Fatty acids and epithelial permeability: effect of conjugated linoleic acid in Caco-2 cells.

Conjugated linoleic acid (CLA) is a collective term referring to the positional and geometric isomers of linoleic acid. This novel fatty acid has been shown to have a number of beneficial actions, including immunomodulatory, anticarcinogenic, and antiatherogenic effects. Tight junctions of epithelial cells determine epithelial membrane integrity and selective paracellular permeability to ions and macromolecules. Occludin and ZO-1 are integral structural components of the tight junction, which are involved in the biogenesis and functional integrity of the epithelial monolayer. This study investigated the effects of two isomers of CLA (cis-9 and trans-10 isomers) on Caco-2 cell transepithelial resistance (TER) development, paracellular epithelial permeability, and occludin and ZO-1 expression. Caco-2 cells were grown in media supplemented with 0.05 mM linoleic acid, cis-9 CLA, or trans-10 CLA for 21 days. The trans-10 CLA isomer delayed Caco-2 cell TER development, which is an in vitro measure of epithelial cell integrity, and increased paracellular epithelial permeability. Immunofluorescent staining of Caco-2 cell epithelial monolayers grown in media supplemented trans-10 CLA showed that the trans-10 CLA isomer altered distribution of occludin and ZO-1. The trans-10 CLA isomer delayed the acquisition of transepithelial resistance and altered the cellular distribution of occludin, which have important implications in relation to epithelial permeability.

Bioactive fatty acids: role in bone biology and bone cell function.

Bone is a unique tissue providing support, movement, and mineral balance for the body. Bone growth is achieved in the young by a process called modeling, and maintained during adulthood by a process termed remodeling. Three types of cells are responsible for the formation of cartilage and bone; the chondrocyte, osteoblast, and osteoclast. These cells are under the influence of a plethora of regulatory molecules, which govern their action to provide an individual optimal bone mass. Interruption of this homeostatic machinery, especially in the elderly, often results in a loss of bone mass (osteoporosis) or cartilage damage (rheumatoid arthritis). Many pharmacological agents have been made available in an effort to prevent or alleviate these pathologies, however, one vector often overlooked is the diet. This review focuses on the relationship between dietary polyunsaturated fatty acids and bone biology, both in vivo and in vitro.

The significance of polyunsaturated fatty acids in cutaneous biology.

The skin epidermis displays a highly active metabolism of polyunsaturated fatty acids (PUFA). Dietary deficiency of linoleic acid (LA) and 18-carbon (n-6) PUFA results in characteristic scaly skin disorder and excessive epidermal water loss. Arachidonic acid, a 20-carbon (n-6) PUFA is metabolized via the cyclooxygenase pathway into predominantly prostaglandin E2 (PGE2) PGF2 alpha, and PGD2 and via the lipoxygenase pathway into predominantly 15-hydroxyeicosatetraenoic acid (15-HETE). The prostaglandins modulate normal skin physiological processes at low concentrations and inflammatory reactions at high concentrations. Similarly, the very active epidermal 15-lipoxygenase transforms dihomogammalinolenic acid (DGLA) into 15-hydroxy eicosatrienoic acid (15-HETrE), eicosapentaenoic acid (EPA) into 15-hydroxyeicosapentaenoic acid (15-HEPE) and docosahexaenoic acid (DHA) into 17-hydroxydocosahexaenoic acid (17-HDoHE), respectively. These monohydroxy acids exhibit anti-inflammatory properties. In contrast, the 18-carbon (n-6) PUFA is transformed into 13-hydroxy-9,11-octadecadienoic acid (13-HODE), which exerts antiproliferative properties in the tissue. Thus, the supplementation of diets with appropriate purified vegetable oils and/or fish oil may generate local cutaneous anti-inflammatory metabolites which could serve as a less toxic in vivo monotherapy or as adjuncts to standard therapeutic regimens for the management of skin inflammatory disorders.

Nutritional modulation of guinea pig skin hyperproliferation by essential fatty acid deficiency is associated with selective down regulation of protein kinase C-beta.

In a previous study we demonstrated that 13-hydroxyoctadecadienoic acid (13-HODE), a 15-lipoxygenase metabolite of linoleic acid is incorporated into epidermal phosphatidyl 4,5-bisphosphate (PtdIns 4,5-P2) and released as 13-HODE-containing-diacylglycerol (13-HODE-DAG). In vitro, 13-HODE-DAG was shown to selectively inhibit epidermal total protein kinase C (PKC-beta) activity. To determine whether these observations are relevant in vivo, guinea pigs were made essential fatty acid deficient (EFAD) by feeding them a basal diet supplemented with 4% hydrogenated coconut oil for 8 wk. Tissue levels of putative 13-HODE-DAG, protein kinase C (PKC) isozymes and tissue hyperproliferation were determined in the epidermal preparations from skin of control safflower oil-fed guinea pigs, those fed EFAD diet and those fed EFAD diet followed by the control diet for 2 wk. Our data revealed that cutaneous 13-HODE and 13-HODE-DAG were significantly lower in EFAD animals than in safflower-fed controls. These reductions were associated with both elevated epidermal hyperproliferation and elevated expressions and activities of PKC-alpha and beta-isozymes. Refeeding the animals with safflower oil for 2 wk replenished tissue levels of 13-HODE-DAG, which inversely correlated with the selective down regulation of PKC-beta expression and activity and the reversal of hyperproliferation. In contrast, although, the expression and activity of PKC-alpha was elevated in the epidermis of the EFAD guinea pigs, this elevated PKC-alpha expression was not down regulated after refeeding the safflower oil diet to the animals.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of dietary n-3 fatty acid deficiency on blood-to-brain transfer of sucrose, alpha-aminoisobutyric acid and phenylalanine in the rat.

