Induction of microsomal 1-acylglycerophosphocholine acyltransferase by peroxisome proliferators in rat kidney; co-induction with peroxisomal beta-oxidation

Induction of microsomal 1-acyl-glycerophosphocholine (GPC) acyltransferase in rat tissues by four peroxisome proliferators, clofibric acid, tiadenol, DEHP and PFOA, was examined. Among the nine tissues examined, kidney, liver and intestinal mucosa responded to the challenges by the peroxisome proliferators to induce the enzyme. The treatment of rats with various dose of clofibric acid, tiadenol, DEHP or PFOA resulted in an induction of kidney microsomal 1-acyl-GPC acyltransferase in a dose-dependent manner. Despite the structural dissimilarity of peroxisome proliferators, the induction of microsomal 1-acyl-GPC acyltransferase was highly correlated with the induction of peroxisomal beta-oxidation. The activity of microsomal 1-acyl-GPC acyltransferase was not affected by changes in hormonal (adrenalectomy, diabetes, hyperthyroidism and hypothyroidism) and nutritional (starvation, starvation-refeeding, fat-free-diet feeding and high-fat-diet feeding) states. The induction of renal microsomal 1-acyl-GPC acyltransferase was seen in mice subsequent to the administration of clofibric acid and tiadenol and in guinea pigs subsequent to the administration of tiadenol. These results may indicate that kidney microsomal 1-acyl-GPC acyltransferase is a highly specific parameter responsive to the challenges by peroxisome proliferators and may suggest that the possibility that the inductions by peroxisome proliferators of microsomal 1-acyl-GPC acyltransferase and peroxisomal beta-oxidation in kidney are co-regulated.     

Inducing effects of clofibric acid on 1-acylglycerophosphorylcholine acyltransferase in kidney and intestinal mucosa of rats

Administration of p-chlorophenoxyisobutyric acid (clofibric acid) markedly increased the activity of microsomal 1-acylglycerophosphorylcholine (1-acyl-GPC) acyltransferase in kidney, intestinal mucosa and liver, but not in brain, heart, lung, spleen, testis or skeletal muscle. In both kidney and liver, a marked dose-dependent increase in the activities of both microsomal 1-acyl-GPC acyltransferase and peroxisomal beta-oxidation was observed. In the rats treated with clofibric acid at a relatively low dose, the increase in the activity of 1-acyl-GPC acyltransferase in kidney was more marked than that in liver. The extent of the relative increase in the activity of 1-acyl-GPC acyltransferase to the activity of peroxisomal beta-oxidation in kidney was more marked than that in liver. The increased activity of 1-acyl-GPC acyltransferase in both kidney and liver lasted throughout the 8-week treatment period of rat with clofibric acid.     

Alterations by peroxisome proliferators of acyl composition of hepatic phosphatidylcholine in rats, mice and guinea-pigs. Role of stearoyl-CoA desaturase.

Rats, mice and guinea-pigs were administered p-chlorophenoxyisobutyric acid (clofibric acid) or 2,2'-(decamethylenedithio)diethanol (tiadenol). The treatments of rats and mice with either clofibric acid or tiadenol increased markedly the activities of stearoyl-CoA desaturase, palmitoyl-CoA chain elongation, 1-acylglycerophosphate (1-acyl-GP) acyltransferase and 1-acylglycerophosphocholine (1-acyl-GPC) acyltransferase, but not 2-acylglycerophosphocholine (2-acyl-GPC) acyltransferase in liver microsomes. The treatment of guinea-pigs with clofibric acid did not cause any change in the activities of these enzymes. The treatment of guinea-pigs with tiadenol caused a slight, but significant, increase in the activities of 1-acyl-GP acyltransferase and 1-acyl-GPC acyltransferase. The treatment of rats and mice with either clofibric acid or tiadenol increased markedly the proportion of 18:1 and decreased greatly the proportion of 18:0 in liver microsomal phosphatidylcholine. However, there is a considerable difference in the effects of the two peroxisome proliferators on the composition of polyunsaturated fatty acids in phosphatidylcholine between rats and mice. The treatment of guinea-pigs with either of the two peroxisome proliferators caused no change in acyl composition of phosphatidylcholine. The possible role of stearoyl-CoA desaturation in the regulation of acyl composition of phosphatidylcholine was discussed.     

