Altered membrane structure in transfected mouse L-cell fibroblasts expressing rat liver fatty acid-binding protein.
Mouse L cell fibroblasts were transfected with cloned cDNA encoding rat liver fatty acid binding protein (L-FABP) also known as sterol carrier protein. Stable transfectant cell lines were selected and expression of L-FABP determined using Western blot analysis. The nontransfected controls and low expression cells did not differ significantly in any of the properties examined. All cell lines showed similar doubling times but cells expressing high levels of L-FABP attained 2-fold higher cell saturation density and differed significantly in their lipid metabolism as indicated by 1) higher cholesterol ester and phospholipid content, and 2) decreased sterol/phospholipid ratio. The observed changes in the lipid composition predicted a lower degree of membrane-lipid order (higher fluidity) in the plasma membranes of cells expressing high levels of L-FABP. Therefore, fluorescent molecule, 1,6-diphenyl-1,3,5-hexatriene, and multifrequency (1-250 MHz) phase and modulation fluorometry were used to probe the effect of L-FABP expression on membrane structure. Steady-state polarization and limiting anisotropy of diphenylhexatriene were significantly lower in the isolated plasma membrane vesicles from the high expression clones. The observed changes in L-cells as a result of de novo expression of L-FABP are consistent with the ability of this protein to bind sterols and fatty acids, stimulate sterol esterification, and stimulate phospholipid biosynthesis. This evidence is supportive of a physiologic role for L-FABP in modulating cellular lipid metabolism and membrane structure.
Jefferson
J R
JR
Department of Pharmacology and Cell Biophysics, University of Cincinnati Medical Center, Ohio 45267-0004.
Powell
D M
DM
Rymaszewski
Z
Z
Kukowska-Latallo
J
J
Lowe
J B
JB
Schroeder
F
F
D000818:Animals; D015153:Blotting, Western; D002352:Carrier Proteins / Q000235:genetics / Q000502:physiology*; D002462:Cell Membrane / Q000032:analysis / Q000378:metabolism*; D003001:Cloning, Molecular; D004247:DNA / Q000235:genetics; D004161:Diphenylhexatriene; D000071438:Fatty Acid-Binding Protein 7; D050556:Fatty Acid-Binding Proteins; D005347:Fibroblasts / Q000378:metabolism / Q000648:ultrastructure*; D005454:Fluorescence Polarization; D005456:Fluorescent Dyes; D015870:Gene Expression; D007739:L Cells; D050356:Lipid Metabolism; D008055:Lipids / Q000032:analysis; D008099:Liver / Q000032:analysis*; D008668:Metallothionein / Q000235:genetics; D051379:Mice; D008970:Molecular Weight; D009363:Neoplasm Proteins; D009419:Nerve Tissue Proteins; D011401:Promoter Regions, Genetic; D051381:Rats; D015183:Restriction Mapping; D014162:Transfection
D002352:Carrier Proteins; C493320:Fabp1 protein, mouse; C493321:Fabp1 protein, rat; C082667:Fabp5 protein, mouse; C086936:Fabp7 protein, mouse; C490174:Fabp7 protein, rat; D000071438:Fatty Acid-Binding Protein 7; D050556:Fatty Acid-Binding Proteins; D005456:Fluorescent Dyes; D008055:Lipids; D009363:Neoplasm Proteins; D009419:Nerve Tissue Proteins; D004161:Diphenylhexatriene; D004247:DNA; D008668:Metallothionein