Possible alterations in blood-to-brain unidirectional transport of sucrose (mol. wt., 342), alpha-aminoisobutyric acid (mol. wt., 104), and L-phenylalanine (mol. wt., 165) induced by a diet deficient in n-3 polyunsaturated fatty acids were studied with respect to blood-brain barrier function. Two groups of rats were for to two generations with a semisynthetic diet. One group of rats was fed a peanut oil+rapeseed oil diet which contained both essential fatty acids: linoleic acid (18:2 n-6) and alpha-linolenic acid, (18:3 n-3). Another group was fed a diet of peanut oil, this diet (containing 18:2 n-6) was deficient in alpha-linolenic acid. The experiments were performed at 6 months of age. Unidirectional transfer rate constants (Ki) of sucrose, alpha-aminoisobutyric acid and L-phenylalanine were measured. The diet based on peanut oil (deficient in n-3) caused a greater blood-to-brain transport of sucrose but not of alpha-aminoisobutyric acid or L-phenylalanine. These observations indicate that regardless of the mechanisms involved, alterations in essential fatty acids induced by diet can modulate to some extent the blood-brain transport of hydrophilic molecules without a carrier.

Essential fatty acids and iron are involved at distinct stages of the proliferative cycle but not in the activation of human T cells.

In the absence of serum, optimal lymphocyte proliferation is obtained when cultures are supplemented with transferrin and an essential fatty acid (EFA). In order to study the effects of iron in conjunction with EFA on T-cell proliferation, we have utilized a chemically defined serum-free culture system to achieve better control of the variables involved. This system includes three different serum-free media (SFM) that differ in total iron content and source of iron: (i) transferrin-free medium containing a high concentration (500 microns) of a soluble iron salt in the form of ferric citrate (Fe-SFM); (ii) iron-saturated human transferrin (5 micrograms/ml) (T-SFM); and (iii) iron-free medium (SFM(-Fe)) without any apparent source of iron. None of these SFM supported proliferation of T cells stimulated by the combination of phorbol 12,13-dibutyrate/ionomycin or phytohemagglutinin. Restoration of the proliferative response was only observed following supplementation of the iron-containing media with linoleic acid (complexed to bovine serum albumin (LA/BSA)). In cultures containing LA/BSA, the addition of iron alone in the absence of transferrin (Fe-SFM) resulted in similar responses to the transferrin-containing medium (T-SFM). Low levels of RNA synthesis in mitogen-stimulated T cells could be demonstrated in the presence or absence of iron and the addition of LA/BSA resulted in marked enhancement of RNA synthesis, regardless of the availability of iron. Cell cycle analysis showed that 91-94% of the cells cultured in SFM were arrested in G0/G1. These cells could progress through the cell cycle following the addition of LA/BSA, but only in the iron-containing media. Unlike DNA or RNA synthesis, activation of T cells could be demonstrated in SFM with or without iron as shown by the normal induction of c-fos and early growth response gene mRNA, normal expression of IL2 and transferrin receptors, and normal IL2 production, despite the arrest of cells in G0/G1. These results suggest that although human T-cell growth is iron and EFA dependent, the early events of T-cell activation are both iron and EFA independent.

Influence of lipid environment on insulin binding in cultured hepatoma cells.

The influence of alterations of plasma membrane physico-chemical properties on insulin binding have been characterized in an insulin-sensitive rat hepatoma cell line adapted to grow for several generations in culture medium enriched with linoleic acid (18:2) or with 25-hydroxycholesterol. The cells took up 18:2 and 25-hydroxycholesterol added to the culture medium, without exhibiting any sign of intolerance or intoxication. These compounds respectively increased and decreased membrane fluidity at 37 degrees C. The cells demonstrated extensive changes in insulin binding parameters in response to experimental modifications of their membrane lipid composition. When determined at 4 degrees C, insulin receptors were present in the control cells at 136,000 sites/cell but this fell to 111,000 (P less than 0.05) in cells enriched in 18:2, and rose to 176,000 (P less than 0.001) in hydroxysterol-grown cells. According to a two-site model, the main effect of 18:2 was a significant increase of the number of high-affinity sites with a concomitant decrease of low-affinity sites. The hydroxysterol had the opposite effects on these parameters. The high-affinity insulin binding capacity of the hepatoma cells was affected by lipid supplementation in a similar way, whether it was determined at 4 degrees C or at 37 degrees C. Assuming a negative cooperativity model, 18:2 enhanced the degree of negative cooperativity among the sites, while 25-hydroxycholesterol reduced it. The time-course of insulin-induced receptor down-regulation was accelerated in the cells enriched in polyunsaturated fatty acids, but reduced in cells exposed to 25-hydroxycholesterol. These insulin-binding alterations cannot be directly related to modifications of cellular growth rate, receptor internalization or membrane fluidity per se, and are discussed as being more likely due to membrane lipid composition than to overall cell metabolism modifications.