Selective increase in acylation of 1-acylglycerophosphorylcholine in livers of rats and mice by peroxisome proliferators

Rats were fed a diet containing p-chlorophenoxyisobutyric acid (clofibric acid). Activity of microsomal 1-acylglycerophosphorylcholine (1-acyl-GPC) acyltransferase in liver was increased approx. 3-fold by the treatment with clofibric acid. The treatment of rats with clofibric acid did not increase activity of microsomal 2-acyl-GPC acyltransferase. Feeding a diet containing 2,2'-(decamethylenedithio)diethanol (tiadenol), di(2-ethylhexyl)phthalate or acetylsalicylic acid also resulted in a selective increase in the activity of 1-acyl-GPC acyltransferase in rat liver. Treatment with clofibric acid increased the activity of 1-acyl-GPC acyltransferase in liver of mouse as well as rat, but did not change the activity in liver of guinea-pig. The relative rate of acylation of 1-acyl-GPC with various acyl-CoAs by hepatic microsomes was not changed by the treatment of rats with clofibric acid.     

Characteristics of dehydroepiandrosterone as a peroxisome proliferator.

Treatment of rats with dehydroepiandrosterone (300 mg/kg body weight, per os, 14 days) caused a remarkable increase in the number of peroxisomes and peroxisomal beta-oxidation activity in the liver. The activities of carnitine acetyltransferase, microsomal laurate 12-hydroxylation, cytosolic palmitoyl-CoA hydrolase, malic enzyme and some other enzymes were also increased. The increases in these enzyme activities were all greater in male rats than in female rats. Immunoblot analysis revealed remarkable induction of acyl-CoA oxidase and enoyl-CoA hydratase/3-hydroxyacyl-CoA dehydrogenase bifunctional enzyme in the liver and to a smaller extent in the kidney, whereas no significant induction of these enzymes was found in the heart. The increase in the hepatic peroxisomal beta-oxidation activity reached a maximal level at day 5 of the treatment of dehydroepiandrosterone and the increased activity rapidly returned to the normal level on discontinuation of the treatment. The increase in the activity was also dose-dependent, which was saturable at a dose of more than 200 mg/kg body weight. All these features in enzyme induction caused by dehydroepiandrosterone correlate well with those observed in the treatment of clofibric acid, a peroxisome proliferator. Co-treatment of dehydroepiandrosterone and clofibric acid showed no synergism in the enhancement of peroxisomal beta-oxidation activity, suggesting the involvement of a common process in the mechanism by which these compounds induce the enzymes. These results indicate that dehydroepiandrosterone is a typical peroxisome proliferator. Since dehydroepiandrosterone is a naturally occurring C19 steroid in mammals, the structure of which is novel compared with those of peroxisome proliferators known so far, this compound could provide particular information in the understanding of the mechanisms underlying the induction of peroxisome proliferation.     

Effects of clofibric acid and tiadenol on cytosolic long-chain acyl-CoA hydrolase and peroxisomal beta-oxidation in liver and extrahepatic tissues of rats

The effects of two peroxisome proliferators, p-chlorophenoxyisobutyric acid (clofibric acid) and 2,2'-(decamethylenedithio)diethanol (tiadenol), on cytosolic long-chain acyl-CoA hydrolase and peroxisomal beta-oxidation were studied in several organs of rat. Among organs of control rats, the brain had the highest activity of long-chain acyl-CoA hydrolase, followed by testis, and a low activity was found in other tissues. Administration of the peroxisome proliferators caused a marked increase in activity of long-chain acyl-CoA hydrolase in both liver and intestinal mucosa and a slight increase in the activity in kidney, but little affected acyl-CoA hydrolase activity in either brain, testis, heart, spleen and skeletal muscle. In accordance with the change in the activity of acyl-CoA hydrolase, the activity of peroxisomal beta-oxidation was markedly increased in liver, intestinal mucosa and kidney, and a slight increase was found in brain and testis, whereas peroxisome proliferators little affected the activity in other organs tested. Gel filtration of cytosol from intestinal mucosa showed that clofibric acid caused an appearance of a new peak in intestinal mucosa. Although cytosol of liver, intestinal mucosa, brain and testis contained two 4-nitrophenyl acetate esterases with different molecular weights (about 105,000 and about 55,000), these esterases are different from cytosolic long-chain acyl-CoA hydrolases of these four organs in respect of molecular weight. The administration of clofibric acid little affected cytosolic 4-nitrophenyl acetate esterases. Comparative studies on cytosolic long-chain acyl-CoA hydrolases from these four organs showed that liver hydrolase I (molecular weight of about 80,000) had properties similar to those of brain and testis enzymes. On the other hand, intestinal mucosa enzyme was different from either hepatic hydrolase I or II (molecular weight of about 40,000). The results from the present study suggest that inductions of peroxisomal beta-oxidation and cytosolic long-chain acyl-CoA hydrolases are essential responses of rats to peroxisome proliferators not only in liver but also in intestinal mucosa and that induced hydrolases are not attributable to non-specific esterases.     

