Hepatic enzymes, CoASH and long-chain acyl-CoA in subcellular fractions as affected by drugs inducing peroxisomes and smooth endoplasmic reticulum

1. The activities of acyl-CoA hydrolase, catalase, urate oxidase and peroxisomal palmitoyl-CoA oxidation as well as the protein content and the level of CoASH and long-chain acyl-CoA were measured in subcellular fractions of liver from rats fed diets containing phenobarbital (0.1% w/w) or clofibrate (0.3% w/w). 2. Whereas phenobarbital administration resulted in increased microsomal protein, the clofibrate-induced increase was almost entirely attributed to the mitochondrial fraction with minor contribution from the light mitochondrial fraction. 3. The specific activity of palmitoyl-CoA hydrolase in the microsomal fraction was only slightly affected while the mitochondrial enzyme was increased to a marked extent (3-4-fold) by clofibrate. 4. Phenobarbital administration mainly enhanced the microsomal palmitoyl-CoA hydrolase. 5. The increased long-chain acyl-CoA and CoASH level observed after clofibrate treatment was mainly associated with the mitochondrial, light mitochondrial and cytosolic fractions, while the slight increase in the levels of these compounds found after phenobarbital feeding was largely of microsomal origin. 6. The findings suggest that there is an intraperoxisomal CoASH and long-chain acyl-CoA pool. 7. The specific activity of palmitoyl-CoA hydrolase, catalase and peroxisomal palmitoyl-CoA oxidation was increased in the lipid-rich floating layer of the cytosol-fraction. 8. The changes distribution of the peroxisomal marker enzymes and microsomal palmitoyl-CoA hydrolase after treatment with hypolipidemic drugs may be related to the origin of peroxisomes.     

The effect of three hypolipidemic drugs on catalase activity and peroxisomal and mitochondrial palmitate oxidation in rat cardiac and skeletal muscle

Catalase activity and peroxisomal and mitochondrial palmitate oxidation have been investigated in cardiac and skeletal muscle from rats fed clofibrate, ciprofibrate or nafenopin in an unrefined diet for different periods of time. Nafenopin was also added to either a high carbohydrate (70% of kilocalories from glucose) or high fat (70% of kilocalories from lard) diet and fed to rats for either 1 or 3 weeks. Catalase activity was elevated in all muscles from rats fed the hypolipidemic drugs. The response of catalase activity in muscle to clofibrate was dose-dependent. The response time of catalase activity was different in individual muscles. Peroxisomal palmitate oxidation was elevated in the heart and soleus muscle from rats fed nafenopin in either the high-carbohydrate or the high-fat diet. There was no change in peroxisomal palmitate oxidation in psoas or extensor digitorum longus muscle from rats fed the drugs. Mitochondrial palmitate oxidation was only slightly increased by nafenopin in the heart and soleus muscles after 3 weeks of nafenopin feeding. The results suggest that the cardiac muscle, like the liver, responds to hypolipidemic drug treatment with an increase in peroxisomal fat oxidation. The skeletal muscle response is less specific and that tissue may not contribute to the hypolipidemic effect of the drugs. The findings also suggest that these drugs do not induce peroxisome proliferation in skeletal muscle.     

Detection of heat-stable delta 3,delta 2-enoyl-CoA isomerase in rat liver mitochondria and peroxisomes by immunochemical study using specific antibody

The subcellular distribution of delta 3,delta 2-enoyl-CoA isomerase [EC 5.3.3.8] and the inducing effect of clofibrate, a peroxisomal proliferator, on the enzyme activity were examined in rat liver. From the results of spectrophotometric investigation of the fractions, which were prepared by sucrose discontinuous gradient centrifugation from the light mitochondrial fraction, the isomerase activity was found in the fractions enriched in mitochondria and those enriched in peroxisomes of the control and the clofibrate treated rat livers. The anti-isomerase antibody reacted with both the mitochondrial isomerase and the peroxisomal isomerase, revealing a single band with an apparent molecular weight of 30,000. However, the isomerase was induced by clofibrate administration mainly in the mitochondrial fraction. These results suggest that delta 3,delta 2-enoyl-CoA isomerase is located in the mitochondria and the peroxisomes of the normal rat liver, and that the isomerase in the mitochondria is induced by clofibrate administration.     

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.     

Studies on peroxisomes. V. Effect of ethyl p-chlorophenoxyisobutyrate on the centrifugal behavior of rat liver peroxisomes.

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.     

