Δ9 desaturase activity in normal mouse liver and hepatoma SS1K

The activity of Δ9 desaturase was determined in the microsomal fraction of normal mouse liver and hepatoma SS1K in the presence of the 105,000 × g supernatant. Neither hepatic nor hepatoma soluble fractions were able to modify the low desaturating capacity. Two enzymes from the microsomal electron transport chain associated with Δ9 desaturase, namely NADH-cytochrom b₅ reductase and NADH-cytochrome C reductase were also measured. The results indicate that the low Δ9 desaturase activity in hepatoma SS1K could be related to the reduced amount of desaturase.     

Effect of ethanol administration on fatty acid desaturation

The effect of ethanol on the fatty acid desaturation by rat liver has been studied using liquid diets of different composition. Acute ethanol administration increased triacylglycerols of total liver lipids, but did not modify significantly the lipidic composition of microsomes. The Δ6 and Δ5 desaturases were inhibited by ethanol whereas the Δ9 desaturase and fatty acid synthetase were apparently modified only by diet composition. NADH-cytochrome (cyt.) c reductase was partially inhibited, whereas NADH-cyt. b₅ reductase remained practically unaltered and NADPH-cyt. c reductase activity was enhanced. Decreased electrons supplied by the microsomal cyt. b₅ electron transport chain would not be the reason for the inhibition of Δ6 and Δ5 desaturases by ethanol.     

Daily variations of the biosynthesis and composition of fatty acids in rats fed on complete and fat-free diets

This report describes the daily changes in fatty acid composition and fatty acid desaturation in rats feeding on a complete diet and a fat-free diet successively. Rats on a complete diet showed a good homeostasis in the percentage of fatty acid in plasma, with a possible palmitic acid rhythm, but the fat-free diet initiated an essential fatty acid-deficient pattern in a few hours. The light-dark period in animals feeding on a complete diet motivates a feeding rhythm that causes changes in linoleic and arachidonic acids in the whole liver and microsomes that are related to Δ6 and Δ5 desaturase activities. The patterns of Δ6 and Δ5 desaturase changes were different. Linoleic acid intake during the dark periods (complete diet feeding) caused a decrease of Δ6 desaturase activity and the activation of Δ5 desaturation that led to an increase of arachidonic acid biosynthesis. The feeding of a fat-free diet eliminated the rhythm observed in linoleic and arachidonic acid composition in the liver and changed the desaturase rhythms. The Δ9 desaturase activity in the liver also showed a daily rhythm in the complete-diet period that disappeared with the change to a fat-free diet, while the activity increased markedly. A negative correlation existed between the percentage of linoleic acid in the liver and the Δ9 desaturase activity. However, no correlation was found between Δ9 desaturase activity and the percentage of 16∶1 and 18∶1 in the complete-diet period.     

Effect of environmental temperature changes on rat liver fatty acid desaturases

Female rats warm-adapted at 30–32 C for 20–25 days and then shifted to 13–15 C for 12, 24, 48, 72 and 120 hr showed that Δ9 desaturase and fatty acid synthetase activity decay after 24 hr of cold exposure, while Δ6 and Δ5 desaturases were increased after this period of time. These results were confirmed by an increase of arachidonic acid of heart and liver microsomes phosphatidylcholine and a decrease of oleic acid. Neither NADH-cyt b₅ reductase nor NADH-cyt c reductase activity of liver microsomes were significantly affected. Male rats warm-adapted under the same conditions and then shifted to 13–15 C for 120 hr did not show significant changes in fatty acid synthetase, Δ9 and Δ6 desaturases and enzymes of the microsomal electron transport chain. Therefore, the desaturase response to environmental temperature changes could be plausibly linked to female hormones.     

Hepatic Δ9 and Δ6 desaturase activities during the recovery period following carbon tetrachloride poisoningdesaturase activities during the recovery period following carbon tetrachloride poisoning

The liver microsomal Δ⁹ and Δ⁶ desaturase activities have been studied in rats with carbon tetrachloride-induced hepatitis. Immediately after poisoning, significant decreases were observed for both types of desaturase activity. However, recovery kinetics were slower for the Δ⁶ desaturase than for the Δ⁹ desaturase. The activities of NADH-ferricyanide and NADH-cytochrome C reductases, proteins involved in the electron transfers associated with microsomal desaturation, were also measured. There was a fall in both activities after poisoning, but this decrease was less than that of the desaturase activities.     