Effects of the tumor promoter 12-O-tetradecanoylphorbol 13-acetate on peroxisomal activities and enzyme activities involved in lipid metabolism in rat liver

The effects of 12-O-tetradecanoylphorbol 13-acetate (TPA) on hepatic lipids and key enzymes involved in esterification, hydrolysis and oxidation of long-chain fatty acids at increasing doses were investigated in rats. TPA administration tended to decrease the mitochondrial activities of palmitoyl-CoA synthetase and carnitine palmitoyltransferase. The microsomal palmitoyl-CoA synthetase activity was increased. TPA administration was also associated with a dose-dependent increase of glycerophosphate acyltransferase activity both in the mitochondrial and microsomal fractions in particular. The data are consistent with a decreased catabolism of long-chain fatty acids at the mitochondrial level, and an increased capacity for esterification of fatty acids in the microsomal fraction. Peroxisomal beta-oxidation was increased about 2-fold in the peroxisome-enriched fraction of TPA-treated rats while the catalase and urate oxidase activities were only marginally affected. TPA administration revealed elevated capacity for hydrolysis of palmitoyl-CoA and palmitoyl-L-carnitine in the microsomal fraction. Neither increased cytosolic palmitoyl-CoA hydrolase activity nor increased hydroxylation of lauric acid nor changes of the hepatic content of cytochrome P-450 isoenzymic forms were observed in the TPA-treated animals. There was no induction of the protein content of the bifunctional enoyl-CoA hydratase. Thus, TPA behaves more like choline-deficient diet and ethionine treatment than well-known peroxisome proliferators. It seems possible that TPA selectively stimulated the peroxisomal activities, i.e., peroxisomal beta-oxidation rather than evoking a peroxisome proliferation capacity.     

Induction of peroxisomal beta-oxidation enzymes in primary cultured rat hepatocytes by clofibric acid

Rat hepatocytes were cultured for 72 h with or without the addition of 0.5 mM clofibric acid. The activities of individual enzymes of the peroxisomal beta-oxidation pathway (acyl-CoA oxidase, enoyl-CoA hydratase-3-hydroxyacyl-CoA dehydrogenase bifunctional protein, and 3-ketoacyl-CoA thiolase) decreased in the control culture, but markedly increased synchronously in the clofibric acid-treated culture. The levels of mRNAs coding for these enzymes and the rates of synthesis of the enzymes were also elevated in the clofibric acid-treated culture, although no proportional relationship was observed between the time-dependent changes of these parameters. The increase in mRNAs was much higher than the increase in the rate of synthesis of the enzymes. The activity of catalase, its mRNA level and the rate of its synthesis were slightly affected. The effects of clofibric acid on the peroxisomal beta-oxidation enzymes and catalase in primary cultured hepatocytes were very similar to those observed in vivo. These results, therefore, suggest that primary culture of hepatocytes should provide a useful means for investigating the mechanism of induction of peroxisomal enzymes and the mechanism of action of peroxisome proliferators.     

