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Fatty-acid synthesis in lactating-goat mammary gland. 2. Medium-chain fatty acid synthesis.

Addition of goat, rat, rabbit and cow mammary microsomal fraction to fatty acid synthetase of goat mammary gland induced C10 fatty acid synthesis. When the microsomal fractions from rat, rabbit and cow mammary gland were incubated with their respective fatty acid synthetases only the cow enzyme synthesized significant amounts of C10 compared to the purified fatty acid synthetases alone. The goat mammary microsomal fraction was not able to induce C10 synthesis by rat and rabbit mammary-gland fatty acid synthetase, but only by the goat and cow enzyme. Both goat and cow mammary-gland fatty acid synthetase have in contrast to the rabbit and rat enzymes the ability to hydrolyse medium-chain acyl-thioesters. We therefore suggest a novel mechanism for medium-chain fatty acid synthesis in lactating-goat mammary gland. This mechanism involves an activation of the medium-chain acyl-thioester hydrolase within the fatty acid synthetase of goat mammary gland by an unknown microsomal factor.

Acetyl Coenzyme A↗

Use of transposon TnphoA to identify genes for cell envelope proteins of Escherichia coli required for long-chain fatty acid transport: the periplasmic protein Tsp potentiates long-chain fatty acid transport.

TnphoA was used to mutagenize the chromosome in an effort to identify membrane-bound and exported components of the long-chain fatty acid transport system of Escherichia coli. This strategy identified three classes of fusions that were unable to grow or grew at reduced rates on minimal agar plates containing the long-chain fatty acid oleate (C18:1), (i) fadL-phoA, (ii) tolC-phoA, and (iii) tsp-phoA, fadL-phoA and tolC-phoA fusions were unable to grow on oleate as the sole carbon and energy source, while the tsp-phoA fusion had a markedly reduced growth rate. As expected, fadL-phoA fusions were unable to grow on oleate plates because the outer membrane-bound fatty acid transport protein FadL was defective. The identification of multiple fadL-phoa fusions demonstrated that this strategy of mutagenesis specifically targeted membrane-bound and exported components required for growth on long-chain fatty acids. tolC-phoA fusions were sensitive to fatty acids (particularly medium chain) and thus unable to grow, whereas the reduced growth rate of tsp-phoA fusions on oleate was apparently due to changes in the energized state of the outer membrane or inner membrane. tsp-phoA fusions transported the long-chain fatty acid oleate at only 50% of wild-type levels when cells were energized with 1 mM DL-lactate. Under conditions in which transport was measured in the absence of lactate, tsp-phoA fusion strains and wild-type strains had the same levels of oleate transport. The tsp+ clone pAZA500 was able to restore wild-type transport activity to the tsp-phoA strain under lactate-energized conditions. These results indicate that the periplasmic protein Tsp potentiates long-chain fatty acid transport.

Alkaline Phosphatase↗

Effects of dietary n-6/n-3 polyunsaturated fatty acid balance on tissue lipid levels, fatty acid patterns, and eicosanoid production in rats.

The effect of varying n-6/n-3 ratios (0.6-10.2) of dietary fats on various lipid parameters was examined in rats under a constant P/S ratio (1.4-1.5) with sardine oil as the source of n-3 polyunsaturated fatty acid (PUFA) by a combination of palm and safflower oils. The concentration of serum cholesterol tended to increase with n-6/n-3 of up to approximately 2, whereas aortic cholesterol decreased. The proportion of arachidonic acid in liver, heart, and aorta phosphatidylcholine increased linearly with increasing n-6/n-3 whereas that of linoleic acid reached a plateau at this ratio of approximately 4. The proportion of n-3 PUFAs decreased with increasing n-6/n-3 in tissue phosphatidylcholine. Although the production of prostacyclin (PGI2) by the thoracic aorta and thromboxane A2 (TXA2) by platelets increased with increasing n-6/n-3, TXA2/PGI2 was maintained at a low level up to n-6/n-3 of approximately 5. These results indicate that, when fish oil is the source of n-3 PUFAs, n-6/n-3 of 2-5 seems to be desirable for the various lipid parameters related to atherosclerosis and thrombosis.

