Activity of human Delta5 and Delta6 desaturases on multiple n-3 and n-6 polyunsaturated fatty acids.

Yeast co-expressing human elongase and desaturase genes were used to investigate whether the same desaturase gene encodes an enzyme able to desaturate n-3 and n-6 fatty acids with the same or different carbon chain length. The results clearly demonstrated that a single human Delta5 desaturase is active on 20:3n-6 and 20:4n-3. Endogenous Delta6 desaturase substrates were generated by providing to the yeast radiolabelled 20:4n-6 or 20:5n-3 which, through two sequential elongations, produced 24:4n-6 and 24:5n-3, respectively. Overall, our data suggest that a single human Delta6 desaturase is active on 18:2n-6, 18:3n-3, 24:4n-6 and 24:5n-3.

Activity and mRNA abundance of Delta-5 and Delta-6 fatty acid desaturases in two human cell lines.

We analyzed fatty acid biosynthesis in Chang and ZR-75-1 cells. Both cell lines could desaturate and further elongate substrates for Delta-5 desaturase. ZR-75-1 but not Chang cells showed Delta-6 desaturation of 18:2n-6, 18:3n-3, 24:4n-6 and 24:5n-3. In both cell lines, the mRNA abundance can be related to Delta-5 or Delta-6 fatty acid desaturase activities. These results suggest that desaturase genes could have, at least in part, independent control mechanisms and that Delta-6 desaturase impairment is not specific to any particular step of the fatty acid metabolic pathways, which may diminish the rationale for the existence of at least two distinct enzymes.

Tissue distribution and metabolism of gamma-linolenoyl-3-eicosapentaenoyl propane diol enterally or intravenously administered to mice bearing human pancreatic carcinomas.

Synthetic propane diol lipids have been proposed as novel compounds to deliver cytocidal polyunsaturated fatty acids (PUFA) such as gamma-linolenic (GLA) and eicosapentaenoic (EPA) acids. To assess the biodistribution and metabolism of these PUFA in immunodeficient mice bearing human pancreatic carcinomas (AsPC-1), gamma-linolenoyl-3-eicosapentaenoyl propane diol (GE diol) was provided in a fat-free diet (5% w:w) for 6 weeks or parentally administered as 14C-GE diol (1 or 3 consecutive doses of 1.66 g/kg/day) in an innovative non-ionic-digalactosyldiacylglycerol emulsion. In tumor, liver, brain, kidney, plasma and fat tissue of mice fed GE diol, PUFA were increased over 25-fold, except for arachidonic acid (AA) levels, which were reduced or remained constant when compared to mice fed control corn oil diet. GLA and EPA were mainly stored in fat tissue. The recovery of radioactivity from the i.v. infected 14C-GE diol was dose and time dependent. Ten days after the i.v. infusion, GLA was only detected in substantial concentrations in tumor and in fat tissue (21 and 202 micrograms/g, respectively). Overall, these studies showed that: GE diol emulsions provide 640-fold higher doses of both GLA and EPA without causing hemolysis or adverse effects in the host mouse when compared to free PUFA infusions; GE diol is metabolized after oral or i.v. administration; tumor concentrations of GLA and EPA from the enterally administered diol were 4 to 13-fold higher than the in vitro cytotoxic levels; EPA, competes with AA and probably inhibits the activity of delta 5 desaturase without affecting the elongation of GLA in the host and tumor tissue; the change in PUFA profile modifies the substrates for eicosanoid synthesis. In short, a potentially desirable cytotoxic PUFA pattern can be achieved in host tissues and, in particular, in a human pancreatic tumor by providing GLA and EPA in the form GE-diol. These findings guarantee further investigations in oncology with this neutral diol lipid.

In vivo and in vitro biotransformation of the lithium salt of gamma-linolenic acid by three human carcinomas.