Rapid stimulation of liver palmitoyl-CoA synthetase, carnitine palmitoyltransferase and glycerophosphate acyltransferase compared to peroxisomal beta-oxidation and palmitoyl-CoA hydrolase in rats fed high-fat diets

Key enzymes involved in oxidation and esterification of long-chain fatty acids were investigated in male rats fed different types and amounts of oil in their diet. A diet with 20% (w/w) fish oil, partially hydrogenated fish oil (PHFO) and partially hydrogenated soybean oil (PHSO) was shown to stimulate the mitochondrial and microsomal palmitoyl-CoA synthetase activity (EC 6.2.1.3) compared to soybean oil-fed animals after 1 week of feeding. Rapeseed oil had no effect. Partially hydrogenated oils in the diet resulted in significantly higher levels of mitochondrial glycerophosphate acyltransferase compared to unhydrogenated oils in the diet. Rats fed 20% (w/w) rapeseed oil had a decreased activity of this mitochondrial enzyme, whereas the microsomal glycerophosphate acyltransferase activity was stimulated to a comparable extent with 20% (w/w) rapeseed oil, fish oil or PHFO in the diet. Increasing the amount of PHFO (from 5 to 25% (w/w)) in the diet for 3 days led to increased mitochondrial and microsomal palmitoyl-CoA synthetase and microsomal glycerophosphate acyltransferase activities with 5% of this oil in the diet. The mitochondrial glycerophosphate acyltransferase was only marginally affected by increasing the oil dose. Administration of 20% (w/w) PHFO increased rapidly the mitochondrial and microsomal palmitoyl-CoA synthetase, carnitine palmitoyltransferase and microsomal glycerophosphate acyltransferase activities almost to their maximum value within 36 h. In contrast, the glycerophosphate acyltransferase and palmitoyl-CoA hydrolase (EC 3.1.2.2) activities of the mitochondrial fraction and the peroxisomal beta-oxidation reached their maximum activities after administration of the dietary oil for 6.5 days. This sequence of enzyme changes (a) is in accordance with the proposal that an increased cellular level of long-chain acyl-CoA species act as metabolic messages for induction of peroxisomal beta-oxidation and palmitoyl-CoA hydrolase, i.e., these enzymes are regulated by a substrate-induced mechanism, and (b) indicates that, with PHFO, a greater part of the activated fatty acids are directed from triacylglycerol esterification and hydrolysis towards oxidation in the mitochondria. It is also conceivable that the mitochondrial beta-oxidation is proceeding before the enhancement of peroxisomal beta-oxidation.     

Induction of overlapping genes by fasting and a peroxisome proliferator in pigs: evidence of functional PPARalpha in nonproliferating species

Peroxisome proliferator-activated receptor alpha (PPARalpha), a key regulator of fatty acid oxidation, is essential for adaptation to fasting in rats and mice. However, physiological functions of PPARalpha in other species, including humans, are controversial. A group of PPARalpha ligands called peroxisome proliferators (PPs) causes peroxisome proliferation and hepatocarcinogenesis only in rats and mice. To elucidate the role of PPARalpha in adaptation to fasting in nonproliferating species, we compared gene expressions in pig liver from fasted and clofibric acid (a PP)-fed groups against a control diet-fed group. As in rats and mice, fasting induced genes involved with mitochondrial fatty acid oxidation and ketogenesis in pigs. Those genes were also induced by clofibric acid feeding, indicating that PPARalpha mediates the induction of these genes. In contrast to rats and mice, little or no induction of genes for peroxisomal or microsomal fatty acid oxidation was observed in clofibric acid-fed pigs. Histology showed no significant hyperplasia or hepatomegaly in the clofibric acid-fed pigs, whereas it showed a reduction of glycogen by clofibric acid, an effect of PPs also observed in rats. Copy number of PPARalpha mRNA was higher in pigs than in mice and rats, suggesting that peroxisomal proliferation and hyperresponse of several genes to PPs seen only in rats and mice are unrelated to the abundance of PPARalpha. In conclusion, PPARalpha is likely to play a central role in adaptation to fasting in pig liver as in rats and mice.     

Effect of Friend virus infection on the biosynthetic enzymes of phosphatidylcholine biosynthesis in spleen microsomes

The peroxisome proliferators clofibric acid and di-(2-ethylhexyl)-phthalate (DEHP) preferentially induced the 12-hydroxylation, compared to the 11-hydroxylation, of lauric acid in rat liver microsomes. A marked increase in the affinity of spectral interaction of this substrate with cytochrome P-450 was also observed. In addition, both clofibric acid and DEHP treatment produced a marked effect on the profile of site- and stereo-specific microsomal metabolites of testosterone. These results demonstrate that both peroxisome proliferators induce similar form(s) of cytochrome P-450 which are active in the metabolism of endogenous substrates of cytochrome P-450. The possible relevance of these findings to the hepatotoxicity of peroxisome proliferators is discussed.     