Essential function of linoleic acid esterified in acylglucosylceramide and acylceramide in maintaining the epidermal water permeability barrier. Evidence from feeding studies with oleate, linoleate, arachidonate, columbinate and alpha-linolenate.

Essential fatty acid-deficient rats were supplemented with 300 mg per day of pure fatty acid esters: oleate (O), linoleate (L), arachidonate (A), and columbinate (C) for 10 days. During this period, the rats in groups L, A, and C all showed a decrease in their initially high trans-epidermal water loss, a classical essential fatty acid-deficiency symptom, to a level seen in non-deficient rats (group N). The trans-epidermal water loss in rats of group O was unaffected by the supplementation. Fatty acid composition of two epidermal sphingolipids, acylglucosylceramide and acylceramide, from the skin were determined. The results indicate that re-establishment of a low trans-epidermal water loss was associated with incorporation of linolenate into the two epidermal sphingolipids. Supplementation with columbinate resulted in relatively high amounts of this fatty acid in the investigated epidermal sphingolipids. Analysis of pooled skin specimens from a previous study in which weanling rats were fed a fat-free diet and supplemented orally with pure alpha-linolenate for 13 weeks (Hansen, H.S. and Jensen, B. (1983) Lipids 18, 682-690) revealed very little polyunsaturated fatty acid in the two sphingolipids. These rats showed increased evaporation which was comparable to that of essential fatty acid-deficient rats. We interpret these results as strong evidence for a very specific and essential function of linoleic acid in maintaining the integrity of the epidermal water permeability barrier. This function of linoleate is independent of its role as precursor for arachidonate and icosanoids.

Dietary fat, prostaglandins and the immune response.

Events leading to the interest in dietary fat and immunity are summarized. This is followed by an account of the possible functions of prostaglandins and other eicosanoids in the immune response. Dietary studies on the influence of quantity and quality of fat on immune responses and the histopathology of lymphoid organs are critically reviewed. The next section deals primarily with work conducted in the authors' laboratories on essential fatty acid deficiency and the immune response and on the effects of varying the ratio of dietary linoleic to linolenic acids on prostaglandin synthesis by immunocompetent cells and immune functions. A brief account of a study on the influence of dietary fat on leukotriene synthesis follows. Finally the problems encountered in research on dietary fat and immunity are discussed and recommendations for design and execution of such studies are given.

Effect of fatty acid modification of cultured hepatoma cells on susceptibility to natural killer cells.

Rat hepatoma (Morris 7777) cells modified with either oleic or linoleic acid exhibited greater susceptibility to normal spleen cell-mediated lysis in a 16-hr 51Cr release assay. At effector:target cell ratios of 300:1, the specific lysis of fatty acid-enriched target cells (cultured for 2 days in fatty acid-supplemented medium) by the normal rat spleen cells was 60% higher than the untreated target cells (P less than 0.01). Prolonging the culture in fatty acid-supplemented medium up to 6 days produced similar effects. Analysis of the fatty acid composition of cellular lipids revealed that an elevation of oleic or linoleic acid was the only significant alteration in the hepatoma cells grown in the oleic or linoleic acid-supplemented medium, respectively. The percentage of the acids was increased in the total cellular phospholipids, the choline, ethanolamine, serine, and inositol phosphoglyceride fractions, and the neutral lipids. In conclusion, we suggest that the elevation of oleic acid and linoleic acid contents in the membranes of the fatty acid-modified hepatoma cells may contribute to the increased susceptibility of these cells to natural killer cell-mediated cytotoxicity.

Visual membranes: specificity of fatty acid precursors for the electrical response to illumination.

Rat electroretinograms were measured as a function of dietary supplements of purified ethyl esters of linolenic acid, linoleic acid, and oleic acid. Polyunsaturated fatty acids derived from precursors of linolenic and linoleic acids appear to be important functional components of photoreceptor cell membranes, although in equal dietary concentrations, linolenic acid precursors affect electroretinogram amplitudes to a greater extent than linoleic acid precursors. The electrical response of photoreceptor cell membranes appears to be a function of the position of the double bonds as well as a function of the total number of double bonds in fatty acid supplements.