Comparative effect of a protein diet on the desaturation,elongation and simultaneous desaturation and elongation of linoleic acid

The effect of a protein diet on the biosynthesis of polyunsaturated fatty acids of the linoleic acid family was studied by incubation of rat liver microsomes with labeled linoleic acid. The incubation was performed in desaturating, elongating and desaturating-elongating conditions. In desaturating conditions, linoleic acid was converted to γ-linolenic acid, whereas in elongating conditions it was converted to 20∶2, 22∶2 and 24∶2. In desaturating-elongating conditions, labeling was found in γ 18∶3, 20∶2, 20∶3, 20∶4 and 22∶2. A protein diet increased the oxidative desaturation of linoleic acid to γ-linolenic and arachidonic acid biosynthesis, whereas the elongating reaction was not enhanced in the experimental conditions tested. It is suggested that the main controllable step in the linoleic acid family is the oxidative desaturation of linoleic acid to γ-linolenic acid.     

Effect of epinephrine on the oxidative desaturation of fatty acids in the rat adrenal gland

Delta-6 and Δ5 desaturation activity of rat adrenal gland microsomes was studied to determine the effect of microsomal protein and the substrate saturation curves. This tissue has a very active Δ6 desaturase for linoleic and α-linoleic acids and a Δ5 desaturase for eicosa-8,11,14-trienoic acid. The administration of epinephrine (1 mg/kg body weight) 12 hr before killing, produced approximately a 50% decrease in desaturation of [1-¹⁴C]linoleic acid to γ-linolenic acid, [1-¹⁴C]α-linolenic acid to octadeca-6,9,12,15-tetraenoic acid and [1-¹⁴C]eicosa-8,11,14-trienoic acid to arachidonic acid. A 30% decrease in Δ5 desaturation activity was also shown after 7 hr of epinephrine treatment. The changes on the oxidative desaturation of the same fatty acids in liver microsomes were similar. No changes were observed in the total fatty acid composition of adrenal microsomes 12 hr after epinephrine treatment. Mechanisms of action of the hormone on the biosynthesis of polyunsaturated fatty acids in the adrenal gland are discussed.     

The activating effect of dietary protein on linoleic acid desaturation

The desaturation of¹⁴C-1-linoleic acid to γ-linolenic acid and their incorporation into the microsomal lipids of rats fed on a balanced diet and a protein diet were measured in vitro. It was shown that a protein diet does not change significantly the distribution of the radioactivity among the different lipidic fractions compared to the animals fed on a balanced diet. However the microsomal desaturation of linoleic acid to γ-linolenic acid increased in the rats maintained on a protein diet. Besides, the amount and composition of the free fatty acids present in the microsomes of the animals fed on both diets were similar enough to discard the hypothesis that they may modify the desaturation of linoleic acid produced by the diet. The enzymic activity of the linoleyl desaturase of liver microsomes of animals fed on a protein diet, measured in substrate saturating conditions, is greater than in animals with balanced diet. Consequently the results support the hypothesis that a protein diet increases specifically the desaturating activity of the microsomes.     

Circadian rhythm of fatty acid desaturation in mouse liver

A study was made of the diurnal changes in liver microsomal desaturation of labeled stearic, linoleic and α-linolenic acids to oleic, γ-linolenic and octadeca-6,9,12,15-tetraenoic acids, respectively. C3H-S mice were used and were exposed to light-dark cycles. A circadian rhythm was observed for stearic acid desaturation, and a different one for linoleic acid. Linoleic and α-linolenic desaturation had similar responses in the day cycle. This would indicate that different mechanisms control the oxidative desaturations of the fatty acids in the 9 and 6 carbons. The fatty acid composition of the whole liver and liver microsomes also showed variations. Remarkable oscillations were observed for stearic and oleic acids. Neither the total protein synthesis nor the free fatty acid concentration in the microsomes followed a rhythm parallel to the desaturation of the studied fatty acids. The injection of cycloheximide 4 hr before measuring the desaturation modified the circadian variation of both the 9 and 6 desaturations. The modification induced by cycloheximide was considered to indicate that both variations are related to the synthesis of specific proteins but not to that of a degradative or inhibitory protein.     