Effect of dietary fatty acids on Δ5 desaturase activity and biosynthesis of arachidonic acid in rat liver microsomes

The effect of different fatty acids supplemented to a fat-free diet on the activity of Δ5 desaturase was studied. Fat-free diet produces a reduction in the conversion of eicosa-8,11,14-trienoic acid to arachidonic acid. The addition of thecis-ω6 acids, linoleic, γ-linolenic or arachidonic to the diet produces an increase of eicosatrienoic acid desaturation, shifting Δ5 desaturase activity towards the controls on a balanced diet. This reactivation is apparently produced by induction of enzyme biosynthesis since linoleate effect was suppressed by simultaneous cycloheximide injection. On the contrary, no changes in Δ5 desaturation activity were found when the diet was supplemented with palmitic or 9-trans,12-trans-linoleic acid. The changes on the activity of Δ5 desaturase were compared with the fatty acid composition of plasma and liver microsomes.     

Stearoyl-coenzyme a desaturase activity in the mammary gland and liver of lactating rats

Stearoyl-CoA desaturase activity in microsomes from lactating rat mammary gland is very low (0.05–0.15 nmol/min/mg of protein) regardless of lactating time. In such microsomes, reductase activities and content of cytochrome b₅ are several-fold lower than in normal rat liver microsomes. Preincubation of the mammary microsomes with purified terminal desaturase gives a 55-fold stimulation of stearoyl-CoA desaturase activity, whereas preincubation with cytochrome b₅ has no effect. However, preincubation of mammary microsomes with both cytochrome b₅ and terminal desaturase results in a 200-fold stimulation of overall desaturation. These observations suggest that negligible stearoyl-CoA desaturase activity in lactating rat mammary microsomes is due to a low cytochrome b₅ content and the absence of terminal enzyme. The hepatic stearoyl-CoA desaturase activity increases 9-fold during lactation. There is little or no change in the NADH-cytochrome c reductase activity or in the concentration of cytochrome b₅ during this period, but the activity of the terminal desaturase increases with the increase of overall desaturation. These results suggest that liver is one of the more important sources of oleic acid for milk triglycerides.     

Effects of zinc deficiency and castration on fatty acid composition and desaturation in rats

The effects of zinc deficiency and testosterone on fatty acid composition of plasma lipids and microsomes of liver, intestine and testes were studied. The activities of fatty acid desaturase (Δ6 and Δ5) in rat liver and testes were also measured. A significant decrease in the level of arachidonic acid was observed in plasma of normal rats fed the zinc-deficient diet. Castration significantly decreased arachidonic acid but increased 20∶3 fatty acid, which is negligible in normal rats. Testosterone and zinc administration restored arachidonic acid to normal values. Zinc deficiency does not significantly change the fatty acid profile in liver, but castration decreased both arachidonic and 22∶6 fatty acid. Intestinal mucosal microsomes showed that the predominant fatty acid in this tissue, palmitic acid, is independent of zinc status, whereas polyunsaturated fatty acids 18∶2 and 20∶4 were decreased by zinc-deficient diet or castration. Zinc deficiency sharply decreased 22∶5 fatty acid and to some extent, other polyunsaturated fatty acids in testis microsomes. These changes in fatty acids are in agreement with increased Δ9 desaturation and decreased Δ5 desaturase activity. In testes, both Δ6 and Δ5 desaturase activities are decreased in zinc deficiency. It appears that zinc influences the conversion of linoleic to arachidonic acid, whereas testosterone influences Δ6 desaturase activity. The data suggest that zinc deficiency may be one of the important factors in the causation of polyunsaturated fatty acid deficiency, which in turn, may induce serum hypertriglyceridemia.     

Fatty acid metabolism in marine fish: Low activity of fatty acyl Δ5 desaturation in gilthead sea bream (Sparus aurata) cells

Marine fish have an absolute dietary requirement for C₂₀ and C₂₂ highly unsaturated fatty acids. Previous studies using cultured cell lines indicated that underlying this requirement in marine fish was either a deficiency in fatty acyl Δ5 desaturase or C_18–20 elongase activity. Recent research in turbot cells found low C_18–20 elongase but high Δ5 desaturase activity. In the present study, the fatty acid desaturase/elongase pathway was investigated in a cell line (SAF-1) from another carnivorous marine fish, sea bream. The metabolic conversions of a range of radiolabeled polyunsaturated fatty acids that comprised the direct substrates for Δ6 desaturase ([1-¹⁴C]18∶2n−6 and [1-¹⁴C]18∶3n−3), C_18–20 elongase ([U-¹⁴C]18∶4n−3), Δ5 desaturase ([1-¹⁴C]20∶3n−6 and [1-¹⁴C]20∶5n−3), and C_20–22 elongase ([1-¹⁴C]20∶4n−6 and [1-¹⁴C]20∶5n−3) were utilized. The results showed that fatty acyl Δ6 desaturase in SAF-1 cells was highly active and that C_18–20 elongase and C_20–22 elongase activities were substantial. A deficiency in the desaturation/elongation pathway was clearly identified at the level of the fatty acyl Δ5 desaturase, which was very low, particularly with 20∶4n−3 as substrate. In comparison, the apparent activities of Δ6 desaturase, C_18–20 elongase, and C_20–22 elongase were approximately 94-, 27-, and 16-fold greater than that for Δ5 desaturase toward their respective n−3 polyunsaturated fatty acid substrates. The evidence obtained in the SAF-1 cell line is consistent with the dietary requirement for C₂₀ and C₂₂ highly unsaturated fatty acids in the marine fish the sea bream, being primarily due to a deficiency in fatty acid Δ5 desaturase activity.     

