On the mechanism of stimulation of peroxisomal beta-oxidation in rat heart by partially hydrogenated fish oil.

By feeding rats a diet containing 20% (w/w) partially hydrogenated fish oil (PHFO), an apparent 6.3-fold increase in the cyanide insensitive palmitoyl-CoA-dependent NAD+ reduction was observed for the heart peroxisomal fractions. This finding was confirmed by a 7.6-fold and 7.9-fold increase in the specific activity of fatty acyl-CoA oxidase, with palmitoyl-CoA and erucoyl-CoA as the substrates, respectively. Immunoblots after SDS-PAGE of rat heart peroxisomal fractions revealed a 12-fold increase in the 52 kDa fatty acyl-CoA oxidase (FAO) subunit for PHFO-fed rats, whereas the 72 kDa subunit of FAO and several other peroxisomal proteins (including the trifunctional enzyme delta 3,delta 2-enoyl-CoA isomerase, 2-enoyl-CoA hydratase, 3-hydroxyacyl-CoA dehydrogenase) increased only 2- to 3-fold. The increase in the 52 kDa subunit was markedly higher than the increase in the steady-state mRNA level of FAO (2.0-fold), and is most likely caused by a rather selective stabilization of the 52 kDa FAO subunit. Interestingly, PHFO feeding caused a larger increase in fatty acyl-CoA oxidase and catalase activities than did clofibrate in the heart. The opposite was the case in the liver, especially for fatty acyl-CoA oxidase. Rats fed a semisynthetic diet containing 6% (w/w) erucic acid (C22:1(n - 9), cis) or brassidic acid (C22:1(n - 9), trans) revealed a 5-fold and 3-fold increase vs. the control (pellet fed) rats in heart FAO activity, respectively, as well as a proportional and selective increase in the specific content of 52 kDa FAO subunit. Thus, the relatively high content of C22 monoene fatty acids appears to be one of the main factors responsible for the increase in rat heart peroxisomal FAO activity during PHFO feeding. However, the PHFO diet increased the heart peroxisomal FAO activity more than diets containing a similar amount of C22:1 in the form of erucic or brassidic acid, and additional compounds of lipid or a more xenobiotic nature may also play a role. SDS-PAGE electrophoresis of highly purified rat liver peroxisomes revealed that the specific content of polypeptides with mobilities corresponding to that of the beta-oxidation enzyme system, increased by a factor of < 2 as a result of feeding the PHFO diet. The 3.1-fold increase in cyanide insensitive palmitoyl-CoA-dependent NAD+ reduction was comparable to the increase (4.1-fold) in the acyl-CoA oxidase activity.(ABSTRACT TRUNCATED AT 400 WORDS)

The peroxisomal beta-oxidation enzyme system of rat heart. Basal level and effect of the peroxisome proliferator clofibrate.

Peroxisomes, isolated from homogenates of rat hearts (myocard), contain a beta-oxidation enzyme system which is indistinguishable from that found in liver, but the total capacity of beta-oxidation is only 0.8% of the liver value (expressed per g of tissue). Fatty acyl-CoA oxidase was assayed by an H2O2 based fluorescent assay avoiding important interfering side reactions. The presence of polypeptides with electrophoretic and immunological properties similar to the beta-oxidation enzymes of liver peroxisomes, was demonstrated by immunoblotting using polyclonal antibodies. The level of 72 and 52 kDa subunits of fatty acyl-CoA oxidase (FAO), quantitated by an anti-FAO1-16 peptide antibody, was only 1% of the level in liver (expressed per g of tissue). Immunoblots of one-dimensional (1-D) SDS-PAGE of rat heart and liver peroxisomal fractions revealed a 60 kDa subunit of the fatty acyl-CoA oxidase in addition to the known 72 and 52 kDa subunits. Immunoblots of two-dimensional (2-D) IEF/SDS-PAGE revealed that all subunits are strongly basic polypeptides, with a microheterogeneity, which probably represents deamidations of the polypeptides. The 2-D immunoblot also revealed another group of polypeptides with M(r) 72 kDa of less basic isoelectric point, possibly representing an isoform of fatty acyl-CoA oxidase. Substrate specificity studies revealed the highest Vmax values with C10-C12. For the very long-chain fatty acids C20-C24, the monoenes revealed much higher Vmax values than the saturated fatty acids. Administration of the classical peroxisome proliferator clofibrate resulted in a similar increase in the fatty acyl-CoA oxidase activity and the 72 and 52 kDa subunits of FAO in the heart. The response (activity) was found to change from 2.2-fold increase in young (34 days) to 11.1-fold increase in adult (76 days) rats. In contrast to liver, where the ratio of the increase in FAO mRNA to the increase in FAO activity was about 4, this ratio in heart was about 0.5.