Lipid metabolism has been considered recently as a novel target for cancer therapy. In this field, lithium gamma-linolenate (LiGLA) is a promising experimental compound for use in the treatment of human tumours. In vivo and in vitro studies allowed us to assess the metabolism of radiolabelled LiGLA by tumour tissue and different organs of the host. In vitro studies demonstrated that human pancreatic (AsPC-1), prostatic (PC-3) and mammary carcinoma (ZR-75-1) cells were capable of elongating GLA from LiGLA to dihomo-gamma-linolenic acid (DGLA) and further desaturating it to arachidonic acid (AA). AsPC-1 cells showed the lowest delta5-desaturase activity on DGLA. In the in vivo studies, nude mice bearing the human carcinomas were given Li[1-(14)C]GLA (2.5 mg kg(-1)) by intravenous injection for 30 min. Mice were either sacrificed after infusion or left for up to 96 h recovery before sacrifice. In general, the organs showed a maximum uptake of radioactivity 30 min after the infusion started (t = 0). Thereafter, in major organs the percentage of injected radioactivity per g of tissue declined below 1% 96 h after infusion. In kidney, brain, testes/ovaries and all three tumour tissues, labelling remained constant throughout the experiment. The ratio of radioactivity in liver to tumour tissues ranged between 16- and 24-fold at t = 0 and between 3.1- and 3.7-fold at 96 h. All tissues showed a progressive increase in the proportion of radioactivity associated with AA with a concomitant decrease in radiolabelled GLA as the time after infusion increased. DGLA declined rapidly in liver and plasma, but at a much slower rate in brain and malignant tissue. Seventy-two hours after the infusion, GLA was only detected in plasma and tumour tissue. The sum of GLA + DGLA varied among tumour tissues, but it remained 2-4 times higher than in liver and plasma. In brain, DGLA is the major contributor to the sum of these fatty acids. Data showed that cytotoxic GLA and DGLA, the latter provided either by the host or by endogenous synthesis, remained in human tumours for at least 4 days.

Metabolism of n-6 fatty acids by NIH-3T3 cells transfected with the ras oncogene.

N-6 fatty acid metabolism was compared in NIH-3T3 cells and DT cells, which differ only in the presence of the v-Ki-ras oncogene. Non-dividing cells were incubated with [1-14C]-labelled fatty acids (18:2n-6, 18:3n-6, 20:3n-6 and 20:4n-6) at different time intervals (2-24 h) and concentration (0-120 microM). In both cells lines, the uptake of different fatty acids from the medium was similar and reached a maximum at 6-8 h. All fatty acids reached the same maximum level in DT cells, whereas, the relative uptake of added fatty acids by NIH-3T3 cells was different: 20:4n-6 > 20:3n-6 > 18:2n-6 = 18:3n-6. Throughout the incubation (2-24 h), desaturation and elongation of n-6 fatty acids was more active in DT cells than in NIH-3T3 cells. However, in both cell lines, incubated with different n-6 fatty acid precursors, the levels of radiolabelled 20:4n-6 were relatively constant. In DT cells, phosphatidylcholine was found to be the major fraction labelled with n-6 fatty acids precursors and those of endogenous synthesis, whereas, in NIH-3T3 cells the neutral lipid fraction, particularly triglycerides, was also strongly labelled. In concentration dependent studies, phospholipid labelling by fatty acids was saturable. At lower concentrations, especially in DT cells, phospholipids were labelled predominantly. As the concentration increased there was an overflow into the triglyceride fraction. Since the differences in fatty acid metabolism between the two cell lines cannot be related to the growth rate, it is suggested that they were a consequence of the expression of the v-Ki-ras oncogene.

Liver delta 5 and delta 6 desaturase activity differs among laboratory rat strains.

