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.     

In vitro study of delta-6- and delta-5-desaturation of linoleic and dihomo-gamma-linolenic acids in WKY rats on a high-sodium diet

Delta 6- and delta 5-desaturation activities of linoleic [1(4)C] and dihomo-gamma-linolenic [2(14)C] acids are increased, in vitro, in liver microsomes of normotensive rats WKY, which receive an hypernatriuric diet (NaCl 3%). These results lead to a best known of delta 6- and delta 5-desaturases, which are involved in fundamental steps of linoleic acid metabolites biosynthesis, implicated in blood pressure regulation.     

Immunochemical evidence for the enzymatic difference of delta 6-desaturase from delta 9- and delta 5-desaturase in rat liver microsomes

The enzymatic properties of the three types of microsomal acyl-CoA desaturases, delta 6-, delta 9- and delta 5-desaturases, were immunologically compared using a monospecific antibody raised against the purified linoleoyl-CoA desaturase (delta 6-desaturase). By the double immunodiffusion technique, the anti-delta 6-desaturase antibody showed a single precipitin line to the purified delta 6-desaturase and microsomes treated with Triton X-100, but no line was observed with the partially purified delta 9-desaturase. The antibody even inhibited definitely delta 6-desaturase activity in microsomes, but neither stearoyl-CoA (delta 9-) nor eicosatrienoic acid (delta 5-) desaturations were inhibited. By these immunological investigations it was confirmed that terminal delta 6-desaturase is different enzyme from desaturases delta 9- and delta 5.     

Dual influence of aging and vitamin B6 deficiency on delta-6-desaturation of essential fatty acids in rat liver microsomes

Delta-6-desaturase (D6D) activity is influenced by many nutritional and non-nutritional factors, among which one of the most important is aging. D6D activity could be susceptible to the dual influence of aging itself and of nutritional deficiencies, due to the reduced intake and/or absorption of essential nutrients. Particularly, vitamin B6 deficiency might be a crucial factor for D6D activity in aged people. Using 20 month old Sprague-Dawley rats fed a diet with a subnormal level of vitamin B6, we evaluated D6D activity for linoleic acid (LA) and alpha-linolenic acid (ALA) in liver microsomes, and the fatty acid composition of microsomal total lipids. We observed a diminished D6D activity for LA and also for ALA in vitamin B6-deficient animals, being approximately 63% and 81% respectively of the corresponding activity in control rats. As a consequence, significant modifications in the relative molar content of microsomal fatty acids were observed. The content of arachidonic and docosahexaenoic acid, the main products of the conversion of LA and ALA respectively, decreased, LA content increased and a decrease in the unsaturation index was observed in liver microsomes of B6-deficient rats. The foregoing results suggest that the impairment of D6D activity by vitamin B6 deficiency might be an important factor in decreasing the synthesis of n-6 and n-3 PUFAs. This may be particularly important in aging, where D6D activity is already impaired.     

Studies on the delta 5-desaturation in ergosterol biosynthesis in yeast.

Studies were carried out on the delta 5-desaturation reaction in ergosterol biosynthesis with a particulate fraction of cell-free extract of yeast. A reduced pyridine nucleotide coenzyme and molecular oxygen were required for the reaction. It was shown that the enzyme activity is located in a fraction corresponding to microsomes. The reaction was inhibited by KCN, but not by CO. Menadione and potassium ferricyanide inhibited the NADPH- and NADH-dependent reactions, respectively, and cytochrome c inhibited both of them. These results suggested an involvement in delta 5-desaturation of a mixed function oxidase system resembling that for the fatty acyl-CoA desaturation reaction.     