Effects of dietary polyamines and clofibrate on metabolism of polyamines in the rat

The activities of catalase, polyamine oxidase, diamine oxidase, ornithine decarboxylase, and peroxisomal β-oxidation were assayed in homogenates from liver and small intestinal mucosa of rats which had been fed either a diet very low in polyamines or a diet containing five times the levels of dietary polyamines (putrescine, spermine, and spermidine) found in a standard rat diet. In rats fed the high polyamine diet, hepatic activities of catalase and polyamine oxidase were significantly decreased. Levels of the other activities were unchanged, except that intestinal ornithine decarboxylase was decreased. In rats treated simultaneously with clofibrate, the high polyamine diet restored activities of catalase, ornithine decarboxylase, and polyamine oxidase back to levels found in rats fed the low polyamine diet. The expected increase in activity of peroxisomal β-oxidation was observed, although this was somewhat diminished in rats fed the high polyamine diet. Intestinal diamine oxidase activity was stimulated by clofibrate, particularly in rats fed the high polyamine diet. For the duration of the experiment (20 days), levels of putrescine, spermine, and spermidine in blood remained remarkably constant irrespective of treatment, suggesting that polyamine homeostasis is essentially independent of dietary supply of polyamines. It is suggested that intestinal absorption/metabolism of polyamines is of significance in this respect. Treatment with clofibrate appeared to alter polyamine homeostasis.     

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.     

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.     

Studies on the intracellular distributions of soluble epoxide hydrolase and of catalase by digitonin-permeabilization of hepatocytes isolated from control and clofibrate-treated mice

Digitonin permeabilization of hepatocytes from control and clofibrate-treated (0.5% by mass, 10 days) male C57bl/6 mice was used to study the intracellular distributions of soluble ('cytosolic') epoxide hydrolase and of catalase. The following conclusions were drawn. (1) About 60% of the total soluble epoxide hydrolase activity in control mouse hepatocytes is situated in the cytosol. (2) The rest is not mitochondrial, but probably peroxisomal. (3) Of the total catalase activity in control mouse hepatocytes, 5-10% is found in the cytosol. (4) Treatment of mice with clofibrate increases the total hepatocyte activity of soluble epoxide hydrolase 4-fold, but does not influence the relative distribution of this enzyme between cytosol and peroxisomes. (5) The total catalase activity is increased 3.5-fold by clofibrate treatment and 15-35% of this activity is shifted from the peroxisomes to the cytosol.     

Dehydrogenases of the pentose phosphate pathway in rat liver peroxisomes

Subcellular distribution of pentose-phosphate cycle enzymes in rat liver was investigated, using differential and isopycnic centrifugation. The activities of the NADP+-dependent dehydrogenases of the pentose-phosphate pathway (glucose-6-phosphate dehydrogenase and phosphogluconate dehydrogenase) were detected in the purified peroxisomal fraction as well as in the cytosol. Both dehydrogenases were localized in the peroxisomal matrix. Chronic administration of the hypolipidemic drug clofibrate (ethyl-alpha-p-chlorophenoxyisobutyrate) caused a 1.5-2.5-fold increase in the amount of glucose-6-phosphate and phosphogluconate dehydrogenases in the purified peroxisomes. Clofibrate decreased the phosphogluconate dehydrogenase, but did not alter glucose-6-phosphate dehydrogenase activity in the cytosolic fraction. The results obtained indicate that the enzymes of the non-oxidative segment of the pentose cycle (transketolase, transaldolase, triosephosphate isomerase and glucose-phosphate isomerase) are present only in a soluble form in the cytosol, but not in the peroxisomes or other particles, and that ionogenic interaction of the enzymes with the mitochondrial and other membranes takes place during homogenization of the tissue in 0.25 M sucrose. Similar to catalase, glucose-6-phosphate dehydrogenase and phosphogluconate dehydrogenase are present in the intact peroxisomes in a latent form. The enzymes have Km values for their substrates in the millimolar range (0.2 mM for glucose-6-phosphate and 0.10-0.12 mM for 6-phosphogluconate). NADP+, but not NAD+, serves as a coenzyme for both enzymes. Glucose-6-phosphate dehydrogenase was inhibited by palmitoyl-CoA, and to a lesser extent by NADPH. Peroxisomal glucose-6-phosphate and phosphogluconate dehydrogenases have molecular mass of 280 kDa and 96 kDa, respectively. The putative functional role of pentose-phosphate cycle dehydrogenases in rat liver peroxisomes is discussed.     

Uncoupling by proteolysis of alpha-adrenergic receptor-mediated inhibition of adenylate cyclase in human platelets

Ethyl 2{5(4-chlorophenyl)pentyl}oxiran-2-carboxylate (POCA) is a new hypoglycaemic compound. The POCA-CoA ester strongly inhibits β-oxidation at carnitine palmitoyltransferase I. Chronic administration of POCA to rats decreases plasma concentrations of cholesterol and triacylglycerol and increases the number of hepatic peroxisomes similarly to hypolipidaemic drugs related to clofibrate. Peroxisomal fractions from rats fed a diet containing 0.2% of POCA for 4 weeks were prepared on self-generated Percoll gradients. POCA induced a 4-fold increase in catalase activity and peroxisomal β-oxidation, agreeing with the morphological data. The increase in peroxisomal β-oxidation caused by POCA feeding does not prevent accumulation of lipid following the inhibition of mitochondrial β-oxidation.     

