Our results indicate that C CoA is formed in the
Our results indicate that C75-CoA is formed in the hypothalamus following stereotaxic injection of C75. Inhibition of hypothalamic CPT1 by C75-CoA in vivo, as seen in our experiments, is independent of the putative inhibition by malonyl-CoA, which may be formed after the action of C75 either on FAS or on AMPK. Hypothalamic CPT1 activity was determined in twice-washed mitochondria. Therefore, malonyl-CoA was unlikely to remain within CPT1, as this metabolite leaves CPT1 freely when mitochondria are washed. In contrast, C75-CoA, as it is a tight-binding inhibitor, remains bound to the enzyme after washing, which means that its inhibition is persistent . Hence, C75-CoA not only inhibits CPT1 in vitro, but it also inhibits CPT1 activity in the hypothalamus after C75 has been converted to its CoA derivative. That C75-CoA is an inhibitor of CPT1A is also supported by the docking analysis of the CPT1A model. Comparison of computer-calculated docking models shows that CoA is bound at the same site, whether it belongs to C75-CoA or malonyl-CoA, or palmitoyl-CoA. The carboxylic contact us bound to the lactone of C75 protrudes into the carnitine site. The inhibitory mechanism of C75-CoA resembles that observed for malonyl-CoA (Fig. 4C) . Several authors reported the competition between malonyl-CoA and carnitine , . The carboxylate group of malonyl-CoA and C75-CoA may partially mimic the interaction between the enzyme and the carboxylate group of carnitine, thus preventing the positioning of this substrate and inhibiting the catalytic activity of the enzyme. Several authors ,  suggest that the presence of two carbonyl groups in close juxtaposition in the malonyl-CoA molecule might be responsible for the interaction and the inhibitory effect on CPT1A. Because C75-CoA also has these two carbonyl groups, it may behave like malonyl-CoA. Indeed the docking models show that C75-CoA could bind to the malonyl-CoA site of the enzyme . Moreover, the hydrocarbon chain of C75 is located at the same site as the hydrocarbon long-chain of palmitic acid. The kinetic experiments of inhibition of C75-CoA against palmitoyl-CoA  are confirmed by the docking studies, and both support the notion that C75-CoA is a strong inhibitor of CPT1A. 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 hydrophobic 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 . 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.