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Physiology & Behavior 85 (2005) 25 – 35 Fatty acid metabolism as a target for obesity treatment Gabriele V. Ronnetta,b,*, Eun-Kyoung Kima, Leslie E. Landreea, Yajun Tua a Department of Neuroscience, The Johns Hopkins University School of Medicine, Baltimore, MD 21205, United States b Department of Neurology, The Johns Hopkins University School of Medicine, Baltimore, MD 21205, United States Abstract Although metabolites and energy balance have long been known to play roles in the regulation of food intake, the potential role of fatty acid metabolism in this process has been considered only recently. Fatty acid synthase (FAS) catalyzes the condensation of acetyl-CoA and malonyl-CoA to generate long-chain fatty acids in the cytoplasm, while the breakdown of fatty acids (h-oxidation) occurs in mitochondria and is regulated by carnitine palmitoyltransferase-1 (CPT-1), the rate-limiting step for the entry of fatty acids into the mitochondria. Inhibition of FAS using cerulenin or synthetic FAS inhibitors such as C75 reduces food intake and induces profound reversible weight loss. Subsequent studies reveal that C75 also stimulates CPT-1 and increases h-oxidation. Hypotheses as to the mechanisms by which C75 and cerulenin mediate their effects have been proposed. Centrally, these compounds alter the expression profiles of feeding-related neuropeptides, often inhibiting the expression of orexigenic peptides. Whether through centrally mediated or peripheral mechanisms, C75 also increases energy consumption, which contributes to weight loss. In vitro and in vivo studies demonstrate that at least part of C75’s effects is mediated by modulation of AMP-activated protein kinase (AMPK), a known peripheral energy-sensing kinase. Collectively, these data suggest a role for fatty acid metabolism in the perception and regulation of energy balance. D 2005 Elsevier Inc. All rights reserved. Keywords: Obesity; C75; Energy expenditure; Weight loss; Fatty acid synthase; Carnitine palmitoyltranseferase-1 1. Introduction Obesity is a global health issue, and affects individuals of all ages in both industrialized and emerging nations [1]. Obesity may be viewed as the dysregulation of two physiological functions, appetite regulation and energy metabolism, which combine to create disordered energy balance [2,3]. The central nervous system (CNS) serves a critical role in the evaluation of energy status, as the hypothalamus and other regions in the brain integrate signals from the periphery and other brain areas to regulate feeding behavior [4 –7]. It has long been recognized that cells in these brain regions register the concentrations of nutrients, such as glucose, to ‘‘sense’’ energy status [8]. * Corresponding author. Department of Neuroscience, 1006B Preclinical Teaching Building, Johns Hopkins University School of Medicine, 725 North Wolfe Street, Baltimore, MD 21205, United States. Tel.: +1 410 614 6482; fax: +1 410 614 8033. E-mail address: gronnett@jhmi.edu (G.V. Ronnett). 0031-9384/$ - see front matter D 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.physbeh.2005.04.014 Only recently has it been appreciated that the hypothalamus may monitor fatty acid metabolism as part of its energy-sensing schema [9 – 12]. While this hypothesis awaits further investigation, it has been shown by a number of laboratories that modulation of fatty acid metabolism may be exploited to inhibit food intake ([9,11– 18]). The present contribution reviews the potential mechanisms by which alterations in fatty acid biosynthesis or metabolism may influence food intake and energy production. 2. Fatty acid metabolism: fatty acid synthase and carnitine palmitoyltransferase-1 In normal lipogenic tissues, such as liver and adipose, the fatty acid pathway facilitates the storage of energy surplus as triglycerides. These triglycerides can later be oxidized to provide energy during times of energy deficiency. Fatty acid synthase (FAS) is a lipogenic 26 G.V. Ronnett et al. / Physiology & Behavior 85 (2005) 25 – 35 enzyme in this pathway that catalyzes the de novo synthesis of long-chain fatty acids in the cytosol. Mammalian FAS is the product of a single non-duplicated gene that generates a ¨250 kDa polypeptide chain, which is modified post-translationally by the addition of a phosphopantotheine group [19]. FAS is active as a homodimer [19]. This enzyme performs a complex seven-step reaction that involves the NADPH-dependent synthesis of the 16-carbon saturated free fatty acid palmitate through the condensation of acetyl-CoA and malonyl-CoA [20]. Malonyl-CoA, the main substrate of FAS, is synthesized from acetyl-CoA by acetyl-CoA carboxylase (ACC) in an ATP-dependent reaction, and is degraded back to acetyl-CoA by malonyl-CoA decarboxylase (MCD), which also requires ATP. The h-ketoacyl synthase moiety of FAS is responsible for the condensation of the three-carbon malonyl-CoA and the two-carbon acetyl-CoA, releasing