This study was designed to examine the variations among rat strains in hepatic fatty acid desaturase activities and to determine the correlations between the activities of these enzymes and the levels of each microsomal fatty acid. Wistar rats from two different sources as well as Long-Evans and Sprague-Dawley rats were selected to assess, under standard and identical experimental conditions, the liver delta 5 and delta 6 desaturase activities. Both desaturase activities were significantly reduced by 56% in Sprague-Dawley rats when compared to BB-Wistar control rats, whereas intermediate reduced values were detected in Wistar (CR) and Long-Evans strains. The activities of delta 5 and delta 6 desaturases were significantly and positively correlated with each other. However, no significant correlations were detected between either delta 5 or delta 6 desaturase activities and levels of any of their fatty acid substrates or any other of the major microsomal fatty acids. Fatty acid composition of microsomal total lipids showed strain dependency. A positive correlation was detected between the microsomal levels of the two major final products of both desaturases, namely 20:4n-6 and 22:6n-3. In general, the sum of n-3 or n-6 fatty acids but not the ratio of one to the other, varied among rat strains. The study demonstrated that delta 6 and delta 5 desaturase activities are strain-related. The data also suggested that (i) the desaturation activity should be measured and not predicted from the fatty acid composition and (ii) different rat strains should be used for lipid metabolic studies before conclusions are drawn for rats in general.

Interaction of dietary fatty acids and cyclosporine A in the borderline hypertensive rat: tissue fatty acids.

In this study we examined (i) the effects of cyclosporine A (CS) on tissue lipid composition and (ii) the effect of changes in dietary n-6 fatty acids on tissue responses to CS. Fatty acid composition of liver, kidney, heart and brain were determined after 4 wk of treatment with CS (10 mg/kg.d p.o.) in male borderline hypertensive rats (BHR, n = 4/group), whose diet was supplemented with either safflower oil or evening primrose oil (EPO). Phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine/phosphatidylinositol, triglyceride and cholesteryl ester fatty acids were measured in kidney, heart, brain and liver. The same parameters were also measured in safflower-fed BHR (n = 4) receiving placebo. The effects of CS on liver microsomal delta 9, delta 6 and delta 5 desaturases in vitro were also followed. CS affected the fatty acid composition of all tissues examined, with the greatest changes seen in the renal phosphatidylcholine and phosphatidylserine/phosphatidylinositol fractions. All CS-induced changes that occurred in the liver, brain and renal fatty acids were reversed by EPO. CS elevated delta 9 desaturase but had no effect on delta 6 and delta 5 desaturase. In light of (i) the observation that EPO normalizes renal function and blood pressure in CS-treated BHR, and (ii) the importance of the kidney in blood pressure regulation, the data suggest that the beneficial effects of EPO on CS toxicity may involve changes in renal phospholipid fatty acid profiles.

Metabolism of radiolabelled 18:2n-6 and 18:3n-6 by NIH-3T3 cells and the DT subclone.

The incorporation and metabolism of delta-6-desaturase substrate and product, [1-14C]-linoleic (18:2n-6) and [1-14C]-gamma-linolenic acid (18:3n-6), was examined in NIH-3T3 cells and the DT subclone which differs only in the presence of the v-Ki-ras oncogene. Similar amounts of post delta-6 and delta-5 desaturase metabolites were found in both cell lines indicating that the activity of these important enzymes of fatty acid metabolism was not affected by the expression of the oncogene. However, measurable quantities of the direct elongation product of 18:2n-6, 20:2n-6, were only found in DT cells. Radiolabel was recovered predominantly from the phospholipid fraction at low fatty acid concentrations, whereas neutral lipid labelling occurred when higher concentrations of exogenous fatty acid were present. This effect was most pronounced in DT cells and may result from the presence of the activated ras oncogene.

Relationship between mouse liver delta 9 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 delta 9 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 delta 9 desaturase activity were inversely correlated with the ratio of the sum of long chain n-6 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 delta 9 desaturase activity. However, a marked reduction in plasma TG content (50-60%) and delta 9 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 delta 9 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 delta 9 desaturase activity, in parallel.

Effect of salt-loading and spontaneous hypertension on in vitro metabolism of [1-14C]linoleic and [2-14C]dihomo-gamma-linolenic acids.

