Tamoxifen-Induced Anorexia Is Associated With Fatty Acid Synthase Inhibition in the Ventromedial Nucleus of the Hypothalamus and Accumulatio
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糖尿病学杂志 2006年第5期
1 Department of Clinical Biochemistry, University of Cambridge, Addenbrooke’s Hospital, Cambridge, U.K
2 Department of Physiology, School of Medicine, University of Santiago de Compostela, Santiago de Compostela, Spain
3 Department of Morphological Sciences, School of Medicine, University of Santiago de Compostela, Santiago de Compostela, Spain
4 Parkside Oncology Clinic, London, U.K
5 Diabetes Research Unit, EBRC-827, Boston Medical Centre, Boston, Massachusetts
ACC, acetyl-CoA carboxylase; AMPK, AMP-activated protein kinase; ARC, arcuate nucleus of the hypothalamus; CART, cocaine- and amphetamine-regulated transcript; FAS, fatty acid synthase; LHA, lateral hypothalamic area; POMC, proopiomelanocortin; PVN, paraventricular nucleus of the hypothalamus; RMH, Royal Marsden Hospital; TMX, tamoxifen; TOFA, 5-(tetradecyloxy)-2-furoic acid; VMN, ventromedial nucleus of the hypothalamus
ABSTRACT
Fatty acid metabolism in the hypothalamus has recently been shown to regulate feeding. The selective estrogen receptor modulator tamoxifen (TMX) exerts a potent anorectic effect. Here, we show that the anorectic effect of TMX is associated with the accumulation of malonyl-CoA in the hypothalamus and inhibition of fatty acid synthase (FAS) expression specifically in the ventromedial nucleus of the hypothalamus (VMN). Furthermore, we demonstrate that FAS mRNA expression is physiologically regulated by fasting and refeeding in the VMN but not in other hypothalamic nuclei. Thus, the VMN appears to be the hypothalamic site where regulation of FAS and feeding converge. Supporting the potential clinical relevance of these observations, reanalysis of a primary breast cancer prevention study showed that obese women treated with TMX gained significantly less body weight over a 6-year period than obese women given placebo. The finding that TMX can modulate appetite through alterations in FAS expression and malonyl-CoA levels suggests a link between hypothalamic sex steroid receptors, fatty acid metabolism, and feeding behavior.
The clinical use of drugs designed to treat specific diseases occasionally reveals unexpected effects of potential therapeutic significance. It has been documented that certain antipsychotic (1) or antidepressant (2) drugs also have effects in food intake and body weight. Although it is unlikely that these drugs might one day be used as treatment for obesity, the discovery of their mechanisms of action may identify important pathways involved in energy homeostasis that could be targeted for antiobesity drug development.
Following a similar rationale, we investigated the molecular mechanisms mediating the anorectic effects of tamoxifen (TMX), a selective estrogen receptor modulator widely used for the treatment of breast cancers (3,4). The effect of TMX on energy homeostasis has been studied in rodents and has been shown to markedly decrease food intake and body weight (5,6). In spite of this, the molecular mechanism for such anorectic effects has not been identified.
Feeding is regulated at hypothalamic levels by orexigenic and anorexigenic neuropeptides. Neuropeptide Y, agouti-related protein, and melanin-concentrating hormone exert orexigenic actions, whereas -melanocyteeCstimulating hormone and cocaine- and amphetamine-regulated transcript (CART) peptides are the main anorexigenic signals. The expression of these neuropeptides is under the control of hormonal and nutritional signals (7eC12). More recently, it has been suggested that the precursors and break-down products of the fatty acid synthesis may be important metabolic signals regulating hypothalamic neuropeptides (10,13,14). The pathway of lipogenesis de novo and the hypothalamic levels of malonyl-CoA have been also identified as a key intermediate controlling food intake. The cellular level of malonyl-CoA depends on its rate of synthesis, catalyzed by acetyl-CoA carboxylase (ACC), relative to its rate of utilization, catalyzed by fatty acid synthase (FAS), and degradation, catalyzed by malonyl-CoA decarboxylase (15,16). While it is well established that the lipogenic enzymes are expressed in the arcuate (ARC), dorsomedial, paraventricular (PVN), and ventromedial (VMN) hypothalamic nuclei (17,18), their nuclei-specific regulation in response to nutrition has not previously been studied. Recent data have linked hypothalamic FAS to feeding regulation. It has been reported that administration of the FAS inhibitors cerulenin and C75 inhibits food intake and induces weight loss (13,19,20). Interestingly, this anorectic effect requires malonyl-CoA accumulation (14). Although it is not likely that these drugs become treatments for obesity, the evidence that selective inhibition of FAS can control feeding raises questions about the physiological involvement of fatty acid biosynthesis controlling food intake.
Using a systems biology approach, we have recently identified a primary action of TMX selectively decreasing the expression and activity of FAS in liver (21). By analogy, we studied whether the anorectic effect of TMX may be mediated through the inhibition of FAS in the hypothalamus. We also investigated the potential relevance of these findings to humans by reanalyzing data from the Royal Marsden Hospital (RMH) trial (3).
RESEARCH DESIGN AND METHODS
Human studies.
We investigated body weight changes in the RMH trial (3) (online appendix [available at http://diabetes.diabetesjournals.org]). The effects of TMX or placebo on body weight were analyzed in 2,329 subjects for whom BMI data were available and who did not develop cancer during the study. The trial was approved by the RMH Ethics Committee (3).
Rat studies.
We used Wistar rats (450eC500 g; Charles River Laboratories), lean Zucker rats [Crl:(ZUC)-faBR, 300eC350 g; The Jackson Laboratories], and obese Zucker rats [Crl:(ZUC)-faBR, 475eC525 g; The Jackson Laboratories] age-matched (11eC12 weeks) with lean Zucker rats. Rats were housed in a temperature-controlled room, with a 12-h light/dark cycle (lights on at 8:00 A.M. and off at 8:00 P.M.). All experiments were conducted in accordance with Home Office Guidelines and the Ethics Committee of the University of Santiago de Compostela. Male rats were used to exclude the confounding effects of the estrous cycle on food intake (22,23).
Subcutaneous TMX treatment.
TMX (Sigma) was dissolved in sesame oil containing 1% benzyl alcohol (5). The control group received a daily subcutaneous injection of vehicle and was provided standard rodent diet ad libitum. The TMX group received daily subcutaneous injections of TMX and was also provided ad libitum food. To monitor the effects of food intake, a pair-fed group was also included; for 5 days, the pair-fed group received the average amount of food eaten by the TMX-treated rats and received vehicle injections. For the hypothalamic studies, a dose of 0.5 mg · kgeC1 · dayeC1 TMX was administered for 5 days. On day 5, rats were killed 4 h after TMX treatment. Zucker lean and obese rats (n = 10eC12 animals/group) were also treated subcutaneously with TMX (0.5 mg · kgeC1 · dayeC1) for 5 days. On the final day of the protocol, rats were given a final dose of TMX (9:00 A.M.) and killed 4 h later. To test the effect of TMX treatment on refeeding, rats were fasted for 24 h before TMX administration. Next, the rats (n = 9eC10 animals/group) were treated with vehicle or TMX (0.5 mg/kg) and allowed to eat ad libitum. In all the protocols, TMX was administered subcutaneously at 9:00 A.M.
Intracerebroventricular TMX citrate treatment.
TMX citrate was used for intracerebroventricular experiments because it can be dissolved in aqueous solutions. Chronic (intracerebroventricular) cannulae were stereotaxically implanted under ketamine-xylacine anesthesia (50 mg/kg i.p.) in the lateral ventricle as previously described (24). Correct localization of the cannulae was confirmed by histological analysis. Before TMX citrate administration, rats (n = 9 animals/group) were fasted for 24 h in order to adjust the rats to the same pretreatment levels of stimulated appetite response. Rats received either a single administration of TMX citrate (10 e蘥 in 10 e蘬 2-hydroxypropyl--cyclodextrin; Sigma) or vehicle (10 e蘬, control rats). Since there are no reported studies about intracerebroventricular treatments with TMX, we selected this dose on the basis of previous literature based upon intracerebroventricular administration of other estrogenic compounds (25). A third group of animals was treated with an equimolar dose of citrate (Sigma) to that of the TMX citrate animals.
Conditioned taste aversion.
Five days before the test, rats were allowed 2 h daytime access to water. On the day of the experiment, rats (n = 16 rats/group) were given access to 0.15% sodium saccharin rather than water for 30 min. Immediately afterward, one experimental group was injected intraperitoneally with 0.15 mol/l lithium chloride (LiCl) in saline (26,27) and subcutaneously with sesame oil containing 1% benzyl alcohol (vehicle). The second experimental group was injected intraperitoneally with saline and subcutaneously with TMX (0.5 mg/kg). A third experimental group (control group) was injected intraperitoneally with saline and subcutaneously with vehicle. Twenty-four hours later, rats were given 2 h access to a two-bottle choice test of 0.15% saccharin versus water. Data were expressed as percent saccharin preference ratio (100 x saccharin intake/[saccharin intake + water intake]).
For the intracerebroventricular-conditioned taste aversion studies, one group was injected intraperitoneally with 0.15 mol/l LiCl in saline and intracerebroventricularlly with vehicle (10% 2-hydroxypropyl--cyclodextrin); the second group was injected intraperitoneally with saline and intracerebroventricularlly with TMX citrate (10 e蘥). A third group (control group) was injected intraperitoneally with saline and intracerebroventricularlly with vehicle. The experiment proceeded as the subcutaneous treatment (n = 16 rats/group).
Intracerebroventricular SHU9119 and 5-(tetradecyloxy)-2-furoic acid treatments.
Rats were fasted for 24 h (n = 8eC12 animals/group). To evaluate the effect of melanocortin receptors on the anorectic actions of TMX, the rats received a single intracerebroventricular administration of saline or the melanocortin-3/-4 receptors antagonist SHU9119 (3 nmol in saline; Phoenix) (28,29). To test the effect of the inhibition of ACC on the anorectic actions of TMX, the rats were treated with a single intracerebroventricular administration of vehicle (DMSO) or an ACC inhibitor: 5-(tetradecyloxy)-2-furoic acid (TOFA; 10 e蘥) (14). Subsequently, these rats received either vehicle or TMX (0.5 mg/kg) administered subcutaneously.
Blood biochemistry.
Kits for the measurement of cholesterol, glucose, and triglycerides were obtainded from Roche (21,30). Free fatty acids were measured using the NEFA-C colorimetric kit (Wako Chemicals) (21,30). Plasma leptin and insulin levels were measured by ELISA kits (Crystal) (21,30).
Real-time quantitative PCR.
