To assess the involvement of the

To assess the involvement of the hydrocarbon chain of C75-CoA positioning in the acyl group binding pocket, we carried out C75-CoA inhibitory experiments with the new mutated protein, CrAT D356A/M564G. This protein has a deep clofibrate pocket for the binding of long-chain instead of short-chain acyl-CoAs and shows CPT1-like behaviour in terms of acyl-CoA specificity, although unlike CPT1A, it is not inhibited by malonyl-CoA [15]. The inhibition by C75-CoA of the mutant CrAT D356A/M564G suggests that C75-CoA fits in the large hydrophobic pocket of this enzyme, as in CPT1A wt, and that the presence of this pocket is necessary for C75-CoA inhibition. However, CrAT D356A/M564G is not as sensitive as CPT1A wt to C75-CoA, since the IC50 for C75-CoA acting on CPT1A wt is 50-fold lower than that observed for the CrAT double mutant. These results indicate that factors other than the presence of a hydrophobic pocket contribute to the inhibitory potency of C75-CoA toward CPT1. CPT1A M593S, which is insensitive to malonyl-CoA inhibition, shows limited sensitivity towards C75-CoA, but its IC50 for C75-CoA is similar to that of CrAT double mutant (25.9μM vs. 12.8μM, respectively). Therefore, the lack of a “malonyl-CoA-like” interaction between CrAT double mutant or CPT1A M593S and the carbonyl groups in the polar head of C75-CoA might explain their limited sensitivity to the inhibitor. We conclude that C75 is converted into C75-CoA and that it strongly inhibits CPT1 in vitro and in vivo. Docking and kinetic analysis revealed the molecular basis by which C75-CoA interacts with the enzyme and its substrates. We also show that C75-CoA is formed in vivo in the hypothalamus, where it inhibits CPT1. Here the inhibition of CPT1 could alter fatty-acid oxidation, thus putatively promoting down-regulation of orexigenic genes and up-regulation of anorexigenic genes, which induces restriction in food intake. These results point to the potential use of drugs to inhibit CPT1 activity, and control food intake in the treatment of obesity and diabetes.
Acknowledgements We thank Jeus Perez-Clausell from the Department of Cell Biology, School of Biology, University of Barcelona, Felipe Casanueva\'s group from the Department of Molecular Endocrinology and Carlos Diéguez\'s group from the Department of Physiology, School of Medicine, University of Santiago de Compostela for their support in stereotaxis experiments, to Olga Jaúregui from the Scientific-Technical Services of the University of Barcelona for her technical assistance in the LC–MS/MS analysis. We also thank Biomol-Informatics SL (http://www.biomol-informatics.com) for bioinformatics consulting. This study was supported by Grant SAF2007-61926 and by grant CTQ2006-13249 from the Ministerio de Educación y Ciencia, Spain; by grant C3/08 from the Fondo de Investigación Sanitaria of the Instituto de Salud Carlos III; by the Activities Program among R&D groups of the Comunidad de Madrid in Biosciences (S-BIO-0260/2006-COMBACT) and by the Ajut de Suport als Grups de Recerca de Catalunya (2005SGR-00733), Spain. Financial support of “Fundación Ramón Areces” to CBMSO is also acknowledged. A.G.C. and D.S. were recipients of fellowships from the University of Barcelona, and A.B. and C.G. from the Ministerio de Educación y Ciencia, Spain.