Effect of malonyl-CoA on Δ6 desaturation activity of rat liver microsomes

The effect of malonyl-CoA on linoleic acid desaturation and elongation reactions of rat liver microsomes was studied. Under strict desaturation conditions, the in vitro microsomal conversion of linoleic acid to γ-linolenic acid is time-dependent. When malonyl-CoA was added to the aforementioned incubation medium, linoleic acid was desaturated to γ-linolenic acid and elongated to its higher homologues. Under these conditions, Δ6 desaturation activity, calculated by adding γ-18∶3, 20∶3 and 20∶4 acids, was neither inhibited nor activated by malonyl-CoA. These results indicate that the elongation of γ-linolenyl-CoA coupled to the desaturation of linoleic acid did not modify Δ6 desaturase activity.     

Age-related changes in Δ6 and Δ5 desaturase activities in rat liver microsomes

Age-related changes in Δ6 desaturation of [1-¹⁴C]α-linolenic acid and [1-¹⁴C]linoleic acid and in Δ5 desaturation of [2-¹⁴C]dihomo-γ-linolenic acid were studied in liver microsomes from Wistar male rats at various ages ranging from 1.5 to 24 mon. Desaturase activities were expressed both as specific activity of liver microsomes and as the capacity of whole liver to desaturate by taking into account the total amount of liver microsomal protein. Δ6 Desaturation of α-linolenic acid increased from 1.5 to 3 mon and then decreased linearly up to 24 mon to reach the same desaturation capacity of liver measured at 1.5 mon. The capacity of liver to desaturate linoleic acid increased up to 6 mon and then remained constant, whereas microsomal specific activity was equal at 1.5 and 24 mon of age. The capacity of liver to convert dihomo-γ-linolenic acid to arachidonic acid by Δ5 desaturation decreased markedly from 1.5 to 3 mon. It then increased to reach, at 24 mon, the same level as that observed at 1.5 mon. Age-related changes in the fatty acid composition of liver microsomal phospholipids at the seven time points studied and of erythrocyte lipids at 1.5 and 24 mon were consistent with the variations in desaturation capacity of liver. In particular, arachidonic acid content in old rats was slightly higher than in young rats whereas contents in linoleic and docosahexaenoic acids varied little throughout the life span. The results suggest that, in liver, the activity of desaturases may be regulated in the course of aging to maintain a constant level of polyunsaturated fatty acids in cellular membranes.     

Effect of low levels of dietary fish oil on fatty acid desaturation and tissue fatty acids in obese and lean rats

The effect of very low levels of dietary long-chain n−3 fatty acids on Δ6 desaturation of linoleic acid (18∶2n−6) and α-linolenic acid (18∶3n−3), and on Δ5 desaturation of dihomo-γ-linolenic acid (20∶3n−6), in liver microsomes and its influence on tissue fatty acids were examined in obese and lean Zucker rats and in Wistar rats. Animals fed for 12 wk a balanced diet containing ca. 200 mg of long-chain polyunsaturated n−3 fatty acids per 100 g of diet were compared to those fed the same amount of α-linoleic acid. Low amounts of long-chain n−3 fatty acids greatly inhibited Δ6 desaturation of 18∶2n−6 and Δ5 desaturation of 20∶3n−6, while Δ6 desaturation of 18∶3n−3 was not inhibited in Zucker rats and was even stimulated in Wistar rats. Inhibition of the biosynthesis of long-chain n−6 fatty acids was reflected in a decrease in arachidonic acid (20∶4n−6) content of serum lipids when fasting, and also in the phospholipid fatty acids of liver microsomes. On the contrary, heart and kidney phospholipids did not develop any decrease in 20∶4n−6 during fish oil ingestion. Docosahexaenoic acid (22∶6n−3), present in the dietary fish oil, was increased in serum lipids and in liver microsome, heart, and kidney phospholipids.     

Effect of catecholamines and β-blockers on linoleic acid desaturation activity

The effect of catecholamines and adrenergic blocking agents on the oxidative desaturation of linoleic acid in rat liver microsomes was studied. Epinephrine (1 mg/kg/body weight) produced a significant decrease on the conversion of [1-¹⁴C]linoleic acid to γ-linolenic acid. The effect of epinephrine was blocked by single injections of the β blockers propranolol (10 mg/kg body weight) or dichloroisoproterenol 30 min before the hormone treatment. Isoproterenol (100 μg/kg body weight) produced a significant decrease on the activity of the linoleyl-CoA desaturase. The effect of the catecholamines was postulated to be mediated through β receptors by an enhancement of the intracellular levels of cyclic AMP.     

