Fatty acid uptake by cultured human keratinocytes grown in medium deficient in or supplemented with essential fatty acids.

Epidermal linoleic acid, i.e. essential fatty acid (EFA), is essential for cutaneous barrier function. Cultured human keratinocytes, routinely used for studies of lipid metabolism, are grown in a keratinocyte serum-free medium (KSFM), under conditions that reveal EFA-deficient cells. Here, fatty acid (FA) uptake was analysed in human adult keratinocytes grown either under EFA-deficient conditions [KSFM supplemented with 10% FCS (A) or 1% UltroserG (B)] or EFA-supplemented conditions [KSFM supplemented with a devised FA cocktail (C) or evening primrose oil (D)]. The FA composition of the total cellular lipid and major lipid fractions was analysed by gas chromatography. Cells grown with supplements A or B balanced their EFA-deficient state primarily with oleic acid. Cells grown with supplements C or D normalized to the epidermal FA composition in vivo with raised linoleic and lower oleic acid contents. When cells were grown longer than 48 h with supplements C or D decreased cell growth was observed. FA uptake was curvilinear with preference for linoleic over oleic acid under all culture conditions. The uptake of linoleic acid by cells cultured with supplement B was twice the uptake of those cultured with supplement A, while the uptake of oleic acid was similar under both culture conditions. Oleic acid uptake of cells cultured with supplement C or D was lower. These results show that the uptake of linoleic, but not that of oleic acid, is influenced by the extracellular FA composition, and that EFA-supplemented keratinocytes compared to EFA-deficient cells might serve as an in vitro model for the study of EFA metabolism.

Quantitative screening of human stratum corneum lipids by sequential one-dimensional thin layer chromatography.

There is a need in many studies on the epidermal permeability barrier for practical and reliable separation of all major fractions of human stratum corneum lipids. Various methods have been described including thin layer chromatography, high performance thin layer chromatography and iatroscan. However, none of these methods seems to be applicable for an inexpensive, rapid and reliable analysis of a large number of samples. Here, such a method for the separation and quantification of all major stratum corneum lipid fractions is presented. This method employs the one-dimensional separation of stratum corneum lipids using thin layer chromatography. The systems used herein are based upon various modifications of the solvents, solvent ratio and developing distance of each system. For quantification of chromatographed and charred lipids a Desaga densitometer is used. The system described here had been successfully applied to 550 samples of human stratum corneum lipids.

Differential utilization of linoleic and arachidonic acid by cultured human keratinocytes.

The essential fatty acid, linoleic acid (LA), is required for the epidermal barrier and LA is also the precursor fatty acid for arachidonic acid (AA). Both fatty acids are imported from systemic sources, because AA is also not synthesized in the epidermis. The present studies were undertaken to compare the uptake and incorporation into cellular lipid of these fatty acids and to determine whether they compete with one another in these processes in relation to keratinocyte differentiation. The incorporation of [14C]LA and/or [14C]AA into phospholipids and triglycerides was examined in keratinocytes cultured under submerged and lifted conditions. In submerged (less well-differentiated) cultures, more LA was incorporated into phospholipids than AA, while AA was incorporated into triglycerides to a greater extent. When given together, neither fatty acid influenced the total and/or relative uptake and lipid distribution of the other. Compared to submerged cultures, the uptake of LA increased 2-fold in lifted (differentiated) cultures, while the uptake of AA was unchanged. Lifting increased the proportion of AA incorporated into phospholipids, but did not alter the distribution of LA among phospholipids or triglycerides. These data suggest that the essential fatty acids, LA and AA, which are destined for different metabolic roles within the keratinocyte do not compete with one another during their uptake and distribution among cellular lipid species. Furthermore, as keratinocytes differentiate in culture, their increased requirement for LA for the synthesis of barrier lipids may be achieved through the preferentially enhanced uptake and lipid incorporation.

Evidence for a novel keratinocyte fatty acid uptake mechanism with preference for linoleic acid: comparison of oleic and linoleic acid uptake by cultured human keratinocytes, fibroblasts and a human hepatoma cell line.

Keratinocytes require the essential fatty acid (FA), linoleic acid (LA), for the synthesis of stratum corneum membrane lipids. A plasma membrane-FA binding protein (PM-FABP), is postulated to mediate cellular FA-uptake in hepatocytes and several other tissues, but the mechanism whereby exogenous FA are taken up by keratinocytes has not been investigated. This study examines the uptake of LA and oleic acid (non-essential) in cultured human keratinocytes, in comparison to dermal fibroblasts and the human hepatoma cell line, HepG2. As previously reported for hepatocytes, FA-uptake in keratinocytes was curvilinear, with an initial (30 s) rapid cellular influx. The initial uptake component was temperature dependent, exhibited saturable kinetics and was significantly inhibited by pretreatment with trypsin. In contrast, fibroblast FA-uptake lacked an initial rapid uptake component, was relatively temperature insensitive, and was not inhibited by trypsin. Keratinocytes differed from both hepatocytes and fibroblasts by more rapid uptake of LA in comparison to oleic acid during the initial influx phase. Moreover, FA-uptake in keratinocytes was not inhibited by preincubation with a anti-rat liver PM-FABP antibody. These data provide evidence for a PM-FA transporter in keratinocytes that is distinct from the hepatic PM-FABP. The apparent preference of the putative keratinocyte FA transporter for LA may function to ensure epidermal capture of sufficient LA for barrier lipid synthesis.

Lipid composition and synthesis of HaCaT cells, an immortalized human keratinocyte line, in comparison with normal human adult keratinocytes.

Cultured keratinocytes are frequently employed for studies of epidermal lipid metabolism. Interpretation of experimental data may be complicated by donor to donor variability, the relatively short culture lifetime and variations between passages, problems that are not encountered to the same extent with immortalized cell lines. The present study was undertaken to compare the lipid composition and synthesis of normal human adult keratinocytes (NHAK) with HaCaT cells, a long-lived, spontaneously immortalized human keratinocyte line, in relation to proliferation and differentiation. No differences between the two cell types were observed: a) in total lipid content; b) in the distribution of major lipid classes during growth at 50%, 75% and 100% confluence; c) in cultures grown at 0.6 mM calcium, at which differentiation is retarded, or at 1.6 mM calcium, at which some differentiation takes place; d) in the incorporation of [14C] acetate into cellular lipids at confluence, or e) in the fatty acid composition of major cellular lipid classes. At 100% confluence NHAK and HaCaT cells differ in their cholesterol metabolism. At all stages of growth, cholesterol synthesis in HaCaT cells is more LDL-dependent than in NHAK. Furthermore, NHAK become less LDL-dependent at confluence whereas HaCaT cells do not. HaCaT cells also revealed a significantly larger fraction of phosphatidyl-ethanolamine, -serine and -inositol at 0.6 mM calcium concentration than NHAK. These findings suggest that HaCaT cells do not differentiate as well as NHAK in vitro and may therefore serve as a model for the study of lipid metabolism in cells defective in terminal differentiation.