The mRNA levels of estrogen receptor and progesterone receptor were studied by using real-time PCR (TaqMan) (online appendix Table 1 and online appendix methods) as previously described (30). We used six to eight rats per group.
In situ hybridization.
Coronal brain sections (16 e蘭) were probed with specific antisense oligos (online appendix Table 2). In situ hybridizations were performed as previously published (24,31,32) (online appendix methods). We used 10eC19 rats per group.
Immunohistochemistry.
Diaminobenzidine immunohistochemistry was performed as described (33) (online appendix methods) using a c-FOS antibody (Santa Cruz). We used six to eight rats per group.
Western blotting.
Hypothalamus total protein lysates were subjected to SDS-PAGE, electrotransferred on a polyvinylidine fluoride membrane, and probed with the indicated antibodies: ACC, pACC-Ser79, AMP-activated protein kinase (AMPK)1, and AMPK2 (Upstate); pAMPK-Thr172 (Cell Signaling); and -actin (Abcam). For protein detection, we used horseradish peroxidaseeCconjugated secondary antibodies and chemiluminescence (Amersham) (online appendix methods). We used 8eC12 rats per group.
Kinase assay.
Hypothalamic protein extracts were incubated at 4°C with the corresponding antibody conjugated to protein G-Sepharose (34). Phosphotransferase activity toward the AMARA-peptide for AMPK activity (35) or LKB1tide-peptide (Upstate) was then measured (36) (online appendix methods). We used 8eC12 rats/group.
Malonyl-CoA assay.
Malonyl-CoA was measured by radioisotopic method in neutralized perchloric acid filtrates (37,38) (online appendix methods). We used 8eC10 rats per group.
Statistical analysis.
Data were expressed as means ± SE and analyzed by using StatView 4.57 (Abacus Concepts). Statistical significance was determined by ANOVA and post hoc Bonferroni test. P < 0.05 was considered significant. Protein and mRNA levels were presented as percentage change in relation to control group (vehicle treated or fed ad libitum).
RESULTS
Subcutaneous administration of TMX reduces food intake and body weight.
Subcutaneous administration of TMX to rats markedly decreased food intake (Fig. 1A and online appendix Fig. 1A) and body weight (Fig. 1B and online appendix Fig. 1B). To determine whether the anorexigenic effect of TMX was dependent on feeding state, subcutaneous TMX was administered to 24-h fasted rats. TMX significantly attenuated refeeding at 6, 12, and 24 h (Fig. 1C).
We detect no differences in plasma glucose, triglycerides, and free fatty acids among the experimental groups (online appendix Table 3). Both TMX-treated and pair-fed rats showed significant decreases in insulin and leptin when compared with vehicle-treated rats (online appendix Table 3). Moreover, as it has been previously demonstrated (21,39), serum total, HDL, and LDL cholesterol were all reduced in the TMX group when compared with the ad libitum and pair-fed groups (online appendix Table 3).
Intracerebroventricular administration of TMX reduces food intake.
Since TMX is insoluble in an aqueous solution, TMX citrate was used for intracerebroventricular experiments. We confirmed that TMX citrate subcutaneous administration, at an equimolar dose to TMX (0.5 mg · kgeC1 · dayeC1), recapitulated the effects observed with TMX (online appendix Fig. 1C and D). Intracerebroventricular administration of TMX citrate to 24-h fasted rats decreased refeeding at 4, 12, and 24 h after treatment (Fig. 1D). This anorectic effect was specific for TMX because administration of citrate alone induced no changes in food intake (4 h: control, 14.2 ± 0.9 vs. citrate, 14.1 ± 1.2 g). Moreover, intracerebroventricular administration of TMX citrate did not affect plasma glucose, insulin, or leptin levels (online appendix Table 4), suggesting that the anorectic effect of TMX was not the result of peripheral secondary effects of TMX. Furthermore, we showed that subcutaneous administration of TMX at an equivalent dose to the intracerebroventricular treatment (10 e蘥 i.c.v. = 25 e蘥 · kgeC1 · dayeC1 s.c.) does not affect feeding, body weight (online appendix Fig. 1E and F), plasma glucose, insulin, or leptin levels (online appendix Table 5). These data make any peripheral effect of centrally administered TMX diffusing from the central nervous system unlikely.
To confirm that TMX was bioactive at hypothalamic levels, we studied the mRNA levels of estrogen receptor and progesterone receptor of TMX-treated rats. As previously reported, the mRNA expression of estrogen receptor (40) and progesterone receptor (41) was increased in the hypothalamus after TMX treatment (online appendix Fig. 2). These results indicate that the anorexic action of intracerebroventricular TMX citrate is mediated by central mechanisms.
The anorectic effect of TMX is not related to aversive effects.
We evaluated whether the anorectic effect of TMX was mediated by conditioned taste aversion (Fig. 1E). As expected, LiCl-treated rats drank significantly less saccharin solution (LiCl-paired flavor), indicating that they developed conditioned taste aversion (26,27,42). Rats treated subcutaneously with TMX or intracerebroventricularly with TMX citrate showed significantly increased saccharine preference ratios compared with LiCl-treated rats. The preference ratios for subcutaneous TMX (55.0 ± 4.4) and intracerebroventricular TMX citrate (86.1 ± 8.5) did not show significant differences when compared with their respective controls (control for the subcutaneous experiment: 65.4 ± 6.0; control for the intracerebroventricular experiment: 96.0 ± 5.1). Additionally, the preference ratios for TMX and TMX citrate were >50%, indicating the absence of aversive effects (26,42).
TMX-treated rats recovered their normal feeding pattern after treatment was discontinued (Fig. 1F), indicating that TMX did not induce a permanent state of wasting (42). Furthermore, we did not observe changes in other commonly used parameters of health status: temperature (Fig. 1G), skin aspect, stool consistency, or obvious abnormal behavior, as was evident in the LiCl-treated animals. These data suggest that the powerful anorectic effect of TMX, when administered intracerebroventricularlly or subcutaneously, was not related to aversive effects, illness, or malaise.
TMX reverses hyperphagia induced by defective leptin receptors.
We examined whether the anorexigenic effect of TMX could overcome the hyperphagic state associated with a defective leptin receptor signaling. Zucker rats were treated with TMX (0.5 mg · kgeC1 · dayeC1) for 5 days. TMX markedly decreased food intake in obese Zucker rats (which lack a functional long form of leptin receptor) and their lean littermates (online appendix Fig. 3A and B) and prevented body weight gain in both animal models (online appendix Fig. 3C and D).
TMX prevents the food restrictioneCinduced decrease in proopiomelanocortin and CART expression.
The levels of CART and proopiomelanocortin (POMC) mRNAs in the ARC were increased in TMX-treated rats when compared with pair-fed animals (Fig. 2A and B). The mRNA levels of both neuropeptides were similar to the levels in untreated fed animals, suggesting that these anorexigenic signals were inappropriately elevated for the level of feeding in TMX-treated animals. Agouti-related protein mRNA was unaffected (Fig. 2C). We detected increased levels of neuropeptide Y mRNA in the ARC (Fig. 2D) and melanin-concentrating hormone mRNA levels in the lateral hypothalamic area (LHA) (Fig. 2E) in the TMX-treated animals. These data suggest a compensatory upregulation of orexigenic neuropeptides against the reduction in feeding induced by TMX. Of note, these effects of TMX were specific since no changes were detected in the mRNA levels of CART in the PVN and in the LHA, corticotrophin-releasing hormone in the PVN, and thyrotrophin-releasing hormone in the PVN (online appendix Fig. 4).
Intracerebroventricular administration of SHU9119 attenuates the anorectic effect of subcutaneous TMX treatment.
Our data suggested that TMX could inhibit feeding through a mechanism involving the POMC neurons in the ARC. To test this hypothesis, we used SHU9119, a specific antagonist of the melanocortin-3 and -4 receptors (28,29). Intracerebroventricular administration of SHU9119 to rats before subcutaneous administration of TMX significantly reversed the anorectic response to TMX (Fig. 2F). SHU9119 did not have any effect on feeding when administered alone. These data suggest a direct effect of SHU9119 blocking TMX action on the melanocortin system, rather than a direct orexigenic effect. Similarly, these data indicate that POMC and melanocortin-4/-3 receptors are likely downstream mediators of TMX anorectic effects.
TMX downregulates the expression of FAS mRNA in the VMN specifically.
It has been recently shown that pharmacological inhibition of hypothalamic FAS inhibits feeding (13,19,20). We have recently reported that TMX decreases the expression and activity of FAS in liver (21). Using in situ hybridization analysis, we analyzed the FAS mRNA levels in hypothalami from rats treated subcutaneously with TMX (0.5 mg · kgeC1 · dayeC1 for 5 days). We also included control groups of fasted rats (24 and 48 h) and refed rats (24 and 48 h). Our data revealed a 40% decrease in FAS mRNA levels in the VMN after TMX treatment (Fig. 3A and B). The expression of FAS in other hypothalamic nuclei (ARC, PVN) (Fig. 3C and D), as well as in the cortex, hippocampus, and thalamus (data not shown), was not affected. Hence, TMX specifically reduced FAS expression in the VMN. Furthermore, TMX exerted this effect centrally since intracerebroventricular administration of TMX citrate also decreased (37%) FAS gene expression in the VMN (Fig. 4A).
Our fasted and refed groups also revealed that FAS gene expression was specifically decreased in the VMN after 24 and 48 h of fasting and that this effect was reverted by refeeding (Fig. 3A and E). This physiological nutritional regulation of FAS expression was specific to the VMN since FAS mRNA expression in the ARC and PVN did not change in response to any of these conditions (Fig. 3F and G).
TMX increases c-fos expression in the VMN specifically.
To further characterize the specificity of the effect of TMX in the VMN, we assessed the hypothalamic levels of c-fos expression, a marker of neuronal activation (13,27). Consistent with our FAS mRNA data (Fig. 3AeCC), TMX-treated rats showed significantly increased c-fos immunoreactivity in the VMN, but not in the ARC, compared with control and pair-fed rats (Fig. 4BeCD).
Decreased FAS gene expression induced by TMX is associated with increased hypothalamic levels of malonyl-CoA.
We found that the anorectic effect of TMX was associated with hypothalamic accumulation of malonyl-CoA (Fig. 5A). This effect was specific for TMX since the hypothalamic malonyl-CoA content in pair-fed and fasted rats was decreased (Fig. 5A and B). Levels of hypothalamic malonyl-CoA recovered after refeeding for 24 or 48 h (Fig. 5B). We also studied if the inhibitory effect of TMX on refeeding (Fig. 1C) correlated with hypothalamic malonyl-CoA content. Refed rats subcutaneously treated with TMX showed a further increase in hypothalamic malonyl-CoA content when compared with refed rats treated with vehicle (Fig. 5C). These results strongly suggest that a rise in malonyl-CoA levels may mediate the inhibitory effect of TMX on refeeding.