ACTH depresses delta 6 and delta 5 desaturation activity in rat adrenal gland and liver

The in vivo and in vitro effect of ACTH on the biosynthesis of polyunsaturated fatty acid of rat adrenal gland and liver was studied. The administration of ACTH to intact rats produced a significant decrease in the conversion of [1-14C]linoleic acid to gamma-linolenic acid, [1-14C]alpha-linolenic acid to octadeca-6,9,12,15-tetraenoic acid, and [1-14C]eicosa-8,11,14-trienoic acid to arachidonic acid in liver and adrenal microsomes. Isolated adrenocortical cells and hepatocytes obtained from animals treated with ACTH showed a decrease in the incorporation and desaturation of exogenous [1-14C]eicosa-8,11,14-trienoic acid. The addition of ACTH to the incubation medium of adrenocortical cells and hepatocytes isolated from untreated rats also caused a decrease in delta 5 desaturation activity. The effect of ACTH on adrenal and liver desaturases could be produced as a consequence of the release of glucocorticoids, already measured in the experiments. However, the in vitro experiments carried out with hepatocytes isolated from untreated rats, where corticosterone was absent, indicated that ACTH can depress delta 5 desaturation per se.     

Gamma-linolenic acid dietary supplementation can reverse the aging influence on rat liver microsome delta 6-desaturase activity.

We have recently demonstrated that in rats the process of delta 6-desaturation of linoleic and alpha-linolenic acids slows with aging. One method of counteracting the effect of slowed desaturation of linoleic acid would be to provide the 6-desaturated metabolite, gamma-linolenic acid (18:3(n-6) GLA) directly. We have here investigated the 6-desaturation of both linoleic and alpha-linolenic acids in liver microsomes of young and old rats given GLA in the form of evening primrose oil (EPO) (B diet) in comparison to animals given soy bean oil alone (A diet), monitoring also the fatty acid composition of liver microsomes and relating this to the microviscosity of the membranes. In young rats the different experimental diets did not produce any difference in delta 6-desaturase (D6D) activity on either substrate suggesting that, when D6D activity is at or near its peak, the variations in diet tested are unable to influence it. In the old animals the rate of 6-desaturation of linoleic and particularly of alpha-linolenic acid was significantly greater in the B diet fed animals than in the A diet fed. The effects of the diets on the fatty acid composition of liver microsomes were consistent with the findings with regard to 6-desaturation. Administration of GLA partially corrected the abnormalities of n-6 essential fatty acid (EFA) metabolism by raising the concentration of 20:4(n-6) and other 6-desaturated EFAs. Furthermore, the GLA rich diet also increased the levels of dihomo-gamma-linolenic acid and of 6-desaturated n-3 EFAs in the liver microsomes. The microviscosity of microsomal membranes as indicated by DPH polarization was correlated with the unsaturation index of the same membranes. There was a very strong correlation between the two. In both young and old rats the B diet reduced the microviscosity and increased the unsaturation index. However, the effect was much greater in the old animals.     

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.     

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.     

Kinetic analysis of delta-6-desaturation in liver microsomes: influence of gamma-linoleic acid dietary supplementation to young and old rats.

Previous experiments demonstrated the ability-of a gamma-linoleic acid (GLA) dietary supplementation (as evening primrose oil--EPO) to counteract the fall off in delta-6-desaturase (D6D) activity of linoleic acid and alpha-linoleic acid in aged rats. Kinetic parameters of the D6D were determined in order to test the possibility that there may be a significant influence of GLA administration to young and aged rats on the Vm and Km values for 6-desaturation of both the substrates. In young rats GLA supplementation did not affect the kinetic parameters, while in old rats it produced an increase of Vm values of 6-desaturation for both the substrates. Thus the administration of small doses of GLA to old rats might offer substantial protection against the loss of D6D affinity observed in aging, enhancing the capacity of the enzyme itself.     

