Induction of peroxisomal beta-oxidation enzymes by dehydroepiandrosterone and its sulfate in primary cultures of rat hepatocytes.

Treatment of cultured rat-hepatocytes with 50 microM dehydroepiandrosterone (DHEA) and its sulfate (DHEAS) for up to 5 days resulted in a progressive increase in peroxisomal beta-oxidation and carnitine acetyltransferase activity. After 5 days, the increases in activity were 2.6- and 4.8-fold for peroxisomal beta-oxidation and 11.7- and 17.1-fold for carnitine acetyltransferase over the initial activity, in DHEA- and DHEAS-treated cells, respectively. The stimulation of the activity of these enzymes by the respective agents was dose-related; it was maximum with 50 to 100 microM DHEA and 50 to 250 microM DHEAS, although DHEAS was more effective for stimulation than DHEA. Western blot analyses revealed the induction of acyl-CoA oxidase, enoyl-CoA hydratase/3-hydroxyacyl-CoA dehydrogenase bifunctional enzyme and carnitine acetyltransferase in the treated cells. Moreover, induction of fatty acid omega-hydroxylase proteins (P-450IVAS) was also revealed. These results indicate that DHEA and DHEAS act directly on hepatocytes. The induction of hepatic peroxisomal beta-oxidation enzymes and several other enzymes in rats administered with DHEA could be accounted for, at least in part, by the direct action of DHEA and its sulfate-conjugate (DHEAS) on liver cells.     

Turnover of enzymes of peroxisomal β-oxidation in rat liver

Male Wistar rats were given a diet containing 2% (w/w) di-(2-ethylhexyl)-phthalate (DEHP), a peroxisomal proliferator, for 4 weeks. The activities of enzymes of peroxisomal β-oxidation and of catalase were markedly increased by the DEHP administration. The time required to reach halfway to the maximal induction for enzymes of peroxisomal β-oxidation was 5–7 days, whereas that for catalase was 3 days. A separate DEHP group was placed on the control diet after 14 days of feeding with the DEHP diet. On the withdrawal of DEHP, activities of enzymes of the β-oxidation system and of catalase decreased to the control levels with a half-life of 2–3 days. Responses of some mitochondrial enzymes involved in fatty acid oxidation are also described.     

Postnatal development of peroxisomal and mitochondrial enzymes in rat liver.

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.     

Freeze-fracture study of rat liver peroxisomes: evidence for an induction of intramembrane particles by agents stimulating peroxisomal proliferation

The membrane ultrastructure of isolated rat liver peroxisomes has been observed by rapid freezing and freeze-fracture techniques. Unidirectional and rotary shadowing allows a clear visualization of the intramembrane particles (IMPs) on both the protoplasmic fracture (PF) leaflet and the endoplasmic fracture (EF) leaflet and reveals an asymmetric distribution of IMPs. Both fracture faces were uniformly studded by IMPs, and the frequency was about seven times higher on the P face (2322 per 1.0 micron2) than on the E face (322 per 1.0 micron2). Administration of the peroxisomal proliferator clofibrate (ethyl-p-chlorophenoxyisobutyrate) induced a marked increase in the frequency of IMPs on both the P face (2.2-fold) and the E face (1.7-fold). The average size decreased (P less than 0.001) from 45.7 +/- 16.5 nm2 to 35.2 +/- 10.8 nm2 on the P face. A similar increase in the frequency of IMPs was observed on the P face (1.8-fold) and the E face (1.8-fold) of peroxisomes from rats fed a semisynthetic diet containing 20% (w/w) of partially hydrogenated fish oil. The average size increased (P less than 0.001) from 36.6 +/- 19.7 to 50.0 +/- 23.5 nm2 on the E face. This study demonstrates alterations both in frequency and size distribution of IMPs in liver peroxisomal membranes on exposure of rats to agents known to induce peroxisomal proliferation. The increase in frequency of IMPs was as expected from the observed increase in one of the major integral membrane polypeptides, with apparent molecular mass of 69 (or 70) kDa, in proliferating rat liver peroxisomes.     