Hypothalamic AMPK/ACC is not affected by TMX.
The control of malonyl-CoA synthesis is regulated to a large extent by ACC. The activity of ACC can in turn be controlled by AMPK (43). Therefore, parallel changes in FAS and AMPK/ACC could explain the presence of altered malonyl-CoA levels in our TMX-treated rats. However, our results showed no differences in the protein levels of AMPK1, AMPK2, or pAMPK (Fig. 6A and B). Similarly, no differences in AMPK1 and AMPK2 activities (Fig. 6D and E) or in ACC, ACC, and pACC levels (Fig. 6A and C) were detected. LKB1 activity was not affected by either TMX or pair-feeding (Fig. 6F). These data suggest that TMX selectively inhibits fatty acid synthesis at the level of FAS activity.
Intracerebroventricular administration of an ACC inhibitor reverses the anorectic effect of subcutaneous TMX treatment by restoring hypothalamic malonyl-CoA levels.
To establish a mechanistic link between the effects of TMX, inhibition of FAS, accumulation of malonyl-CoA, and anorectic effects, we investigated the effect of TOFA, an inhibitor of ACC. Intracerebroventricular administration of TOFA completely prevented the anorectic effect of subcutaneous TMX on food intake during a 24-h refeeding experiment (Fig. 7A). This dose of TOFA, when administered alone, did not have any effect on feeding, excluding the possibility of a direct orexigenic effect. Furthermore, administration of TOFA prevented a TMX-induced increase in malonyl-CoA, suggesting that alterations in malonyl-CoA levels mediate the anorectic effect of TMX (Fig. 7B).
TMX administration prevents weight gain in human obese patients.
The potential relevance of our data were assessed by reanalyzing data from the RMH trial (3). When subjects were stratified according to their body weight at the beginning of the treatment, we found that obese (BMI 30 kg/m2) women treated with TMX initially lost weight and remained significantly lighter than those receiving placebo at 3, 24, 48, and 72 months (Fig. 8).
DISCUSSION
TMX-treated rats experience anorexia and weight loss (5,6). However, the mechanism for this anorectic effect has not been identified. We have recently reported that TMX inhibits FAS gene expression and activity in rat liver (21). As pharmacological inhibition of hypothalamic FAS elicits an anorectic response through a malonyl-CoAeCdependent mechanism (13,19,20), we hypothesized that decreased food intake in response to TMX may be the result of FAS inhibition and hypothalamic malonyl-CoA accumulation. Here, we demonstrated that TMX administration decreases FAS mRNA expression, specifically in the VMN, despite FAS being globally expressed in the brain in addition to other hypothalamic nuclei, like the ARC or PVN. This nucleus specificity of TMX was further supported by c-fos expression data that show specific VMN-neuron activation in response to TMX treatment. These data represent the first evidence of a nucleus-specific regulation of hypothalamic FAS mRNA associated with changes in food intake. Our fasting-refeeding experiments further supported a specific role for VMN and FAS expression in the regulation of feeding, since mRNA levels were only reduced in the VMN during fasting. These observations suggest that FAS in the VMN may act as a nutritional sensor to modulate food intake.
Next, we investigated whether TMX induced changes in hypothalamic malonyl-CoA. Although the decrease in FAS mRNA was restricted to just one nucleus, changes in malonyl-CoA levels were detectable when we analyzed the whole hypothalamus. It may be possible that inhibition of FAS activity could be a more generalized effect than mRNA regulation; however, it is also possible that the changes in malonyl-CoA levels in the VMN are significant enough to be detectable when the whole hypothalamus is assayed.
If FAS expression within the VMN has a role in regulating food intake, then the fact that FAS mRNA levels decrease in response to both fasting and TMX treatment is apparently contradictory. The explanation for this apparent paradox is found by examining malonyl-CoA levels. We demonstrate that the anorectic effect induced by TMX is similar to that of C75 in that it requires the accumulation of hypothalamic malonyl-CoA (14). The anorectic effect of TMX was reverted by pharmacological inhibition of ACC that prevented accumulation of malonyl-CoA. Thus, specific inhibition of FAS activity by TMX in the presence of maintained ACC activity (see below) facilitates accumulation of malonyl-CoA. Conversely, during fasting the decrease in FAS mRNA expression is associated with decreased levels of malonyl-CoA, suggesting that early steps of the hypothalamic lipogenic pathway are physiologically inhibited during fasting. Supporting this argument are recent reports demonstrating that during fasting, activation of hypothalamic AMPK phosphorylates and inhibits ACC (44,45).
We also investigated the effects of TMX on other components of the fatty acid biosynthetic pathway. AMPK plays a critical role in the hypothalamic regulation of feeding through its effects on the lipogenic pathway and neuropeptides (44,45). In situations of energy demand such as fasting, AMPK is phosphorylated by LKB1 at Thr172 (and then activated), resulting in phosphorylation and subsequent inhibition of ACC, thereby inactivating the fatty acid biosynthetic pathway (43). Under conditions of energy surplus, ACC is not phosphorylated, facilitating production of malonyl-CoA (43,45). Our data showed that FAS inhibition induced by TMX is not associated with changes in either AMPK protein levels/activity or phosphorylated ACC levels. These data suggest that the hypothalamic AMPK/ACC axis is not affected by TMX. Hence, the accumulation of malonyl-CoA induced by TMX is the result of specific FAS inhibition in the presence of normal ACC activity (online appendix Fig. 5).
Food restriction is associated with reduction in the mRNA levels of CART and POMC in the ARC (7eC9,12). This expected reduction in CART and POMC levels was blocked by TMX. Furthermore, pharmacological inhibition of melanocortin-3/-4 receptors by SHU9119 prevented the TMX anorectic effects, indicating that inhibition of feeding by TMX involves the melanocortin system. CART/POMC neurons receive neural projections and excitatory inputs from neurons in the VMN (46,47); therefore, changes in FAS/malonyl-CoA in the VMN might be linked to CART/POMC changes in the ARC. However, this mechanism still needs to be clarified.
TMX is a drug widely used for the treatment of estrogen receptoreCpositive breast cancers (3,4). Despite its widespread use, the action of TMX on human body weight is controversial. Data from the WHEL (Women’s Healthy Eating and Living) clinical trial show that obese women with breast cancer under TMX treatment had a decreased BMI (48). However, measurement of BMI in this study occurred at varying times after breast cancer diagnosis and thus did not necessarily reflect prediagnosis BMI. The results of this study may also have been confounded by the concurrent diagnosis of cancer. Hence, we reanalyzed the body weight data from the RMH trial (3). Since these subjects were free of cancer during the study, changes in body weight are more likely to be a direct effect of TMX. We found that obese (BMI 30 kg/m2) women treated with TMX were significantly lighter than those receiving placebo. Although this is a post hoc analysis of the data, we suggest that the large size of the cohort studied strongly supports the finding that low-dose TMX can induce a sustainable body weight decrease in obese humans. Although the RMH trial was not an obesity prevention/treatment trial, the finding that the obese women lost weight initially for 4 years and on average had maintained their initial weight by the end of the study is a highly significant deviation from the expected (49,50).
In conclusion, this study demonstrates that the anorectic actions of TMX involve a specific decrease in FAS mRNA levels in the VMN and hypothalamic malonyl-CoA accumulation. Specific regulation of FAS in the VMN by TMX or fasting suggests that fatty acid metabolism in this nucleus may play a physiological role in feeding control. Altogether, our data indicate that altering FAS in the VMN may be a suitable strategy for altering food intake and body weight homeostasis in experimental animals and obese subjects.
ACKNOWLEDGMENTS
This work has been supported by grants from the Medical Research Council (to A.J.V.-P.), the Welcome Trust (to A.J.V.-P.), the Spanish Ministry of Education (to C.D.), the Xunta de Galicia (to C.D.), and the European Union (LSHM-CT-2003-503041) (to C.D.). A.K.S. is funded by the U.S. Public Health Service (DK 19514) and a grant from the Juvenile Diabetes Research Foundation. M.L. is funded by the Marie Curie Program (QLK6-CT-2002-51671).
We are grateful to Keith Burling, Mark Campbell, and Janice Carter for their technical assistance, to Dr. Anthony P. Coll (University of Cambridge, Cambridge, U.K.), to Dr. Benjamin Challis (University of Cambridge, Cambridge, U.K.), to Prof. Manuel Ros (University Rey Juan Carlos, Madrid, Spain), and to Prof. Manuel Tena-Sempere (University of Cordoba, Cordoba, Spain) for their useful comments and criticism. We thank Prof. Len Storlien (AstraZeneca, Mondal, Sweden) for supplying us TOFA and Prof. Dario Alessi (University of Dundee, Dundee, U.K.) for the LKB1 antibody.
FOOTNOTES
M.L. and C.J.L. contributed equally to this work.