Differential responses to fasting and subsequent feeding by microsomal systems of rat liver: 6- and 9-desaturation of fatty acids

Hepatic microsomal preparations from nonfasted, fasted, and fasted-fed rats were employed, together with cofactors, in studies of 9-desaturation of stearate-1-(14)C and 6-desaturation of linolenate-1-(14)C. Prior fasting sharply reduced 9-desaturation but did not affect 6-desaturation; feeding restored 9-desaturation. Position of desaturation was determined by permanganate-periodate oxidation and separation of the dicarboxylic acids. Feeding after fasting stimulated both desaturase systems but either dl-ethionine or actinomycin D prevented this. Dietary carbohydrate or saturated fat increased 9-desaturation and dietary protein increased 6-desaturation. Insulin treatment of nonfasted rats increased 9-desaturation but not 6-desaturation. High dietary unsaturated fat (20% safflower oil) stimulated 6-desaturation but inhibited the 9-desaturation response to feeding. The results indicate that the two desaturases are distinct and are inducible in response to different substances.     

Delta-6-desaturation of linoleic and alpha-linolenic acids in aged rats: a kinetic analysis

Kinetic parameters of the delta-6-desaturation reaction were determined using both cis-linoleic and alpha-linolenic acid as substrates in liver microsomes of rats of different ages. The Km value for delta-6-desaturation of linoleic acid increased proportionally to the animal age, while the Vm did not change until 25 months of age. The Km values for alpha-linolenic acid were similar in young and senescent rats; on the contrary there was a significant aging influence on the Vm values. The affinity of the enzyme for the (n-3) series substrate was not so influenced by aging as the affinity for the (n-6) series substrate. This loss of affinity may be a key factor in aging through altering the polyunsaturated fatty acid content and distribution into cellular phospholipids.     

Metabolism of 3 alpha, 7 alpha-dihydroxy-5 beta-cholestanoic acid by rat liver in vivo and in vitro.

The metabolism of 3 alpha, 7 alpha-dihydroxy-5 beta-cholestanoic acid was studied in the bile fistula rats and in preparations from rat liver homogenates. In the bile fistula rats, the main products were chenodeoxycholic acid, alpha-muricholic acid, and beta-muricholic acid. Only small amounts of cholic acid were formed. Incubations of 3 alpha, 7 alpha-dihydroxy-5 beta-cholestanoic acid with microsomes and NADPH yielded as the main product 3 alpha, 6 beta, 7 alpha-trihydroxy-5 beta-cholestanoic acid. The formation of small amounts of 3 alpha, 7 alpha, 12 alpha-trihydroxy-5 beta-cholestanoic acid was shown. The major product in incubations of 3 alpha, 7 alpha-dihydroxy-5 beta-cholestanoic acid with microsomes and the 100,000 g supernatant fluid fortified with ATP was identified as 3 alpha, 7 alpha, 24 xi-trihydroxy-5 beta-cholestanoic acid. This compound was converted into chenodeoxycholic acid and its metabolites in the bile fistula rat.     

Hormonal modulation of delta6 and delta5 desaturases: case of diabetes

Animal biosynthesis of high polyunsaturated fatty acids from linoleic, alpha-linolenic and oleic acids is mainly modulated by the delta6 and delta5 desaturases through dietary and hormonal stimulated mechanisms. From hormones, only insulin activates both enzymes. In experimental diabetes mellitus type-1, the depressed delta6 desaturase is restored by insulin stimulation of the gene expression of its mRNA. However, cAMP or cycloheximide injection prevents this effect. The depression of delta6 and delta5 desaturases in diabetes is rapidly correlated by lower contents of arachidonic acid and higher contents of linoleic in almost all the tissues except brain. However, docosahexaenoic n-3 acid enhancement, mainly in liver phospholipids, is not explained yet. In experimental non-insulin dependent diabetes, the effect upon the delta6 and delta5 desaturases is not clear. From all other hormones glucagon, adrenaline, glucocorticoids, mineralocorticoids, oestriol, oestradiol, testosterone and ACTH depress both desaturases, and a few hormones: progesterone, cortexolone and pregnanediol are inactive.     

Hepatic microsomal delta 6 and delta 5 desaturations of linoleic acid in lactating rats fed with a low-protein diet supplemented or not with methionine

In the lactating rat, a low protein diet (8% casein) decreases strongly liver microsomal delta 6 and delta 5 desaturase activities. The supplementation of the diet with 0.4% methionine improves partly delta 6 desaturase activity but has no effect on delta 5 desaturase activity.     

