title: fat-1 transgene expression prevents cell culture-induced loss of membrane n-3 fatty acids in activated CD4+ T-cells.
abstract: In order to evaluate the effects of fatty acids on immune cell membrane structure and function, it is often necessary to maintain cells in culture. However, cell culture conditions typically reverse alterations in polyunsaturated fatty acid (PUFA) composition achieved by dietary lipid manipulation. Therefore, we hypothesized that T-cells from transgenic mice expressing the Caenorhabditis elegans n-3 desaturase (fat-1) gene would be resistant to the culture-induced loss of n-3 PUFA and, therefore, obviate the need to incorporate fatty acids or homologous serum into the medium. CD4+ T-cells were isolated from (i) control wild type (WT) mice fed a safflower oil-n-6 PUFA enriched diet (SAF) devoid of n-3 PUFA, (ii) fat-1 transgenic mice (enriched with endogenous n-3 PUFA) fed a SAF diet, or (iii) WT mice fed a fish oil (FO) based diet enriched in n-3 PUFA. T-cell phospholipids isolated from WT mice fed FO diet (enriched in n-3 PUFA) and fat-1 transgenic mice fed a SAF diet (enriched in n-6 PUFA) were both enriched in n-3 PUFA. As expected, the mol% levels of both n-3 and n-6 PUFA were decreased in cultures of CD4+ T-cells from FO-fed WT mice after 3d in culture. In contrast, the expression of n-3 desaturase prevented the culture-induced decrease of n-3 PUFA in CD4+ T-cells from the transgenic mice. Carboxyfluorescein succinidyl ester (CFSE) -labeled CD4+ T-cells from fat-1/SAF vs. WT/SAF mice stimulated with anti-CD3 and anti-CD28 for 3d, exhibited a reduced (P<0.05) number of cell divisions. We conclude that fat-1-containing CD4+ T-cells express a physiologically relevant, n-3 PUFA enriched, membrane fatty acid composition which is resistant to conventional cell culture-induced depletion.
authors.affiliation: Department of Nutrition and Food Science, Texas A&M University, College Station, TX 77843-2253, USA.
authors.forename: Yang-Yi
authors.initials: YY
authors.lastname: Fan
authors.forename: Wooki
authors.initials: W
authors.lastname: Kim
authors.forename: Evelyn
authors.initials: E
authors.lastname: Callaway
authors.forename: Roger
authors.initials: R
authors.lastname: Smith
authors.forename: Qian
authors.initials: Q
authors.lastname: Jia
authors.forename: Lan
authors.initials: L
authors.lastname: Zhou
authors.forename: David N
authors.initials: DN
authors.lastname: McMurray
authors.forename: Robert S
authors.initials: RS
authors.lastname: Chapkin
mesh_terms: D000818:Animals; D015496:CD4-Positive T-Lymphocytes / Q000166:cytology / Q000378:metabolism*; D029742:Caenorhabditis elegans Proteins / Q000235:genetics / Q000502:physiology*; D002462:Cell Membrane / Q000378:metabolism*; D049109:Cell Proliferation; D044943:Fatty Acid Desaturases / Q000235:genetics / Q000502:physiology*; D015525:Fatty Acids, Omega-3 / Q000378:metabolism*; D005260:Female; D005434:Flow Cytometry; D051379:Mice; D008810:Mice, Inbred C57BL; D008822:Mice, Transgenic; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014886:Weaning
chemical_list: D029742:Caenorhabditis elegans Proteins; D015525:Fatty Acids, Omega-3; C466741:fat-1 protein, C elegans; D044943:Fatty Acid Desaturases