Reciprocal interactions in the desaturation of linoleic acid into γ-linolenic and eicosa-8,11,14-trienoic into arachidonic

Variable concentrations of [I¹⁴C] linoleic acid and [I¹⁴C] eicosa-8,11,14-trienoic acid were incubated with liver microsomes in a medium containing the necessary cofactors for fatty acid desaturation. The conversion of linoleic into γ-linolenic acid and eicosatrienoic into arachidonic acid were mutually inhibited and the inhibition depended on the concentration of the fatty acids incubated.     

Dietary arachidonic acid and hepatic desaturation of fatty acids in obese zucker rats

The effect of low levels of dietary arachidonic acid (20:4n-6) on Δ6 desaturation of linoleic acid (18:2n-6) and α-linolenic acid (18:3n-3), and on Δ5 desaturation of dihomo-γ-linolenic acid (20:3n-6) were studied in liver microsomes of obese Zucker rats, in comparison with their lean littermates. Fatty acid composition of serum total lipids and of phospholipids from liver microsomes and from total heart and kidney was determined to see whether modifications of desaturation rate, if any, were reflected in the tissue fatty acid profiles. Animals fed for 12 wk on a balanced diet, containing 20:4n-6 and 18:2n-6, were compared to those fed 18:2n-6 only. The low amount of dietary 20:4n-6 greatly inhibited Δ6 desaturation of 18:2n-6 and Δ5 desaturation of 20:3n-6, whereas Δ6 desaturation of 18:3n-3 was slightly increased in obese rats. Inhibition of the biosynthesis of long-chain n-6 fatty acids by dietary arachidonic acid was only slightly reflected in the 20:4n-6 content of liver microsome phospholipids. On the contrary, the enrichment of serum total lipids and heart and kidney phospholipids in this fatty acid was pronounced, more in obese than in lean animals. Our results show that, although the desaturation rate of the n-6 fatty acids in liver microsomes was greatly decreased by the presence of arachidonic acid in the diet, the tissue phospholipid content in arachidonic acid was not depressed. The potentiality of synthesis of eicosanoids of the 2 family from this fatty acid is consequently not lower, especially in obese rats, in which certain tissues are deficient in arachidonic acid, in comparison with their lean littermates.     

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.     

The Production of Conjugated α-Linolenic, γ-Linolenic and Stearidonic Acids by Strains of Bifidobacteria and Propionibacteria

Conjugated fatty acids are regularly found in nature and have a history of biogenic activity in animals and humans. A number of these conjugated fatty acids are microbially produced and have been associated with potent anti-carcinogenic, anti-adipogenic, anti-atherosclerotic and anti-diabetogenic activities. Therefore, the identification of novel conjugated fatty acids is highly desirable. In this study, strains of bifidobacteria and propionibacteria previously shown by us and others to display linoleic acid isomerase activity were assessed for their ability to conjugate a range of other unsaturated fatty acids during fermentation. Only four, linoleic, α-linolenic, γ-linolenic and stearidonic acids, were converted to their respective conjugated isomers, conjugated linoleic acid (CLA), conjugated α-linolenic acid (CLNA), conjugated γ-linolenic acid (CGLA) and conjugated stearidonic acid (CSA), each of which contained a conjugated double bond at the 9,11 position. Of the strains assayed, Bifidobacterium breve DPC6330 proved the most effective conjugated fatty acid producer, bio-converting 70% of the linoleic acid to CLA, 90% of the α-linolenic acid to CLNA, 17% of the γ-linolenic acid to CGLA, and 28% of the stearidonic acid to CSA at a substrate concentration of 0.3 mg mL⁻¹. In conclusion, strains of bifidobacteria and propionibacteria can bio-convert linoleic, α-linolenic, γ-linolenic and stearidonic acids to their conjugated isomers via the activity of the enzyme linoleic acid isomerase. These conjugated fatty acids may offer the combined health promoting properties of conjugated fatty acids such as CLA and CLNA, along with those of the unsaturated fatty acids from which they are formed.     