Animals↗

Comparison of the fatty acid compositions in intraepithelial and infiltrating lesions of the cervix: part II, free fatty acid profiles.

In the second part of this study, the emphasis is on the free fatty acids during cervical carginogenesis, since they may reflect active cell metabolism during this disease process. Lipids were extracted from biopsies of normal epithelial tissue (N) (n=36), cervical intraepithelial lesions (CIL) (n=47), and infiltrating lesions (Ca) (n=47) of the cervix. Samples, from which the free fatty acid compositions were determined, were saponified, methylated and analysed by GLC. In accordance with results obtained on total fatty acid compositions, essential fatty acid deficiency (EFAD) in the intraepithelial lesions, compared with normal tissue (linoleic acid, P< 0.01), and infiltrating lesions compared with intraepithelial lesions (linoleic acid and arachidonic acid, P< 0.01) were observed. High levels of oleic acid were also observed when infiltrating lesions were compared with normal tissue (P < 0.01). As previously mentioned by us in part I of this study, with regard to possible disturbances in metabolic pathways based on the total fatty acid profiles during stages of cervical cancer, EFAD is prevalent during cervical carcinogenesis. This EFAD in cancer cells may result in many defective cell mechanisms, since fatty acids are associated with biochemical events such as lipid peroxidation, signal transduction and immune responses. The high level of oleic acid in cancer cells is known to activate PKC and thus contribute to the continous growth stimulus thought to exist in malignant cells. From a therapeutic viewpoint, substantial changes in the fatty acid composition of the membranes can be produced in cancer cells by selective fatty acid supplementation strategies. At present, modifications of the fatty acid compositions of cell membranes represent an experimental model that has promoted increased understanding of lipid transportation, membrane remodelling, and the relationship between membrane lipids and membrane function. By addressing factors responsible for insufficient essential fatty acid levels, carginogenesis may be prevented or treated. The clinical feasibility of using modification of fatty acids in tumours or cancer by diet or perfusion as an adjunct to standard therapies should be tested.

Cervix Uteri↗

Serum lipids, hepatic glycerolipid metabolism and peroxisomal fatty acid oxidation in rats fed omega-3 and omega-6 fatty acids.

Rats were fed, for 3 weeks, high-fat (20% w/w) diets containing sunflower-seed oil, linseed oil or fish oil. Chow-fed rats were used as a low-fat reference. The high-fat diets markedly reduced non-fasting-rat serum triacylglycerol as compared with the low-fat reference, and the highest reduction (85%) was observed with the fish-oil group, which was significantly lower than that of the other high-fat diets. The serum concentration of phospholipids was significantly reduced (30%) only in the fish-oil-fed animals, whereas serum non-esterified fatty acids were reduced 40-50% by both the fish-oil- and linseed-oil-fed groups. The liver content of triacylglycerol showed a 1.7-fold increase with the fish-oil diet and 2-2.5-fold with the other dietary groups when compared with rats fed a low-fat diet, whereas the hepatic content of phospholipids was unchanged. Peroxisomal fatty acid oxidation (acyl-CoA oxidase) was 2-fold increased for the rats fed fish oil; however this was not significantly higher when comparison was made with rats fed the linseed-oil diet. There was no difference in phosphatidate hydrolysis (microsomal and cytosolic fractions) among animals fed the various diets. Acyl-CoA:diacylglycerol acyltransferase activity was increased by all high-fat diets, but the fish-oil-diet-fed group showed a significantly lower enzyme activity than did rats fed the other high-fat diets. A linear correlation between acyl-CoA:diacylglycerol acyltransferase activity and liver triacylglycerol was observed, and the microsomal enzyme activity was decreased 40-50% by incubation in the presence of eicosapentaenoyl-CoA. CoA derivatives of arachidonic, linolenic and linoleic acid had no inhibitory effect when compared with the control. These results indicate that dietary fish oil may have greater triacylglycerol-lowering effect than other polyunsaturated diets, owing to decreased triacylglycerol synthesis caused by inhibition of acyl-CoA:diacylglycerol acyltransferase. In addition, increased peroxisomal fatty acid oxidation and decreased availability of non-esterified fatty acids could also contribute by decreasing the amounts of fatty acids as substrates for triacylglycerol synthesis and secretion.

