumption, catalase inhibitor 3-amino-1,2,4-triazole (amino-triazole) and peroxisome proliferator clofibrate on the level of Fe/ADP-ascorbate-induced lipid peroxidation has been studied in the rat myocardium. The intensity of lipid peroxidation was measured using chemiluminescence technique and malondialdehyde formation. 2. Combined us well as separate treatment with ethanol (36% of dietary calories) and aminotriazole caused elevation of the rate of lipid peroxidation in the nuclear-free homogenate or total particulate fraction of the rat heart. The most pronounced effect was noted during combined application of ethanol and aminotriazole. 3. Prolonged clofibrate treatment significantly increased the level of nonenzymatic lipid peroxidation in the rat myocardium. 4. Peroxidative alteration of the myocardial lipids in vivo was evaluated by measurement of conjugated dienes (absorbance at 233 nm). Separate ethanol, aminotriazole or clofibrate treatment did not affect the level of u.v. absorption of lipids from the total particulate fraction. However, when ethanol and aminotriazole were administered simultaneously an increase of conjugated diene formation was observed. 5. The data obtained confirm the hypothesis that ethanol or clofibrate-induced activation of the myocardial lipid peroxidation may be due to the increase of hydrogen peroxide-generating capacity of the heart microperoxisomes.     

Restoration of antioxidants in liver by methionine feeding in experimental rat urolithiasis.

The effect of methionine or citrate on antioxidant defense system has been studied in urolithic rat. Liver weight and its protein concentration did not change in the rats fed with calculi producing diet (CPD) when compared to normal diet fed rats. Feeding rats along with citrate (c-CPD) or methionine (m-CPD) improved their body weight gain. Liver microsomes and mitochondria fractions of CPD and c-CPD fed groups showed increased susceptibility for lipid peroxidation in presence of ascorbate and t-butyl hydroperoxide when compared to either control or m-CPD fed groups. Increased superoxide dismutase and xanthine oxidase activities, decreased catalase, glutathione peroxidase and glucose-6-phosphate dehydrogenase activities, decreased concentrations of reduced glutathione, total thiols, ascorbic acid and vitamin-E and increased formation of hydroxyl radical, hydroperoxides and diene conjugates were observed in the liver of both CPD fed group as well as c-CPD fed group. Except SOD and xanthine oxidase, all other parameters were normalized in m-CPD fed group. This suggested that feeding methionine reduced the susceptibility for lipid peroxidation by restoration of the level of free radical scavengers.     

Morphometric and cytochemical evaluation of clofibrate-induced peroxisomal proliferation in adult rat hepatocytes cultured on floating collagen gels

The present ultrastructural morphometric and cytochemical studies demonstrate clofibrate induced changes in peroxisomes in adult rat hepatocytes maintained for 14 days in primary culture on floating collagen gels. Catalase activity and the number and diameter of peroxisomes were reduced in hepatocytes cultured for between 2/3 and 7 days. However, hepatocytes cultured for 7-14 days had well-developed peroxisomes containing crystalloid nucleoids. The number of anucleoid peroxisomes in hepatocytes treated with 2 mM Na clofibrate increased with culture age, and by day 14 the number was 2.9 times greater than in freshly isolated hepatocytes. Catalase activity, as well as the number of nucleoid-containing peroxisomes were much greater in treated hepatocytes than in untreated controls, but decreased slightly with culture age. The diameter of peroxisomes was not reduced in the treated cells. These results suggest that the treatment with Na clofibrate is effective both for proliferation and maintenance of peroxisomes and for enhancing catalase activity. In treated hepatocytes, matrical plates were formed in peroxisomes from days 5 to 14 and the number of plate-containing peroxisomes increased with culture age.     

Structure and Function in the Grass Ligule: Mitochondria and Microbodies in the Membranous Ligule of Lolium temulentum L., an Ultrastructural and Cytochemical Study