Enhancement of choline dehydrogenase activity in rat liver mitochondria by clofibrate feeding

Male rats were fed a diet containing 0.75% clofibrate. After three weeks, the specific activity of choline dehydrogenase of a liver mitochondrial fraction was more then doubled. However, the activity of two other NAD-independent flavoenzymes of the inner mitochondrial membrane namely proline and succinate dehydrogenases were not altered significantly. Thus changes of the inner mitochondrial membrane induced by clofibrate seem to be restricted to some particular enzymes.     

Quantification and regulation of the subcellular distribution of bile acid coenzyme A:amino acid N-acyltransferase activity in rat liver

Bile acid coenzyme A:amino acid N-acyltransferase (BAT) is responsible for the amidation of bile acids with the amino acids glycine and taurine. To quantify total BAT activity in liver subcellular organelles, livers from young adult male and female Sprague-Dawley rats were fractionated into multiple subcellular compartments. In male and female rats, 65-75% of total liver BAT activity was found in the cytosol, 15-17% was found in the peroxisomes, and 5-10% was found in the heavy mitochondrial fraction. After clofibrate treatment, male rats displayed an increase in peroxisomal BAT specific activity and a decrease in cytosolic BAT specific activity, whereas females showed an opposite response. However, there was no overall change in BAT specific activity in whole liver homogenate. Treatment with rosiglitazone or cholestyramine had no effect on BAT activity in any subcellular compartment. These experiments indicate that the majority of BAT activity in the rat liver resides in the cytosol. Approximately 15% of BAT activity is present in the peroxisomal matrix. These data support the novel finding that clofibrate treatment does not directly regulate BAT activity but does alter the subcellular localization of BAT.     

Hypolipidemic drug clofibrate induces hepatic dedifferentiation

The effect of clofibrate on rat liver enzymes and metabolites was compared with that produced by partial hepatectomy and an extrahepatic tumor. Clofibrate administration produced decrease in gamma-glutamyltranspeptidase (GGT) activity with concomitant increase in glutathione concentration. The drug was able to exert its GGT-lowering effect even when fed to tumor-bearing animals. Presence of an extrahepatic neoplasm as well as administration of clofibrate resulted in marked decrease in activities of hepatic arginase and ornithine transaminase. Administration of clofibrate to the tumor-bearing rat produced a further decrease in activities of these two enzymes. These results suggest that clofibrate causes hepatic dedifferentiation and simulates an extrahepatic tumor. However, clofibrate did not induce any significant increase in polyamine profile unlike the other two experimental conditions.     

Effect of clofibrate treatment on aconitase activity in rat liver and other tissues

The activity of both mitochondrial and cytosolic aconitases was significantly increased in the livers of male rats following treatment with the hypolipidemic drug clofibrate. Cycloheximide or puromycin administration to rats inhibited the inducing effect of clofibrate on the enzyme activity. Aconitase activity in small intestine homogenate was also increased by clofibrate. The drug did not affect the enzyme activity in rat kidney, heart and brain.     

Effects of fat content in the diet on hepatic peroxisomes of the rat

Effects of fat content in the diet on rat liver peroxisomes was examined. In the livers of rats fed for one week on the high-fat diet containing 30% fat, the cyanide-insensitive palmitoyl-CoA oxidation was accelerated to eight times that of control and the enzymic activities of catalase, carnitine acetyltransferase and carnitine palmitoyltransferase were elevated by the factors of 1.3, 5 and 2, respectively. In contrast, the activities of D-amino acid oxidase in addition to the three enzymes mentioned above were all lowered by 20% when the animals were maintained on a fat-free diet for the same period of time. It appears that the high-fat diet-induced increase in the activity of carnitine palmitoyltransferase is a result of the raised activity of this enzyme in mitochondria only while the apparent high activity reflects stimulation of carnitine acetyltransferase in all the subcellular fractions. Another notable effect of the high-fat diet was a remarkable increase in the quantity of a peroxisome-associated polypeptide which was separable by sodium dodecyl sulfate polyacrylamide gel electrophoresis. It is noteworthy that this effect of the high-fat diet resemble that of clofibrate. If the diet was deprived of fat, however, this polypeptide species, with an estimated molecular weight of 80 000, decreased to a level slightly lower than normal. On the basis of the electron micrographic criteria, the high-fat diet provoked a marked proliferation of hepatic peroxisomes.     

