Modulation of delta 6 and delta 5 rat liver microsomal desaturase activities by dexamethasone-induced factor

This report supports evidence for the existence of a dexamethasone-induced factor that modulates fatty acid desaturase activities. Dexamethasone at a dose of 1 mg/rat produced a significant decrease in microsomal delta 6 and delta 5 desaturation activity 12 h after the injection. Both desaturase activities were depressed by a soluble factor present in the cytosolic fraction of cells, since the supernatant of microsomes separated at 110,000 X g from hormonal-treated rat liver homogenates, added to crude or washed control microsomes, was able to inhibit in vitro linoleic and homo-gamma-linolenic conversion to gamma-linolenic and arachidonic acids, respectively. The inhibitory factor was loosely bound to microsomes, since it was also present in a soluble fraction obtained after washing crude microsomes from dexamethasone-treated rats with a low-ionic-strength solution. Besides, trypsin digestion deactivates the dexamethasone-induced factor. Therefore, the depressing effect of glucocorticoids on delta 6 and delta 5 desaturation capacity depends on an unchanged protein structure present in the cytosolic fraction of the cell and whose biosynthesis is brought about by hormonal induction.     

The role of cytochrome b5 in delta 12 desaturation of oleic acid by microsomes of safflower (Carthamus tinctorius L.)

The electron donors for the membrane-bound fatty acid desaturases of higher plants have not previously been identified. In order to assess the participation of cytochrome b5 in microsomal fatty acid desaturation, the cytoplasmic domain of microsomal cytochrome b5 was purified from Brassica oleracea, and murine polyclonal antibodies were prepared. The IgG fraction from ascites fluid inhibited 62% of NADH-dependent cytochrome c reduction in safflower (Carthamus tinctorius L.) microsomes. These antibodies also blocked desaturation of oleic acid to linoleic acid in lipids of C. tinctorius microsomes by 93%, suggesting that cytochrome b5 is the electron donor for the delta 12 desaturase.     

Oxidative desaturation of eicosa-8,11-dienoic acid to eicosa-5,8,11-trienoic acid: comparison of different diets on oxidative desaturation at the 5,6 and 6,7 positions

The oxidative desaturation of [1-(14)C]eicosa-8,11-dienoic acid to eicosa-5,8,11-trienoic acid by rat liver microsomes was studied, and the kinetic conditions appropriate to measure the specific activity of the enzyme were determined. A comparative study of the effects of a balanced diet and essential fatty acid-free diets on the oxidative desaturation of oleic and linoleic acids at the 6,7 position and the oxidative desaturation of eicosadienoic acid at the 5,6 position were made. Eicosadienoic acid showed a higher conversion than oleic acid for all the diets. The conversion of oleic and linoleic acids to Delta6 acids was equally increased by fat-free diets with or without added methyl palmitate, whereas the oxidative 5-desaturation of eicosadienoic acid at the 5,6 position was not changed. The effect was apparently independent of the amount of endogenous free fatty acids. The results suggest that the rate-limiting and principal regulatory step in the biosynthesis of eicosa-5,8,11-trienoic acid is the 6-desaturation of oleic acid. The 5-desaturation of eicosadienoic acid was increased by a protein diet and decreased by alloxan diabetes to a lesser extent than the 6-desaturation of linoleic acid. The 5-desaturation of eicosadienoic acid would constitute a secondary regulatory step.     

Evidence for participation of cytochrome b5 in microsomal delta-6 desaturation of fatty acids.

The delta-6 desaturation of linoleic acid to gamma-linolenic acid and oleic acid to 6,9-octadecadienoic acid by rat liver microsomes was investigated. Using a specific antibody prepared against purified rat liver cytochrome b5, we demonstrated that cytochrome b5 participated in delta-6 desaturation of both fatty acids. The reaction products were identified as their methyl ester derivatives by argentation thin-layer chromatography, gas-liquid chromatography, and reductive ozonolysis followed by gas-liquid chromatography.     

Influence of environmental temperature on the fatty acid desaturation and elongation activity of fish (Pimelodus maculatus) liver microsomes.