Additional information for this article can be found in an online appendix at http://diabetes.diabetesjournals.org.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
REFERENCES
Wirshing DA: Schizophrenia and obesity: impact of antipsychotic medications. J Clin Psychiatry 65 (Suppl. 18):13eC26, 2004
Korner J, Aronne LJ: Pharmacological approaches to weight reduction: therapeutic targets. J Clin Endocrinol Metab 89:2616eC2621, 2004
Powles T, Eeles R, Ashley S, Easton D, Chang J, Dowsett M, Tidy A, Viggers J, Davey J: Interim analysis of the incidence of breast cancer in the Royal Marsden Hospital tamoxifen randomised chemoprevention trial. Lancet 352:98eC101, 1998
Jordan VC: Selective estrogen receptor modulation: concept and consequences in cancer. Cancer Cell 5:207eC213, 2004
Wade GN, Heller HW: Tamoxifen mimics the effects of estradiol on food intake, body weight, and body composition in rats. Am J Physiol 264:R1219eCR1223, 1993
Gray JM, Schrock S, Bishop M: Estrogens and antiestrogens: actions and interactions with fluphenazine on food intake and body weight in rats. Am J Physiol 264:R1214eCR1218, 1993
Meister B: Control of food intake via leptin receptors in the hypothalamus. Vitam Horm 59:265eC304, 2000
Schwartz MW, Woods SC, Porte D Jr, Seeley RJ, Baskin DG: Central nervous system control of food intake. Nature 404:661eC671, 2000
Saper CB, Chou TC, Elmquist JK: The need to feed: homeostatic and hedonic control of eating. Neuron 36:199eC211, 2002
Obici S, Feng Z, Morgan K, Stein D, Karkanias G, Rossetti L: Central administration of oleic acid inhibits glucose production and food intake. Diabetes 51:271eC275, 2002
Cota D, Marsicano G, Lutz B, Vicennati V, Stalla GK, Pasquali R, Pagotto U: Endogenous cannabinoid system as a modulator of food intake. Int J Obes Relat Metab Disord 27:289eC301, 2003
Flier JS: Obesity wars: molecular progress confronts an expanding epidemic. Cell 116:337eC350, 2004
Gao S, Lane MD: Effect of the anorectic fatty acid synthase inhibitor C75 on neuronal activity in the hypothalamus and brainstem. Proc Natl Acad Sci U S A 100:5628eC5633, 2003
Hu Z, Cha SH, Chohnan S, Lane MD: Hypothalamic malonyl-CoA as a mediator of feeding behavior. Proc Natl Acad Sci U S A 100:12624eC12629, 2003
Ruderman NB, Saha AK, Kraegen EW: Minireview: malonyl CoA, AMP-activated protein kinase, and adiposity. Endocrinology 144:5166eC5171, 2003
Lam TK, Schwartz GJ, Rossetti L: Hypothalamic sensing of fatty acids. Nat Neurosci 8:579eC584, 2005
Kim EK, Miller I, Landree LE, Borisy-Rudin FF, Brown P, Tihan T, Townsend CA, Witters LA, Moran TH, Kuhajda FP, Ronnett GV: Expression of FAS within hypothalamic neurons: a model for decreased food intake after C75 treatment. Am J Physiol Endocrinol Metab 283:E867eCE879, 2002
Sorensen A, Travers MT, Vernon RG, Price NT, Barber MC: Localization of messenger RNAs encoding enzymes associated with malonyl-CoA metabolism in mouse brain. Brain Res Gene Expr Patterns 1:167eC173, 2002
Loftus TM, Jaworsky DE, Frehywot GL, Townsend CA, Ronnett GV, Lane MD, Kuhajda FP: Reduced food intake and body weight in mice treated with fatty acid synthase inhibitors. Science 288:2379eC2381, 2000
Clegg DJ, Wortman MD, Benoit SC, McOsker CC, Seeley RJ: Comparison of central and peripheral administration of C75 on food intake, body weight, and conditioned taste aversion. Diabetes 51:3196eC3201, 2002
Lelliott CJ, Le畃ez M, Curtis RK, Parker N, Laudes M, Yeo G, Jimeenez-Lian M, Grosse J, Saha AK, Wiggins D, Hauton D, Brand MD, O’Rahilly S, Griffin JL, Gibbons GF, Vidal-Puig A: Transcript and metabolite analysis of the effects of tamoxifen in rat liver reveals inhibition of fatty acid synthesis in the presence of hepatic steatosis. FASEB J 19:1108eC1119, 2005
Mystkowski P, Seeley RJ, Hahn TM, Baskin DG, Havel PJ, Matsumoto AM, Wilkinson CW, Peacock-Kinzig K, Blake KA, Schwartz MW: Hypothalamic melanin-concentrating hormone and estrogen-induced weight loss. J Neurosci 20:8637eC8642, 2000
Tritos NA, Segal-Lieberman G, Vezeridis PS, Maratos-Flier E: Estradiol-induced anorexia is independent of leptin and melanin-concentrating hormone. Obes Res 12:716eC724, 2004
Le畃ez M, Seoane LM, Tovar S, Nogueiras R, Dieeguez C, Seare猻 R: Orexin-A regulates growth hormone releasing hormone mRNA content in a nucleus specific manner and somatostatin mRNA content in a growth hormone-dependent fashion in the rat hypothalamus. Eur J Neurosci 19:2080eC2088, 2004
Liang YQ, Akishita M, Kim S, Ako J, Hashimoto M, Iijima K, Ohike Y, Watanabe T, Sudoh N, Toba K, Yoshizumi M, Ouchi Y: Estrogen receptor beta is involved in the anorectic action of estrogen. Int J Obes Relat Metab Disord 26:1103eC1109, 2002
Kelly JF, Elias CF, Lee CE, Ahima RS, Seeley RJ, Bjorbaek C, Oka T, Saper CB, Flier JS, Elmquist JK: Ciliary neurotrophic factor and leptin induce distinct patterns of immediate early gene expression in the brain. Diabetes 53:911eC920, 2004
Halatchev IG, Cone RD: Peripheral administration of PYY3-36 produces conditioned taste aversion in mice. Cell Metab 1:159eC168, 2005
Kask A, Mutulis F, Muceniece R, Pahkla R, Mutule I, Wikberg JE, Rago L, Schioth HB: Discovery of a novel superpotent and selective melanocortin-4 receptor antagonist (HS024): evaluation in vitro and in vivo. Endocrinology 139:5006eC5014, 1998
Heisler LK, Cowley MA, Tecott LH, Fan W, Low MJ, Smart JL, Rubinstein M, Tatro JB, Marcus JN, Holstege H, Lee CE, Cone RD, Elmquist JK: Activation of central melanocortin pathways by fenfluramine. Science 297:609eC611, 2002
Medina-Ge甿ez G, Virtue S, Lelliott C, Boiani R, Campbell M, Christodoulides C, Perrin C, Jimeenez-Lian M, Blount M, Dixon J, Zahn D, Thresher RR, Aparicio S, Carlton M, Colledge WH, Kettunen MI, Seppanen-Laakso T, Sethi JK, O’Rahilly S, Brindle K, Cinti S, Oresic M, Burcelin R, Vidal-Puig A: The link between nutritional status and insulin sensitivity is dependent on the adipocyte-specific peroxisome proliferator-activated receptor-2 isoform. Diabetes 54:1706eC1716, 2005
Coll AP, Challis BG, Le畃ez M, Piper S, Yeo GS, O’Rahilly S: Proopiomelanocortin-deficient mice are hypersensitive to the adverse metabolic effects of glucocorticoids. Diabetes 54:2269eC2276, 2005
Le畃ez M, Seoane LM, Tovar S, Garce MC, Nogueiras R, Dieeguez C, Seare猻 RM: A possible role of neuropeptide Y, agouti-related protein and leptin receptor isoforms in hypothalamic programming by perinatal feeding in the rat. Diabetologia 48:140eC148, 2005
Nogueiras R, Gallego R, Gualillo O, Caminos JE, Garce-Caballero T, Casanueva FF, Dieeguez C: Resistin is expressed in different rat tissues and is regulated in a tissue- and gender-specific manner. FEBS Lett 548:21eC27, 2003
Sakamoto K, Goransson O, Hardie DG, Alessi DR: Activity of LKB1 and AMPK-related kinases in skeletal muscle: effects of contraction, phenformin, and AICAR. Am J Physiol Endocrinol Metab 287:E310eCE317, 2004
Dale S, Wilson WA, Edelman AM, Hardie DG: Similar substrate recognition motifs for mammalian AMP-activated protein kinase, higher plant HMG-CoA reductase kinase-A, yeast SNF1, and mammalian calmodulin-dependent protein kinase I. FEBS Lett 361:191eC195, 1995
Lizcano JM, Goransson O, Toth R, Deak M, Morrice NA, Boudeau J, Hawley SA, Udd L, Makela TP, Hardie DG, Alessi DR: LKB1 is a master kinase that activates 13 kinases of the AMPK subfamily, including MARK/PAR-1. EMBO J 23:833eC843, 2004
McGarry JD, Stark MJ, Foster DW: Hepatic malonyl-CoA levels of fed, fasted and diabetic rats as measured using a simple radioisotopic assay. J Biol Chem 253:8291eC8293, 1978
Saha AK, Kurowski TG, Ruderman NB: A malonyl-CoA fuel-sensing mechanism in muscle: effects of insulin, glucose, and denervation. Am J Physiol 269:E283eCE289, 1995
Gold E, Stapley S, Goulding A: Tamoxifen and norethisterone: effects on plasma cholesterol and total body calcium content in the estrogen-deficient rat. Horm Metab Res 26:100eC103, 1994
Patisaul HB, Aultman EA, Bielsky IF, Young LJ, Wilson ME: Immediate and residual effects of tamoxifen and ethynylestradiol in the female rat hypothalamus. Brain Res 978:185eC193, 2003
Tena-Sempere M, Navarro VM, Mayen A, Bellido C, Sanchez-Criado JE: Regulation of estrogen receptor (ER) isoform messenger RNA expression by different ER ligands in female rat pituitary. Biol Reprod 70:671eC678, 2004
Kim MS, Park JY, Namkoong C, Jang PG, Ryu JW, Song HS, Yun JY, Namgoong IS, Ha J, Park IS, Lee IK, Viollet B, Youn JH, Lee HK, Lee KU: Anti-obesity effects of alpha-lipoic acid mediated by suppression of hypothalamic AMP-activated protein kinase. Nat Med 10:727eC733, 2004
Kahn BB, Alquier T, Carling D, Hardie DG: AMP-activated protein kinase: ancient energy gauge provides clues to modern understanding of metabolism. Cell Metab 1:15eC25, 2005
Minokoshi Y, Alquier T, Furukawa N, Kim YB, Lee A, Xue B, Mu J, Foufelle F, Ferre P, Birnbaum MJ, Stuck BJ, Kahn BB: AMP-kinase regulates food intake by responding to hormonal and nutrient signals in the hypothalamus. Nature 428:569eC574, 2004
Andersson U, Filipsson K, Abbott CR, Woods A, Smith K, Bloom SR, Carling D, Small CJ: AMP-activated protein kinase plays a role in the control of food intake. J Biol Chem 279:12005eC12008, 2004
Pinto S, Roseberry AG, Liu H, Diano S, Shanabrough M, Cai X, Friedman JM, Horvath TL: Rapid rewiring of ARC nucleus feeding circuits by leptin. Science 304:110eC115, 2004
Sternson SM, Shepherd GM, Friedman JM: Topographic mapping of VMH ARC nucleus microcircuits and their reorganization by fasting. Nat Neurosci 8:1356eC1363, 2005
Wasserman L, Flatt SW, Natarajan L, Laughlin G, Matusalem M, Faerber S, Rock CL, Barrett-Connor E, Pierce JP: Correlates of obesity in postmenopausal women with breast cancer: comparison of genetic, demographic, disease-related, life history and dietary factors. Int J Obes Relat Metab Disord 28:49eC56, 2004
Finer N: Pharmacotherapy of obesity. Best Pract Res Clin Endocrinol Metab 16:717eC742, 2002
Li Z, Maglione M, Tu W, Mojica W, Arterburn D, Shugarman LR, Hilton L, Suttorp M, Solomon V, Shekelle PG, Morton SC: Meta-analysis: pharmacologic treatment of obesity. Ann Intern Med 142:532eC546, 2005(Miguel Leez, Christopher )
2 Department of Physiology, School of Medicine, University of Santiago de Compostela, Santiago de Compostela, Spain
3 Department of Morphological Sciences, School of Medicine, University of Santiago de Compostela, Santiago de Compostela, Spain
4 Parkside Oncology Clinic, London, U.K
5 Diabetes Research Unit, EBRC-827, Boston Medical Centre, Boston, Massachusetts
ACC, acetyl-CoA carboxylase; AMPK, AMP-activated protein kinase; ARC, arcuate nucleus of the hypothalamus; CART, cocaine- and amphetamine-regulated transcript; FAS, fatty acid synthase; LHA, lateral hypothalamic area; POMC, proopiomelanocortin; PVN, paraventricular nucleus of the hypothalamus; RMH, Royal Marsden Hospital; TMX, tamoxifen; TOFA, 5-(tetradecyloxy)-2-furoic acid; VMN, ventromedial nucleus of the hypothalamus
ABSTRACT
Fatty acid metabolism in the hypothalamus has recently been shown to regulate feeding. The selective estrogen receptor modulator tamoxifen (TMX) exerts a potent anorectic effect. Here, we show that the anorectic effect of TMX is associated with the accumulation of malonyl-CoA in the hypothalamus and inhibition of fatty acid synthase (FAS) expression specifically in the ventromedial nucleus of the hypothalamus (VMN). Furthermore, we demonstrate that FAS mRNA expression is physiologically regulated by fasting and refeeding in the VMN but not in other hypothalamic nuclei. Thus, the VMN appears to be the hypothalamic site where regulation of FAS and feeding converge. Supporting the potential clinical relevance of these observations, reanalysis of a primary breast cancer prevention study showed that obese women treated with TMX gained significantly less body weight over a 6-year period than obese women given placebo. The finding that TMX can modulate appetite through alterations in FAS expression and malonyl-CoA levels suggests a link between hypothalamic sex steroid receptors, fatty acid metabolism, and feeding behavior.