Acyltransferases↗

Feeding infant piglets formula with long-chain polyunsaturated fatty acids as triacylglycerols or phospholipids influences the distribution of these fatty acids in plasma lipoprotein fractions.

Several sources of long-chain polyunsaturated fatty acids (LCP) are currently available for infant formula supplementation. These oils differ in their fatty acid composition, the chemical form of the fatty acid esters [triacylglycerols (TG) or phospholipids (PL)] and presence of other lipid components. These differences may affect LCP absorption, distribution and metabolic fate after ingestion. The purpose of the present study was to evaluate the influence of different chemical forms of dietary LCP on the composition of plasma, plasma lipoproteins, liver and jejunum in infant piglets. Thirty pigs (5 d old) were bottle-fed different diets for 4 wk: a control diet (C), a diet containing LCP as TG from tuna and fungal oils (TF-TG) or a diet containing LCP as PL from egg yolk (E-PL). We measured lipid and fatty acid composition of plasma and lipoproteins, as well as lipid composition of liver and intestinal mucosa. The arachidonic and docosahexaenoic acids in HDL-PL were significantly higher in piglets fed the E-PL diet than in those fed the TF-TG diet. Opposite results were found in the LDL-PL diet. No significant differences were found between groups in TG or cholesterol concentrations of plasma or lipoproteins. Arachidonic acid in plasma PL and cholesteryl esters was significantly higher in the E-PL group than in the TF-TG group. The chemical form in which LCP esters are present in different dietary sources influences their distribution in plasma lipoproteins. This may be important for infant nutrition and suggests that not all LCP sources may be biologically equivalent.

Animal Feed↗

Levels of polyunsaturated fatty acids in lymphocytes of rats fed on diets varying in polyunsaturated fatty acid content.

1. When weanling rats were fed on a diet containing 0.1 g/kg of the diet as polyunsaturated fatty acid, it was found that after 2 weeks the level of linoleate in the lymphocyte lipids was 56 mg/g total fatty acids, as compared with a level of 138 mg/g in rats on a normal diet (P less than 0.005). Similar levels were obtained from rats which had been fed for up to 16 weeks on the deficient diet, but in a group killed after 28 weeks on the diet level was found to be only 20 mg/g total fatty acids. The arachidonate level was found to be approximately 220 mg/g total fatty acids, regardless of whether the rats were fed on a diet deficient in linoleate for up to 16 weeks or on a normal diet. In the group of rats killed after 28 weeks on the linoleate deficient diet, however, the arachidonate level was only 60 mg/g total fatty acids. 2. Percentage values for total fatty acids are given for plasma, adipose tissue, and lymphocytes for rats on normal and experimental diets. 3. Scatter diagrams of the levels of linoleate v. arachidonate in the lymphocyte total fatty acids showed no correlation between the levels of the two acids (r 0.05), but similar plots of linoleate and oleate levels showed an inverse correlation (r -0.68).

Adipose Tissue↗

Essential fatty acid metabolism in cardiomyocytes grown in media enriched with different N-6/N-3 fatty acid combinations.

We have evaluated the effects of three different 18:3n-6, 20:5n-3 and 22:6n-3 fatty acid combinations on essential fatty acid (EFA) metabolism in rat cultured cardiomyocytes. The desaturating/elongating activities for linoleic (LA) and alpha-linolenic acid (ALA) were evaluated by radiolabeling the cells with 1-[14C]LA or 1-[14C]ALA and the fatty acid pattern of cardiomyocytes was assessed by gas chromatography. LA and ALA conversion to more unsaturated metabolites was reduced by increasing respectively n-3 and n-6 fatty acid concentration in the media. The all three combinations used reduced the saturated and increased the polyunsaturated fatty acid content of cardiomyocytes. The n-6/n-3 fatty acid ratio did not change compared to control cells in cardiomyocytes receiving the highest amount of 18:3n-6 and the lowest amounts of n-3 fatty acids. This combination may be suitable for modifying EFA desaturating/elongating activities without altering the physicochemical parameters which are related to the correct balance between n-6 and n-3 fatty acid content.