The ultrastructure and distribution of the mitochondria andmicrobodies within the tissues of the ligule of mature leavesof Lolium temulentum L. are described. Staining of the mitochondriaand microbodies with diaminobenzidine (DAB) was observed. Mitochondrialstaining was totally inhibited by potassium cyanide and sodiumazide but not by aminotriazole, and did not occur in the presenceof added hydrogen peroxide; it was concluded that cytochromec oxidase (EC 1.9.1.3 [EC] ) was responsible. In the microbodies stainingwas reduced in the absence of added hydrogen peroxide and eliminatedin the presence of aminotriazole; it was concluded that catalase(EC 1.11.1.6 [EC] ) was responsible. Presence of succinic dehydrogenase(EC 1.3.99.1 [EC] ) activity was demonstrated in the mitochondriausing the copper-ferricyanide reaction; presence of sodium malonateand absence of sodium succinate greatly reduced mitochondrialdeposition of reaction product. The results are discussed inrelation to possible functions of these organelles in the ligule. Key words: Ligule, Lolium temulentum L., Enzyme ultracytochemistry     

Preventive effects of dehydroepiandrosterone acetate on the fatty liver induced by orotic acid in male rats.

Preventive effects of dehydroepiandrosteone acetate (DHEA-A) and clofibrate (positive control substance) on the fatty liver induced by orotic acid (OA) were examined on the male Sprague-Dawley rats fed a high sucrose based diet containing 1% OA and this diet further mixed with 0.5% DHEA-A or 0.5% clofibrate for 2 weeks. Numerous lipid droplets were observed in the hepatocytes of the rats treated with OA alone, but not in those treated with DHEA-A or clofibrate. In comparison to the group with OA alone, the DHEA-A or clofibrate treated rats showed a larger relative liver weight (to body weight) which was accompanied by increased peroxisomes in the hepatocytes. These results indicate that DHEA-A, as well as clofibrate, may prevent OA-induced fatty liver.     

Clofibrate feeding increases cytoplasmic but not mitochondrial malic enzyme activity in rat kidney cortex

Administration of clofibrate for 21 days to rats increased the malic enzyme activity in the kidney cortex by about 80 per cent. This effect seems to be specific since the drug did not alter significantly the activity either of lactate dehydrogenase, citrate synthase or total mitochondrial protein content in this organ. The increase in activity of malic enzyme in the 13,000 g supernatant (extramitochondrial) fraction in rats treated with the drug was about 80 per cent, whereas in the pellet (mitochondrial fraction) it was about 40 per cent. The specific activity of malic enzyme in the kidney cortex cytosol from clofibrate-treated rats was about twice that in controls. In contrast clofibrate treatment did not affect its specific activity in isolated mitochondria. Calculations showed that 0.57 and 0.53 mumoles min-1 g-1 wet tissue of mitochondrial malic enzyme was obtained in control and clofibrate-treated rats respectively. Thus, clofibrate feeding increases the amount of cytoplasmic but not mitochondrial malic enzyme activity.     

Specific repression of transthyretin gene expression in rat liver by a peroxisome proliferator clofibrate.

The effect of clofibrate, a hypolipidemic drug and a potent peroxisome proliferator, on expression of a nonperoxisomal transthyretin (prealbumin) gene was studied using rats fed clofibrate for various periods. Upon feeding a diet containing clofibrate, the level of transthyretin mRNA was down-regulated, reaching 20% of the control level, in an almost reciprocal time course to that of increases in the levels of peroxisomal mRNAs. Studies on expression of other steroid hormone regulated genes suggest that clofibrate may down-regulate several but not all types of steroid hormone regulated mRNA expression.     

Effect of clofibrate feeding on some lipogenic enzyme activities induced by high carbohydrate diet

Clofibrate administration by stomach tube or intraperitoneally for 3 successive days to rats fed standard diet or starved for 72 hr caused about 2-fold increase of malic enzyme activity in the liver and adipose tissue. The drug administered by stomach tube (but not intraperitoneally) to the rats fed fat free-high carbohydrate diet significantly blocked the inducing effect of the diet on malic enzyme activity in both tissues. Clofibrate blocked the induction by fat free-high carbohydrate diet of hexose monophosphate shunt dehydrogenases and ATP-citrate lyase in the liver. The amount of fat free-high carbohydrate diet consumed by rats received clofibrate by stomach tube was much less than by untreated animals. It is concluded therefore that the significant decrease of food consumption by rats receiving clofibrate by stomach tube is responsible for the inhibitory effect of the drug on some lipogenic enzymes activity induced by fat free-high carbohydrate diet.     

