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While the brain does not utilize fatty acids as a primary energy source, recent evidence shows that intermediates of fatty acid metabolism serve as hypothalamic sensors of energy status. Increased hypothalamic malonyl-CoA, an intermediate in fatty acid synthesis, is indicative of energy surplus and leads to the suppression of food intake and increased energy expenditure. Malonyl-CoA functions as an inhibitor of carnitine palmitoyl-transferase 1 (CPT1), a mitochondrial outer membrane enzyme that initiates translocation of fatty acids into mitochondria for oxidation. The mammalian brain expresses a unique homologous CPT1, CPT1c, that binds malonyl-CoA tightly but does not support fatty acid oxidation in vivo, in hypothalamic explants or in heterologous cell culture systems. CPT1c knockout (KO) mice under fasted or refed conditions do not exhibit an altered CNS transcriptome of genes known to be involved in fatty acid metabolism. CPT1c KO mice exhibit normal levels of metabolites and of hypothalamic malonyl-CoA and fatty acyl-CoA levels either in the fasted or refed states. However, CPT1c KO mice exhibit decreased food intake and lower body weight than wild-type littermates. In contrast, CPT1c KO mice gain excessive body weight and body fat when fed a high-fat diet while maintaining lower or equivalent food intake. Heterozygous mice display an intermediate phenotype. These findings provide further evidence that CPT1c plays a role in maintaining energy homeostasis, but not through altered fatty acid oxidation.
Cytokines and endocrine hormones control food intake and energy expenditure and are critical for maintaining homeostasis and averting serious health problems such as those associated with obesity. In addition, a recently identified system for maintaining energy balance utilizes an ancient nutrient-sensing pathway with which the CNS monitors energy needs by assessing current energy surplus/deficit and responding by modulating appetite and peripheral energy expenditure (Wolfgang and Lane 2006b). It was recently found that key regulatory enzymes and intermediates in the fatty acid biosynthetic pathway act as hypothalamic sensors that monitor energy status and adjust food intake and increase energy expenditure accordingly. Specifically, changes in the level of malonyl-CoA, a well-characterized intermediate in fatty acid synthesis, was found to modulate energy balance in the hypothalamus (Wolfgang and Lane 2006a). Thus, increasing hypothalamic malonyl-CoA provokes anorexia and increased peripheral energy expenditure leading to a leaner phenotype (Loftus et al. 2000; Hu et al. 2003; Cha et al. 2005). Conversely, lowering malonyl-CoA by over-expressing malonyl-CoA decarboxylase in the hypothalamus increases food intake and produces obesity in rodent models showing that this system is both sufficient and required for the suppression of food intake (He et al. 2006). The physiological relevance of this mode of regulation is supported by the demonstration that fluctuation of hypothalamic malonyl-CoA level caused by fasting (0.3 μM) and refeeding (1.2 μM) produce concomitant changes in food intake, energy expenditure, and neuropeptide expression in the hypothalamus (Shimokawa et al. 2002; Gao and Lane 2003; Hu et al. 2003).
The molecular mechanism by which hypothalamic malonyl-CoA transmits satiety signals has not been elucidated. The brain-specific carnitine palmitoyl-transferase 1c (CPT1c) is a candidate as a downstream target of malonyl-CoA because it possesses appropriate characteristics (Price et al. 2002; Wolfgang et al. 2006; Dai et al. 2007). Like CPT1a (liver) and CPT1b (muscle), CPT1c is localized in the outer membrane of mitochondria (Dai et al. 2007). Malonyl-CoA, which is known to inhibit both CPT1a and CPT1b, binds tightly to CPT1c (Kd = ∼0.3 μM) within its dynamic range in the hypothalamus (Wolfgang et al. 2006). Furthermore, CPT1c (Dai et al. 2007) and its mRNA (Lein et al. 2007) is enriched in areas of the brain that are known to regulate feeding behavior. Finally, we have shown that a mouse knockout (KO) of CPT1c results in a phenotype consistent with a malonyl-CoA target protein. CPT1c KO mice exhibit reduced food intake and body weight on a standard low-fat diet (Wolfgang et al. 2006). Thus, CPT1c possesses biochemical and physiological characteristics to place it downstream of malonyl-CoA in the hypothalamic signaling pathway that regulates food intake and energy expenditure.