Effect of different acids with Δ9,12-dienoic structures on Δ9 desaturation activity in rat liver microsomes

The effect of oral administration, for 24 or 48 hr, of different octadeca fatty acids containing a 9,12-dienoic structure on the fatty acid composition and Δ9 desaturation activity of liver microsomes of rat fed a fat-free diet was studied. The ethyl esters of linoelaidic and γ-linolenic acids, the methyl ester of linoleic acid and free columbinic acid were administered to rats maintained on a fat-free diet. The supplementation of the fat-free diet with linoelaidate produced no relevant changes in the fatty acid composition pattern of liver microsomes and did not modify the percentage of conversion of palmitic to palmitoleic acid. The addition of linoleate or γ-linolenate to the fat-free diet returned liver microsome Δ9 desaturation activity toward the control and partially restored the liver microsome fatty acid spectrum found in the fat-free diet. Columbinic acid (5-trans-9-cis,12-cis-18∶3), which cannot be transformed into arachidonic acid, also decreased the Δ9 desaturation activity enhanced by the fat-free diet and evoked changes in the microsomal fatty acid composition similar to those produced by the ω6 fatty acids. These results suggest that the modulation of Δ9 desaturase activity evoked by dietary administration of unsaturated acids of ω6 series would depend on thecis double bond configuration of these acids.     

Effect of L-triiodothyronine on Δ9 desaturase activity in liver microsomes of male rats

Male rats injected with a single saturating dose of L-triiodothyronine (T₃) showed, after a lag time of approximately eight hr, a sharp rise in Δ9 desaturase activity. Desaturase activity reached a plateau which was 1–1.2 times above the base line levels of rats which were not hormone-treated. The plateau was maintained for five days in animals which were kept on daily hormone-treatment. The increase in Δ9 desaturase activity by T₃ required ongoing protein synthesis, because the increase in enzymatic activity due to hormone treatment was completely abolished in the presence of cycloheximide. These findings suggest that cycloheximide may block the induction of Δ9 desaturase by T₃ and/or inhibit the synthesis of protein(s) essential to the desaturation-response to T₃. Modifications observed in liver microsomal fatty acid composition in T₃ treated rats were independent of the effect on desaturation. It is suggested that other factors, such as diet, membrane lipid synthesis and degradation, as well as fatty acid turnover and oxidation, could be involved in affecting the fatty acid composition of thyroid hormone-treated rats.     

Identification and Functional Expression of a Δ9-Fatty Acid Desaturase from <Emphasis Type="Italic">Psychrobacter urativorans</Emphasis> in <Emphasis Type="Italic">Escherichia coli</Emphasis>

The Δ9-fatty acid desaturase is a key enzyme in the synthesis of unsaturated fatty acids. The fatty acid composition of membrane phospholipids in Psychrobacter urativorans is characterized by a high degree of desaturation at Δ9 position. Based on CODEHOP-mediated PCR strategy, a novel gene designated as PuFAD9, putatively encoding a Δ9-fatty acid desaturase (PuFAD9), was isolated from P. urativorans. The gene consists of 1,455 bp and codes for 484 amino acids. Analysis of the amino acid sequence reveals three histidine clusters and a hydropathy profile, typical for membrane-bound desaturases. Activity of the PuFAD9 protein, recombinantly expressed in Escherichia coli was confirmed by GC-MS analysis of the cellular fatty acid composition. It was found that the ratio between palmitoleic and palmitic acid in E. coli cells heterologously expressing the PuFAD9 gene was significantly affected by IPTG induction and the growth temperature.     

