The enzyme carnitine palmitoyltransferase-1 (CPT1) regulates long-chain fatty acid (LCFA) entry into mitochondria, where the LCFAs undergo beta-oxidation. To investigate the mechanism(s) by which central metabolism of lipids can modulate energy balance, we selectively reduced lipid oxidation in the hypothalamus. We decreased the activity of CPT1 by administering to rats a ribozyme-containing plasmid designed specifically to decrease the expression of this enzyme or by infusing pharmacological inhibitors of its activity into the third cerebral ventricle. Either genetic or biochemical inhibition of hypothalamic CPT1 activity was sufficient to substantially diminish food intake and endogenous glucose production. These results indicated that changes in the rate of lipid oxidation in selective hypothalamic neurons signaled nutrient availability to the hypothalamus, which in turn modulated the exogenous and endogenous inputs of nutrients into the circulation.

Short-term overfeeding blunts the central effects of fatty acids on food intake and glucose production. This acquired defect in nutrient sensing could contribute to the rapid onset of hyperphagia and insulin resistance in this model. Here we examined whether central inhibition of lipid oxidation is sufficient to restore the hypothalamic levels of long-chain fatty acyl-CoAs (LCFA-CoAs) and to normalize food intake and glucose homeostasis in overfed rats. To this end, we targeted the liver isoform of carnitine palmitoyltransferase-1 (encoded by the CPT1A gene) by infusing either a sequence-specific ribozyme against CPT1A or an isoform-selective inhibitor of CPT1A activity in the third cerebral ventricle or in the mediobasal hypothalamus (MBH). Inhibition of CPT1A activity normalized the hypothalamic levels of LCFA-CoAs and markedly inhibited feeding behavior and hepatic glucose fluxes in overfed rats. Thus central inhibition of lipid oxidation is sufficient to restore hypothalamic lipid sensing as well as glucose and energy homeostasis in this model and may be an effective approach to the treatment of diet-induced obesity and insulin resistance. IntroductionWorldwide prevalence of obesity has been on the rise, along with its dreaded health consequences: diabetes, vascular diseases, and hypertension (1, 2). Obesity is due to a mismatch between caloric intake and energy expenditure in susceptible individuals. Redundant molecular and neural mechanisms control energy and glucose homeostasis (3-9). Among them, the integration of nutritional cues within the hypothalamus is emerging as a critical step in the metabolic and behavioral adaptation to changes in energy balance. The hypothalamic metabolism of fatty acids can profoundly modify feeding behavior and glucose metabolism (10-13) and has been proposed to function as a biochemical sensor for nutrient availability that in turn exerts negative feedback on nutrient intake (13, 14) and glucose production (10,(15)(16)(17). In this regard, the hypothalamic levels of long-chain fatty acyl-CoAs (LCFA-CoAs) can be increased by enhanced esterification of circulating or central lipids (10, 16) and/or by the local inhibition of lipid oxidation (18). These interventions also result in marked inhibition of feeding behavior and liver glucose fluxes (10,15,16,18) (Figure 1A).Sprague-Dawley rats presented with a highly palatable (lardsupplemented) diet promptly double their daily caloric intake and develop severe hepatic insulin resistance within just 3 days (19,20). In this model the central administration of the long-chain fatty acid (LCFA) oleic acid fails to decrease food intake and glucose production (21). Is defective sensing of lipids within the hypothalamus partly responsible for the defective behavioral and metabolic adap-

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PERSPECTIVEhomology in the active site. These data would point a medicinal chemist to expect that it should be relatively easy to gain selectivity between CPT2 and CPT1 inhibitors, while it might prove to be a daunting task for a competitive inhibitor to differentiate CPT1A from CPT1B. An alternative option is to achieve tissue selective distribution of an unspecific inhibitor via permeability and pharmacodynamic properties.The homology between human, rat, and mouse enzymes, on the other hand, is rather high (see Table 3b), and species differences at the in vitro level are not likely to be significant (the rate of FAO in different species and the level of CPT activity control can be considerably different). 22 CPT1 is subject to multiple controlling factors. Expression level and basal activity of CPT1 depend on fasting state, 23 exercise, 24 type of diet, 25 exposure to cold, infections, metabolic disease, and enzyme inhibition. 9 Diabetes does not affect CPT1B activity 26 but increases CPT1A activity. 27 Malonyl-CoA, the first committed step of fatty acid synthesis, whose concentration is in turn highly regulated by multiple mechanisms, is a potent inhibitor of CPT1B (∼0.03 μM IC 50 for the rat enzyme) and a less potent inhibitor (by about 2 orders of magnitude) of CPT1A. 28,29 The sensitivity of CPT1A to allosteric inhibition by malonyl-CoA varies under different physiological conditions, amplifying the effect of the cellular malonyl-CoA concentration. In particular, declining malonyl-CoA concentrations reduce the sensitivity of the enzyme to allosteric inhibition (and vice versa). Mitochondrial outer membrane composition also affects sensitivity of CPT1A to malonyl-CoA. Studies regarding the binding of malonyl-CoA and other active-site directed inhibitors have shown that there are two separate binding sites for malonyl-CoA: a high-affinity binding site located on the cytoplasmic side of the protein and a second low-affinity site that corresponds to the catalytic site and where also CoA exerts a product-inhibition action. 30 It is postulated that one of these binding sites is a contact interaction between the N-and C-terminal segments of the protein, which explains the sensitivity to membrane fluidity as well as the loss of sensitivity to malonyl-CoA if the N-terminal portion of the protein is deleted (which also leads to loss of function) or by specific single point mutations in this region. The N-terminal domain is also responsible for the targeting of the enzyme to the outer mitochondrial membrane. 31 There is evidence that CPT activity is controlled by phosphorylation 32 and nitration, 33 although the hypothesis has been advanced that CPT is constitutively phosphorylated. 28