Fatty acid metabolism in liver of rats treated with hypolipidemic sulphur-substituted fatty acid analogues

The purpose of this study was to investigate early biochemical changes and possible mechanisms via which alkyl(C12)thioacetic acid (CMTTD, blocked for beta-oxidation), alkyl(C12)thiopropionic acid (CETTD, undergo one cycle of beta-oxidation) and a 3-thiadicarboxylic acid (BCMTD, blocked for both omega- (and beta-oxidation) influence the peroxisomal beta-oxidation in liver of rats. Treatment of rats with CMTTD caused a stimulation of the palmitoyl-CoA synthetase activity accompanied with increased concentration of hepatic acid-insoluble CoA. This effect was already established during 12-24 h of feeding. From 2 days of feeding, the cellular level of acid-insoluble CoA began to decrease, whereas free CoASH content increased. Stimulation of [1-14C]palmitoyl-CoA oxidation in the presence of KCN, palmitoyl-CoA-dependent dehydrogenase (termed peroxisomal beta-oxidation) and palmitoyl-CoA hydrolase activities were revealed after 36-48 h of CMTTD-feeding. Administration of BCMTD affected the enzymatic activities and altered the distribution of CoA between acid-insoluble and free forms comparable to what was observed in CMTTD-treated rats. It is evident that treatment of peroxisome proliferators (BCMTD and CMTTD), the level of acyl-CoA esters and the enzyme activity involved in their formation precede the increase in peroxisomal and palmitoyl-CoA hydrolase activities. In CMTTD-fed animals the activity of cyanide-insensitive fatty acid oxidation remained unchanged when the mitochondrial beta-oxidation and carnitine palmitoyltransferase operated at maximum rates. The sequence and redistribution of CoA and enzyme changes were interpreted as support for the hypothesis that substrate supply is an important factor in the regulation of peroxisomal fatty acid metabolism, i.e., the fatty acyl-CoA species appear to be catabolized by peroxisomes at high rates only when uptake into mitochondria is saturated. Administration of CETTD led to an inhibition of mitochondrial fatty acid oxidation accompanied with a rise in the concentration of acyl-CoA esters in the liver. Consequently, fatty liver developed. The peroxisomal beta-oxidation was marginally affected. Whether inhibition of mitochondrial beta-oxidation may be involved in regulation of peroxisomal fatty acid metabolism and in development of fatty liver should be considered.     

Proliferation of peroxisomes and induction of peroxisomal beta-oxidation enzymes in rat hepatoma H4IIEC3 by ciprofibrate

For the analysis of the molecular mechanism of the action of peroxisome proliferators, we attempted to establish the optimal conditions for obtaining the effects of the chemicals in vitro, employing an established cell line, Reuber rat hepatoma H4IIEC3. Histochemical analyses revealed a marked increase in the number, size, and catalase content of peroxisomes in the cells cultured on a medium containing 0.5 mM ciprofibrate, a peroxisome proliferator. The activity of acyl-CoA oxidase, the initial enzyme of the peroxisomal beta-oxidation system, was increased by more than 10-fold by the same treatment. Catalase was also induced significantly, whereas the activities of glutamate dehydrogenase and lactate dehydrogenase, mitochondrial and cytosolic marker enzymes, did not change upon the treatment. Immunoblotting and RNA-blotting analyses confirmed the increases in the amount of protein and mRNA for all the three enzymes of the peroxisomal beta-oxidation system. Cell fractionation experiments gave a partial separation of peroxisomes from other organelles for the induced culture. Thus, H4IIEC3 cells offer a good in vitro model system of the induction of peroxisomes and peroxisomal beta-oxidation enzymes by peroxisome proliferators.     