Changes in fatty acid desaturation in hepatic and intestinal tissues induced by intestinal resection

We have studied the activities of Δ9, Δ6 and Δ5 desaturases in rat liver and jejunum when the entero-hepatic circulation was interrupted by either 50 or 75% intestinal resection. Desaturase activities in caecal mucosa were also determined in control and operated rats. Distal small bowel resection significantly altered the activities of desaturase enzymes in liver, jejunum and caecum. Thus, the Δ9 desaturase activity was lower in hepatic microsomes from operated animals, and this decrease was not related to the extent by which the intestine was removed. However, a significant increase in both Δ6 and Δ5 desaturases was found in these animals compared to shamoperated rats, the increase in both desaturases being higher after 75% than after 50% intestinal resection. The activities of Δ9 and Δ5 desaturases were significantly increased in jejunal mucosa of resected rats. The activity of Δ6 desaturase was increased only in 50% resected-animals. An increase in Δ6 desaturase activity was observed in caecal mucosa after resection, together with a decrease in Δ9 desaturase and no change in Δ5 desaturase activities. Enzyme activities were highest in the liver relative to the activity in jejunal and caecal homogenates. In sham rats, the caecal mucosa showed higher Δ9 and Δ6 but lower Δ5 desaturase activities than in jejunal mucosa.     

Effect of cold environment on hepatic microsomal Δ6 and Δ9 desaturase activity of male rats

Male rats maintained at 24 C and then shifted to 5 C for 5 days increased food intake and decreased in growth rate and food conversion. No modification was observed in Δ6 desaturase activity, while Δ9 desaturase activity decreased after this period of time. These results were confirmed by liver microsomal and mitochondrial fatty acid composition. The phospholipid composition of liver microsomes was unaltered, whereas in mitochondria, phosphatidylcholine and sphingomyelin decreased and phosphatidylethanolamine increased due to the cold environment. The influence of food intake and weight changes on fatty acid metabolism was studied using (i) rats maintained at 5 C with restricted food intake to match the food intake of those kept at 24 C with food ad libitum and (ii) rats maintained at 24 C whose food intake was also restricted so that their growth rate would be the same as that of rats maintained at 5 C with food ad libitum, respectively. These results indicate that the negative metabolic balance state of these cold conditions is not an active factor modifier of Δ6 desaturase activity, whereas it decreases Δ9 desaturase activity, reflecting the lipogenic characteristics of the latter enzyme.     

Studies on the inhibition of the desaturases by cyclopropenoid fatty acids

Unwashed rat liver microsomes were used to study the inhibition of the Δ⁶ and Δ⁹ desaturases by cyclopropenoid fatty acids with the ring structure about the 9,10 or 6,7 carbon atoms. The 9,10 cyclopropenoid acid (sterculic acid) is shown to be an effective inhibitor of only Δ⁹ desaturase and then only in the presence of MgCl₂ and coenzyme A (prepresence of MgCl₂ and coenzyme A (presumably due to the formation of sterculoyl-CoA). Two 6,7 cyclopropenoid acids of different chain lengths showed no marked inhibition of either the Δ⁶ or Δ⁹ desaturase. By the use of [³H]-sterculic acid, it has been shown that under conditions of high inhibition of the Δ⁹ desaturase the inhibitor is not covalently attached to the enzyme at any point. This disproves older ideas on the mechanism of inhibition that assumed reaction between the cyclopropenoid ring and sulphydryl groups on the enzymes. Cyclopropenoid fatty acids are named according to the number of carbon atoms in the chain; thus sterculic acid is a C₁₈ cyclopropenoid fatty acid.     

Calcium deficiency modifies polyunsaturated fatty acid metabolism in growing rats

Fatty acid desaturase activities were determined in liver microsomes from calcium-deficient rats and compared to calcium-sufficient ones. The calcium-deprived diet (0.5 g/kg) administered for 60 d caused a 30% inhibition in the Δ5 desaturase activity and a 45–55% decrease in Δ6 and Δ9, respectively, facts that cannot be attributed to a reduction in food intake. In vitro addition of calcium, ethyleneglycol-bis(β-aminoethyl ether)N,N-tetraacetic acid, and/or cytosol fractions from control or calcium-deficient rats to microsomes from both groups of animals indicates that the reduced desaturase capacities would be the consequence of an indirect effect of calcium deprivation. The present work shows that the reduced unsaturated fatty acid biosynthesis might be the result of modifications in the physicochemical properties of microsomal membranes. Such changes could also be derived from the inhibition of phospholipase A₂ activity induced by calcium deficiency.     

