3 Molecular and Cellular Biochemistry 192: 3–8, 1999. © 1999 Kluwer Academic Publishers. Printed in the Netherlands. CD36 antisense expression in 3T3-F442A preadipocytes Zeina Sfeir, 1 Azeddine Ibrahimi, 1 Ez-zoubir Amri, 2 Paul Grimaldi 2 and Nada Abumrad 1 1 Department of Physiology and Biophysics, State University of New York at Stony Brook, NY, USA; 2 Department of Biochemistry, University of Nice, France Abstract An adipocyte membrane glycoprotein, FAT, homologous to CD36, has been implicated in the binding/transport of long-chain fatty acids. FAT/CD36 was identified by reaction with reactive long chain fatty acids derivatives under conditions where they inhibited FA uptake. Expression of CD36 in fibroblasts lacking the protein led to induction of a saturable high affinity, phloretin- sensitive component of oleate uptake. In this report, we have examined the effects of FAT/CD36 antisense expression in 3T3-F442A preadipocyte cells, on FA uptake and cell differentiation. Cells were transfected with pSG5-TAF vector obtained by insertion of antisense coding sequence of FAT/CD36 into the BamH 1 site of pSG5. Four clones were selected based on expression of antisense CD36 mRNA. Levels of CD36 protein were determined by flow cytometry and correlated with rates of oleate uptake. Three clones, TAF13, TAF25, and TAF38 exhibited low CD36 expression and one clone TAF 18 had expression comparable to that of F442A control cells. FA uptake rates in clones TAF13, TAF25 and TAF3 8 were lower than those observed in TAF18. At confluence, adipocyte differentiation could be promoted by addition of insulin and triiodothyronine only in TAF18 cells but not in TAF13, TAF25 or TAF38. Addition of fatty acids to clones TAF13, TAF25 and TAF38 lead to an induction of CD36 expression, an enhancement of FA uptake and better cell differentiation. The data support a role of CD36 in the membrane uptake of long chain FA. CD36 expression and FA uptake appear to be closely linked to preadipocyte differentiation. (Mol Cell Biochem 192: 3–8, 1999) Key words: fatty acid transport, CD36-antisense RNA, transfection, adipocyte differentiation Abbreviations: FA – fatty acids; GAPDH – glyceraldehyde phosphate dehydrogenase; BSA – bovine serum albumin; ALBP – Adipose Lipid Binding Protein; LPL – Lipoprotein lipase Introduction Kinetic characterization of membrane permeation of long- chain fatty acid (FA) by rat adipocytes identified a saturable component and suggested the involvement of a high affinity carrier system [1–3]. Later studies, using reactive sulfo-N- succinimidyl derivatives of oleate (SSO), implicated a mem- brane protein with an apparent molecular weight of 88 kDa in the uptake process [4, 5]. The protein was specifically labeled under conditions where FA uptake into the cell was inhibited by about 75%. The isolated protein (FAT) had an amino Address for offprints: N.A. Abumrad, Department of Physiology and Biophysics, State University of New York at Stony Brook, New York 11794-8661, USA terminal sequence similar to that of human glycoprotein IV or CD36 [6, 7]. The complete protein sequence, deduced from the cDNA, was 85% similar to that of CD36 [8]. Multiple lines of evidence were consistent with a role for the protein in FA uptake. Purified CD36, in vitro, bound FA at nM concentrations and low ligand to protein ratio [9]. Distribution of FAT/CD36 mRNA favored tissues with a high metabolic capacity for FA [8, 10]. It was high in the heart and in muscle tissues with a predominance of red oxidative fibers and was upregulated during heart development when FA utilization increases [10]. In culture, CD36 mRNA was a