Differential induction of peroxisomal and microsomal fatty-acid-oxidising enzymes by peroxisome proliferators in rat liver and kidney. Characterisation of a renal cytochrome P-450 and implications for peroxisome proliferation

The induction of renal fatty-acid-oxidising enzymes has been investigated following short-term exposure to a group of structurally diverse peroxisome proliferators and compared to the more extensively documented hepatic responses in the rat. There was a marked compound dependence on induction of both cytochrome P-450-IVA1-dependent omega-hydroxylation of lauric acid and enzymes of the peroxisomal fatty acid beta-oxidation pathway (measured as cyanide-insensitive palmitoyl-CoA oxidation and enoyl-CoA hydratase). Cytochrome P-450 IVA1 (or a very closely related isoenzyme in the same gene family) was a major constitutive haemoprotein in rat kidney microsomes and actively supported the omega-hydroxylation of lauric acid. This activity was induced 2-3-fold by peroxisome proliferators such as clofibrate, di-(2-ethylhexyl)phthalate, bezafibrate and nafenopin. By using a cDNA probe to the cytochrome P-450 IVA1 gene in Northern blot analysis, we have shown that increased renal and hepatic omega-hydroxylation of lauric acid, after treatment with peroxisome proliferators is a consequences of a substantial increase in the mRNA coding for this haemoprotein. In addition, programming of an in vitro rabbit reticulocyte translation system with both renal and hepatic RNA resulted in the synthesis of similar (if not identical) cytochrome-P-450-IVA1-related polypeptides. Furthermore, we have provided Western blot evidence that both rat liver and kidney microsomes contain two closely related cytochrome P-450 IVA1 polypeptides, the major one characterised by a monomeric molecular mass of 51.5 kDa (identical to authentic, purified hepatic cytochrome P-450 IVA1) and a minor one of 52 kDa. The kidney-supported fatty acid omega-hydroxylase activity was refractory to inhibition by a polyclonal antibody to liver cytochrome P-450 IVA1, which may be related to the existence of two closely related (but immunochemically distinct) fatty acid hydroxylases in this tissue. Our studies have also demonstrated that certain of the compounds tested (including clofibrate, bezafibrate and nafenopin) induced renal fatty acid beta-oxidation, mirroring the increased omega-hydroxylase activity in the endoplasmic reticulum. Our studies have also indicated that the kidney was more refractory to induction of the endoplasmic reticulum and peroxisomal fatty-acid-oxidising enzymes than the liver. Taken collectively, our data is strongly suggestive of a possible linkage of the renal fatty acid oxidative enzymes in these two organelles, a situation that also occurs in the liver. In addition, our studies have provided a possible conceptual framework that may rationalise the decreased susceptibility of the k     

Differential induction of peroxisomal populations in subcellular fractions of rat liver

In rat liver, peroxisome proliferators induce profound changes in the number and protein composition of peroxisomes, which upon subcellular fractionation is reflected in heterogeneity in sedimentation properties of peroxisome populations. In this study we have investigated the time course of induction of the peroxisomal proteins catalase, acyl-CoA oxidase (ACO) and the 70 kDa peroxisomal membrane protein (PMP70) in different subcellular fractions. Rats were fed a di(2-ethylhexyl)phthalate (DEHP) containing diet for 8 days and livers were removed at different time-points, fractionated by differential centrifugation into nuclear, heavy and light mitochondrial, microsomal and soluble fractions, and organelle marker enzymes were measured. Catalase was enriched mainly in the light mitochondrial and soluble fractions, while ACO was enriched in the nuclear fraction (about 30%) and in the soluble fraction. PMP70 was found in all fractions except the soluble fraction. DEHP treatment induced ACO, catalase and PMP70 activity and immunoreactive protein, but the time course and extent of induction was markedly different in the various subcellular fractions. All three proteins were induced more rapidly in the nuclear fraction than in the light mitochondrial or microsomal fractions, with catalase and PMP70 being maximally induced in the nuclear fraction already at 2 days of treatment. Refeeding a normal diet quickly normalized most parameters. These results suggest that induction of a heavy peroxisomal compartment is an early event and that induction of 'small peroxisomes', containing PMP70 and ACO, is a late event. These data are compatible with a model where peroxisomes initially proliferate by growth of a heavy, possibly reticular-like, structure rather than formation of peroxisomes by division of pre-existing organelles into small peroxisomes that subsequently grow. The various peroxisome populations that can be separated by subcellular fractionation may represent peroxisomes at different stages of biogenesis.     