In vivo induction of 4-enoyl-CoA reductase by clofibrate in liver mitochondria and its effect on pent-4-enoate metabolism

Feeding of clofibrate to male rats leads to a 4–7 fold increase in the activity of the 4-enoyl-CoA reductase in the liver. Concomitantly the inhibition of fatty acid oxidation by pent-4-enoate is abolished, and an increased glucose formation in the presence of pent-4-enoate is observed. It is suggested that pent-4-enoate is converted to propionyl-CoA via the reaction sequence pent-4-enoyl-CoA→pent-2,4-dienoyl-CoA→pent-2-enoyl-CoA→propionyl-CoA + acetyl-CoA.     

Cytochemical localization of catalase in leaf microbodies (peroxisomes)

Segments of mature tobacco leaves were fixed in glutaraldehyde, incubated in medium containing 3,3''-diaminobenzidine (DAB) and hydrogen peroxide, and postfixed in osmium tetroxide. Electron microscopic observation of treated tissues revealed pronounced deposition of a highly electron-opaque material in microbodies but not in other organelles. The coarsely granular reaction product is presumably osmium black formed by reaction of oxidized DAB with osmium tetroxide. Reaction of the microbodies with DAB was completely inhibited by 0.02 M 3-amino-1,2,4-triazole and was considerably reduced by 0.01 M potassium cyanide. These results, when considered in light of recent biochemical studies, strongly suggest that catalase is responsible for the reaction. Sharp localization of this enzyme in microbodies establishes that they are identical to the catalase-rich "peroxisomes" recently isolated from leaf cell homogenates. A browning reaction that occurred in leaves during the incubation step was inhibited by cyanide but not by aminotriazole and therefore could not have been caused by the same enzyme. This reaction and a slight deposition of dense material within primary and secondary walls are ascribed to oxidation of DAB by soluble and wall-localized peroxidases.     

Organic aciduria in rats made resistant to hypoglycin toxicity by pretreatment with clofibrate.

1. The lethal, hypoglycaemic and hypothermic effects of hypoglycin in fasted rats are prevented if the rats had been fed on a diet containing clofibrate (0.5% w/w). 2. Injection of hypoglycin into fasted rats maintained on a standard diet caused severe prostration, hypothermia and a massive dicarboxylic aciduria [Tanaka (1972) J. Biol. Chem. 247, 7465-7478]. 3. Rats maintained on a diet containing clofibrate appeared normal after injection of hypoglycin, but had a marked dicarboxylic aciduria which was less than that induced in rats on a normal diet. 4. After administration of hypoglycin, butyryl-CoA and decanoyl-CoA, but not palmitoyl-CoA, dehydrogenase activities were strongly inhibited (80-95%) in the livers of animals on a standard diet. 5. Clofibrate feeding decreased the inhibition of these dehydrogenases to about 40-60%. 6. It was concluded that although clofibrate protects against the toxic effects of hypoglycin, some enzyme inhibitions as indicated by dicarboxylic aciduria are only partly prevented.     

Effect of clofibrate treatment on lipid peroxidation in rat liver homogenate and subcellular fractions

1. A study was made of the effect of hypolipidemic drug clofibrate on the level of lipid peroxidation in homogenates and subcellular fractions of rat liver. The intensity of lipid peroxidation was measured using chemiluminescence technique and malondialdehyde formation. 2. It was shown that under the action of clofibrate the levels of Fe/ADP-ascorbate-, as well as t-butyl hydroperoxide (Bu'OOH)-induced lipid peroxidation were decreased in the whole and "post-nuclear" liver homogenates. Dilution of the homogenates prevented depressing effect of clofibrate on lipid peroxidation. 3. Clofibrate significantly decreased the level of the Bu'OOH-dependent lipid peroxidation, but did not affect the activity of the Fe/ADP-ascorbate-induced reaction in rat liver mitochondria and microsomes. 4. Peroxidative alteration of membrane lipids in vivo was evaluated by determining the extent of conjugated dienes formation (absorption at 233 nm). It was shown that clofibrate did not increase the level of ultraviolet absorption of lipids from rat liver subcellular fractions. 5. The data obtained indicate that cytosol from the clofibrate treated rat liver contains a factor(s) which prevents lipid peroxidation in the mitochondria and microsomes.     

A light- and electron-microscope study of hepatocytes of rats fed different diets

Ketogenic diets are used in the treatment of epilepsy in children refractory to drug therapy. This study identifies changes in liver morphology in rats fed four different diets: a normal rodent chow diet, a calorie-restricted high-fat (ketogenic) diet and each diet supplemented with clofibric acid. Hepatocytes of rats fed the ketogenic diet show many lipid droplets and these are reduced to control levels when clofibrate is present in the diet. Mitochondria are enlarged in the livers of rats fed the ketogenic diet and further enlarged if clofibrate is present. Alterations in the appearance or numbers of other organelles are also found.     