CO2 in the process. The phosphopantotheine group tethers the growing fatty acyl chain as it proceeds through subsequent reduction and dehydration steps. This series of steps is repeated seven times, after which the fatty acyl chain is hydrolyzed from the enzyme to yield one palmitate. The production of one palmitate costs the cell 7 ATP and 14 NADPH. While FAS and ACC are both regulated transcriptionally, it is ACC that is regulated rapidly during times of energy fluctuation by phosphorylation; citrate enhances its activity [21], while long-chain acyl-CoA’s, either generated by de novo synthesis or of dietary origin, competitively inhibit ACC [22]. Although previous studies [23] suggested that although FAS is expressed in the brain, it was thought to localize predominantly to non-neuronal cells. Immunohistochemical studies done by our group demonstrated that FAS was expressed predominantly in neurons in many regions of the brain, along with other enzymes in the fatty acid pathway, including ACC and MCD [9]. FAS displayed a similar distribution in human hypothalamus. In lipogenic tissues in the periphery, FAS activity is regulated by transcriptional control, mainly in response to diet, glucose, thyroxine and SREBP1C [24]. Within 24 h of starvation, FAS message in liver is highly reduced, as is its protein expression [9]. This is not the case in brain; FAS mRNA and protein are essentially unaffected by 24 h of starvation [9]. These data suggested that FAS serves a different function in the brain from its energy storage role in the periphery. Fatty acid degradation occurs in the mitochondria through the process of h-oxidation. Carnitine palmitoyltransferase-1 (CPT-1) is the rate-limiting step for the entry of fatty acids into the mitochondria [25 – 27]. Aside from its role as a substrate for FAS, malonyl-CoA is a competitive inhibitor of CPT-1. When energy is surfeit and the rate of fatty acid synthesis is high, high levels of malonyl-CoA prevent the oxidation of newly synthesized fatty acids by inhibiting CPT-1. Alternatively, when acetyl-CoA levels are reduced or when ACC activity is diminished and malonyl- CoA levels fall, CPT-1 is activated. CPT-1 activity is measured using permeabilized cells or isolated mitochondria [28 – 30]. These studies have revealed that the regulation of CPT-1 is complex and involves membrane interactions. As with other metabolic pathways, enzymes within the fatty acid pathway must shift from anabolic to catabolic states in response to changes in energy availability. Hence, FAS and CPT-1 function at a metabolic crossroads between energy storage (anabolism) and consumption of stored energy (catabolism), and as such, activity through the fatty acid metabolic pathway reflects the energetic state of the cell (Fig. 1). 3. Cerulenin and C75: tools for exploring the roles of FAS and CPT-1 in feeding and energy balance Interest in the role of FAS in feeding and energy balance originated in the field of cancer biology [31]. The observation by Kuhajda et al. that many human cancers express high levels of FAS [32 –34] raised the possibility that inhibition of FAS might offer a therapeutic approach in cancer treatment. The first compound to be studied was cerulenin [31]. Cerulenin ([2S,3R]2,3-epoxy-4-oxo7E,10E-dodecadienamide) is a natural antibiotic product of the fungus Cephalosporium ceruleans and a broadspectrum FAS inhibitor [35,36]. It was the first reagent used for in vivo studies, despite its limited solubility in aqueous solutions. While cerulenin induced apoptosis in cancer cells, it did not affect non-transformed cells. However, the doselimiting ‘‘toxicity’’ was weight loss. Cerulenin treatment has since been shown to induce profound and reversible dosedependent weight loss in lean [37] and ob/ob mice [38]. However, in some studies, cerulenin has no significant effect on food intake and the expression of feeding-related neuropeptides in lean mice [39,40]. Strain differences between mice may influence the response to cerulenin. Aside from solubility issues, cerulenin has also been reported to interfere with protein acylation [41], which may be due to its highly reactive epoxide group. To investigate the weight loss caused by FAS inhibition, it was decided to create synthetic FAS inhibitors, the strategy was to design compounds that resembled malonylCoA, the natural substrate for FAS [42]. Of the seven enzyme activities on FAS, the h-ketoacyl synthase that covalently joins acetate and malonate together is unique to FAS. This h-ketoacyl synthase moiety was therefore chosen for the design of several families of inhibitors [31,33,42]. Indeed, C75, an a-methylene-g-butyrolactone, interferes with the binding of malonyl-CoA to the active site of FAS. While malonyl-CoA forms a covalent adduct with FAS via the pantotheine arm, more recent inhibitors have been designed which do not appear to bind covalently, making them more acceptable compounds for potential drug development (Townsend and