Effect of triton WR-1339 on peroxisomal enzymes of rat liver

The effect of Triton WR-1339 on peroxisomal enzymes of rat liver was studied. The dose vs. response relationships of peroxisomal enzyme activities to Triton WR-1339 were first examined 3.5 days after injection. Catalase activity was reduced to 50% of that of the control at a dose of 200 mg per 100 g body weight; it was found that the decrease depended on the dose of this compound. Urate oxidase activity was not significantly affected. D-Amino acid oxidase activity showed intermediate behavior. The activities of these enzymes were found to be reduced more markedly at 2 days than at 3.5 days after injection, and subsequently the levels of the activities recovered. At 2 days after injection of a dose of 200 mg per 100 g body weight, the activities of catalase, D-amino acid oxidase and urate oxidase had decreased to 40, 60 and 60%,respectively, of the control values.It was found that the decreases in the activities of these enzymes caused by Triton WR-1339 had occurred in the large granule fraction, but not in the cytoplasm.Measurement of the specific activity, Ouchterlony gel diffusion and quantitative immunoprecipitation suggested that there was a similarity between the Triton WR-1339-treated and untreated rats in the nature of purified catalases.These results suggest that Triton WR-1339 depresses the activities of liver peroxisomal enzymes, especially the catalase activity.     

Effect of thyroid hormone on peroxisomal flavin enzymes in rat liver and kidney

The effect of thyroid hormone on peroxisomal enzyme activity was studied in thyroidectomized- and T4-administered-thyroidectomized rats. In liver, the activities of isozyme A of L-alpha-hydroxyacid oxidase, D-amino acid oxidase, urate oxidase and catalase were decreased by thyroidectomy, and the diminished enzyme activities were restored by T4 administration to rats. These modifications induced by thyroidectomy or by T4 administration, however, were prominent only in immature animals (20-day-old rats). Although the changes in-alpha-hydroxyacid oxidase and D-amino acid oxidase activities, induced by thyroidectomy or by T4 administration, were also observed in 40-day-old rats, those in urate oxidase and catalase activities were not significant in 40-day-old rats. Acyl CoA oxidase activity was not affected by thyroidectomy or by T4 administration in either 20- or 40-day-old rats. In the kidney, isozyme B of L-alpha-hydroxyacid oxidase activity was reduced by thyroidectomy and the diminished enzyme activity was restored by T4 administration in both 20- and 40-day-old rats. D-Amino acid oxidase and catalase activities in kidney, however, were not significantly modified by thyroidectomy or by T4 administration in either 20- or 40-day-old rats. The results suggest that thyroid hormone can modify the peroxisomal enzyme activity, which is prominent in immature animals.     

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.     

Influence of subtotal hepatectomy on peroxisomes and peroxisomal enzymes of rat liver and isolated liver cell fractions.