The clinical use of drugs designed to treat specific diseases occasionally reveals unexpected effects of potential therapeutic significance. It has been documented that certain antipsychotic (1) or antidepressant (2) drugs also have effects in food intake and body weight. Although it is unlikely that these drugs might one day be used as treatment for obesity, the discovery of their mechanisms of action may identify important pathways involved in energy homeostasis that could be targeted for antiobesity drug development.
Following a similar rationale, we investigated the molecular mechanisms mediating the anorectic effects of tamoxifen (TMX), a selective estrogen receptor modulator widely used for the treatment of breast cancers (3,4). The effect of TMX on energy homeostasis has been studied in rodents and has been shown to markedly decrease food intake and body weight (5,6). In spite of this, the molecular mechanism for such anorectic effects has not been identified.
Feeding is regulated at hypothalamic levels by orexigenic and anorexigenic neuropeptides. Neuropeptide Y, agouti-related protein, and melanin-concentrating hormone exert orexigenic actions, whereas -melanocyteeCstimulating hormone and cocaine- and amphetamine-regulated transcript (CART) peptides are the main anorexigenic signals. The expression of these neuropeptides is under the control of hormonal and nutritional signals (7eC12). More recently, it has been suggested that the precursors and break-down products of the fatty acid synthesis may be important metabolic signals regulating hypothalamic neuropeptides (10,13,14). The pathway of lipogenesis de novo and the hypothalamic levels of malonyl-CoA have been also identified as a key intermediate controlling food intake. The cellular level of malonyl-CoA depends on its rate of synthesis, catalyzed by acetyl-CoA carboxylase (ACC), relative to its rate of utilization, catalyzed by fatty acid synthase (FAS), and degradation, catalyzed by malonyl-CoA decarboxylase (15,16). While it is well established that the lipogenic enzymes are expressed in the arcuate (ARC), dorsomedial, paraventricular (PVN), and ventromedial (VMN) hypothalamic nuclei (17,18), their nuclei-specific regulation in response to nutrition has not previously been studied. Recent data have linked hypothalamic FAS to feeding regulation. It has been reported that administration of the FAS inhibitors cerulenin and C75 inhibits food intake and induces weight loss (13,19,20). Interestingly, this anorectic effect requires malonyl-CoA accumulation (14). Although it is not likely that these drugs become treatments for obesity, the evidence that selective inhibition of FAS can control feeding raises questions about the physiological involvement of fatty acid biosynthesis controlling food intake.
Using a systems biology approach, we have recently identified a primary action of TMX selectively decreasing the expression and activity of FAS in liver (21). By analogy, we studied whether the anorectic effect of TMX may be mediated through the inhibition of FAS in the hypothalamus. We also investigated the potential relevance of these findings to humans by reanalyzing data from the Royal Marsden Hospital (RMH) trial (3).
RESEARCH DESIGN AND METHODS
Human studies.
We investigated body weight changes in the RMH trial (3) (online appendix [available at http://diabetes.diabetesjournals.org]). The effects of TMX or placebo on body weight were analyzed in 2,329 subjects for whom BMI data were available and who did not develop cancer during the study. The trial was approved by the RMH Ethics Committee (3).
Rat studies.
We used Wistar rats (450eC500 g; Charles River Laboratories), lean Zucker rats [Crl:(ZUC)-faBR, 300eC350 g; The Jackson Laboratories], and obese Zucker rats [Crl:(ZUC)-faBR, 475eC525 g; The Jackson Laboratories] age-matched (11eC12 weeks) with lean Zucker rats. Rats were housed in a temperature-controlled room, with a 12-h light/dark cycle (lights on at 8:00 A.M. and off at 8:00 P.M.). All experiments were conducted in accordance with Home Office Guidelines and the Ethics Committee of the University of Santiago de Compostela. Male rats were used to exclude the confounding effects of the estrous cycle on food intake (22,23).
Subcutaneous TMX treatment.
TMX (Sigma) was dissolved in sesame oil containing 1% benzyl alcohol (5). The control group received a daily subcutaneous injection of vehicle and was provided standard rodent diet ad libitum. The TMX group received daily subcutaneous injections of TMX and was also provided ad libitum food. To monitor the effects of food intake, a pair-fed group was also included; for 5 days, the pair-fed group received the average amount of food eaten by the TMX-treated rats and received vehicle injections. For the hypothalamic studies, a dose of 0.5 mg · kgeC1 · dayeC1 TMX was administered for 5 days. On day 5, rats were killed 4 h after TMX treatment. Zucker lean and obese rats (n = 10eC12 animals/group) were also treated subcutaneously with TMX (0.5 mg · kgeC1 · dayeC1) for 5 days. On the final day of the protocol, rats were given a final dose of TMX (9:00 A.M.) and killed 4 h later. To test the effect of TMX treatment on refeeding, rats were fasted for 24 h before TMX administration. Next, the rats (n = 9eC10 animals/group) were treated with vehicle or TMX (0.5 mg/kg) and allowed to eat ad libitum. In all the protocols, TMX was administered subcutaneously at 9:00 A.M.
Intracerebroventricular TMX citrate treatment.
TMX citrate was used for intracerebroventricular experiments because it can be dissolved in aqueous solutions. Chronic (intracerebroventricular) cannulae were stereotaxically implanted under ketamine-xylacine anesthesia (50 mg/kg i.p.) in the lateral ventricle as previously described (24). Correct localization of the cannulae was confirmed by histological analysis. Before TMX citrate administration, rats (n = 9 animals/group) were fasted for 24 h in order to adjust the rats to the same pretreatment levels of stimulated appetite response. Rats received either a single administration of TMX citrate (10 e蘥 in 10 e蘬 2-hydroxypropyl--cyclodextrin; Sigma) or vehicle (10 e蘬, control rats). Since there are no reported studies about intracerebroventricular treatments with TMX, we selected this dose on the basis of previous literature based upon intracerebroventricular administration of other estrogenic compounds (25). A third group of animals was treated with an equimolar dose of citrate (Sigma) to that of the TMX citrate animals.
Conditioned taste aversion.
Five days before the test, rats were allowed 2 h daytime access to water. On the day of the experiment, rats (n = 16 rats/group) were given access to 0.15% sodium saccharin rather than water for 30 min. Immediately afterward, one experimental group was injected intraperitoneally with 0.15 mol/l lithium chloride (LiCl) in saline (26,27) and subcutaneously with sesame oil containing 1% benzyl alcohol (vehicle). The second experimental group was injected intraperitoneally with saline and subcutaneously with TMX (0.5 mg/kg). A third experimental group (control group) was injected intraperitoneally with saline and subcutaneously with vehicle. Twenty-four hours later, rats were given 2 h access to a two-bottle choice test of 0.15% saccharin versus water. Data were expressed as percent saccharin preference ratio (100 x saccharin intake/[saccharin intake + water intake]).
For the intracerebroventricular-conditioned taste aversion studies, one group was injected intraperitoneally with 0.15 mol/l LiCl in saline and intracerebroventricularlly with vehicle (10% 2-hydroxypropyl--cyclodextrin); the second group was injected intraperitoneally with saline and intracerebroventricularlly with TMX citrate (10 e蘥). A third group (control group) was injected intraperitoneally with saline and intracerebroventricularlly with vehicle. The experiment proceeded as the subcutaneous treatment (n = 16 rats/group).
Intracerebroventricular SHU9119 and 5-(tetradecyloxy)-2-furoic acid treatments.
Rats were fasted for 24 h (n = 8eC12 animals/group). To evaluate the effect of melanocortin receptors on the anorectic actions of TMX, the rats received a single intracerebroventricular administration of saline or the melanocortin-3/-4 receptors antagonist SHU9119 (3 nmol in saline; Phoenix) (28,29). To test the effect of the inhibition of ACC on the anorectic actions of TMX, the rats were treated with a single intracerebroventricular administration of vehicle (DMSO) or an ACC inhibitor: 5-(tetradecyloxy)-2-furoic acid (TOFA; 10 e蘥) (14). Subsequently, these rats received either vehicle or TMX (0.5 mg/kg) administered subcutaneously.
Blood biochemistry.
Kits for the measurement of cholesterol, glucose, and triglycerides were obtainded from Roche (21,30). Free fatty acids were measured using the NEFA-C colorimetric kit (Wako Chemicals) (21,30). Plasma leptin and insulin levels were measured by ELISA kits (Crystal) (21,30).
Real-time quantitative PCR.
The mRNA levels of estrogen receptor and progesterone receptor were studied by using real-time PCR (TaqMan) (online appendix Table 1 and online appendix methods) as previously described (30). We used six to eight rats per group.
