Characterization of the Murine Fatty Acid Transport Protein Gene and Its Insulin Response Sequence*

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 ∼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.

[1]  J. Sambrook,et al.  Molecular Cloning: A Laboratory Manual , 2001 .

[2]  D. Stump,et al.  Uptake of Long Chain Free Fatty Acids Is Selectively Up-regulated in Adipocytes of Zucker Rats with Genetic Obesity and Non-insulin-dependent Diabetes Mellitus* , 1997, The Journal of Biological Chemistry.

[3]  T. Aoyama,et al.  Molecular Cloning of cDNA Encoding Rat Very Long-chain Acyl-CoA Synthetase* , 1996, The Journal of Biological Chemistry.

[4]  R. O’Brien,et al.  Regulation of gene expression by insulin. , 1996, The Biochemical journal.

[5]  Thomas Shenk,et al.  The Serotonin 1a Receptor Gene Contains a TATA-less Promoter that Responds to MAZ and Sp1 (*) , 1996, The Journal of Biological Chemistry.

[6]  T. Ince,et al.  A Conserved Downstream Element Defines a New Class of RNA Polymerase II Promoters (*) , 1995, The Journal of Biological Chemistry.

[7]  E. Wahle,et al.  3'-end cleavage and polyadenylation of mRNA precursors. , 1995, Biochimica et biophysica acta.

[8]  J. Wang,et al.  Hepatic nuclear factor 3- and hormone-regulated expression of the phosphoenolpyruvate carboxykinase and insulin-like growth factor-binding protein 1 genes , 1995, Molecular and cellular biology.

[9]  H. Lodish,et al.  Expression cloning and characterization of a novel adipocyte long chain fatty acid transport protein , 1994, Cell.

[10]  M. Hediger,et al.  Structure, function and evolution of solute transporters in prokaryotes and eukaryotes. , 1994, The Journal of experimental biology.

[11]  T. Unterman,et al.  Hepatocyte nuclear factor-3 (HNF-3) binds to the insulin response sequence in the IGF binding protein-1 (IGFBP-1) promoter and enhances promoter function. , 1994, Biochemical and biophysical research communications.

[12]  M. Lane,et al.  CCAAT/enhancer binding protein alpha is sufficient to initiate the 3T3-L1 adipocyte differentiation program. , 1994, Proceedings of the National Academy of Sciences of the United States of America.

[13]  C. Kahn,et al.  Insulin stimulates phosphorylation of c-Jun, c-Fos, and Fos-related proteins in cultured adipocytes. , 1994, The Journal of biological chemistry.

[14]  M. Mueckler.  Facilitative glucose transporters. , 1994, European journal of biochemistry.

[15]  H. Westerhoff,et al.  Movement of fatty acids, fatty acid analogues, and bile acids across phospholipid bilayers. , 1993, Biochemistry.

[16]  P. Grimaldi,et al.  Cloning of a rat adipocyte membrane protein implicated in binding or transport of long-chain fatty acids that is induced during preadipocyte differentiation. Homology with human CD36. , 1993, The Journal of biological chemistry.

[17]  D. Powell,et al.  Identification of an insulin-responsive element in the promoter of the human gene for insulin-like growth factor binding protein-1. , 1993, The Journal of biological chemistry.

[18]  G. Schütz,et al.  Activation of the tyrosine aminotransferase gene is dependent on synergy between liver-specific and hormone-responsive elements. , 1993, Proceedings of the National Academy of Sciences of the United States of America.

[19]  F. Kamp,et al.  pH gradients across phospholipid membranes caused by fast flip-flop of un-ionized fatty acids. , 1992, Proceedings of the National Academy of Sciences of the United States of America.

[20]  A. Jochemsen,et al.  Molecular and functional analysis of the XPBC/ERCC-3 promoter: transcription activity is dependent on the integrity of an Sp1-binding site. , 1992, Nucleic Acids Research.

[21]  G. Gerber,et al.  Photoaffinity labeling and fatty acid permeation in 3T3-L1 adipocytes. , 1991, The Journal of biological chemistry.