The activities of peroxisomal enzymes of rat liver were followed 1 to 10 days after subtotal (60-70%) hepatectomy in homogenates prepared from regenerating livers and in cell fractions isolated from them. Catalase activity was found to be depressed in the total liver homogenate (H) as well as in the mitochondrial (M) and soluble (S) fractions, while it did not change appreciably in the microsomal (Mc) and lysosomal (L) fractions. Alpha-hydroxyacid oxidase behaved in a similar fashion. In contrast to these enzymes, urate oxidase activity remained unchanged in H, whereas it was decreased in M and increased in L and Mc during the first 5 days after operation. These results agree well with the assumption that microbody proliferation is initiated by the fragmentation of large peroxisomes. The different relations of peroxisomal enzyme activities during regeneration time are discussed with respect to the possible existence of various kinds of peroxisomes with different enzyme equipments and with different turnover rates. Biochemical examinations ions were paralleled to morphological and histochemical studies. An early increase in number of peroxisomes was found to occur during the first day after partial hepatectomy, which is accompanied by decrease in particle size. During the first mitotic wave (24-36 hrs post op.) the number of peroxisomes per cell was reduced to about the half. After this time number and size of the particles began to increase. Positive staining of ribosomes was frequently observed in the vicinity of peroxisomes after the application of the cytochemical catalase reaction (alkaline diaminobenzidine medium). This phenomenon is interpreted to represent rather a diffusion artifact than the cytochemical identification of newly synthesized catalase.     

In vivo induction of tight junction proliferation in rat liver

The chronic administration of phalloidin induces an extensive development of tight junctions between rat hepatocytes. The junctional strands lose their predominantly parallel orientation with respect to the canalicular lumen and extend abluminally in irregular patterns which cover large membrane areas at considerable distance from the bile canaliculi. These changes indicate both proliferation and provide further evidence that these junctions are not permanent differentiations of the cell membrane.     

Dehydroepiandrosterone feeding and protein phosphorylation, phosphatases, and lipogenic enzymes in mouse liver

Dehydroepiandrosterone (DHEA) treatment is effective in preventing or delaying the onset of various genetic and induced disorders of mice and rats. Associated with the beneficial therapeutic effects exerted by action of this steroid is the development of hepatomegaly. To determine whether the changes associated with hepatomegaly also involve alterations in activities of tissue enzymes, we evaluated the effects of DHEA (0.45% in food, w/w) on hepatic protein kinases, phosphatases, and lipogenic enzymes in mice of various strains. The rates of fatty acid and cholesterol syntheses also were evaluated. DHEA administration resulted in profound changes in the sodium dodecylsulfate-polyacrylamide gel electrophoresis patterns of endogenous radiophosphorylated proteins obtained by incubation of liver homogenates with (gamma-32P]ATP. These changes were dependent upon the medium used for homogenization. Thus, when homogenates of liver tissue of DHEA-treated mice were prepared in Tris buffer containing sucrose (0.25 M) there was a marked decrease in phosphorylation of the proteins of relative molecular weight approximately 116,000 (Mr approximately 116,000), approximately 82,000, approximately 80,000, approximately 58,000, approximately 56,000, approximately 48,000, approximately 34,000, and approximately 31,000 compared with controls. With liver homogenates of DHEA-treated mice prepared in Tris buffer alone, there was a marked increase in phosphorylation of the proteins of Mr approximately 70,000, approximately 49,000, approximately 34,000, approximately 31,000, and 28,000 compared with controls. Moreover, the specific activity of kinases for endogenous protein acceptors in liver of control mice was higher than that in liver of DHEA-treated animals. The specific activities of casein kinase, cAMP-dependent protein kinase, and cGMP-dependent protein kinase remained unchanged with DHEA treatment, but the specific activity of histone kinase was increased approximately 30%. Long-term administration of DHEA also was associated with increases in the specific activities of liver AMPase and GTPase (approximately two times), but not of other nucleotidases, alkaline phosphatase, acid phosphatase, glucose-6-phosphatase, or phosphotyrosine phosphatase. The activity of hepatic NADP-linked malic enzyme was increased significantly (two to three times) by DHEA treatment of female mice of three different strains, but was unchanged in male C57BL/6 mice. The specific activities of hepatic glucose-6-phosphate dehydrogenase, NADP-linked isocitrate dehydrogenase, and ATP-citrate lyase were not affected significantly by DHEA treatment of mice. The rate of hepatic lipogenesis, determined by incorporation of tritium from 3H2O into fatty acids, was decreased approximately 70% in DHEA-treated mice, while the rate of cholesterol synthesis was increased approximately 44% compared with controls.