Effects of lactation on L-leucine metabolism in the rat. Studies in vivo and in vitro.

1. The turnover rate of L-[1-14C]leucine was increased by 35% in lactating rats compared with virgin rats. Starvation or removal of pups (24 h) returned the value to that of the virgin rat. 2. Incorporation of L-[U-14C]leucine into lipid and protein of mammary glands of lactating rats in vivo increased 7-fold and 6-fold respectively compared with glands of virgin rats. Lactation caused no change in the incorporation of L-[U-14C]leucine into hepatic lipid and protein. 3. The production of 14CO2 from L[l-14C]leucine (in the presence of glucose) was similar in isolated acini from glands of fed (chow) and starved lactating rats. Feeding with a 'cafeteria' diet caused a slight decrease, and removal of pups a large decrease, in the oxidative decarboxylation of leucine. 4. Oxidation of L-[2-14C]leucine to 14CO2 was increased about 3-fold in acini from starved lactating rats or lactating rats fed on a 'cafeteria' diet compared with rats fed on a chow diet. Insulin decreased the formation of 14CO2 in all three situations. 5. Incorporation of L-[U-14C]- and [2-14C]-leucine into lipid was decreased in acini from starved lactating rats and lactating rats fed on a 'cafeteria' diet. Insulin tended to increase the conversion of [2-14C]leucine into lipid, but this was significant only in the case of the acini from 'cafeteria'-fed rats. 6. Experiments with (-)-hydroxycitrate indicate that the major route for conversion of leucine carbon into lipid in acini is via citrate translocation from the mitochondria. 7. The physiological implications of these findings are discussed.     

Allopurinol promotes and butylated hydroxy toluene prevents sugar-induced cataractogenesis

Rats fed a high galactose (30% galactose) diet (w/w) or made diabetic by injecting streptozotocin developed mature cataracts in approximately 45 and 90 days, respectively. Addition of allopurinol, a commonly used drug in the therapy of gout, to the high galactose diet or to the normal diet fed to diabetic rats advanced cataractogenesis in both the groups by approximately 50%. Allopurinol fed to control rats did not cause cataract formation. Feeding butylated hydroxy toluene (BHT), an antioxidant, prevented the allopurinol-induced advancement of cataract formation in galactosemic and diabetic rats. Assuming that these results are applicable in human subjects, there is need for caution in using allopurinol for the therapy of gout in diabetic subjects.     

Properties of a collagenase inhibitor partially purified from cultures of smooth muscle cells

1. The metabolism of [14-14C]erucate and [U-14C]palmitate has been investigated in perfused heart from rats fed 0.3% clofibrate for 10 days and from control rats. 2. The total uptake of fatty acids in the heart increased in the clofibrate fed group. Clofibrate increased the oxidation of [14-14C]erucic acid by 100% and the oxidation of [U-14C]palmitic acid by 30% compared to controls. 3. The chain-shortening of erucate to C20:1 and C18:1 fatty acids in the perfused heart was stimulated at least two-fold by clofibrate feeding. 4. The activity of the peroxisomal marker enzyme catalase increased 60%, the activity of cytochrome oxidase increased approx. 16% and the content of total coenzyme A increased 30% in heart homogenates from rats fed clofibrate compared to controls. 5. The isolated mitochondrial fraction from clofibrate fed rats showed an increased capacity for oxidation of palmitoylcarnitine and decanoylcarnitine, while the oxidation of erucoylcarnitine showed little change. 6. It is suggested that clofibrate increases the oxidation of [14-14C]erucic acid in the perfused heart by increasing the capacity for chain-shortening of [14-14C]erucate in the peroxisomal beta-oxidation system.     

Evidence for two classes of microbodies in meiocytes of the red algaPalmaria palmata

Summary Microbodies, usually spherical and about 0.2 m in diameter, were found to be associated with prophase nuclei in vegetative cells and meiocytes of the red algaPalmaria palmata. Nucleus-associated microbodies in meiocytes were numerous, but they did not react to the DAB cytochemical test for catalase and peroxidase activity. Microbodies not associated with nuclei in the same cells were intensely DAB-positive. Neither aminotriazole nor potassium cyanide inhibited the DAB reaction.