Enhancement of rat liver catalase activity dietary cholesterol

The effects of a fat-supplemented diet and clofibrate (ethylchlorophenoxyisobutirate) upon serum lipids and liver catalase activity were studied in male rats. A butter-supplemented diet produced a striking increase of serum triglycerides but did not affect the liver catalase activity. Cholesterol (1%, w/w), added to the butter supplemented diet markedly increased liver catalase activity. This diet produced a hypercholesterolemic state higher than that induced by a butter-supplemented diet only, although the hypertriglyceridemic effect was less pronounced. Clofibrate given a butter-supplemented diet produced a marked increase of liver catalase activity (about four-fold). When clofibrate is administered with the cholesterol-supplemented diet, the increment observed in the liver catalase activity was the same as that induced with the cholesterol supplemented diet alone. Clofibrate, in either lipid-rich diet, failed to induce a hypocholesterolemic response, although a clear hypotrigliceridemic effect was evident. This effect appears to be potentiated with clofibrate and the cholesterol supplemented diet. Thus the increment in liver catalase activity induced by dietary cholesterol and clofibrate seems to be related to a hypotriglyceridemic effect which gives support to a role of liver peroxisomes in lipid metabolism. The role that liver catalase would play, in this regard, remains unclear from these results.     

Peroxisomal proliferation induced by treatment with clofibrate in a patient with a peroxisomal disease

The induction of peroxisomal proliferation in liver parenchymal cells of rats fed a diet containing clofibrate, a hypolipidemic drug, is a well-established event. However, the available data on human hepatocytes in vivo and in vitro indicate that agents that induce peroxisomal proliferation in rats and mice have no effect on human liver cells. The authors are reporting the case of a patient with clinical and laboratory diagnosis of X-linked-adrenoleukodistrophy. In an initial liver biopsy, a reduced volume fraction of peroxisomes was found (Vv.=.012) after a morphometric analysis, initiating treatment with clofibrate at a dose of 1.5 g/d. The administration of clofibrate was maintained for 7 yr. Liver biopsies were taken after 2, 4, and 7 yr, to follow the peroxisomal response. Results demonstrated a 500% increase in peroxisomal Vv. (.060) after 2 yr of treatment, compared with the pretreatment Vv. In subsequent biopsies, the peroxisomal Vv. value was maintained at 225 and 183% increases above the pretreatment biopsy (.027 and .022, respectively).     

Carnitine octanoyltransferase of mouse liver peroxisomes: properties and effect of hypolipidemic drugs

Carnitine octanoyltransferase (COT) in 500g supernatant fluids from mouse liver has a specific activity at least twice that of carnitine acetyltransferase (CAT) or carnitine palmitoyltransferase (CPT). When mice are fed diets containing the lipid-lowering drugs, clofibrate or nafenopin, the specific activity of COT increases 4- and 11-fold, respectively. Liver homogenates from mice fed a control diet, and diets containing clofibrate, nafenopin, or Wy-14,643 were fractionated by sucrose gradient centrifugation, and the subcellular distribution of carnitine acyltransferases was determined. In the controls, peroxisomes contained about 70% of the total COT. The specific activity of COT in the peroxisomal peak was 12-fold greater than either CAT or CPT, and 20-fold greater than the COT activity in the mitochondrial fraction. Treatment with hypolipidemic drugs increased the specific activity of peroxisomal COT 2- to 3-fold and CAT 6- to 12-fold, while mitochondrial COT increased 5- to 11-fold and CAT 19- to 54-fold. COT was purified to homogeneity from livers of mice treated with Wy-14,643. It had an apparent Mr of 60,000 by Sephadex G-100 and sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis, and a maximum activity for octanoyl-CoA with acetyl-CoA and palmitoyl-CoA having activities of 2 and 10%, respectively, when 100 microM acyl-CoA substrates were used. The Km's for 1-carnitine, octanoyl-CoA, palmitoyl-CoA, and acetyl-CoA were 130, 15, 69, and 155 microM, respectively, in the forward direction; and in the reverse direction were 110, 100, 104, and 783 microM for CoASH, octanoylcarnitine, palmitoylcarnitine, and acetylcarnitine, respectively. With Vmax conditions, acetyl-CoA and palmitoyl-CoA had activities of 8 and 26% of the activity for octanoyl-CoA, and acetylcarnitine and palmitoylcarnitine had activities of 7 and 22%, respectively, of the activity for octanoylcarnitine. It is concluded that COT is a separate enzyme present in large amounts in the matrix of mouse liver peroxisomes, with kinetic properties that greatly favor medium-chain acylcarnitine formation.