Animals↗

Brain astrocyte synthesis of docosahexaenoic acid from n-3 fatty acids is limited at the elongation of docosapentaenoic acid.

The phospholipids, particularly phosphatidylethanolamine, of brain gray matter are enriched with docosahexaenoic acid (22:6n-3). The importance of uptake of preformed 22:6n-3 from plasma compared with synthesis from the alpha-linolenic acid (18:3n-3) precursor in brain is not known. Deficiency of 18:3n-3 results in a compensatory increase in the n-6 docosapentaenoic acid (22:5n-6) in brain, which could be formed from the precursor linoleic acid (18:2n-6) in liver or brain. We studied n-3 and n-6 fatty acid incorporation in brain astrocytes cultured in chemically defined medium using delipidated serum supplemented with specific fatty acids. High performance liquid chromatography with evaporative light scattering detection and gas liquid chromatography were used to separate and quantify cell and media lipids and fatty acids. Although astrocytes are able to form 22:6n-3, incubation with 18:3n-3 or eicosapentaenoic acid (20:5n-3) resulted in a time and concentration dependent accumulation of 22:5n-3 and decrease in 22:6n-3 g/g cell fatty acids. Astrocytes cultured with 18:2n-6 failed to accumulate 22:5n-6. Astrocytes secreted cholesterol esters (CE) and phosphatidylethanolamine containing saturated and monounsaturated fatty acids, and arachidonic acid (20:4n-6) and 22:6n-3. These studies suggest conversion of 22:5n-3 limits 22:6n-3 synthesis, and show astrocytes release fatty acids in CE.

Animals↗

[Nature, origin and role of fatty acids of the nervous system: an essential fatty acid, an alpha-linolenic acid, changing the structure and the cerebral function].

Saturated and monounsaturated fatty acids are synthetized in cytosol, endoplasmic reticulum and mitochondria. Their mechanisms of synthesis and fate are different according to the organelle. For some of them, a nutritional origin is not excluded. Minimum dietary linoleic and alpha-linolenic acid to ensure normal brain structure and function are respectively 2.4% and 0.4% of the calories. Alpha-linolenic acid deficiency alters membrane composition and fluidity, changes the fluidizing effect of ethanol, reduces some enzymatic activities (ATP ase, 5' nucleotidase), diminishes the efficiency of the blood-brain barrier, affects electroretinogram, reduces learning capacities and makes the animals more fragile in the presence of neurotoxins. Very long polyunsaturated chains, which could be the essential fatty acids for the brain, must be considered at the level of nutrition, pharmacology and toxicology (alteration of structures and peroxidations).

Animals↗

The membrane-associated 40 KD fatty acid binding protein (Berk's protein), a putative fatty acid transporter is present in human skeletal muscle.

Muscle tissue (1.1 +/- 0.1 grams) was obtained from seven healthy individuals (3 males, 4 females) using an open incision approach before and after ingestion of either 75 grams of dextrose (N=5) or water (N=2). Purified sarcolemmal membranes from the muscle were prepared using a sucrose step gradient. A polyclonal antibody raised against the purified (99%) rat hepatocyte 40 KD membrane fatty acid binding protein (mFABP-L) was used to probe for this putative transporter in the muscle membranes using Western blot. A single band at the 40 KD MW band was identified which reacted antigenically with the protein purified from rat livers. These response of Berk's protein 60-75 minutes after dextrose ingestion (or water) was erratic and no specific trend could be identified. Our data demonstrate that the 40 KD mFABP-L originally isolated from rat liver is also present in human skeletal muscle membrane. This protein may be involved in transport of fatty acids across the membrane of skeletal muscle, however its physiological role in human fatty acid metabolism remains to be established.

Adult↗