Abstract
Fatty acid transport protein (FATP) was identified by expression cloning strategies (Schaffer, J. E., and Lodish, H. F. (1994) Cell 79, 427-436) and shown by transfection analysis to catalyze the transfer of long-chain fatty acids across the plasma membrane of cells. It is expressed highly in tissues exhibiting rapid fatty acid metabolism such as skeletal muscle, heart, and adipose. FATP mRNA levels are down-regulated by insulin in cultured 3T3-L1 adipocytes and up-regulated by nutrient depletion in murine adipose tissue (Man, M. Z., Hui, T. Y., Schaffer, J. E., Lodish, H. F., and Bernlohr, D. A. (1996) Mol. Endocrinol. 10, 1021-1028). To determine the molecular mechanism of insulin regulation of FATP transcription, we have isolated the murine FATP gene and its 5'-flanking sequences. The FATP gene spans approximately 16 kilobases and contains 13 exons, of which exon 2 is alternatively spliced. S1 nuclease and RNase protection assays revealed the presence of multiple transcription start sites; the DNA sequence upstream of the predominant transcription start sites lacks a typical TATA box. By transient transfection assays in 3T3-L1 adipocytes, the inhibitory action of insulin on FATP transcription was localized to a cis-acting element with the sequence 5'-TGTTTTC-3' from -1347 to -1353. This sequence is very similar to the insulin response sequence found in the regulatory region of other genes negatively regulated by insulin such as those encoding phosphoenolpyruvate carboxykinase, tyrosine aminotransferase, and insulin-like growth factor-binding protein 1. Fluorescence in situ hybridization analysis revealed that the murine FATP gene is localized to chromosome 8, band 8B3.3. Interestingly, this region of chromosome 8 contains a cluster of three other genes important for fatty acid homeostasis, lipoprotein lipase, the mitochondrial uncoupling protein 1 (UCP1) and sterol regulatory element-binding protein 1. These results characterize the murine FATP gene and its insulin responsiveness as well as present a framework for future studies of its role in lipid metabolism, obesity, and type II diabetes mellitus.
Highlights
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EBI Data Bank with accession number(s) AF023256, AF023257, and AF023258
These results suggest that the fatty acid transport protein (FATP) gene is unique in the genome and 10 –30 kb in size
Insulin Responsive Sequences—We have previously demonstrated that insulin represses FATP transcription in 3T3-L1 adipocytes [18]
Summary
Cell Culture and Transfection—3T3-L1 preadipocytes were grown to confluence and induced to differentiate to adipocytes as described previously [27]. Fragments containing various lengths of the FATP promoter were amplified by PCR with restriction sites engineered for subcloning into the promoterless pGL3-Basic luciferase expression vector (Promega, Madison, WI). These PCR generated fragments were fully sequenced to check for mutations introduced by Taq polymerase. PC1F and pC1R, containing the sequence between Ϫ1189 and ϩ74, were constructed by ligating the fragment into the HindIII site of the vector in forward and reverse orientations, respectively. The two plasmids (C1F⌬G and C1F⌬S) that contain sequences Ϫ273/ϩ84 and ϩ53/ϩ84 were generated by digesting C1F with BglII and SacI, respectively, followed by self-ligation. Competitors were added to the reaction mixtures in 20-, 50-, and 200-fold molar excess
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