The effect of environmental temperature on the activity of liver microsomes of fish (Pimelodus maculatus) to desaturate and elongate oleic, linoleic and alpha-linolenic acids was studied. It was found that: 1. Fish kept at 14-15 degrees C had higher desaturation and elongation activity than animals kept at 29-30 degrees C. The ratio of activity was the same for the three fatty acids. 2. A decrease of the environmental temperature increased the V of linoleic acid desaturation to gamma-linolenic acid, but did not modify the approximate Km of the reaction. 3. The inactivation of the delta6-desaturase of microsomes separated from fish kept at 29-30 degrees C and 14-15 degrees C was the same when heated at 40 degrees C. However, the enzyme was deactivated faster when heated at 29-30 degrees C than at 14-15 degrees C. 4. The increase of the delta6-desaturation activity of the microsomes evoked by the decrease of the temperature of the aquarium was mostly compensated for by the correlative decrease of the specific reaction rate of the reaction. For this reason it is assumed that the adaptive change of the desaturation activity of the microsomes with the environmental temperature does not greatly modify the fatty acid composition of the fish.     

Regulation of linoleic acid delta 6-desaturation by a cytosolic lipoprotein-like fraction in isolated rat liver microsomes

A peripheral component of the delta 6-fatty acid-desaturase system of rat liver microsomes has been isolated from the cytosol by ultracentrifugation at a saline density of 1.26 g/ml. It exhibited lipoprotein characteristics with an approximate protein/lipid ratio of 1.22 and free fatty acids and phosphatidylcholine as its main lipid components. Linoleic acid desaturation activity diminished in washed microsomes, since they lost the adsorbed cytosolic fraction. Addition of the factor reactivated the reaction and the recovery was dependent on the concentration of the factor in the medium. Linoleic acid and linoleyl-CoA were bound by the cytosolic fraction. However, the transport of substrate to the desaturase was not apparently a main function of the cytosolic fraction, since transport occurred equally in the absence of the factor. Moreover, the solubilization of linoleyl-CoA was not enhanced and the free monomeric concentration was not altered by the presence of the cytosolic fraction. In addition, the factor did not divert delta 6-desaturase substrate to or from other metabolic pathways such as esterification to phospholipids. gamma-Linolenic acid produced by delta 6-desaturation of linoleic acid in the microsomes inhibited the desaturase, but it was removed by the factor from the membrane towards the cytosol, preventing the inhibition. The anti-inhibitory effect of the cytosolic factor was blockaded by addition of columbinic acid or gamma-linolenic acid to the factor. Moreover, the inhibitory effect of arachidonic acid was not prevented by addition of the cytosolic fraction. These results suggest that the cytosolic fraction studied would optimize the delta 6-desaturation of linoleic acid in vitro in rat liver microsomes by removal of the product, gamma-linolenic acid, as it is formed.     

Mutant and immunochemical studies on the involvement of cytochrome b5 in fatty acid desaturation by yeast microsomes.

The involvement of cytochrome b5 in palmitoyl-CoA desaturation by yeast microsomes was studied by using yeast mutants requiring unsaturated fatty acids and an antibody to yeast cytochrome b5. The mutants used were an unsaturated fatty acid auxotroph (strain E5) and a pleiotropic mutant (strain Ole 3) which requires either Tween 80 and ergosterol or delta-aminolevulinic acid for growth. Microsomes from the wild-type strain possessed both the desaturase activity and cytochrome b5, whereas those from mutant E5 contained the cytochrome but lacked the desaturase activity. Microsomes from mutant Ole 3 grown with Tween 80 plus ergosterol were devoid of both the desaturase activity and cytochrome b5, but those from delta-aminolevulinic acid-grown mutant Ole 3 contained cytochrome b5 and catalyzed the desaturation. The cytochrome b5 content in microsomes from mutant Ole 3 could be varied by changing the delta-aminolevulinic acid concentration in the growth medium, and the desaturase activity of the microsomes increased as their cytochrome b5 content was increased. The antibody to yeast cytochrome b5, but not the control gamma-globulin fraction, inhibited the NADH-cytochrome c reductase and NADH-dependent desaturase activities of the wild-type microsomes. It is concluded that cytochrome b5 is actually involved in the desaturase system of yeast microsomes. The lack of desaturase activity in mutant Ole 3 grown with Tween 80 plus ergosterol seems to be due to the absence of cytochrome b5 in microsomes, whereas the genetic lesion in mutant E5 appears to be located at ther terminal desaturase.     