It is firmly established that CPT1a and CPT1b, homologs of CPT1c, catalyze the initiating step of fatty acid oxidation by which long-chain acyl-CoA are translocated from the cytoplasm into the mitochondrial matrix, the site containing the enzymes of the β-oxidation pathway (McGarry et al. 1977, 1978; McGarry and Foster 1980; McGarry 1995a,b). The enzymatic reaction catalyzed by CPT1a and CPT1b involves the transfer of the fatty acyl group from acyl-CoA to carnitine. The fatty acyl-carnitine then traverses the inner mitochondrial membrane via a cation transporter into the matrix. Despite extensive investigations in our (Wolfgang et al. 2006) and another (Price et al. 2002) laboratory and the high degree of amino acid sequence identity/similarity (> 50% or > 66%) of CPT1c to CPT1a, it has not been possible to demonstrate an enzymatic activity for CPT1c (Price et al. 2002) (Wolfgang et al. 2006). It is surprising that this unique ‘enzyme’ is expressed in the CNS, as neural tissue does not normally use fatty acids as a major physiological fuel. Rather, the CNS relies on glucose as the primary fuel when carbohydrate is available, or ketones during fasting and high-fat feeding. These facts suggest a unique, perhaps regulatory, function for CPT1c.
In this study, we explore the biochemical properties of CPT1c ex vivo and in vivo and verify that CPT1c does not facilitate the oxidation of long-chain fatty acids under a variety of conditions. Moreover, we show that disruption of the CPT1c gene produces a phenotype that is consistent with a regulatory role.
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Carnitine palmitoyl-transferase 1c, a recently discovered mitochondrial protein, which is expressed in neurons of the CNS and possesses enigmatic characteristics (Price et al. 2002). It is not required for life nor is it essential for neuronal survival. CPT1c KO mice are viable, fertile, and do not suffer dire consequences upon fasting or feeding a high fat diet, conditions that lead to a shift in energy source from glucose to ketones (Wolfgang et al. 2006). CPT1c KO mice have no obvious histological abnormalities even in later life. Unlike its counterparts in liver (CPT1a) and muscle (CPT1b), CPT1c does not appear to participate directly in fatty acid oxidation in so far as we have determined to date. However, disruption of the CPT1c gene reveals a complex metabolic phenotype (e.g. the response to feeding a high fat diet) suggesting that CPT1c plays a regulatory, rather than a direct metabolic role. These and other observations are consistent with the hypothesis that CPT1c is a target of the metabolic intermediate, malonyl-CoA, to control body weight.
The expression of CPT1c is restricted to the CNS and is enriched in neural feeding centers within the hypothalamus. Therefore, CPT1c is located appropriately to play a role in energy homeostasis. However, CPT1c is ubiquitously expressed in neurons throughout the brain with highest expression in hippocampal neurons suggesting it also serves a broader role (Price et al. 2002; Lein et al. 2007).
The biochemical reaction that CPT1c catalyzes has been difficult to elucidate, if indeed it functions as an enzyme. CPT1c has a striking amino acid similarity to other CPTs and it is unclear from structural considerations why CPT1c does not behave as a putative member of this family. We have performed 3D modeling based on the crystal structure coordinates of the carnitine octanoyl transferase (Jogl and Tong 2003), which suggests that CPT1c possesses a binding pocket large enough to accommodate a long-chain fatty acyl group. However, CPT1c does not catalyze acyl transfer to carnitine when assayed with a large array (∼50) of potential acyl-CoA substrate donors (Wolfgang et al. 2006) and here we report no evidence, either in vitro or in vivo, for a role in fatty acid oxidation. In addition, we have tested several other potential acceptor substrates including serine, ethanolamine, choline, and sphingosine derivatives none of which served as acceptors. It is still possible, of course, that a unique substrate exists for a CPT1c-catalyzed enzymatic reaction. However, we believe it unlikely that we have missed a CPT1c substrate involved in fatty acid oxidation given the extensive in vitro and ex vivo analyses reported in the present paper. More likely explanations are that CPT1c functions in a non-enzymatic process or that the reaction carried out has unique or restrictive activation requirements (e.g. allosteric or covalent modification) that were not satisfied.