The present study compared the effect of spontaneous hypertension and salt-loading on in vitro metabolism of 18:2n-6 (linoleic acid) and 20:3n-6 (dihomo-gamma-linolenic acid). Ten weanling spontaneously hypertensive rats (SHR) and 10 normotensive Wistar-Kyoto rats (WKY) maintained on a rodent lab chow were given tap water with (n = 5) or without (n = 5) addition of 1% NaCl for 4 weeks. Thereafter, animals were killed and liver microsomes were prepared. Aliquots of microsomes suspended in the phosphate-sucrose buffer containing MgCl2, ATP, CoA, and NADPH were incubated with 0.3 microCi of [1-14C]-18:2n-6 or [2-14C]-20:3n-6 at 37 degrees C for 15 min. The activity of delta 6- and delta 5-desaturases, and the distribution of radioactivity in different lipid fractions and in phospholipid fatty acids were determined. Results showed that both spontaneous hypertension and salt-loading suppressed the desaturation of radiolabeled 18:2n-6 and of 20:3n-6. Incubation of microsomes with [1-14C]-18:2n-6 resulted in 29% of radioactivity being associated with phospholipid fraction, of which 3% was associated with 18:3n-6. Incubation with radiolabeled 20:3n-6 resulted in 30% of the radioactivity being incorporated into phospholipids, of which 50% was associated with 20:4n-6 (arachidonic acid). Salt-loading suppressed the incorporation of radiolabeled fatty acids into phospholipids, more so in SHR than in WKY. Thus, salt-loading not only suppressed the desaturation of 18:2n-6 and 20:3n-6, but also interfered with the acylation of n-6 fatty acids into the phospholipid fraction.

Effect of n-3 and n-6 fatty acids on hepatic microsomal lipid metabolism: a time course study.

The present study examines the time dependent effects of n-6 and n-3 polyunsaturated fatty acids on liver microsomal lipid metabolism in FVB mice fed a diet supplemented with a mixture of free fatty acids (mainly 18:3n-6 and 20:5n-3) at 25 mg/g diet. Significant changes in the fatty acid composition of total liver and microsomal lipids were observed after 7 days on the diets. Thereafter, some animals remained on the same diet while others were fed a diet supplemented with hydrogenated coconut oil (HCO). With the exception of 20:5n-3 which showed a slower recovery, establishment of the HCO pattern was rapid indicating that the diet-induced changes could be easily reversed. The unsaturation index, the cholesterol/phospholipid ratio and the microviscosity of the microsomal membranes were not affected by these dietary manipulations. Unsaturated fatty acid supplementation reduced the activity of delta 9 desaturase by 50%. Feeding the HCO diet to mice previously fed the EPA/GLA diet led to a progressive increase in delta 9 desaturase activity, reaching 80% of the day zero values after 14 days. The monoene content of hepatic total lipids reflected, in most cases, the changes in enzyme activity. This study shows that a low dose of a n-3 and n-6 free fatty acid mixture increases the quantities of members of the n-3 family, without loss of n-6 fatty acids in microsomal membranes and modifies the activity of delta 9 desaturase without altering the microsome physicochemical parameters.

The effect of n-6 fatty acids on normal and v-Ki ras transformed NIH-3T3 cells.

The effect of exogenous gamma-linolenic acid (18:3n-6) was examined on NIH-3T3 and a subclone expressing the v-Ki-ras oncogene (DT). 18:3n-6 inhibited DT cell growth more readily than NIH-3T3 cell growth. In comparison, linoleic acid (18:2n-6) had no effect on the growth of either cell line. DT cells elongated and desaturated both 18:2n-6 and 18:3n-6 to dihomo-gamma-linolenic acid (20:3n-6) and arachidonic acid (20:4n-6) to a much greater extent than NIH-3T3 cells and had a much higher membrane fluidity. The presence of the ras gene or its product appears to increase the metabolism of polyunsaturated fatty acids and potentiate the cytostatic actions of 18:3n-6.

Effects of an eicosapentaenoic and docosahexaenoic acid concentrate on a human lung carcinoma grown in nude mice.