McFadden, personal communication). G.V. Ronnett et al. / Physiology & Behavior 85 (2005) 25 – 35 4. The central effects of alterations in fatty acid metabolism on feeding and body weight Since the initial observations in the Kuhajda and Ronnett laboratories [37], other investigators have explored the effects of altering fatty acid metabolism on food intake [10,14,16,38,39,43 – 45]. While the effect is striking, several hypotheses that appear to conflict have been proposed to explain the mechanisms of these effects on food intake. Hypotheses differ as to the roles of alterations in FAS and CPT-1 activities, and malonyl-CoA, energy (ATP) and NADPH levels in mediating changes in food intake and body weight. C75 was initially designed as a FAS inhibitor [46], and similar to cerulenin, reduced food intake and caused significant weight loss when given peripherally (intraperitoneally, i.p.) or centrally (intracerebroventricularly, i.c.v.) at a hundredth of the peripheral dose, suggesting that at least part of the effect of C75 was mediated centrally [13]. The anorexia caused by C75 in lean and ob/ob mice leading to weight loss was associated with a reduction of orexigenic neuropeptide NPY message in the hypothalamus compared to NPY levels found in fasted animals [13]. By double labeling in situ hybridization, FAS and NPY were found to co-localize to a subset of neurons with the arcuate nucleus, suggesting that C75 could be acting in a cell-autonomous manner to affect NPY expression [9]. An effect of C75 on the expression levels of NPY as well as on the expression of other neuropeptides has been shown by other groups [12,14,15]. In two of our recent studies that utilized dietinduced obese (DIO) mice that had received chronic treatment with C75 (i.p.), weight loss was accompanied by an increase in cocaine and amphetamine-related transcript (CART) expression [18,47]. Taken together, these data suggested that C75 could act centrally to alter feeding and initiated a number of further experiments by us and others. Although behavior was not apparently affected by the doses used in the initial studies, the effect of C75 on feeding could have been due to malaise. To assess this possibility formally, we determined whether C75 produced a conditioned taste aversion [9]. C75 administration i.p. had no effect on saccharin preference relative to vehicle control. Thus, C75 did not appear to reduce food intake secondary to the production of illness or malaise. These results are in contrast to other studies that demonstrated an aversive response to peripherally administered C75, although not to centrally administered compound [16]. More recent studies indicate that chemical impurities in some preparations of C75 can contribute to sickness behavior, but the volume and perhaps pH in which C75 is administered i.p. are major factors in producing a sickness response (Kuhajda and Thupari, personal communication). Our studies have always used minimal injection volumes and avoided this issue. Many regions of the brain contribute to the regulation of food intake [6,48]. We [49] and others [44] have surveyed 27 the areas that C75 may influence by examining the effect of C75 on c-Fos-immunoreactivity. Within several hours, C75 treatment caused a striking and specific increase in the number of c-Fos-immunoreactive cells in hindbrain feedingrelated nuclei as well as in the paraventricular nucleus (PVN), lateral aspects of the arcuate nucleus (ARC) and the central amygdala. In contrast, C75 prevented the normal 24 h fasting-induced increases in c-Fos-immunoreactivity in the medial ARC and three of its targets: lateral magnocellular PVN, lateral hypothalamus and dorsomedial hypothalamus. Compared to C75, cerulenin increased c-Fosimmunoreactivity in POMC-expressing neurons even though it similarly increased c-Fos-immunoreactivity in the lateral peri-ARC and decreased fasting-induced c-Fosimmunoreactivity in medial ARC [50]. One group has suggested that C75 is a non-specific neuronal activator, based upon in vitro assays [51]. The reproducible and highly specific changes in c-Fos expression in vivo in regions related to feeding behavior strongly argue against this conclusion. Taken together, these studies utilizing C75 and cerulenin suggested that hypothalamic mechanisms, specifically alterations in the expression of hypothalamic peptides that influence food intake, could mediate part of the anorectic responses to C75 and cerulenin. The mechanism by which neuropeptide expression is modulated was investigated. Several pieces of evidence supported our initial hypothesis that hypothalamic malonylCoA (which was hypothesized to increase with FAS inhibition upon C75 administration) was the metabolic mediator that regulated feeding [52]. This was a logical speculation, as malonyl-CoA is a key regulator of fatty acid oxidation in peripheral tissues such as muscle [25,26,53]. FAS inhibition would increase malonyl-CoA levels and shut down CPT-1 