Short-chain aliphatic alcohols increase rat-liver microsomal membrane fluidity and affect the activities of some microsomal membrane-bound enzymes

n-Butyl and isoamyl alcohols decrease the steady-state fluorescence anisotropy of 1,6-diphenyl-1,3,5-hexatriene and enhance the efficiency of pyrene excimer formation when these probes are incorporated in rat-liver microsomal membrane, suggesting an increase in rotational and translational mobilities. Neither alcohol modifies NADH-ferricyanide reductase activity but both increase NADH-cytochrome c reductase activity. This was interpreted as an increase in the rate of lateral diffusion of the proteins cytochrome b5 and cytochrome b5 reductase as a consequence of the enhanced membrane lipid phase fluidity. Microsomal delta 9 and delta 6 desaturase activities in the presence of isoamyl alcohol were also studied. This alcohol decreases delta 9 desaturation when it is measured at a low substrate concentration (13 microM palmitic acid), but it is not modified when it is measured at a high substrate concentration (66 microM palmitic acid). delta 6 desaturation is diminished by isoamyl alcohol when it is measured with both 13 microM and 66 microM linoleic acid. The influence of isoamyl alcohol on the glucose-6-phosphatase system activity was also studied. In non-detergent-treated microsomes, isoamyl alcohol enhances glucose-6-phosphatase activity. However, if microsomes are previously treated with 0.1% Triton X-100 isoamyl alcohol does not modify this activity. The enhancement of the glucose 6-phosphate transport rate is not due to membrane permeability barrier disruption, since isoamyl alcohol does not modify mannose-6-phosphohydrolase latency. This would suggest that an increase in membrane lipid phase fluidity specifically activates glucose 6-phosphate transport across the membrane.     

Effects of Dietary Fish Oil on Polyunsaturated Fatty Acid Desaturation in Fetal and Postnatal Rats

The effects were determined of dietary fish oil on the polyunsaturated fatty acid desaturation in rats fed on fish oil-containing diets (FS group) and on non-fish oil diets (CN group) during the fetal to postnatal periods. Although the desaturase activity in the liver microsomes of the FS group was higher than that of the CN group before birth, this was not altered by dietary fish oil after birth. However, a lower 20:4n-6 concentration before and after birth, and lower linoleic acid desaturation index ((dihomo-γ-linolenic acid + arachidonic acid)/linoleic acid)) at 10 wk of age in the FS group than in the CN group were observed in the liver microsomal phospholipids. The Δ6-desaturase activity in the brain microsomes of the FS group was lower than that of the CN group. These findings suggest that an intake of dietary fish oil by dams and postnatal rats affected the arachidonic acid concentration due to the decreased desaturase activity in the rats’ microsomes.     

Increased activity of stearoyl-CoA desaturation in liver from rat fed clofibric acid

Male rats were fed a diet containing 0.5% (w/w) p-chlorophenoxyisobutyric acid (clofibric acid), a hypolipidemic drug. Activities of stearoyl-CoA desaturation in hepatic microsomes were increased approx. 4 times following the administration of clofibric acid for 7 days. An increase in the activity of desaturation of stearic acid was also observed in the liver of clofibric acid-fed rats in vivo. The increase in the activity of microsomal stearoyl-CoA desaturation by clofibric acid-feeding was due to the increase in the activity of terminal desaturase as measured by the rate constant for cytochrome b5 reoxidation, but not due to the changes in cytochrome b5 content and NADH-cytochrome b5 reductase activity. Increases in the activity of stearoyl-CoA desaturation by clofibric acid-feeding were also observed in rats of hormonally altered state, such as diabetic rats, hyperthyroid rats and hypothyroid rats. Percentages of octadecenoic acid in total fatty acid of hepatic lipid were increased with the increase in the activity of stearoyl-CoA desaturation.     