The effects of the n-3 fatty acids, eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), on the growth of a human lung mucoepidermoid carcinoma (HLMC) in athymic mice were studied. The mice were divided into three groups which were given either a control chow diet (C), a chow diet supplemented with EPA/DHA (P) (25 or 50 mg of free n-3 fatty acids/g of pellet/day), or chow diet supplemented with palmitic acid (S) (isocaloric with P). Two independent experimental schedules were followed: i) host mice bearing either tumors that were allowed to reach 4000 mm3, or only 35 mm3, were fed C, P or S for 21 or 41 days; ii) animals were fed C, P and S for 9 days before tumor implant and were maintained on these diets throughout tumor growth. Food consumption, mouse weight and liver/body weight ratio showed no significant differences between supplemented diets and chow. Tumor growth was markedly inhibited (45%) in both experiments by the EPA/DHA supplemented diet. In Experiment 2, only 60% of mice fed diet P had tumors. The fatty acid composition of neutral and polar lipids of host liver and tumor reflected the dietary intake of n-3 fatty acids; the content of arachidonic acid was reduced by 50%, and EPA/DHA was increased 3- to 5-fold. Tumor prostaglandin E2 levels were reduced 7.4-fold in the P group. The reduced PGE2 content may be a factor in tumor growth inhibition.

Neuroendocrine alterations in nude mice with a human lung carcinoma producing pro-opiomelanocortin, corticotrophin-releasing hormone and arginine vasopressin.

A mucoepidermoid carcinoma of the lung (ICD classification 8430/3) resected from a patient with no clinical signs of pituitary-adrenal alterations was transplanted into 2-month-old athymic nu/nu nude mice, with the purpose of studying the effects exerted by the human tumour on the host hypothalamo-pituitary-adrenal axis. The tumour produces peptides derived from different regions of pro-opiomelanocortin (POMC: ACTH, 7.6 +/- 0.7; N-terminal POMC, 6.6 +/- 0.6; beta-LPH/endorphin, 7.3 +/- 0.7; and alpha-MSH;3.8 +/- 0.5 pmol/g wet tissue) and the neuropeptides corticotrophin-releasing hormone and arginine vasopressin (CRH: 3.6 +/- 0.4 and AVP: 1.1 +/- 0.2 pmol/g wet tissue). Immunohistochemical staining of consecutive sections of the tumour indicated that staining of tumour cells for the different peptides was not uniform and although some cells co-stained with CRH and AVP, POMC-positive cells appeared to be distinct from CRH and AVP cells. Tumour extracts were chromatographed on Sephadex G-75 and fractions monitored for POMC-derived peptides. A single peak with characteristics of alpha-MSH was detected. The ACTH, N-POMC and beta-LPH/endorphin radioimmunoassays (RIA) detected a peak at large molecular weight, eluting at the position expected for POMC. These RIA systems also revealed an ACTH(1-39) peak and another peak which probably correspond to 13 kDa ACTH, a peak eluting at the position of hN-POMC(1-48), a beta-LPH-like peak, and a smaller sized peak which may represent alpha- or gamma-endorphin. The ACTH, N-POMC and beta-LPH/endorphin contents of anterior lobe (AL) extracts, but not neutrointermediate lobe (NIL) extracts, showed a striking decrease in tumour-bearing (TB) nude mice. However, while no difference was seen in the alpha-MSH content of AL extract between TB and control (C) nude mice, it decreased in NIL extracts of TB animals. The contents of CRH and AVP in stalk-median eminence extracts of TB nude mice was significantly lower than that of C nude mice. Basal plasma corticosteroids were raised in TB nude mice at levels comparable to those in stressed C nude mice, and although adrenal weights did not vary between TB and C nude mice, morphological changes indicating hypertrophy were found in the adrenal glands of the host animals. It was concluded that the tumour dramatically alters the hypothalamo-pituitary-adrenal axis of the host, and that it may be a useful model for studying tumour-host interactions in ectopic hormone-producing tumours.

In vitro effect of eicosapentaenoic and docosahexaenoic acids on prostaglandin E2 synthesis in a human lung carcinoma.