Differential induction of peroxisomal beta-oxidation enzymes by clofibric acid and aspirin in piglet tissues

Peroxisomal beta-oxidation (POX) of fatty acids is important in lipid catabolism and thermogenesis. To investigate the effects of peroxisome proliferators on peroxisomal and mitochondrial beta-oxidation in piglet tissues, newborn pigs (1-2 days old) were allowed ad libitum access to milk replacer supplemented with 0.5% clofibric acid (CA) or 1% aspirin for 14 days. CA increased ratios of liver weight to body weight (P < 0.07), kidney weight to body weight (P < 0.05), and heart weight to body weight (P < 0.001). Aspirin decreased daily food intake and final body weight but increased the ratio of heart weight to body weight (P < 0.01). In liver, activities of POX, fatty acyl-CoA oxidase (FAO), total carnitine palmitoyltransferase (CPT), and catalase were 2.7-, 2.2-, 1.5-fold, and 33% greater, respectively, for pigs given CA than for control pigs. In heart, these variables were 2.2-, 4.1-, 1.9-, and 1.8-fold greater, respectively, for pigs given CA than for control pigs. CA did not change these variables in either kidney or muscle, except that CPT activity was increased approximately 110% (P < 0.01) in kidney. Aspirin increased only hepatic FAO and CPT activities. Northern blot analysis revealed that CA increased the abundance of catalase mRNA in heart by approximately 2.2-fold. We conclude that 1) POX and CPT in newborn pigs can be induced by peroxisomal proliferators with tissue specificity and 2) the relatively smaller induction of POX in piglets (compared with that in young or adult rodents) may be related to either age or species differences.     

Role of stearoyl-CoA desaturase and 1-acylglycerophosphorylcholine acyltransferase in the regulation of the acyl composition of phosphatidylcholine in rat liver

The role of stearoyl-CoA desaturase and 1-acylglycerophosphorylcholine (1-acylGPC) acyltransferase in regulating acyl composition of microsomal phosphatidylcholine was investigated in rat liver, using rats in five different kinds of physiological state: clofibric acid-fed rats, diabetic rats, insulin-treated diabetic rats, starved rats and starved-refed rats. There was a reverse linear correlation between 18:1 and 18:2 in the C-2 position, and a similar correlation was found between 18:1 and 18:2 in microsomal free fatty acids. The proportion of 18:1 or 18:2 in the C-2 position of phosphatidylcholine correlated with the proportion of the respective fatty acids in microsomal free unsaturated fatty acids which could be incorporated effectively, except for the group of clofibric acid-fed rats. In this group alone, 1-acylGPC acyltransferase was induced markedly. The proportion of 18:1 in microsomal free fatty acids correlated well with the activity of stearoyl-CoA desaturase. The physiological significance of stearoyl-CoA desaturase and 1-acylGPC acyltransferase was discussed in relation to the regulation of the acyl composition of phosphatidylcholine.     

Increased response to peroxisomal proliferators in starved rats

The induction of liver peroxisomal beta-oxidation activities by bezafibrate or Wy 14,643 was 2-4-fold higher in starved rats than in fed animals. The increased response to peroxisomal proliferators in starved rats was independent of the mode of administration of the proliferator, given either orally or by intraperitoneal injection. Inhibitors of carnitine acyltransferase I could prevent the induction of peroxisomal activities in starved rats but not in fed animals. In contrast to fasted rats, the induction of liver peroxisomal activities in streptozotocin-diabetic rats was not susceptible to bezafibrate. The higher sensitivity to peroxisomal proliferators under conditions of starvation may allow for the detection of xenobiotic peroxisomal proliferators of low proliferative potency.     

Sex-related difference in the inductions by perfluoro-octanoic acid of peroxisomal beta-oxidation, microsomal 1-acylglycerophosphocholine acyltransferase and cytosolic long-chain acyl-CoA hydrolase in rat liver.

Inductions by perfluoro-octanoic acid (PFOA) of hepatomegaly, peroxisomal beta-oxidation, microsomal 1-acylglycerophosphocholine acyltransferase and cytosolic long-chain acyl-CoA hydrolase were compared in liver between male and female rats. Marked inductions of these four parameters were seen concurrently in liver of male rats, whereas the inductions in liver of female rats were far less pronounced. The sex-related difference in the response of rat liver to PFOA was much more marked than that seen with p-chlorophenoxyisobutyric acid (clofibric acid) or 2,2'-(decamethylenedithio)diethanol (tiadenol). Hormonal manipulations revealed that this sex-related difference in the inductions is strongly dependent on sex hormones, namely that testosterone is necessary for the inductions, whereas oestradiol prevented the inductions by PFOA.