Activities of liver microsomal fatty acid desaturases in zinc-deficient rats force-fed diets with a coconut oil/safflower oil mixture of linseed oil

The present study was conducted to investigate the effect of zinc deficiency on fatty acid desaturation in rats fed two different types of dietary fat, a mixture of coconut oil and safflower oil (7∶1, w/w, “coconut oil diet”) or linseed oil (“linseed oil diet”). In order to ensure an adequate food intake, all rats were force-fed by gastric tube. Zinc deficiency caused statistical significant reducion of Δ9-desaturase activity in liver microsomes of rats fed coconut oil diet and tendencial reduction (p<0.15) in rats fed linseed oil diet compared with control rats fed diets with the same type of fat. In agreement with this effect, zinc deficiency in the rats fed both types of dietary fat increased the ratio between total saturated and total monounsaturated fatty in liver phospholipids and liver microsomes. Zinc deficient rats on the coconut oil diet had unchanged Δ6-desaturase activity with linoleic acid as substrate and lowered activity with α-linolenic acid as substrate. In contrast, zinc deficient rats on the linseed oil diet had increased Δ6-desaturase activity with linoleic acid as substrate and unchanged activity with α-linolenic acid. Because linoleic acid is the main substrate for Δ6-desaturase in the rats fed coconut oil diet, and α-linolenic acid is the main substrate in the rats fed linseed oil diet, it is concluded that in vivo Δ6-desaturation was not changed by zinc deficiency in the rats fed both types of dietary fat. Activity of Δ5-desaturase was also not changed by zinc deficiency in the rats fed both dietary fats. Levels of fatty acids in liver phospholipids and microsomes derived by Δ4-, Δ5-, and Δ6-desaturation were not consistently changed by zinc deficiency in the rats fed both types of dietary fat. Thus, the enzyme studies and also fatty acid composition data of liver phospholipids and microsomes indicate that zinc deficiency does not considerably disturb desaturation of linoleic and α-linolenic acid. Therefore, it is suggested that similarities between deficiencies of zinc and essential fatty acids described in literature are not due to disturbed desaturation of linoleic acid in zinc deficiency. The present study also indicates that zinc deficiency enhances incorporation of eicosapentaenoic acid into phosphatidylcholine of rats fed diets with large amounts ofn-3 polyunsaturated fatty acids.     

Effect of diet on linoleic acid desaturation and on some enzymes of carbohydrate metabolism

The effect of diet on the desaturation of linoleic acid to gamma-linolenic acid by liver microsomal preparations, on blood glucose and insulin levels, and on activities of glucokinase, hexokinase, pyruvate kinase, and alpha-glycerophosphate dehydrogenase have been studied. The female rats used in these experiments were maintained on one of the following dietary regimes: (a) fasted, (b) fasted for 96 hr and refed glucose, (c) balanced diet, (d) carbohydrate-free diet, (e) lipid-free diet, or (f) protein-free diet. Fasting for 96 hr caused a decrease of both linoleic acid desaturation and glucokinase and pyruvate kinase activity together with a slight decrease of the blood insulin level. Alpha-glycerophosphate dehydrogenase activity was not modified. Refeeding of glucose for 50 hr increased the conversion of linoleic acid to linolenic acid as well as the activities of all the enzymes studied except alpha-glycerophosphate dehydrogenase. The increase in desaturation, however, was transient. The feeding of a lipid-free diet did not modify the tested parameters. Feeding a carbohydrate-free diet for 96 hr resulted in increased linoleic acid desaturation but decreased glucokinase and pyruvate kinase activity, thus apparently eliminating a putative correlation between the fatty acid desaturating activity and glycolytic activity or blood insulin levels under these experimental conditions. The findings suggest that dietary proteins may play an important role in determining the level of fatty acid desaturation.     

Spontaneous diabetes in BB rats: evidence for insulin dependent liver microsomal delta 6 and delta 5 desaturase activities

We studied linoleic acid delta 5 and dihomo-gamma-linolenic acid delta 5 desaturations, and fatty acid composition, of liver microsomes in the insulin-dependent spontaneously diabetic adult female BB rat. These desaturations were defective along the normo- and hyper-glycemic period and restored during the hypoglycemic period which followed the insulin injection to the diabetic rats. The fatty acid composition of BB rats microsomes was not consistent with the desaturase activities at the different periods of glycemia, probably because other factors than desaturation impairments were involved in the evolution of fatty acid composition.     

Assay for the terminal enzyme of the stearoyl coenzyme A desaturase system using chick embryo liver microsomes.