activity. Furthermore, TOFA, an inhibitor of ACC [54] that decreases malonyl-CoA levels, interfered with C75’s effects. It was reasonable to propose that longchain fatty acyl-CoA’s that accumulated due to decreased fatty acid oxidation could signal increased nutrient availability and decrease food intake. In study using inhibitors of CPT-1 in the hypothalamus [10], the inhibition of the hypothalamic CPT-1 resulted in reduced food intake. However, it was not demonstrated that CPT-1 inhibition had any effect on body weight, suggesting that other actions might contribute to C75’s effects. The other effects of C75 were revealed in subsequent studies. If C75 elevated malonyl-CoA levels, thereby shutting down fatty acid oxidation, one might predict that C75 would lead to an accumulation of fat in the liver, an undesirable outcome. Surprisingly, however, C75 treatment resulted in the removal of fat accumulated in liver as well as adipose tissues of DIO mice [18]. If FAS inhibition causes an increase in malonyl-CoA levels, how could the disappearance of fat occur above that seen in pair fed group? This paradox has been investigated in a series of experiments that demonstrates that C75 stimulates CPT-1 activity and, more importantly, fatty acid oxidation. 28 G.V. Ronnett et al. / Physiology & Behavior 85 (2005) 25 – 35 The effects of C75 on fatty acid oxidation were studied using in vitro cellular models and in vivo using diet-induced obese (DIO) mice [18,47,55,56]. Whole animal calorimetry revealed that C75-treated DIO mice had a greater weight loss and an increased production of energy due to fatty acid oxidation compared to pair-fed animals. Etomoxir, a potent inhibitor of CPT-1 [57], was able to reverse the increased energy expenditure and reduced the C75-induced weight loss by inhibiting fatty acid oxidation. This was mechanistically studied using several cell lines, including rodent adipocytes, hepatocytes and human breast cancer cells, all displayed C75-induced increased fatty acid oxidation and elevated ATP levels due to increased CPT-1 activity, even in the presence of high concentrations of malonyl-CoA [55]. Consistent with these findings, two other studies demonstrated that C75 can activate CPT-1 and -2 and overcome the inactivation of CPT-1 by malonyl-CoA [58,59]. Thus, C75 may cause weight loss not only centrally by reduction in food intake, but also peripherally by stimulating CPT-1 and increasing fatty acid oxidation, leading to a loss of adipose tissue and a resolution of fatty liver in addition to profound weight loss. While these studies concerned themselves predominantly with the effects of C75 on peripheral tissues, C75 also stimulates CPT-1 activity in neuron in vitro [56]. The question remains as to how modulation of CPT-1 (inhibition or stimulation) in hypothalamic regions influences food intake. It may be important to acknowledge that the measurement of CPT-1 activity in isolation may not reflect the activity of the fatty acid oxidation pathway, which may be the relevant parameter. If CPT-1 is inhibited and levels of fatty acyl-CoA’s increase, ACC will be inactivated, causing malonyl-CoA levels to fall. Furthermore, malonyl-CoA levels may be tightly controlled, as MCD is highly expressed in neurons. Therefore, CPT-1 activity may increase to favor fatty acid oxidation. These variables are complex and make a case for measuring fatty acid oxidation as a ‘‘readout of pathway activity’’. Additionally, ex vivo or in vitro assays of CPT-1 may not accurately reflect CPT-1 activity in vivo, as cell permeabilization and mitochondrial isolation alter malonyl-CoA levels and disrupt membrane components that influence CPT-1 activity [28 – 30]. However, elegant studies had already considered the role of fatty acid oxidation in feeding behavior. Studies from many laboratories, most notably those of Ritter and Scharrer, used a variety of pharmacological fatty acid oxidation inhibitors for multiple enzyme targets to demonstrate that systemic inhibition of fatty acid oxidation stimulates food intake in rodents [60 –65]. Fatty acid oxidation inhibition increased food intake in animals fed a fat enriched diet (40% of metabolizable energy as fat), but was ineffective in animals consuming a low fat (7% of metabolizable energy as fat) diet [66], suggesting that a dependence on fatty acid metabolism was necessary for the feeding effect. Beverly et al. investigated the role of fatty acid oxidation in food intake centrally [67]. Rats treated with a fatty acid oxidation inhibitor into the ventrolateral hypothalamus displayed transient decreased food intake, which resolved with continued administration such that there were no significant changes in weight or carcass composition after 2 weeks of central fatty acid oxidation inhibition. In other work, central injection of oleic acid had an anorectic effect, accompanied by decreased NPY expression [17]. Central administration of another short-chain fatty acid, a-lipoic acid, reduced food intake without a change in NPY levels [68]. These differences may reflect differences in dietary composition or physiological versus supraphysiological alterations in fatty acid concentrations during treatments. Thus, it is likely that fatty acid pathways contribute to the regulation of energy balance and metabolic homeostasis by distinct mechanisms in the hypothalamus. Nonetheless, the finding that C75 can successfully overcome the inhibitory effect of malonyl-CoA raised issues concerning the hypothesis that malonyl-CoA is the mediator of C75’s effects. This issue shall be addressed using novel compounds that either inhibit FAS or stimulate CPT-1, permitting clarification of mechanism. 