In situ hybridization.
Coronal brain sections (16 e蘭) were probed with specific antisense oligos (online appendix Table 2). In situ hybridizations were performed as previously published (24,31,32) (online appendix methods). We used 10eC19 rats per group.
Immunohistochemistry.
Diaminobenzidine immunohistochemistry was performed as described (33) (online appendix methods) using a c-FOS antibody (Santa Cruz). We used six to eight rats per group.
Western blotting.
Hypothalamus total protein lysates were subjected to SDS-PAGE, electrotransferred on a polyvinylidine fluoride membrane, and probed with the indicated antibodies: ACC, pACC-Ser79, AMP-activated protein kinase (AMPK)1, and AMPK2 (Upstate); pAMPK-Thr172 (Cell Signaling); and -actin (Abcam). For protein detection, we used horseradish peroxidaseeCconjugated secondary antibodies and chemiluminescence (Amersham) (online appendix methods). We used 8eC12 rats per group.
Kinase assay.
Hypothalamic protein extracts were incubated at 4°C with the corresponding antibody conjugated to protein G-Sepharose (34). Phosphotransferase activity toward the AMARA-peptide for AMPK activity (35) or LKB1tide-peptide (Upstate) was then measured (36) (online appendix methods). We used 8eC12 rats/group.
Malonyl-CoA assay.
Malonyl-CoA was measured by radioisotopic method in neutralized perchloric acid filtrates (37,38) (online appendix methods). We used 8eC10 rats per group.
Statistical analysis.
Data were expressed as means ± SE and analyzed by using StatView 4.57 (Abacus Concepts). Statistical significance was determined by ANOVA and post hoc Bonferroni test. P < 0.05 was considered significant. Protein and mRNA levels were presented as percentage change in relation to control group (vehicle treated or fed ad libitum).
RESULTS
Subcutaneous administration of TMX reduces food intake and body weight.
Subcutaneous administration of TMX to rats markedly decreased food intake (Fig. 1A and online appendix Fig. 1A) and body weight (Fig. 1B and online appendix Fig. 1B). To determine whether the anorexigenic effect of TMX was dependent on feeding state, subcutaneous TMX was administered to 24-h fasted rats. TMX significantly attenuated refeeding at 6, 12, and 24 h (Fig. 1C).
We detect no differences in plasma glucose, triglycerides, and free fatty acids among the experimental groups (online appendix Table 3). Both TMX-treated and pair-fed rats showed significant decreases in insulin and leptin when compared with vehicle-treated rats (online appendix Table 3). Moreover, as it has been previously demonstrated (21,39), serum total, HDL, and LDL cholesterol were all reduced in the TMX group when compared with the ad libitum and pair-fed groups (online appendix Table 3).
Intracerebroventricular administration of TMX reduces food intake.
Since TMX is insoluble in an aqueous solution, TMX citrate was used for intracerebroventricular experiments. We confirmed that TMX citrate subcutaneous administration, at an equimolar dose to TMX (0.5 mg · kgeC1 · dayeC1), recapitulated the effects observed with TMX (online appendix Fig. 1C and D). Intracerebroventricular administration of TMX citrate to 24-h fasted rats decreased refeeding at 4, 12, and 24 h after treatment (Fig. 1D). This anorectic effect was specific for TMX because administration of citrate alone induced no changes in food intake (4 h: control, 14.2 ± 0.9 vs. citrate, 14.1 ± 1.2 g). Moreover, intracerebroventricular administration of TMX citrate did not affect plasma glucose, insulin, or leptin levels (online appendix Table 4), suggesting that the anorectic effect of TMX was not the result of peripheral secondary effects of TMX. Furthermore, we showed that subcutaneous administration of TMX at an equivalent dose to the intracerebroventricular treatment (10 e蘥 i.c.v. = 25 e蘥 · kgeC1 · dayeC1 s.c.) does not affect feeding, body weight (online appendix Fig. 1E and F), plasma glucose, insulin, or leptin levels (online appendix Table 5). These data make any peripheral effect of centrally administered TMX diffusing from the central nervous system unlikely.
To confirm that TMX was bioactive at hypothalamic levels, we studied the mRNA levels of estrogen receptor and progesterone receptor of TMX-treated rats. As previously reported, the mRNA expression of estrogen receptor (40) and progesterone receptor (41) was increased in the hypothalamus after TMX treatment (online appendix Fig. 2). These results indicate that the anorexic action of intracerebroventricular TMX citrate is mediated by central mechanisms.
The anorectic effect of TMX is not related to aversive effects.
We evaluated whether the anorectic effect of TMX was mediated by conditioned taste aversion (Fig. 1E). As expected, LiCl-treated rats drank significantly less saccharin solution (LiCl-paired flavor), indicating that they developed conditioned taste aversion (26,27,42). Rats treated subcutaneously with TMX or intracerebroventricularly with TMX citrate showed significantly increased saccharine preference ratios compared with LiCl-treated rats. The preference ratios for subcutaneous TMX (55.0 ± 4.4) and intracerebroventricular TMX citrate (86.1 ± 8.5) did not show significant differences when compared with their respective controls (control for the subcutaneous experiment: 65.4 ± 6.0; control for the intracerebroventricular experiment: 96.0 ± 5.1). Additionally, the preference ratios for TMX and TMX citrate were >50%, indicating the absence of aversive effects (26,42).
TMX-treated rats recovered their normal feeding pattern after treatment was discontinued (Fig. 1F), indicating that TMX did not induce a permanent state of wasting (42). Furthermore, we did not observe changes in other commonly used parameters of health status: temperature (Fig. 1G), skin aspect, stool consistency, or obvious abnormal behavior, as was evident in the LiCl-treated animals. These data suggest that the powerful anorectic effect of TMX, when administered intracerebroventricularlly or subcutaneously, was not related to aversive effects, illness, or malaise.
TMX reverses hyperphagia induced by defective leptin receptors.
We examined whether the anorexigenic effect of TMX could overcome the hyperphagic state associated with a defective leptin receptor signaling. Zucker rats were treated with TMX (0.5 mg · kgeC1 · dayeC1) for 5 days. TMX markedly decreased food intake in obese Zucker rats (which lack a functional long form of leptin receptor) and their lean littermates (online appendix Fig. 3A and B) and prevented body weight gain in both animal models (online appendix Fig. 3C and D).
TMX prevents the food restrictioneCinduced decrease in proopiomelanocortin and CART expression.
The levels of CART and proopiomelanocortin (POMC) mRNAs in the ARC were increased in TMX-treated rats when compared with pair-fed animals (Fig. 2A and B). The mRNA levels of both neuropeptides were similar to the levels in untreated fed animals, suggesting that these anorexigenic signals were inappropriately elevated for the level of feeding in TMX-treated animals. Agouti-related protein mRNA was unaffected (Fig. 2C). We detected increased levels of neuropeptide Y mRNA in the ARC (Fig. 2D) and melanin-concentrating hormone mRNA levels in the lateral hypothalamic area (LHA) (Fig. 2E) in the TMX-treated animals. These data suggest a compensatory upregulation of orexigenic neuropeptides against the reduction in feeding induced by TMX. Of note, these effects of TMX were specific since no changes were detected in the mRNA levels of CART in the PVN and in the LHA, corticotrophin-releasing hormone in the PVN, and thyrotrophin-releasing hormone in the PVN (online appendix Fig. 4).
Intracerebroventricular administration of SHU9119 attenuates the anorectic effect of subcutaneous TMX treatment.
Our data suggested that TMX could inhibit feeding through a mechanism involving the POMC neurons in the ARC. To test this hypothesis, we used SHU9119, a specific antagonist of the melanocortin-3 and -4 receptors (28,29). Intracerebroventricular administration of SHU9119 to rats before subcutaneous administration of TMX significantly reversed the anorectic response to TMX (Fig. 2F). SHU9119 did not have any effect on feeding when administered alone. These data suggest a direct effect of SHU9119 blocking TMX action on the melanocortin system, rather than a direct orexigenic effect. Similarly, these data indicate that POMC and melanocortin-4/-3 receptors are likely downstream mediators of TMX anorectic effects.
TMX downregulates the expression of FAS mRNA in the VMN specifically.
It has been recently shown that pharmacological inhibition of hypothalamic FAS inhibits feeding (13,19,20). We have recently reported that TMX decreases the expression and activity of FAS in liver (21). Using in situ hybridization analysis, we analyzed the FAS mRNA levels in hypothalami from rats treated subcutaneously with TMX (0.5 mg · kgeC1 · dayeC1 for 5 days). We also included control groups of fasted rats (24 and 48 h) and refed rats (24 and 48 h). Our data revealed a 40% decrease in FAS mRNA levels in the VMN after TMX treatment (Fig. 3A and B). The expression of FAS in other hypothalamic nuclei (ARC, PVN) (Fig. 3C and D), as well as in the cortex, hippocampus, and thalamus (data not shown), was not affected. Hence, TMX specifically reduced FAS expression in the VMN. Furthermore, TMX exerted this effect centrally since intracerebroventricular administration of TMX citrate also decreased (37%) FAS gene expression in the VMN (Fig. 4A).
Our fasted and refed groups also revealed that FAS gene expression was specifically decreased in the VMN after 24 and 48 h of fasting and that this effect was reverted by refeeding (Fig. 3A and E). This physiological nutritional regulation of FAS expression was specific to the VMN since FAS mRNA expression in the ARC and PVN did not change in response to any of these conditions (Fig. 3F and G).
TMX increases c-fos expression in the VMN specifically.
To further characterize the specificity of the effect of TMX in the VMN, we assessed the hypothalamic levels of c-fos expression, a marker of neuronal activation (13,27). Consistent with our FAS mRNA data (Fig. 3AeCC), TMX-treated rats showed significantly increased c-fos immunoreactivity in the VMN, but not in the ARC, compared with control and pair-fed rats (Fig. 4BeCD).
Decreased FAS gene expression induced by TMX is associated with increased hypothalamic levels of malonyl-CoA.
We found that the anorectic effect of TMX was associated with hypothalamic accumulation of malonyl-CoA (Fig. 5A). This effect was specific for TMX since the hypothalamic malonyl-CoA content in pair-fed and fasted rats was decreased (Fig. 5A and B). Levels of hypothalamic malonyl-CoA recovered after refeeding for 24 or 48 h (Fig. 5B). We also studied if the inhibitory effect of TMX on refeeding (Fig. 1C) correlated with hypothalamic malonyl-CoA content. Refed rats subcutaneously treated with TMX showed a further increase in hypothalamic malonyl-CoA content when compared with refed rats treated with vehicle (Fig. 5C). These results strongly suggest that a rise in malonyl-CoA levels may mediate the inhibitory effect of TMX on refeeding.