The NADH-dependent stearoyl CoA desaturase of hepatic microsomes (EC 1.14.99.5) is an enzyme system consisting of cytochrome b5 reductase (EC 1.6.2.2), cytochrome b5, and the terminal desaturase. We have developed a simple method for routine assay of the terminal enzyme based on complementation of the enzyme with chick embryo liver microsomes lacking desaturase activity. Desaturation of [1-14C]stearoyl CoA by the enzyme-microsome mixture is then assayed by thin-layer chromatography of the reaction products and determination of the amount of oleate formed. Microsomes from the livers of starved-refed rats were used as the source of the stearoyl CoA desaturase. The enzyme alone, solubilized and free from cytocrome b5 reductase and cytochrome b5, was unable to catalyze the desaturation of stearoyl CoA. However, after preincubation with chick embryo liver microsomes in the presence of 1% Triton X-100, the enzyme was active. The enzyme activity was linear with time and desaturase protein under the conditions described and depended on the concentrations of Triton X-100 present in the preincubation and the assay. The optimum concentrations of Triton X-100 were 1% for the preincubation and 0.1-0.15% in the assay. The desaturation activity was dependent on NADH and O2, and was inhibited 95% by 1 mM KCN. The use of chick embryo liver microsomes in this method eliminates the need to use purified cytochrome b5 reductase, cytochrome b5, and liposomes for routine assays and greatly reduces the complexities of timing and order of addition encountered in the existing assays.     

The effects of unsaturated fatty acids on hepatic microsomal drug metabolism and cytochrome P-450

1. The effects of unsaturated fatty acids on drug-metabolizing enzymes in vitro were measured by using rat and rabbit hepatic 9000g supernatant fractions. 2. Unsaturated fatty acids inhibited the hepatic microsomal metabolism of ;type I' drugs with inhibition increasing with unsaturation: arachidonic acid>linolenic acid>linoleic acid>oleic acid. Inhibition was independent of lipid peroxidation. Linoleic acid competitively inhibited the microsomal O-demethylation of p-nitroanisole and the N-demethylation of (+)-benzphetamine. 3. The hepatic microsomal metabolism of ;type II' substrates, aniline and (-)-amphetamine, was not affected by unsaturated fatty acids. 4. The rate of reduction of p-nitrobenzoic acid and Neoprontosil was accelerated by unsaturated fatty acids. 5. Linoleic acid up to 3.5mm did not decelerate the generation of NADPH by rat liver soluble fraction, nor the activity of NADPH-cytochrome c reductase of rat liver microsomes. Hepatic microsomal NADPH oxidase activity was slightly enhanced by added linoleic acid. 6. No measurable disappearance of exogenously added linoleic acid occurred when this fatty acid was incubated with rat liver microsomes and an NADPH source. 7. The unsaturated fatty acids used in this study produced type I spectra when added to rat liver microsomes, and affected several microsomal enzyme activities in a manner characteristic of type I ligands.     

Novel Plant Growth Inhibitors and an Insect Antifeedant from Chrysanthemum coronarium (Japanese Name: Shungiku)

The effect of overnight fasting on the dietary protein-dependent change in the fatty acid composition of tissue lipids was studied in rats fed with casein or soybean protein (20%) diets containing 5 or 2% corn oil. The activity of the Δ6-desaturase of liver microsomes, a key enzyme of linoleate metabolism to arachidonate, was depressed significantly by overnight fasting, and the protein effect disappeared, irrespective of the level of dietary fat. The proportion of linoleate in liver phosphatidylcholine was decreased, whereas that of arachidonate was increased after overnight fasting in rats fed with a low fat diet, resulting in an elevation of the linoleate desaturation index. Although the effect of fasting became obscure on a high fat diet, the protein effects were maintained even after fasting. A similar trend was also observed in various lipid fractions. Thus, the effect of dietary protein on the polyunsaturated fatty acid profile was not modulated by overnight fasting, particularly when a minimal amount of linoleic acid was supplied.     

Characterization of fatty acid desaturase activity in rat lung microsomes.