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 ATP on the microsomal desaturation of unsaturated fatty acids

The effect of ATP on the microsomal desaturation of linoleic acid to γ-linolenic acid was studied in a system in vitro with the following results: (1) preincubation of rat liver microsomes with ATP alone in N₂ or in the presence of CoA and Mg⁺⁺ followed by subsequent incubation with 1-¹⁴C-linoleic acid plus NADH in O₂ resulted in enhancement of 1-¹⁴C-linoleic acid desaturation when compared with control samples in which no preincubation was performed; (2) the preincubation of the microsomes with ATP, Mg⁺⁺ and CoA in the presence of 1-¹⁴C-linoleic acid decreased the desaturation of the labeled acid to γ-linolenic acid upon subsequent incubation with NADH, as a consequence of incorporation of the acid into the microsomal lipids; (3) the increase of linoleic acid desaturation depended on the ATP concentration during preincubation and followed a sigmoidal curve. It was specific for ATP, and neither GTP, CTP, ADP nor AMP produced a similar effec. However, GTP or CTP could replace ATP as a cofactor in the microsomal desaturation of free linoleic acid to γ-linolenic, suggesting that directly or indirectly they may activate conversion of the free acid to linoleyl-CoA; (4) preincubation of microsomes with ATP activated the acylation of CoA. However, this activation showed no quantitative correlation with enhancement of the desaturation reaction; (5) addition of ATP also stimulated conversion of linoleyl-CoA to γ-linolenic acid. This enhancement was not related to inhibition of the linoleyl-CoA hydrolase; (6) however, in spite of these results, preincubation with ATP did not increase the initial velocity of linoleic acid or linoleyl-CoA desaturation; (7) preincubation of microsomes with ATP also increased the 6-desaturation of oleic acid and α-linolenic acid but did not increase the 9-desaturation of plamitic and stearic acid.     

Tissue phospholipid fatty acid composition in the diabetic rat

Tissue phospholipid fatty acid compositions in streptozotocin-induced diabetic rats were studied. The major changes in liver, plasma, erythrocyte and heart were increased proportions of linoleic and dihomo-γ-linolenic acids and a decreased proportion of aracchidonic acid. The latter was not significantly changed in phospholipids of kidney, adrenal gland and testis. Skin fatty acids in diabetic rats showed an increase in the proportion of arachidonic acid and a reduction in the proportion of linoleic acid. The fatty acid desaturating activity in diabetes may be regulated differently in different tissues.     

Borago officinalis oil: Fatty acid fractionation by immobilized Candida rugosa lipase

γ-Linolenic acid (Z,Z,Z-6,9,12-octadecatrienoic acid), a very important polyunsaturated fatty acid is found in the free fatty acid fraction prepared by the hydrolysis of borage oil. Our aim was to enrich this fraction in γ-linolenic acid using selective esterification. Candida rugosa lipase was used as catalyst after immobilization on the following ion-exchange resins: Amberlite IRC50, IRA35, IRA93, and Duolite A7, A368, A568. In every case, immobilization modified the lipae’s specificity: palmitic, stearic, oleic, and linoleic acids were preferentially esterified compared to γ-linolenic acid, thus allowing a γ-linolenic acid enrichment of 3.0.     

The desaturation step in the animal biosynthesis of polyunsaturated fatty acids

The aerobic desaturation of unsaturated fatty acids in the microsomes has been systematically explored and some of the relevant experimental works have been assembled. The contribution of the microsomal electron transport chain and lipoproteic structure of the microsomes is analyzed. Evidences of linoleyl-CoA and α-linolenyl-CoA being desaturated by the same enzyme are presented. The linoleic acid desaturation is shown to be different in different tissues and to decrease with aging. The effect of competitive reactions with acids of the same or different series, the competition of desaturation and transesterification, and dietary and hormonal contributions to unsaturated fatty acids desaturation are summarized. Alltrans linoleic acid and elaidic acid were not desaturated in our experimental conditions by rat liver microsomes. From the bulk of data collected, a hypothetical model of 6-olefinase is drawn. The main features of the model are: The existence of both binding and desaturating sites; the binding of acyl-CoA and enzyme through hydrophobic forces of the Van der Waals type and weak polar attractions due to double bonds; the orienting binding characteristics of double bond proximate to the place where olefination will take place; and the importance of enzyme conformation that requires that the orienting double bond must have acis structure.     

