Permeation of long-chain fatty acid into adipocytes. Kinetics, specificity, and evidence for involvement of a membrane protein.
This study extends our earlier work (Abumrad, N. A., Perkins, R.C., Park, J.H., and Park, C.R. J. Biol. Chem. 256, 9183-9191) which showed that oleate permeates the plasma membrane of the rat adipocyte principally by a transport process with the characteristics of facilitated diffusion. In the present study, fatty acid (FA) transport is characterized with regard to its specificity and susceptibility to inhibition by protein modifiers. The kinetics of competitive inhibition for transport of oleate and stearate are shown under conditions where complications due to competition for binding of FAs to the albumin in the medium are minimized. Stearate inhibits influx of tracer oleate with a Ki that closely approximates its Km and, conversely, oleate inhibits similarly the influx of tracer stearate. Specificity of the FA transport system is shown in studies using a variety of natural FAs of different chain length, or FA analogues. Oleate (Km = 0.06 microM), stearate (Km = 0.16 microM), linoleate (Km = 0.22 microM), palmitate, (Km = 0.2 microM), and laurate (Km = 1.5 microM) are good substrates, but octanoate is not transported. An oxazolidine ring on C-5 but not on C-16 of stearate blocks binding to the transporter. Methylation of the carboxyl function but not alpha-bromination inhibits transport. These studies suggest that a FA must have a hydrocarbon chain of at least nine carbons and a free carboxyl function to be recognized by the transporter. FA transport does not require Na or ATP. Pronase but not trypsin treatment of intact cells reduces fatty acid influx. Transport is insensitive to maleimides. It is strongly and irreversibly blocked by pretreatment of the cells with the stilbene compounds, 4,4'-diisothiocyanostilbene-2,2'-disulfonate and 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid, but only slightly inhibited by dipyridamole. Polyacrylamide gel electrophoresis of plasma membrane proteins from cells treated with [3H] 4,4'-diisothiocyanostilbene-2,2'-disulfonate shows a peak of radioactivity at about Mr = 85,000. When cells are incubated in various concentrations of this agent, the counts recovered in the peak reach a maximum coincident with maximum inhibition of transport. We conclude that permeation of the plasma membrane of the adipocyte by long-chain FAs at physiological concentrations is mediated by a protein transporter with distinct specificity requirements.
D000273:Adipose Tissue / Q000378:metabolism*; D000818:Animals; D002210:Caprylates / Q000378:metabolism; D002250:Carbon Radioisotopes; D002352:Carrier Proteins / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism; D002463:Cell Membrane Permeability; D000071438:Fatty Acid-Binding Protein 7; D050556:Fatty Acid-Binding Proteins; D005227:Fatty Acids / Q000378:metabolism*; D066298:In Vitro Techniques; D007700:Kinetics; D007850:Lauric Acids / Q000378:metabolism; D008297:Male; D009363:Neoplasm Proteins; D009419:Nerve Tissue Proteins; D019301:Oleic Acid; D009829:Oleic Acids / Q000378:metabolism; D019308:Palmitic Acid; D010169:Palmitic Acids / Q000378:metabolism; D051381:Rats; D011919:Rats, Inbred Strains; D013229:Stearic Acids / Q000378:metabolism; D013379:Substrate Specificity; D014316:Tritium
D002210:Caprylates; D002250:Carbon Radioisotopes; D002352:Carrier Proteins; C490174:Fabp7 protein, rat; D000071438:Fatty Acid-Binding Protein 7; D050556:Fatty Acid-Binding Proteins; D005227:Fatty Acids; D007850:Lauric Acids; D009363:Neoplasm Proteins; D009419:Nerve Tissue Proteins; D009829:Oleic Acids; D010169:Palmitic Acids; D013229:Stearic Acids; D014316:Tritium; C030358:lauric acid; D019301:Oleic Acid; D019308:Palmitic Acid; C031183:stearic acid; C031492:octanoic acid