[22]  R. Umek,et al.  Regulated expression of three C/EBP isoforms during adipose conversion of 3T3-L1 cells. , 1991, Genes & development.

[23]  Ian J.Jackson,et al.  A reappraisal of non-consensus mRNA splice sites. , 1991, Nucleic acids research.

[24]  I. Jackson,et al.  The tyrosinase-related protein-1 gene has a structure and promoter sequence very different from tyrosinase. , 1991, Nucleic acids research.

[25]  J. Azizkhan,et al.  Transcriptional initiation is controlled by upstream GC-box interactions in a TATAA-less promoter , 1990, Molecular and cellular biology.

[26]  R. O’Brien,et al.  Regulation of phosphoenolpyruvate carboxykinase gene expression by insulin. Use of the stable transfection approach to locate an insulin responsive sequence. , 1990, Molecular endocrinology.

[27]  J. L. Messina,et al.  Insulin's regulation of c-fos gene transcription in hepatoma cells. , 1990, The Journal of biological chemistry.

[28]  E. Mira,et al.  Insulin short-term control of rat liver alpha 2-microglobulin gene transcription. , 1989, The Journal of biological chemistry.

[29]  H. Gallinaro,et al.  The 5' splice site: phylogenetic evolution and variable geometry of association with U1RNA. , 1989, Nucleic acids research.

[30]  W. Stremmel.  Fatty acid uptake by isolated rat heart myocytes represents a carrier-mediated transport process. , 1988, The Journal of clinical investigation.

[31]  M. Aebi,et al.  5′ cleavage site in eukaryotic pre-mRNA splicing is determined by the overall 5′ splice region, not by the conserved 5′ GU , 1987, Cell.

[32]  P. Chomczyński,et al.  Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. , 1987, Analytical biochemistry.

[33]  S. Fujii,et al.  Isolation and partial characterization of an amphiphilic 56-kDa fatty acid binding protein from rat renal basolateral membrane. , 1987, Journal of biochemistry.

[34]  D. Straus,et al.  Insulin negatively regulates albumin mRNA at the transcriptional and post-transcriptional level in rat hepatoma cells. , 1987, The Journal of biological chemistry.

[35]  J. McLauchlan,et al.  The consensus sequence YGTGTTYY located downstream from the AATAAA signal is required for efficient formation of mRNA 3' termini. , 1985, Nucleic acids research.

[36]  C. Park,et al.  Permeation of long-chain fatty acid into adipocytes. Kinetics, specificity, and evidence for involvement of a membrane protein. , 1984, The Journal of biological chemistry.

[37]  R. Roeder,et al.  Accurate transcription initiation by RNA polymerase II in a soluble extract from isolated mammalian nuclei. , 1983, Nucleic acids research.

[38]  C. Park,et al.  Mechanism of long chain fatty acid permeation in the isolated adipocyte. , 1981, The Journal of biological chemistry.

[39]  M. Lane,et al.  Induction of fatty acid synthetase synthesis in differentiating 3T3-L1 preadipocytes. , 1980, The Journal of biological chemistry.

[40]  H. Pownall,et al.  Mechanism and kinetics of transfer of a fluorescent fatty acid between single-walled phosphatidylcholine vesicles. , 1980, Biochemistry.

[41]  F. Sauer,et al.  Effect of trypsin, phospholipases, and membrane-impermeable reagents on the uptake of palmitic acid by isolated rat liver cells. , 1974, Archives of biochemistry and biophysics.

[42]  E. Wahle,et al.  The biochemistry of 3'-end cleavage and polyadenylation of messenger RNA precursors. , 1992, Annual review of biochemistry.

[43]  N L Harris,et al.  Splice junctions, branch point sites, and exons: sequence statistics, identification, and applications to genome project. , 1990, Methods in enzymology.

[44]  W. Stremmel,et al.  Isolation and partial characterization of a fatty acid binding protein in rat liver plasma membranes. , 1985, Proceedings of the National Academy of Sciences of the United States of America.

[45]  P Chambon,et al.  Organization and expression of eucaryotic split genes coding for proteins. , 1981, Annual review of biochemistry.