5. The peripheral effects of alterations of fatty acid metabolism While the consequences of altered fatty acid metabolism centrally have generated interest, the effects of peripheral and perhaps central administration of C75 or cerulenin on fatty acid oxidation in the periphery also contribute to the effects of these compounds on body weight. It is reasonable that decreased FAS activity would decrease fat accumulation in peripheral tissues [43]. However, the dramatic decrease in hepatic fat accumulation in C75treated DIO mice compared to pair fed mice instigated studies that demonstrated that C75, likely through its stimulation of CPT-1, increases fatty acid oxidation thus contributing to the profound weight loss seen with C75 [55]. Although this effect may be mediated directly by peripheral mechanisms, a recent study raises the possibility that central FAS inhibition may play a role in increasing peripheral fatty acid oxidation. Jin et al. demonstrated that central administration of cerulenin increased peripheral CPT-1 activity in isolated soleus muscle and liver and elevated core temperature [40]. Peripheral administration of cerulenin initially decreased CPT-1 activity, but with an hour, CPT-1 activity was increased. These investigators suggest that cerulenin altered sympathetic activity to cause the increase in CPT-1 activity and the elevation of core temperature. Clarification of these observations awaits determination of the effect of cerulenin on fatty acid oxidation, not just CPT-1 activity. Additionally, malonyl-CoA levels were not fixed in these ex vivo assays, raising the possibility that fluctuations in malonyl-CoA levels might account for the biphasic changes in CPT-1 activity. A previous study found that cerulenin did not alter G.V. Ronnett et al. / Physiology & Behavior 85 (2005) 25 – 35 fatty acid oxidation [55], and it would be important to extend these types of studies to the model used by Jin et al. The relationship between fatty acid oxidation inhibition and the sympathetic nervous system has been studied by Ritter and colleagues [64,69 –72]. Fatty acid oxidation inhibition using mercaptoacetate (MA) stimulated the release of norepinephrine from the sympathetic system, but did not induce a release of epinephrine from the adrenal medulla [64,73]. Importantly, the effects of fatty acid oxidation inhibition (lipoprivic feeding) are also quite specific, differing significantly from those of 2-deoxyglucose administration (glucoprivic feeding) [64,72,74]. Therefore, systemic fatty acid oxidation inhibition reduces hepatic ATP, stimulates hepatic vagal afferents to the NTS, where signals are transmitted to the hypothalamus leading to increased food consumption. What is unclear, but is investigated in the studies by Jin et al. [40] and Thupari et al. [55], is the role of fatty acid oxidation stimulation. Again, these issues will be clarified by future studies that no doubt will evaluate the role of diet, strain and assay type in outcomes. 6. Molecular mediators of C75’s effects Although malonyl-CoA is the substrate of FAS, other molecules are required for palmitate synthesis, including ATP (required by ACC to generate malonyl-CoA) and NADPH. Perhaps C75 affects cellular energy balance by inhibiting FAS and/or stimulating CPT-1, which could be sensed in specific neurons within regions concerned with appetite or energy homeostasis to alter food intake. We considered several candidates that might be affected by a change in energy availability mediated by C75. AMPK is well known as a sensor of peripheral energy balance and a member of a metabolite-sensing protein kinase family [75 – 78]. Increases in the cellular AMP/ATP ratio, changes in pH and redox status, and increases in the creatine/phosphocreatine ratio phosphorylated and activate AMPK [76,79 – 81]. In turn, AMPK alters cellular metabolism and gene expression to collectively inhibit anabolic processes and stimulate catabolic processes in an attempt to restore ATP levels [78,82]. AMPK is a heterotrimeric protein consisting of an a catalytic subunit and regulatory h and g subunits [82]. There are two a isoforms, a1 and 2 [83]. In the adult brain, the a2 subunit is the predominantly expressed isoform in neurons [83,84]. In the periphery, AMPK activity is regulated by