FAT/CD36-mediated long-chain fatty acid uptake in adipocytes requires plasma membrane rafts.
We previously reported that lipid rafts are involved in long-chain fatty acid (LCFA) uptake in 3T3-L1 adipocytes. The present data show that LCFA uptake does not depend on caveolae endocytosis because expression of a dominant negative mutant of dynamin had no effect on uptake of [3H]oleic acid, whereas it effectively prevented endocytosis of cholera toxin. Isolation of detergent-resistant membranes (DRMs) from 3T3-L1 cell homogenates revealed that FAT/CD36 was expressed in both DRMs and detergent-soluble membranes (DSMs), whereas FATP1 and FATP4 were present only in DSMs but not DRMs. Disruption of lipid rafts by cyclodextrin and specific inhibition of FAT/CD36 by sulfo-N-succinimidyl oleate (SSO) significantly decreased uptake of [3H]oleic acid, but simultaneous treatment had no additional or synergistic effects, suggesting that both treatments target the same mechanism. Indeed, subcellular fractionation demonstrated that plasma membrane fatty acid translocase (FAT/CD36) is exclusively located in lipid rafts, whereas intracellular FAT/CD36 cofractionated with DSMs. Binding assays confirmed that [3H]SSO predominantly binds to FAT/CD36 within plasma membrane DRMs. In conclusion, our data strongly suggest that FAT/CD36 mediates raft-dependent LCFA uptake. Plasma membrane lipid rafts might control LCFA uptake by regulating surface availability of FAT/CD36.
Pohl
Jürgen
J
Department of Internal Medicine IV, Ruprechts-Karls-University, D-69117 Heidelberg, Germany.
Ring
Axel
A
Korkmaz
Umine
U
Ehehalt
Robert
R
Stremmel
Wolfgang
W
D041721:3T3-L1 Cells; D017667:Adipocytes / Q000378:metabolism*; D000818:Animals; D015153:Blotting, Western; D018955:CD36 Antigens / Q000378:metabolism / Q000502:physiology*; D021941:Caveolae / Q000378:metabolism; D002462:Cell Membrane / Q000378:metabolism; D002478:Cells, Cultured; D002772:Cholera Toxin / Q000378:metabolism / Q000494:pharmacology; D003902:Detergents / Q000494:pharmacology; D034285:Dynamin II / Q000235:genetics; D004705:Endocytosis; D050612:Fatty Acid Transport Proteins; D005227:Fatty Acids / Q000378:metabolism; D005799:Genes, Dominant; D006801:Humans; D008055:Lipids / Q000737:chemistry; D021962:Membrane Microdomains / Q000378:metabolism*; D026901:Membrane Transport Proteins / Q000378:metabolism; D051379:Mice; D008856:Microscopy, Fluorescence; D009154:Mutation; D019301:Oleic Acid / Q000378:metabolism; D009829:Oleic Acids / Q000494:pharmacology; D017434:Protein Structure, Tertiary; D013388:Succinimides / Q000494:pharmacology; D013997:Time Factors
D018955:CD36 Antigens; D003902:Detergents; D050612:Fatty Acid Transport Proteins; D005227:Fatty Acids; D008055:Lipids; D026901:Membrane Transport Proteins; D009829:Oleic Acids; C493568:SLC27A1 protein, human; C493571:SLC27A4 protein, human; C493569:Slc27a1 protein, mouse; C493572:Slc27a4 protein, mouse; D013388:Succinimides; C120556:sulfo-N-succinimidyl oleate; D019301:Oleic Acid; D002772:Cholera Toxin; D034285:Dynamin II