Acyl group distributions in tissue lipids of rats fed evening primrose oil (λ-linolenic plus linoleic acid) or soybean oil (α-linolenic plus linoleic acid)

Three groups of rats were fed diets with either 10 weight percent (wt%) of evening primrose oil, safflower oil or soybean oil for 11 weeks. Diets contained 7.1 wt% linoleic acid +0.8 wt% γ-linolenic acid, 7.6 wt% linoleic acid, or 5.3 wt% linoleic acid +0.7 wt% α-linolenic acid, respectively. In liver mitochondria as well as in heart, dietary γ-linolenic acid did not affect the fatty acid profiles of phosphatidylcholnes (PC), phosphatidylethanolamines (PE) or cardiolipins (CL), whereas dietary α-linolenic acid caused an increased formation of (n−3) polyunsaturated fatty acids (PUFA). The liver Δ6− and Δ5-desaturase activities determined in vitro were not affected by the dietary fats. In brain PE, which are rich in C22− and C20-(n−3) PUFA, as well as in testes PC and PE, which are rich in (n−6) PUFA, no effects were found from a partial replacement of dietary linoleic acid with γ-linolenic acid or α-linolenic acid. In kidney PC, PE, phosphatidylinositol (PI) and CL, 20∶3(n−6) was moderately elevated to ca. 1% following intake of γ-linolenic acid, whereas partial replacement of linoleic acid with α-linolenic acid was followed by increased deposition of 22∶6(n−3) in PC and PE of testes and kidney. Thus, no general effect of evening primrose oil on the content of (n−6) PUFA in rat tissue phospholipids was observed, wheras a significant incorporation of γ-linolenic acid into liver and adipose tissue triglycerides was found.     

Δ6 and Δ5 desaturase activities in liver from obese zucker rats at different ages

Δ6 Desaturation of linoleic acid (18∶2 n−6) and Δ5 desaturation of dihomo-γ-linolenic acid (20∶3 n−6) were measured in liver microsomes from genetically obese Zucker rats (fa/fa) and from their lean littermates (Fa/−). Both groups were fed a balanced commercial diet. The rats were 6, 9 and 12 weeks old, which corresponded to stages in their active growth period. The content of total fatty acids and n−6 polyunsaturated fatty acids in whole liver and liver microsomes was also determined in order to ascertain how the desaturase activities measuredin vitro reflected regulation of essential fatty acid metabolismin vivo. Contrary to values obtained for Δ6 desaturation, Δ5 desaturation at nonsaturating substrate levels were lower in obese rats than in lean controls. In contrast, at saturating substrate level, the maximal Δ5 desaturase activities were the same in both phenotypes and they increased with age. Study of Δ5 desaturation kinetics (1/V vs 1/S) showed that V_m did not differ between 12-week-old obese and lean rats, whereas K_M in obese rats was much lower than in controls, expressing the very low affinity of the enzyme for the substrate in obese animals. The fatty acid composition of liver lipids reflected the results of desaturase activitiesin vitro. In particular, the ratios 20∶4 n−6/20∶3 n−6 were lower in obese rats than in lean rats, which can be explained by the lower conversion of 20∶3 n−6 into 20∶4 n−6 by Δ5 desaturation. However, the total amount of 20∶4 n−6 in the whole liver did not differ between phenotypes, whatever their age. This work presents evidence for a relationship between the changes in fatty acid compositional data in hepatic total lipids, total lipids of liver microsomes and modifications of fatty acid desaturase activities in the genetically obese Zucker rat.     

Cloning of Δ12- and Δ6-desaturases from Mortierella alpina and recombinant production of γ-linolenic acid in Saccharomyces cerevisiae

Two cDNA clones with homology to known desaturase genes were isolated from the fungus Mortierella alpina. The open reading frame in one clone encoded 399 amino acids and exhibited Δ12-desaturase activity when expressed in Saccharomyces cerevisiae in the presence of endogenous fatty acid substrate oleic acid. The insert in another clone contained an open reading frame encoding 457 amino acids and exhibited Δ6-desaturase activity in S. cerevisiae in the presence of exogenous fatty acid substrate linoleic acid. Expression of the Δ12-desaturase gene under appropriate media and temperature conditions led to the production of linoleic acid at levels up to 25% of the total fatty acids in yeast. When linoleic acid was provided as an exogenous substrate to the yeast cultures expressing the Δ6-desaturase activity, the level of γ-linolenic acid reached 10% of the total yeast fatty acids. Co-expression of both the Δ6- and Δ12-desaturase cDNA resulted in the endogenous production of γ-linolenic acid. The yields of γ-linolenic acid reached as high as 8% of total fatty acids in yeast.