Hypothalamic AMPK/ACC is not affected by TMX.
The control of malonyl-CoA synthesis is regulated to a large extent by ACC. The activity of ACC can in turn be controlled by AMPK (43). Therefore, parallel changes in FAS and AMPK/ACC could explain the presence of altered malonyl-CoA levels in our TMX-treated rats. However, our results showed no differences in the protein levels of AMPK1, AMPK2, or pAMPK (Fig. 6A and B). Similarly, no differences in AMPK1 and AMPK2 activities (Fig. 6D and E) or in ACC, ACC, and pACC levels (Fig. 6A and C) were detected. LKB1 activity was not affected by either TMX or pair-feeding (Fig. 6F). These data suggest that TMX selectively inhibits fatty acid synthesis at the level of FAS activity.
Intracerebroventricular administration of an ACC inhibitor reverses the anorectic effect of subcutaneous TMX treatment by restoring hypothalamic malonyl-CoA levels.
To establish a mechanistic link between the effects of TMX, inhibition of FAS, accumulation of malonyl-CoA, and anorectic effects, we investigated the effect of TOFA, an inhibitor of ACC. Intracerebroventricular administration of TOFA completely prevented the anorectic effect of subcutaneous TMX on food intake during a 24-h refeeding experiment (Fig. 7A). This dose of TOFA, when administered alone, did not have any effect on feeding, excluding the possibility of a direct orexigenic effect. Furthermore, administration of TOFA prevented a TMX-induced increase in malonyl-CoA, suggesting that alterations in malonyl-CoA levels mediate the anorectic effect of TMX (Fig. 7B).
TMX administration prevents weight gain in human obese patients.
The potential relevance of our data were assessed by reanalyzing data from the RMH trial (3). When subjects were stratified according to their body weight at the beginning of the treatment, we found that obese (BMI 30 kg/m2) women treated with TMX initially lost weight and remained significantly lighter than those receiving placebo at 3, 24, 48, and 72 months (Fig. 8).
DISCUSSION
TMX-treated rats experience anorexia and weight loss (5,6). However, the mechanism for this anorectic effect has not been identified. We have recently reported that TMX inhibits FAS gene expression and activity in rat liver (21). As pharmacological inhibition of hypothalamic FAS elicits an anorectic response through a malonyl-CoAeCdependent mechanism (13,19,20), we hypothesized that decreased food intake in response to TMX may be the result of FAS inhibition and hypothalamic malonyl-CoA accumulation. Here, we demonstrated that TMX administration decreases FAS mRNA expression, specifically in the VMN, despite FAS being globally expressed in the brain in addition to other hypothalamic nuclei, like the ARC or PVN. This nucleus specificity of TMX was further supported by c-fos expression data that show specific VMN-neuron activation in response to TMX treatment. These data represent the first evidence of a nucleus-specific regulation of hypothalamic FAS mRNA associated with changes in food intake. Our fasting-refeeding experiments further supported a specific role for VMN and FAS expression in the regulation of feeding, since mRNA levels were only reduced in the VMN during fasting. These observations suggest that FAS in the VMN may act as a nutritional sensor to modulate food intake.
Next, we investigated whether TMX induced changes in hypothalamic malonyl-CoA. Although the decrease in FAS mRNA was restricted to just one nucleus, changes in malonyl-CoA levels were detectable when we analyzed the whole hypothalamus. It may be possible that inhibition of FAS activity could be a more generalized effect than mRNA regulation; however, it is also possible that the changes in malonyl-CoA levels in the VMN are significant enough to be detectable when the whole hypothalamus is assayed.
If FAS expression within the VMN has a role in regulating food intake, then the fact that FAS mRNA levels decrease in response to both fasting and TMX treatment is apparently contradictory. The explanation for this apparent paradox is found by examining malonyl-CoA levels. We demonstrate that the anorectic effect induced by TMX is similar to that of C75 in that it requires the accumulation of hypothalamic malonyl-CoA (14). The anorectic effect of TMX was reverted by pharmacological inhibition of ACC that prevented accumulation of malonyl-CoA. Thus, specific inhibition of FAS activity by TMX in the presence of maintained ACC activity (see below) facilitates accumulation of malonyl-CoA. Conversely, during fasting the decrease in FAS mRNA expression is associated with decreased levels of malonyl-CoA, suggesting that early steps of the hypothalamic lipogenic pathway are physiologically inhibited during fasting. Supporting this argument are recent reports demonstrating that during fasting, activation of hypothalamic AMPK phosphorylates and inhibits ACC (44,45).
We also investigated the effects of TMX on other components of the fatty acid biosynthetic pathway. AMPK plays a critical role in the hypothalamic regulation of feeding through its effects on the lipogenic pathway and neuropeptides (44,45). In situations of energy demand such as fasting, AMPK is phosphorylated by LKB1 at Thr172 (and then activated), resulting in phosphorylation and subsequent inhibition of ACC, thereby inactivating the fatty acid biosynthetic pathway (43). Under conditions of energy surplus, ACC is not phosphorylated, facilitating production of malonyl-CoA (43,45). Our data showed that FAS inhibition induced by TMX is not associated with changes in either AMPK protein levels/activity or phosphorylated ACC levels. These data suggest that the hypothalamic AMPK/ACC axis is not affected by TMX. Hence, the accumulation of malonyl-CoA induced by TMX is the result of specific FAS inhibition in the presence of normal ACC activity (online appendix Fig. 5).
Food restriction is associated with reduction in the mRNA levels of CART and POMC in the ARC (7eC9,12). This expected reduction in CART and POMC levels was blocked by TMX. Furthermore, pharmacological inhibition of melanocortin-3/-4 receptors by SHU9119 prevented the TMX anorectic effects, indicating that inhibition of feeding by TMX involves the melanocortin system. CART/POMC neurons receive neural projections and excitatory inputs from neurons in the VMN (46,47); therefore, changes in FAS/malonyl-CoA in the VMN might be linked to CART/POMC changes in the ARC. However, this mechanism still needs to be clarified.
TMX is a drug widely used for the treatment of estrogen receptoreCpositive breast cancers (3,4). Despite its widespread use, the action of TMX on human body weight is controversial. Data from the WHEL (Women’s Healthy Eating and Living) clinical trial show that obese women with breast cancer under TMX treatment had a decreased BMI (48). However, measurement of BMI in this study occurred at varying times after breast cancer diagnosis and thus did not necessarily reflect prediagnosis BMI. The results of this study may also have been confounded by the concurrent diagnosis of cancer. Hence, we reanalyzed the body weight data from the RMH trial (3). Since these subjects were free of cancer during the study, changes in body weight are more likely to be a direct effect of TMX. We found that obese (BMI 30 kg/m2) women treated with TMX were significantly lighter than those receiving placebo. Although this is a post hoc analysis of the data, we suggest that the large size of the cohort studied strongly supports the finding that low-dose TMX can induce a sustainable body weight decrease in obese humans. Although the RMH trial was not an obesity prevention/treatment trial, the finding that the obese women lost weight initially for 4 years and on average had maintained their initial weight by the end of the study is a highly significant deviation from the expected (49,50).
In conclusion, this study demonstrates that the anorectic actions of TMX involve a specific decrease in FAS mRNA levels in the VMN and hypothalamic malonyl-CoA accumulation. Specific regulation of FAS in the VMN by TMX or fasting suggests that fatty acid metabolism in this nucleus may play a physiological role in feeding control. Altogether, our data indicate that altering FAS in the VMN may be a suitable strategy for altering food intake and body weight homeostasis in experimental animals and obese subjects.
ACKNOWLEDGMENTS
This work has been supported by grants from the Medical Research Council (to A.J.V.-P.), the Welcome Trust (to A.J.V.-P.), the Spanish Ministry of Education (to C.D.), the Xunta de Galicia (to C.D.), and the European Union (LSHM-CT-2003-503041) (to C.D.). A.K.S. is funded by the U.S. Public Health Service (DK 19514) and a grant from the Juvenile Diabetes Research Foundation. M.L. is funded by the Marie Curie Program (QLK6-CT-2002-51671).
We are grateful to Keith Burling, Mark Campbell, and Janice Carter for their technical assistance, to Dr. Anthony P. Coll (University of Cambridge, Cambridge, U.K.), to Dr. Benjamin Challis (University of Cambridge, Cambridge, U.K.), to Prof. Manuel Ros (University Rey Juan Carlos, Madrid, Spain), and to Prof. Manuel Tena-Sempere (University of Cordoba, Cordoba, Spain) for their useful comments and criticism. We thank Prof. Len Storlien (AstraZeneca, Mondal, Sweden) for supplying us TOFA and Prof. Dario Alessi (University of Dundee, Dundee, U.K.) for the LKB1 antibody.
FOOTNOTES
M.L. and C.J.L. contributed equally to this work.