Preparations of rat lung microsomes containing 0.030-0.050 nmole of cytochromes P-450 and b5 per mg microsomal protein have been observed to contain significant levels of fatty acid desaturase activity. Both stearoyl CoA and palmitoyl CoA are desaturated to their monounsaturated analogues, oleic acid and palmitoleic acid, respectively. Activity (per mg microsomal protein) of the lung preparations varied according to the diet of the animals prior to killing in the order: fat free diet greater than normal rat chow greater than starvation. All preparations exhibited approximately 50% inhibition when incubated in the presence of 0.10 mM CN-. Maximal activity was obtained with the 0.50 mM NADH less activity with equal amounts of NADPH, and there was no synergistic interaction of NADH and NADPH together. The rate of desaturation was linear with protein concentrations between 0.15-1.5 mg microsomal protein/incubation at incubation times up to 8 min. A pH optimum range of 7.0-7.4 was observed. For all variables of fatty acid desaturase activity which were examined, the rate of desaturation of stearoyl CoA was approximately twice that for palmitoyl CoA. These results indicate that the same fatty acid desaturation system which is functional in the liver is also present in significant amounts in mammalian lungs.     

Effect of epinephrine on the oxidative desaturation of fatty acids in the rat.

The effect of epinephrine on the oxidative desaturation of fatty acids by liver microsomal preparations of rats has been studied. Administration of epinephrine (1 mg/kg body weight) produced a significant decrease in desaturation of [l-14C]=linoleic acid to gamma-linolenic acid and of [L-14C]alpha-linolenic acid to actadeca-6,9,12,15-tetraenoic acid 12 hr after the infection. Lower doses produced a lesser effect on the delta6-desaturation activity. Epinephrine administration modified the V max of linoleic acid desaturation but not the K m. There was also a slight increase in palmityl desaturation activity. The effect of epinephrine on delta6-desaturation activity was postulated to be mediated through an enhancement of the intracellular cyclic AMP levels that lead to an increase of a glucose metabolite. This metabolite would inhibit delta6-desaturation activity.     

Effect of glucocorticoids on the oxidative desaturation of fatty acids by rat liver microsomes.

The effect of glucocorticoids on the oxidative desaturation of fatty acids by liver microsomal preparations of rats has been studied. Hydrocortisone produced a significant decrease in the conversion of [1-14C]linoleic acid to gamma-linolenic acid and [1-14C]eicosa-8, 11, 14-trienoic acid to arachidonic acid. Triamcinolone and dexamethasaone were more active than hydrocortisone in depressing delta 6 and delta 5 fatty acid desaturating activity in liver microsomes. The glucocorticoids evoked a maximal response approximately 24 hr after admission. Palmitic acid conversion to palmitoleic acid showed no statistically significant changes by any of the glucocorticoids. The mechanism of action of glucocorticoids is apparently different from other hyperglycemic hormones that produce similar effects.     

Zinc deficiency and the desaturation of linoleic acid in rats force-fed fat-free diets

Recent studies with rats force-fed zinc-deficient diets containing various types of fat failed to demonstrate a role of zinc in desaturation of linoleic acid. The present study was conducted to investigate the effect of zinc deficiency on desaturation of linoleic acid in rats that were initially force-fed fat-free diets to stimulate activity of desaturases. Therefore, rats were fed zinc-adequate and zinc-deficient fat-free diets for 6 d. After that period, the groups were divided and half of the rats continued feeding the fat-free diet for another 3.5 d whereas the other half was switched to a fat diet by supplementing the fat-free diet with 5% safflower oil. In order to assess desaturation of linoleic acid, fatty acid compositions of liver phosphatidylcholine, ethanolamine, and-serine were considered, particularly levels of individual (n-6) polyunsaturated fatty acids (PUFA). Levels of total and individual (n-6) PUFA were similar in zinc-adequate and zinc-deficient rats fed the fat-free diet throughout the experiment. Addition of 5% safflower oil increased levels of total and individual (n-6) PUFA in both zinc-adequate and zinc-deficient rats. However, total (n-6) PUFA in all types of phospholipids were higher in zinc-adequate rats than in zinc-deficient rats. Additionally, in zinc-deficient rats there were changes of (n-6) PUFA levels typical for impaired Δ5 and Δ6 desaturation: linoleic acid and dihomo-γ-linolenic acid were elevated; arachidonic acid, docosatetraenoic acid, and docosapentaenoic were lowered by zinc deficiency. Therefore, the study shows that zinc deficiency impairs desaturation of linoleic acid in rats force-fed fat-free diets and therefore supports results from former convential zinc deficiency experiments suggesting a role of zinc for desaturation of linoleic acid.