The capacity to synthesize both prostaglandins E1 (PGE1) and E2 (PGE2) has been determined in human lung mucoepidermoid carcinoma homogenates when [14C]-fatty acid precursors were added to the incubation medium. Only 10% of the total radioactivity recovered in PGs was found in PGF1 alpha and PGF2 alpha. The experiments were principally focused to inhibit the PGE2 synthesis either with pure eicosapentaenoic (EPA) and docosahexaenoic (DHA) acids or with mixtures of both n-3 fatty acids obtained from fish oil. The results demonstrated that significant inhibitions were found when using 25 microM or a higher concentration of pure EPA or DHA in the incubation medium; however, 5 microM of mixtures of different EPA/DHA ratio caused the same inhibition. The results suggest that EPA and DHA, when added together, may enforce their inhibitory effect on PGE2 synthesis.

Microsomal fatty acid desaturation and elongation in a human lung carcinoma grown in nude mice.

Since tumor cells show abnormal fatty acid composition, it is likely that their desaturase systems were affected to some extent. Although desaturase activities in experimental tumors have been evaluated, to our knowledge, fatty acid desaturases in human neoplasms and particularly in human tumors grown in nude mice have not been assessed yet. We have therefore, chosen a rapidly growing human lung mucoepidermoid carcinoma (HLMC) grown in nude mice to study microsomal fatty acid desaturation and chain elongation activities. Tumor microsomal proteins were incubated with unlabeled malonyl-CoA and one of the following fatty acids: [1-14C]palmitic (16:0), [1-14C]linoleic (18:2), alpha-[1-14C]linolenic (alpha-18:3), and unlabeled gamma-linolenic (gamma-18:3) plus [2-14C]malonyl-CoA. Data show that HLMC microsomes were capable to desaturate 16:0, alpha-18:3, and dihomogammalinolenic acids (20:3) by delta 9, delta 6 and delta 5 desaturase, respectively; however, delta 6 desaturase activity on [14C]18:2 was not detected. The microsomal elongation system was active in all fatty acid series tested except for 18:2. These findings show that the undetectable activity for 18:2 desaturation is not exclusively found in experimental tumors.

Tumor lipids and liver lipid metabolism in the model human lung carcinoma/nude mice.

Tumor lipids were studied in the experimental model Human Lung Carcinoma/nude mice as well as the effect of this human neoplasm on the host liver lipid metabolism. Fatty acid profiles from tumoral lipids revealed the loss of specificity for fatty acid composition in triglycerides. Host liver fatty acid composition and cholesterol metabolism were affected by the implanted human lung tissue. A noticeable increase ratio between saturated/unsaturated fatty acids was observed in host liver fatty acid phospholipids (1.17 +/- 0.17) in comparison to control liver (0.84 +/- 0.04). Cholesterol synthesis was assessed "in vivo" by means of [14C]acetate incorporation. The specific radioactivity of [14C] cholesterol was increased by a factor of about 6 in host liver as compared with control liver. This observation along with the marked decrease in the cholesterol content of host liver and the hypocholesterolemia detected in the host mice led us to suggest an increase in the liver cholesterol catabolism promoted by the presence of the tumor.

In vitro conversion of saturated to monounsaturated fatty acid by Ehrlich ascites cells.

In this paper, evidence is presented on the capacity of Ehrlich ascites cells to synthesize in vitro monounsaturated fatty acids from radioactive palmitate. Localization of the double bond was determined by ozonolysis and subsequent reduction of the ozonides to aldesters followed by gas liquid chromatography. These results proved that Ehrlich ascites cells have a delta 9 desaturase that catalyzes the biosynthesis of palmitoleic acid from palmitic acid and oleic and vaccenic acid via elongation-desaturation and desaturation-elongation, respectively, using palmitic acid as substrate. Furthermore, it is shown that, as in the hepatic cells, delta 9 desaturase enzyme activity of the tumoral cells is associated with the endoplasmic reticulum. The electron transport components involved in the desaturase system, i.e., NADH-cytochrome b5 reductase and NADH-cytochrome c reductase, were also measured. The activities of these enzymes do not appear to be rate-limiting in the desaturase activity of these tumoral cells.