vigorous exercise, nutrient starvation and ischemia –hypoxia [76,78]. AMPK is activated when the AMP/ATP ratio increased; AMP activates AMPK kinase (AMPKK), which phosphorylates AMPK on Thr172 on the a subunit [79]. High concentrations of ATP inhibit AMPK activity [85]. The changes incurred with AMPK activation in the periphery are complex, and include acute regulation of important metabolic pathways, followed by chronic transcriptional changes. AMPK inhibits anabolism (fatty acid 29 synthesis, triglyceride synthesis) and stimulates catabolism (fatty acid oxidation, glycolysis and glucose uptake). As concerns fatty acid synthesis, AMPK phosphorylates and inactivates ACC, thus inhibiting fatty acid synthesis by decreasing malonyl-CoA availability [86]. AMPK also phosphorylates and inactivates HMG (3-hydroxy-3-methylglutaryl)-CoA reductase, thus affecting the biosynthesis of isoprenoids and cholesterol [87,88]. 6-Phosphofructo-2 kinase (PFK) is phosphorylated by AMPK, thus increasing fructose-2,6-bisphosphate activity, thereby stimulating 6phosphofructo-1-kinase (PFK-1), and increasing glycolysis. AMPK knockout mice indicate distinct roles for the two a subunits. While AMPKa1-deficient mice that have no apparent metabolic defect, AMPKa2-deficient mice display metabolic defects, including reduced glycogen synthesis, glucose intolerance and insulin resistance [89]. Beyond its role as a peripheral energy sensor, recent studies have focused on the role of AMPK in neuronal energy metabolism. Using a primary neuronal culture system, we demonstrated a role for FAS, CPT-1 and AMPK in neuronal energy metabolism in vitro [56]. C75 inhibits FAS, and increases CPT-1 activity, fatty acid oxidation and glucose oxidation in neurons, similar to its effect in peripheral tissues [56]. Concomitantly, ATP levels increase following a brief (15 min) drop. This biphasic effect on ATP levels (a brief drop and then a significant increase) was seen with C75 and cerulenin, but not with TOFA. TOFA treatment actually resulted in the largest increase in ATP levels, but was not preceded by an initial drop in ATP levels. For cerulenin and C75, these fluctuations in ATP levels were accompanied by changes in AMPK phosphorylation and activity: the initial drop in ATP paralleled the increase in AMPK phosphorylation and activity, whereas the subsequent prolonged increase in ATP correlates with a significant decrease in phosphorylation and AMPK activity. Most surprisingly, TOFA treatment, which caused the largest increase in ATP levels, did not alter AMPK activity. It is intriguing to speculate that this is why TOFA treatment interfered with the effect of C75 on food intake: TOFA would disrupt the biphasic fluctuation in ATP levels, thereby interfering with AMPK inhibition, negating C75’s effects. It would be the effect of TOFA on ATP levels and not on decreasing malonyl-CoA levels, which is the key event. Nonetheless, it is clear that pharmacological alterations in neuronal fatty acid metabolism can influence neuronal energy balance and AMPK activity. Could these effects of C75 on AMPK and neuronal energy balance mediate some of the effects of C75 seen in vivo? During the past year, studies from several laboratories have revealed that hypothalamic AMPK serves as a neuronal energy sensor in the regulation of food intake [11,90,91]. We investigated a role for hypothalamic AMPK in the regulation of feeding using a pharmacological approach [11]. AICAR (5-aminoimidazole-4-carboxamide-1-h-dribofuranoside) is a compound that is taken up into cells and phosphorylated to form ZMP [92], which mimics the 30 G.V. Ronnett et al. / Physiology & Behavior 85 (2005) 25 – 35 Fig. 1. Working model for the effects of C75 on the FAS pathway. (A) During states of energy surplus, acetyl-CoA carboxylase (ACC) catalyzes the synthesis of malonyl-CoA from acetyl-CoA in an ATP-dependent reaction. Fatty acid synthase (FAS) then synthesizes palmitate by a series of condensation, reduction and dehydration cycles that utilizes NADPH. Acyl-CoA synthase (ACS) adds the CoA group to generate palmitoyl-CoA. Under conditions of energy surplus, malonyl-CoA levels are high and inhibit carnitine palmitoyltransferase-1 (CPT-1) from transporting long-chain acyl-CoA’s into mitochondria for oxidation, as this would constitute a futile cycle. (B) During states of energy deficiency, ambient levels of malonyl-CoA fall, permitting long-chain acyl-CoA’s to enter the mitochondria for oxidation. (C) We hypothesize that C75 inhibits FAS and stimulates CPT-1 to alter cellular energy balance, even when malonyl-CoA levels are elevated. effects of AMP on AMPK activation [93], thus stimulating AMPK activity. AICAR increases food intake, whereas C75 and compound C, an inhibitor of AMPK, decrease food intake. C75 rapidly (minutes) reduces the level of the phosphorylated AMPKa (pAMPKa) subunit in the hypothalamus, even in fasted mice that had elevated hypothalamic pAMPKa levels. AICAR is able to reverse both