Relationship between mouse liver Δ9 desaturase activity and plasma lipids

This study was undertaken to investigate the total plasma fatty acid composition and the relationship between plasma triacylglycerol (TG) levels and liver Δ9 desaturase activity in mice fed n−3 and/or n−6 fatty acid or hydrogenated coconut oil (HCO) (maximum 25 mg/g) supplemented diets. Generally, plasma TG levels and Δ9 desaturase activity were inversely correlated with the ratio of the sum of long chain n−3 fatty acids to 18∶2n−6 and to the ratio of the sum of long chain n−3 fatty acids to 18∶n−3, but they were positively correlated with the ratio of products and substrates (18∶1/18∶0) of the enzyme in plasma total lipids. The n−3 fatty acid (mainly 20∶5n−3) enriched diet, when compared to the HCO diet at 21 d, caused a significant reduction in plasma TG levels but not in Δ9 desaturase activity. However, a marked reduction in plasma TG content (50–60%) and Δ9 desaturase activity (55–70%) was observed when both 20∶5n−3 and 18∶3n−6 were supplemented in the diet. The plasma TG levels and Δ9 desaturase activity rose again when the animals were fed the HCO diet or chow. The results suggest that low dose supplementation of a mixture of n−3 (mainly 20∶5n−3) and n−6 (18∶3n−6) fatty acids modified both plasma TG content and liver Δ9 desaturase activity, in parallel.     

Sesamin is a potent and specific inhibitor of Δ5 desaturase in polyunsaturated fatty acid biosynthesis

Incubation with sesame oil increases the mycelial dihomo-γ-linolenic acid content of an arachidonic acid-producing fungus,Mortierella alpina, but decreases its arachidonic acid content [Shimizu, S., K. Akimoto, H. Kawashima, Y. Shinmen and H. Yamada (1989)J. Am. Oil Chem. Soc. 66, 237–241]. The factor causing these effects was isolated and identified to be (+)-sesamin. The results obtained in experiments with both a cell-free extract of the fungus and with rat liver microsomes demonstrated that (+)-sesamin specifically inhibits Δ5 desaturase at low concentrations, but does not inhibit Δ6, Δ9 and Δ12 desaturases. Kinetic analysis showed that (+)-sesamin is a noncompetitive inhibitor (K_i for rat liver Δ5 desaturase, 155 μM). (+)-Sesamolin, (+)-sesaminol and (+)-episesamin, also inhibited only Δ5 desaturases of the fungus and liver. These results demonstrate that (+)-sesamin and related lignan compounds present in sesame seeds or its oil are specific inhibitors of Δ5 desaturase in polyunsaturated fatty acid biosynthesis in both microorganisms and animals. On leave from Suntory Ltd.     

Effects of dietary triolein and sunflower oil on insulin release and lipid metabolism in zucker rats

Obese and lean male Zucker rats were fed ad libitum on diets containing either 50 (L) or 200 (H) g/kg diet of either triolein (T) or sunflowerseed oil (S). The specific activity of the hepatic microsomal Δ9 desaturase enzyme was depressed in both lean and obese rats fed the HS diet compared with the other three diets. The fatty acid composition of liver and subcutaneous white adipose tissue lipids were consistent with a lower Δ9 desaturation activity in rats fed the H diets, particularly for the HS diet. In both genotypes, microsomal Δ9 desaturase activity and the ratio of 16∶1/(16∶0+16∶1) fatty acids in liver lipids were inversely related to the proportion of 18∶2 in liver lipid. Plasma insulin concentrations and rates of glucose-stimulated insulin release in vivo were higher in obese rats compared with lean rats, and plasma insulin levels were higher in rats fed S compared with T. There was no relationship between Δ9 desaturase activity and either plasma insulin concentration or rates of insulin release in vitro. These findings suggest that hepatic Δ9 desaturase activity of Zucker rats is responsive to changes in the proportion of 18∶2 in liver lipids but is not affected by changes in insulin secretion.     

Suppression by dexamethasone of isoproterenol-mediated changes in fatty acyl-CoA desaturase activity ofTetrahymena microsomes

Preincubation ofTetrahymena pyriformis cells with dexamethasone inhibited the microsomal fatty acyl-CoA desaturase activities of isoproterenol-induced modulation; that is, an increase in Δ⁹-desaturase activity accompanied by a decrease in Δ¹²-desaturase activity. Although isoproterenol caused an increase in Δ¹²-terminal component activity with decreased Δ¹²-terminal component activity, dexamethasone reduced these isoproterenol-mediated activity changes. In cells treated with dexamethasone prior to isoproterenol administration, stimulation of cyclic AMP accumulation by isoproterenol was inhibited. These results suggest that dexamethasone may repress isoproterenol modulation of the activity of terminal components (cyanide-sensitive factor) in the fatty acyl-CoA desaturase system by reducing the cyclic AMP level.