In vitro desaturation or elongation of monotrans isomers of linoleic acid by rat liver microsomes

Several nutritional studies have shown the in vivo conversion of the 9c,12t-18:2 and 9t,12c-18:2 into long chain polyunsaturated fatty acids (PUFA) containing 20 carbons (geometrical isomers of eicosadienoic and eicosatetraenoic acids). In the present work, some in vitro studies were carried out in order to have precise information on the conversion of these two isomers.In a first set of experiments, studies were focused on the in vitro 6 desaturation, the first regulatory step of the biosynthesis of n-6 long chain PUFA, from 9c,12c-18:2. Rat liver microsomes were prepared and incubated under desaturation conditions with [1-¹⁴C]-9c,12c-18:2 in presence of unlabelled 9c,12t-, 9t,12c- or 9t,12t-18:2. The data show that each trans isomer induced a decrease of the 6 desaturation of the [1-¹⁴C]-9c,12c-18:2, but the 9c,12t-18:2 was the most potent inhibitor (up to 63%). Rat liver microsomes were also incubated with [1-¹⁴C]-9c,12c-18:2, [1-¹⁴C]-9c,12t-18:2 or [1-¹⁴C]-9t,12c-18:2 under desaturation conditions. The results indicated that 18:2 9c,12t is a much better substrate for desaturase than 9t,12c-18:2. Moreover, the conversion levels of [1-¹⁴C]-9c,12t-18:2 was similar to what was observed for its all cis homologue, at low substrate concentration only. In a second set of experiments, in vitro elongation studies of each mono-trans 18:2 isomers and 9c,12c-18:2 were carried out. For that purpose, rat liver microsomes were incubated with [1-¹⁴C]-9c,12c-18:2, [1-¹⁴C]-9c,12t-18:2 or [1-¹⁴C]-9t,12c-18:2 under elongation conditions. The data show that [1-¹⁴C]-9t,12c-18:2 is better elongated than 9c,12c-18:2 while the amount of product formed from [1-¹⁴C]-9c,12t-18:2 was lower than was produced from the 9c,12c-18:2.Thus, the desaturation enzymes presented a higher affinity for the 9c,12t-18:2 whereas the elongation enzyme presented a higher affinity for the 9t,12c-18:2.     

Comparison of the effects of carbon tetrachloride and of 2,3,7,8-tetrachlorodibenzo-p-dioxin on the disposition of linoleic acid in rat liver in vitro

Both 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) and carbon tetrachloride (CCl4) have conspicuous effects on lipid metabolism in rat liver. Although it is generally accepted that CCl4 administration leads to hepatic lipid peroxidation in vivo, conflicting reports from different laboratories make it unclear whether or not lipid peroxidation is involved in the mechanism of toxicity of TCDD. The present study involved pretreating F344 rats with CCl4 or TCDD, then at predetermined times thereafter, giving [U-14C]linoleic acid. A variety of compound classes were monitored in extracts of liver taken 30 min after the label was given. A previously unreported effect of CCl4 was a conspicuous increase in turnover of 1,2-diglycerides. That CCl4 did cause lipid peroxidation was evident from the presence of allylic hydroxyacids not seen in vehicle-treated controls, greatly increased radioactivity in protein-bound material, and decreased levels of arachidonate without decreased synthesis from linolate. Where effects of TCDD pretreatment could be seen, they were much less than the corresponding effects of CCl4. No allylic hydroxyacids were detected in livers of TCDD-treated rats. The concentration of arachidonate was not reduced, and elongation of linolate was not stimulated, indicating that TCDD did not cause extensive-but-repaired peroxidation. It is concluded that while TCDD may slightly increase hepatic lipid peroxidation in rats in vivo, the extent of such stimulation appears to be too slight to account for the toxicity of TCDD.