The inhibition of FAS by C produces an accumulation of

The inhibition of FAS by C75 produces an accumulation of malonyl-CoA which is difficult to reconcile with the activation of CPT1 reported by others [2], [16], [17], [18]. To unravel this paradox the mechanism of action of C75 needs to be examined. We recently demonstrated that C75 is converted in vitro to C75-CoA, a potent inhibitor of CPT1 [19]. CPT1 activity was also inhibited in mitochondria from pancreas-, muscle-, and kidney-derived cell lines incubated with C75, which indicates that C75-CoA is produced in these cells. This inhibition was followed by a decrease in fatty-acid oxidation. The role of CPT1 in heart, liver and pancreatic β-cells makes it a potential target in the treatment of diabetes, obesity, and other human diseases.
Materials and methods
Results
Discussion Energy balance is monitored by the hypothalamus, where inhibitors of FAS have been described to suppress food intake [40], [41]. C75 acts on fatty-acid metabolism by inhibiting FAS activity. Inhibition of FAS produces accumulation of malonyl-CoA, which, as a physiological inhibitor of CPT1, prevents the oxidation of newly synthesized fatty acids. Moreover, C75 may activate CPT1 [2], [18]. The effect of C75 on FAS is difficult to reconcile with the C75-activation of CPT1 as they have opposite effects: inhibition by malonyl-CoA and direct activation of CPT1. The simultaneous activation and inhibition of CPT1 by C75 appears paradoxical, and it has not been addressed satisfactorily. Kuhajda et al. suggested that C75 could modulate the inhibition of AMP-activated protein kinase (AMPK) which would lead to an increase in acetyl-CoA carboxylase (ACC) activity and a subsequent increase in malonyl-CoA levels [42]. This does not solve the paradox, since there would be an increase in malonyl-CoA (putatively inhibiting CPT1 activity) and a simultaneous activation of CPT1 produced by C75 itself (Fig. 7). Here we attempt to explain the anorectic effects of C75 in terms of its inhibitory action on CPT1. Inhibition of CPT1 could prevent the oxidation of fatty acids of the ARC nucleus in the 7-Nitroindazole leading to a local accumulation of LCFA-CoAs. The increase in LCFA-CoA is a central signal of ‘nutrient abundance’ which in turn activates a chain of neuronal events, via up-regulation of anorexigenic genes and down regulation of orexigenic genes, that would promote a switch in fuel sources from carbohydrates to lipids and limit food intake [8]. The detailed mechanism of this up- or down-regulation has not been addressed yet. Likewise, central administration of fatty acid suppresses food intake. Here we demonstrated that C75 is transformed into C75-CoA in the hypothalamus where it inhibits CPT1 activity. We propose that this direct effect in vivo of C75-CoA on CPT1 would explain by itself the inhibition of CPT1 activity. However, FAS could be inhibited by C75 in the hypothalamus. Therefore, malonyl-CoA could be in excess, inhibiting CPT1, together with C75-CoA. The anorectic effects of C75 are similar to those produced by other CPT1 inhibitors such as ST1326 and tetradecylglycidic acid, and to those of a riboprobe that specifically cleaves CPT1A mRNA [8]. The CPT1 inhibitor etomoxir also decreased feeding and reduced body weight in rats supporting those previous results [8]. It was important to discern whether C75 was an inhibitor or an activator of CPT1. Our results indicate that C75 is neither an activator nor an inhibitor of CPT1 when incubated with yeast-expressed CPT1A. C75 did not overcome the inhibition caused by C75-CoA. This agrees with the observed net effect of inhibition of CPT1 activity. Results shown here and elsewhere [19] demonstrate that C75-CoA inhibits CPT1A in vitro more strongly than etomoxiryl-CoA (IC50 values are 0.24μM and 4.1μM, respectively). The finding that C75 forms a CoA adduct before it can inhibit CPT1 is reminiscent of the case of etomoxir. McGarry and co-workers [43], [44] showed that the CPT1 inhibitory molecules were neither tetradecylglycidic acid nor etomoxir themselves, but rather their CoA esters produced in the presence of acyl-CoA synthetase. The CoA group may direct and fix the drug molecule in the cavities of CPT1, as it does with the natural physiological inhibitor malonyl-CoA and with the substrate palmitoyl-CoA. The synthesis of C75-CoA is produced stereo specifically through the electrophilic methylene group of C75. Moreover, our finding that C75-CoA is produced in absence of acyl-CoA synthetase indicated that it could be synthesised in the hypothalamus, irrespective of whether it is expressed in hypothalamic neurons.