Thor S. Eikhom

On the mechanism of induction of the enzyme systems for peroxisomal β-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.

cAMP inhibits translation by inducing Ca2+/calmodulin-independent elongation factor 2 kinase activity in IPC-81 cells

Treatment of IPC‐81 cells led to inhibition of protein synthesis, which was accompanied by an increase in the average size of polysomes and a decreased rate of elongation, indicating that it involved inhibition of peptide chain elongation. This inhibition was also associated with increased phosphorylation of elongation factor eEF2 (which inhibits its activity) and enhanced Ca2+/calmodulin‐independent activity of eEF2 kinase. Previous work has shown that phosphorylation of eEF2 kinase by cAMP‐dependent protein kinase (cAPK) in vitro induces such activator‐independent activity, and the present data show that such a mechanism can occur in intact cells to link physiological levels of cAPK activation with inhibition of protein synthesis.

The peroxisomal β-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.

On the mechanism of stimulation of peroxisomal β-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)

Ribosomal rna metabolism in synchronized plasmacytoma cells.

Abstract Ribosome synthesis and metabolism has been studied in a plasmacytoma cell line synchronized by isoleucine deprivation. Ribosomal RNA (rRNA) was characterized by gel electrophoresis. The rate of ribosome synthesis (as measured by the appearance of labelled rRNA in the cytoplasm) varied greatly during the cell cycle. It was low during the G l phase, increased rapidly during the S phase, remained high during part of the G 2 phase, and dropped to a minimum during mitosis. A slowdown in the increasing rate of RNA synthesis was observed during the middle of the S phase. No significant decrease in the total nucleotide pool per cell could be observed during the S phase. The accumulation of RNA (as determined by absorbance measurements) was highest during the S and G 2 phases. Pulse labelling of rRNA and pulse chase experiments demonstrated that newly synthesized ribosomal subunits entered into free polysomes to the highest extent during the S phase. The percentage of membrane-bound polysomes of total polysomes increased during the G 1 phase, as did the percentage of labelled rRNA in the membrane-bound fraction.

Effect of the growth conditions on the ratio between native 40 S and 60 S ribosomal subunits in various cell types

Abstract A non-stoichiometric ratio of the native ribosomal subunits was observed in rapidly growing transformed cells in suspension culture. Both the plasmacytoma cell line MPC-11 and mouse L cells (line 929) demonstrated a great excess of the small ribosomal subunits in the cytoplasm. These cells contained a large quantity of native 40 S subunits and a small quantity of native 60 S subunits, and the difference was more pronounced for cells having a generation time of 16 h than for cells having a generation time of 24 h. Ribosomes isolated from starved cells did not contain this great excess of the small subunits. This was true both for L cells grown in Falcon flasks and for Krebs II ascites cells isolated shortly before the tumour became fatal to the host. Under these growth conditions the pool of native 60 S subunits was greatly increased compared to the same pool in rapidly growing cells in suspension culture. Liver cells isolated from mice infected with Krebs II ascites cells or MPC-11 cells apparently contained a lower ratio between native 40 S and 60 S ribosomal subunits (as judged from sucrose gradients) than did liver cells isolated from uninfected animals. These experiments indicate that the ability to display a non-stoichiometric ratio between the ribosomal subunits may not be restricted to transformed cells only.

A growth-dependent excess of 40 S ribosomal subunits in plasmacytoma cells grown in suspension culture.

Abstract When the plasmacytoma cell line MPC-11 was grown at high cell density in suspension culture (limited feeding) an approximately stoichiometric ratio between ribosomal 60S and 40S subunits was found in the cytoplasm. When grown in suspension culture at low cell density (well-fed cells) the ratio of 40–60S ribosomal subunits was higher than stoichiometric. A transfer from high cell density (poor growth conditions) to low cell density (good growth conditions) by addition of fresh medium strongly favoured accumulation of native 40S subunits in the cytoplasm compared with the accumulation of native 60S subunits. Both absorbance measurements and rRNA labelling experiments suggested the presence of an excess of 40S subunits under these conditions. The ratio between the ribosomal subunits in the cytoplasm of this transformed cell line, therefore, is strongly influenced by the growth conditions.