A fluorometric assay of acyl-CoA oxidase activity by a coupled peroxidatic reaction: elimination of interfering side reactions.

We have developed a simple, reliable, and sensitive method for the assay of peroxisomal fatty acyl-CoA oxidase (FAO, EC 1.13.-) in subcellular fractions. It is based on a peroxidase-linked oxidation of 4-hydroxyphenylacetic acid to a fluorescent compound [M.S. Poosch and R.K. Yamasaki (1986) Biochim. Biophys. Acta 884, 585-593]. Our method eliminates the contribution of important interfering side reactions, notably those due to the presence of reducing agents, which function as competitive substrates to 4-hydroxyphenylacetic acid. Rapidly reacting thiol groups are of particular importance, notably CoASH present endogenously (e.g. in peroxisomes and mitochondria) or formed by enzymatic hydrolysis of acyl-CoA. Alkylation of the thiol compounds by N-ethylmaleimide eliminates this disturbing side reaction, and increases the amount of fluorescent product in the coupled peroxidatic reaction. The method is suitable for routine assay of FAO activity in a wide range of tissues, notably in those with a low specific peroxisomal beta-oxidation activity and/or a high content of reducing agents. As an example of this we have included data from rat heart peroxisomal fractions. The effect of alkylation of sulfhydryl groups in the incubation mixture also applies to other oxidase reactions based on H2O2-coupled peroxidatic reactions, if the oxidase itself does not contain functional sulfhydryl groups.

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.

On the mechanism of induction of the enzyme systems for peroxisomal beta-oxidation of fatty acids in rat liver by diets rich in partially hydrogenated fish oil.

In this paper, we describe the early biochemical changes in liver cells that occur in rats fed a semisynthetic diet containing 20% (w/w) partially hydrogenated fish oil. Within hours the level of ornithine decarboxylase (ODC) increased, peaked at about 24 h (11-fold increase) and returned to subnormal levels within 48 h. The diet evoked a similar rapid increase in the cellular level of mRNA for the bifunctional enzyme of peroxisomal beta-oxidation (enoyl-CoA hydratase: beta-hydroxyacyl-CoA dehydrogenase (HD)) (12-fold), followed by increases in the specific content of HD protein (3-fold) and the capacity for beta-oxidation in peroxisomes (5.3-fold). The cellular level of long-chain acyl-CoA increased 2.1-fold. By contrast, no significant changes were observed in the specific activities of ornithine decarboxylase, peroxisomal beta-oxidation activity and microsomal omega-hydroxylation as well as the level of long-chain acyl-CoA in livers of rats fed (1 week) diets containing 20% (w/w) soybean oil with added 3 or 6% (w/w) of either elaidic acid (18:1(11) (trans)), brassidic acid (22:1(13) (trans)) or erucic acid (22:1(13) (cis)). Expression of normal levels of mRNA for the bifunctional enzyme was also found. Morphometric analyses revealed no proliferation of peroxisomes in these fatty acid-supplemented diets, in contrast to that observed with the partially hydrogenated fish oil diet. These results are consistent with the proposal (Flatmark, T., Christiansen, E.N. and Kryvi, H. (1983) Biochim. Biohys. Acta 753, 460-466) that components in dietary oils, different from C22:1 cis and trans fatty acids, are responsible for the pleiotropic responses evoked in target cells. Thus, the pattern of response induced by partially hydrogenated fish oil mimics those induced by xenobiotic compounds collectively termed peroxisome proliferators.