Additional information for this article can be found in an online appendix at http://diabetes.diabetesjournals.org.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
REFERENCES
Wirshing DA: Schizophrenia and obesity: impact of antipsychotic medications. J Clin Psychiatry 65 (Suppl. 18):13eC26, 2004
Korner J, Aronne LJ: Pharmacological approaches to weight reduction: therapeutic targets. J Clin Endocrinol Metab 89:2616eC2621, 2004
Powles T, Eeles R, Ashley S, Easton D, Chang J, Dowsett M, Tidy A, Viggers J, Davey J: Interim analysis of the incidence of breast cancer in the Royal Marsden Hospital tamoxifen randomised chemoprevention trial. Lancet 352:98eC101, 1998
Jordan VC: Selective estrogen receptor modulation: concept and consequences in cancer. Cancer Cell 5:207eC213, 2004
Wade GN, Heller HW: Tamoxifen mimics the effects of estradiol on food intake, body weight, and body composition in rats. Am J Physiol 264:R1219eCR1223, 1993
Gray JM, Schrock S, Bishop M: Estrogens and antiestrogens: actions and interactions with fluphenazine on food intake and body weight in rats. Am J Physiol 264:R1214eCR1218, 1993
Meister B: Control of food intake via leptin receptors in the hypothalamus. Vitam Horm 59:265eC304, 2000
Schwartz MW, Woods SC, Porte D Jr, Seeley RJ, Baskin DG: Central nervous system control of food intake. Nature 404:661eC671, 2000
Saper CB, Chou TC, Elmquist JK: The need to feed: homeostatic and hedonic control of eating. Neuron 36:199eC211, 2002
Obici S, Feng Z, Morgan K, Stein D, Karkanias G, Rossetti L: Central administration of oleic acid inhibits glucose production and food intake. Diabetes 51:271eC275, 2002
Cota D, Marsicano G, Lutz B, Vicennati V, Stalla GK, Pasquali R, Pagotto U: Endogenous cannabinoid system as a modulator of food intake. Int J Obes Relat Metab Disord 27:289eC301, 2003
Flier JS: Obesity wars: molecular progress confronts an expanding epidemic. Cell 116:337eC350, 2004
Gao S, Lane MD: Effect of the anorectic fatty acid synthase inhibitor C75 on neuronal activity in the hypothalamus and brainstem. Proc Natl Acad Sci U S A 100:5628eC5633, 2003
Hu Z, Cha SH, Chohnan S, Lane MD: Hypothalamic malonyl-CoA as a mediator of feeding behavior. Proc Natl Acad Sci U S A 100:12624eC12629, 2003
Ruderman NB, Saha AK, Kraegen EW: Minireview: malonyl CoA, AMP-activated protein kinase, and adiposity. Endocrinology 144:5166eC5171, 2003
Lam TK, Schwartz GJ, Rossetti L: Hypothalamic sensing of fatty acids. Nat Neurosci 8:579eC584, 2005
Kim EK, Miller I, Landree LE, Borisy-Rudin FF, Brown P, Tihan T, Townsend CA, Witters LA, Moran TH, Kuhajda FP, Ronnett GV: Expression of FAS within hypothalamic neurons: a model for decreased food intake after C75 treatment. Am J Physiol Endocrinol Metab 283:E867eCE879, 2002
Sorensen A, Travers MT, Vernon RG, Price NT, Barber MC: Localization of messenger RNAs encoding enzymes associated with malonyl-CoA metabolism in mouse brain. Brain Res Gene Expr Patterns 1:167eC173, 2002
Loftus TM, Jaworsky DE, Frehywot GL, Townsend CA, Ronnett GV, Lane MD, Kuhajda FP: Reduced food intake and body weight in mice treated with fatty acid synthase inhibitors. Science 288:2379eC2381, 2000
Clegg DJ, Wortman MD, Benoit SC, McOsker CC, Seeley RJ: Comparison of central and peripheral administration of C75 on food intake, body weight, and conditioned taste aversion. Diabetes 51:3196eC3201, 2002
Lelliott CJ, Le畃ez M, Curtis RK, Parker N, Laudes M, Yeo G, Jimeenez-Lian M, Grosse J, Saha AK, Wiggins D, Hauton D, Brand MD, O’Rahilly S, Griffin JL, Gibbons GF, Vidal-Puig A: Transcript and metabolite analysis of the effects of tamoxifen in rat liver reveals inhibition of fatty acid synthesis in the presence of hepatic steatosis. FASEB J 19:1108eC1119, 2005
Mystkowski P, Seeley RJ, Hahn TM, Baskin DG, Havel PJ, Matsumoto AM, Wilkinson CW, Peacock-Kinzig K, Blake KA, Schwartz MW: Hypothalamic melanin-concentrating hormone and estrogen-induced weight loss. J Neurosci 20:8637eC8642, 2000
Tritos NA, Segal-Lieberman G, Vezeridis PS, Maratos-Flier E: Estradiol-induced anorexia is independent of leptin and melanin-concentrating hormone. Obes Res 12:716eC724, 2004
Le畃ez M, Seoane LM, Tovar S, Nogueiras R, Dieeguez C, Seare猻 R: Orexin-A regulates growth hormone releasing hormone mRNA content in a nucleus specific manner and somatostatin mRNA content in a growth hormone-dependent fashion in the rat hypothalamus. Eur J Neurosci 19:2080eC2088, 2004
Liang YQ, Akishita M, Kim S, Ako J, Hashimoto M, Iijima K, Ohike Y, Watanabe T, Sudoh N, Toba K, Yoshizumi M, Ouchi Y: Estrogen receptor beta is involved in the anorectic action of estrogen. Int J Obes Relat Metab Disord 26:1103eC1109, 2002
Kelly JF, Elias CF, Lee CE, Ahima RS, Seeley RJ, Bjorbaek C, Oka T, Saper CB, Flier JS, Elmquist JK: Ciliary neurotrophic factor and leptin induce distinct patterns of immediate early gene expression in the brain. Diabetes 53:911eC920, 2004
Halatchev IG, Cone RD: Peripheral administration of PYY3-36 produces conditioned taste aversion in mice. Cell Metab 1:159eC168, 2005
Kask A, Mutulis F, Muceniece R, Pahkla R, Mutule I, Wikberg JE, Rago L, Schioth HB: Discovery of a novel superpotent and selective melanocortin-4 receptor antagonist (HS024): evaluation in vitro and in vivo. Endocrinology 139:5006eC5014, 1998
Heisler LK, Cowley MA, Tecott LH, Fan W, Low MJ, Smart JL, Rubinstein M, Tatro JB, Marcus JN, Holstege H, Lee CE, Cone RD, Elmquist JK: Activation of central melanocortin pathways by fenfluramine. Science 297:609eC611, 2002
Medina-Ge甿ez G, Virtue S, Lelliott C, Boiani R, Campbell M, Christodoulides C, Perrin C, Jimeenez-Lian M, Blount M, Dixon J, Zahn D, Thresher RR, Aparicio S, Carlton M, Colledge WH, Kettunen MI, Seppanen-Laakso T, Sethi JK, O’Rahilly S, Brindle K, Cinti S, Oresic M, Burcelin R, Vidal-Puig A: The link between nutritional status and insulin sensitivity is dependent on the adipocyte-specific peroxisome proliferator-activated receptor-2 isoform. Diabetes 54:1706eC1716, 2005
Coll AP, Challis BG, Le畃ez M, Piper S, Yeo GS, O’Rahilly S: Proopiomelanocortin-deficient mice are hypersensitive to the adverse metabolic effects of glucocorticoids. Diabetes 54:2269eC2276, 2005
Le畃ez M, Seoane LM, Tovar S, Garce MC, Nogueiras R, Dieeguez C, Seare猻 RM: A possible role of neuropeptide Y, agouti-related protein and leptin receptor isoforms in hypothalamic programming by perinatal feeding in the rat. Diabetologia 48:140eC148, 2005
Nogueiras R, Gallego R, Gualillo O, Caminos JE, Garce-Caballero T, Casanueva FF, Dieeguez C: Resistin is expressed in different rat tissues and is regulated in a tissue- and gender-specific manner. FEBS Lett 548:21eC27, 2003
Sakamoto K, Goransson O, Hardie DG, Alessi DR: Activity of LKB1 and AMPK-related kinases in skeletal muscle: effects of contraction, phenformin, and AICAR. Am J Physiol Endocrinol Metab 287:E310eCE317, 2004
Dale S, Wilson WA, Edelman AM, Hardie DG: Similar substrate recognition motifs for mammalian AMP-activated protein kinase, higher plant HMG-CoA reductase kinase-A, yeast SNF1, and mammalian calmodulin-dependent protein kinase I. FEBS Lett 361:191eC195, 1995
Lizcano JM, Goransson O, Toth R, Deak M, Morrice NA, Boudeau J, Hawley SA, Udd L, Makela TP, Hardie DG, Alessi DR: LKB1 is a master kinase that activates 13 kinases of the AMPK subfamily, including MARK/PAR-1. EMBO J 23:833eC843, 2004
McGarry JD, Stark MJ, Foster DW: Hepatic malonyl-CoA levels of fed, fasted and diabetic rats as measured using a simple radioisotopic assay. J Biol Chem 253:8291eC8293, 1978
Saha AK, Kurowski TG, Ruderman NB: A malonyl-CoA fuel-sensing mechanism in muscle: effects of insulin, glucose, and denervation. Am J Physiol 269:E283eCE289, 1995
Gold E, Stapley S, Goulding A: Tamoxifen and norethisterone: effects on plasma cholesterol and total body calcium content in the estrogen-deficient rat. Horm Metab Res 26:100eC103, 1994
Patisaul HB, Aultman EA, Bielsky IF, Young LJ, Wilson ME: Immediate and residual effects of tamoxifen and ethynylestradiol in the female rat hypothalamus. Brain Res 978:185eC193, 2003
Tena-Sempere M, Navarro VM, Mayen A, Bellido C, Sanchez-Criado JE: Regulation of estrogen receptor (ER) isoform messenger RNA expression by different ER ligands in female rat pituitary. Biol Reprod 70:671eC678, 2004
Kim MS, Park JY, Namkoong C, Jang PG, Ryu JW, Song HS, Yun JY, Namgoong IS, Ha J, Park IS, Lee IK, Viollet B, Youn JH, Lee HK, Lee KU: Anti-obesity effects of alpha-lipoic acid mediated by suppression of hypothalamic AMP-activated protein kinase. Nat Med 10:727eC733, 2004
Kahn BB, Alquier T, Carling D, Hardie DG: AMP-activated protein kinase: ancient energy gauge provides clues to modern understanding of metabolism. Cell Metab 1:15eC25, 2005
Minokoshi Y, Alquier T, Furukawa N, Kim YB, Lee A, Xue B, Mu J, Foufelle F, Ferre P, Birnbaum MJ, Stuck BJ, Kahn BB: AMP-kinase regulates food intake by responding to hormonal and nutrient signals in the hypothalamus. Nature 428:569eC574, 2004
Andersson U, Filipsson K, Abbott CR, Woods A, Smith K, Bloom SR, Carling D, Small CJ: AMP-activated protein kinase plays a role in the control of food intake. J Biol Chem 279:12005eC12008, 2004
Pinto S, Roseberry AG, Liu H, Diano S, Shanabrough M, Cai X, Friedman JM, Horvath TL: Rapid rewiring of ARC nucleus feeding circuits by leptin. Science 304:110eC115, 2004
Sternson SM, Shepherd GM, Friedman JM: Topographic mapping of VMH ARC nucleus microcircuits and their reorganization by fasting. Nat Neurosci 8:1356eC1363, 2005
Wasserman L, Flatt SW, Natarajan L, Laughlin G, Matusalem M, Faerber S, Rock CL, Barrett-Connor E, Pierce JP: Correlates of obesity in postmenopausal women with breast cancer: comparison of genetic, demographic, disease-related, life history and dietary factors. Int J Obes Relat Metab Disord 28:49eC56, 2004
Finer N: Pharmacotherapy of obesity. Best Pract Res Clin Endocrinol Metab 16:717eC742, 2002
Li Z, Maglione M, Tu W, Mojica W, Arterburn D, Shugarman LR, Hilton L, Suttorp M, Solomon V, Shekelle PG, Morton SC: Meta-analysis: pharmacologic treatment of obesity. Ann Intern Med 142:532eC546, 2005(Miguel Leez, Christopher )