the C75-induced anorexia and the decrease in hypothalamic G.V. Ronnett et al. / Physiology & Behavior 85 (2005) 25 – 35 pAMPKa levels. As shown in vitro, C75 elevates hypothalamic neuronal ATP levels, which we hypothesize contributes to the mechanism through which C75 decreases AMPK activity. C75 also reduces the level of phosphorylated cAMP response element binding protein (pCREB) in the arcuate nucleus, suggesting a mechanism for the reduction in NPY expression seen with C75 treatment. These data indicate that modulation of fatty acid activity in the hypothalamus can alter neuronal energy perception via AMPK, which may function as a physiological energy sensor in the hypothalamus. Additional studies on the role of AMPK in the sensing of neuronal energy balance have emerged. Leptin inactivates hypothalamic AMPK, leading to anorexia [91], whereas it activates AMPK in skeletal muscle [94]. Intriguingly, the central administration of anorexigenic factors including insulin, glucose or MC3, and 4 agonists also inactivate AMPK [90]. In contrast, AGRP, ghrelin or AICAR activate hypothalamic AMPK, perhaps contributing to the resulting hyperphagia [90]. Our studies suggest a mechanism by which C75 can affect feeding behavior at least in part by modulating AMPK activity. By inhibiting FAS and stimulating CPT-1, C75 increases ATP levels in hypothalamic neurons. This would signal a positive energy balance, inactivating AMPK, and contribute to a decrease in NPY expression. When energy stores are depleted or decreased by fasting or increased activity, AMPK is activated, activating several downstream signals, including the CREB-NPY pathway to influence food intake. Under physiological conditions (normal feeding), it appears that there is relatively little change in the level of phosphorylated AMPK in the hypothalamus; a prolonged period of decreased food intake appears to be required before hypothalamic pAMPK levels increase. Thus, AMPK may function as a ‘‘fuel sensor’’ in the CNS, as it does in peripheral tissues such as muscle [88,94]. Although several pathways have been proposed to function upstream of AMPK pathway [95], the contributions of these pathways remain to be elucidated. An intriguing study by Wortman et al. hypothesized that the anorexic action of C75 could be mediated by a change in glucose uptake or utilization in the CNS [45]. Thus, feeding rats an essentially carbohydrate-free diet abrogated the effects of C75 on food intake. The effect of C75 was restored by the addition of other nutrient sources. While it has been shown, at least in vitro, that C75 increases glucose oxidation, this effect was transient when compared to the increase in fatty acid oxidation [56]. The hypothesis posed by Wortman et al. did not account for fatty acids as an energy source, as it was assumed C75 would inhibit fatty acid oxidation. However, the contribution of increased glucose utilization may still be significant and explain the results obtained using a carbohydrate-free diet. Alternatively, it must be considered that a high fat diet will essentially shut down the FAS pathway; therefore, C75 may have had no effect as FAS was already inactive due to a lack 31 of available malonyl-CoA. Yet, the results obtained by Wortman et al. are striking and clearly warrant further in vivo investigations. 7. FAS and CPT-1 modulation also influence gene expression Initial studies on the effects of C75 on weight loss and energy production utilized an acute treatment paradigm. Chronic exposure to C75 could potentially act through another mechanism in the CNS and in peripheral tissues, due to its effects on fatty acid synthesis and oxidation. A 2week chronic C75 treatment model was used to treat both DIO and lean mice to examine the effects of C75 on body weight, food intake and energy expenditure [47]. Over this 2-week period, C75 treatment was more efficacious in DIO mice compared to lean mice as measured by increased weight loss, decreased food intake and increased energy expenditure in the DIO mice compared to the lean mice. It was hypothesized that changes in gene expression in the DIO mice might account for this observation. Overall, analysis of the gene expression changes in hypothalamus in the 2-week DIO C75 treatment paradigm demonstrate the inhibition of orexigenic neuropeptide expression and induction of anorexigenic neuropeptide expression. Despite differences in the method of measurement of mRNA levels, the overall patterns of expression are similar to our prior study [18] and reflect the decreased food consumption seen in C75-treated mice. However, studies have shown differences in hypothalamic neuropeptide responses between DIO and lean rodents [96 – 100], and consistent with those studies, C75 treatment had a qualitatively different effect on hypothalamic neuropeptide expression in DIO and lean mice. C75 inhibited orexigenic neuropeptide (NPY, AGRP) expression and increased anorexigenic neuropeptide (POMC and CART) expression