Malonyl-CoA is an allosteric inhibitor of CPT1a and CPT1b, which catalyze the initiating step of fatty acid oxidation (McGarry et al. 1977, 1978; McGarry and Foster 1980; McGarry 1995a,b). Malonyl-CoA is the product of the acetyl-CoA carboxylase catalyzed carboxylation of acetyl-CoA and serves as the basic chain elongation unit for fatty acid synthase (FAS). Inhibition of CPT1 by malonyl-CoA ensures that fatty acid oxidation and fatty acid synthesis does not occur concomitantly in cell types where both processes coexist. Therefore, we reasoned that CPT1c might be inhibited by malonyl-CoA in an analogous manner. Indeed, CPT1c does bind to malonyl-CoA tightly within the dynamic concentration range that occurs in the hypothalamus (Wolfgang et al. 2006), but since an enzymatic reaction has not been discovered, it is possible that malonyl-CoA binding has another role. Nevertheless, it is of great interest that the CPT1c KO produces a phenotype expected of a target protein.
The fatty acid biosynthetic pathway in the hypothalamus has clearly been shown to be involved in body weight regulation from both a pharmacological and genetic perspective (Wolfgang and Lane 2006b). The nexus to these systems appears to be the regulation of malonyl-CoA level. Malonyl-CoA concentration is regulated in large part by the rate of synthesis by acetyl-CoA carboxylase, which is in turn regulated/inhibited by phosphorylation/inhibition by 5′-AMPK, a cellular sensor of changes in the [AMP]/[ATP] ratio which inhibits several biosynthetic enzymes in times of energy deficit (Hardie 2004; Kahn et al. 2005). Consistent with malonyl-CoA as a mediator of energy homeostasis (Wolfgang and Lane 2006a) several laboratories have now shown that modulation of AMPK leads to coordinated changes in food intake (Andersson et al. 2004; Minokoshi et al. 2004).
Elevation of hypothalamic malonyl-CoA by the inhibition of FAS rapidly inhibits food intake and increases energy expenditure (Wolfgang and Lane 2006a). Another non-lipogenic tissue that utilizes malonyl-CoA as a signaling intermediate is skeletal muscle. Muscle utilizes malonyl-CoA not for the elongation of fatty acids, as muscle has little to no FAS, but rather to regulate CPT1b and thereby, fatty acid oxidation (Cha et al. 2006). This constitutes a precedent for malonyl-CoA as a signaling intermediate.
Another signaling protein that requires anchorage to the mitochondrial outer membrane to be active is mitochondrial anti-viral signaling protein (MAVS). MAVS functions to enable interferon signaling via nuclear factor-kappa B activation after viral infection (Seth et al. 2005). Several viruses produce peptidases to evade this response by clipping MAVS from the mitochondria (Li et al. 2005). It is not known why this signaling protein requires association with the mitochondria, but it is clear that anchorage specifically to mitochondria is required.
We suggest that CPT1c is anchored to the mitochondria because it plays a role in fatty acid metabolism, however, to date we have been unable to identify biochemical alterations in fatty acid metabolism in CPT1c KO animals. CPT1c KO animals do not appear to accumulate long-chain fatty acyl-CoA in the hypothalamus or the CNS (Table 2) or free fatty acids (data not shown). Elucidation of the biochemical reaction(s) in which CPT1c participates will be necessary to understanding how nutrient signaling is mediated.