in DIO mice. In contrast, in lean mice, C75 decreased expression of anorexigenic neuropeptides (POMC and CART) without a change in orexigenic neuropeptides (NPY, AGRP) expression. DIO mice continued to demonstrate reduced food intake and a strongly anorexigenic neuropeptide profile throughout the duration of treatment. In contrast, by the conclusion of treatment, lean mice were eating an amount nearly equivalent to controls and they had only a modestly orexigenic hypothalamic profile. Interestingly, chronic C75 treatment induced a number of changes in gene expression in peripheral tissues [47]. A number of these changes were limited to the C75-treated DIO group and most commonly involved WAT. In combination, many of these changes favored the oxidation of fatty acid over their incorporation into triglyceride. Specifically, ACCh was down regulated by C75 along with glycerol-3-phosphate acyltransferase (GPAT), while expression of the liver isoform of CPT-1 was increased. GPAT is the initial committed step for fatty acid incorporation into 32 G.V. Ronnett et al. / Physiology & Behavior 85 (2005) 25 – 35 structural lipids or triglycerides [101]. These alterations would favor the entrance of fatty acid into the mitochondria for oxidation, and direct fatty acids away from storage and toward oxidation. C75 also increased the expression of UCP2 in WAT, liver and muscle. In addition to promoting uncoupling of mitochondrial oxidative phosphorylation, UCP2 has a role in limiting free radical formation during fatty acid oxidation [102]. Through its mitochondrial uncoupling, increased UCP2 expression could allow for increased fatty acid oxidation without the necessity of producing excess ATP. The peroxisome proliferator-activated receptors (PPARs) are a group of three nuclear receptor isoforms, PPARa, PPARg and PPARy, regulate a variety of functions related to energy metabolism [103]. C75 affected the expression of PPARa and PPARg. In DIO mice, C75 reduced the expression of PPARg in WAT, further promoting the reduction of lipid storage. In the liver, C75 reduced the expression of PPARa despite the increased expression of both L-CPT-1 and ACO. These data suggest that the pattern of gene expression favoring fatty acid oxidation was not due to increased PPARa activity, but via a yet undetermined mechanism. Accomplishing increased fatty acid oxidation without enhancing PPARa expression could have a particular advantage to cardiac muscle. Increased PPARa expression increases fatty acid transport beyond the capacity of increased fatty acid oxidation leading to fatty acid deposition in cardiac muscle. This is thought to be the mechanism responsible for diabetic cardiomyopathy [104]. Real-time RT-PCR measurements on cardiac muscle after 1 month of C75 treatment failed to show any increase in PPARa expression (data not shown). Several interesting observations can be made from the chronic treatment paradigm. The results of our fatty acid metabolism gene expression analysis advance our understanding of the selectivity of C75 in reducing adipose tissue mass, as opposed the lean mass. It appears reasonable that the dramatic increase in fatty acid oxidation in C75-treated DIO mice would likely require more than competitive stimulation of CPT-1. Interestingly, additional studies have shown that the weight loss effect of C75 treatment persists for nearly 2 weeks beyond cessation of therapy (Thupari and Kuhajda, personal communication), further supporting the importance of these gene expression changes on C75 weight maintenance. The exploration of the mechanism of action of C75 serves to further our understanding of the biological consequences of fatty acid synthesis inhibition and fatty acid oxidation stimulation in vivo. 8. Conclusions Collectively, the results presented here demonstrate that C75 treatment has several effects on neuronal energy metabolism and on neuronal activity. By altering glucose and fatty acid metabolism, and thus neuronal energy levels, C75 may influence energy perception, at least partially through the modulation of AMPK activity. We believe that the effect of C75 on neuronal energy is physiologically relevant due to the robust effect on neuronal activity and on downstream energy-sensing molecules such as AMPK, followed by alterations in pathways that are regulated by AMPK (ACC phosphorylation and glucose metabolism). This shift in the energy state of the neuron could signal the CNS, via altering neuronal activity, to inhibit feeding. Understanding the effect of modulating fatty acid metabolism on neuronal energy perception could lead to a better understanding of the mechanisms underlying feeding behavior and could potentially lead to new therapeutic targets for weight loss. Acknowledgements This work was supported by grants from the NINDS and NIDDK (GVR), and an NINDS F32 Fellowship to LEL. Compounds C and C75, described in this report, were provided by FASgen. 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