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APPETITE, OBESITY, DIGESTION, AND METABOLISM

Pharmacological stimulation of brain carnitine palmitoyl-transferase-1 decreases food intake and body weight

Departments of ¹Psychiatry and Behavioral Sciences, ²Neuroscience, and ⁵Pathology, Johns Hopkins University School of Medicine, Baltimore; ³FASgen, Baltimore; and ⁴Department of Chemistry, Johns Hopkins University, Baltimore, Maryland

Submitted 11 December 2006 ; accepted in final form 4 December 2007

	ABSTRACT

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Inhibition of brain carnitine palmitoyl-transferase-1 (CPT-1) is reported to decrease food intake and body weight in rats. Yet, the fatty acid synthase (FAS) inhibitor and CPT-1 stimulator C75 produces hypophagia and weight loss when given to rodents intracerebroventricularly (icv). Thus roles and relative contributions of altered brain CPT-1 activity and fatty acid oxidation in these phenomena remain unclarified. We administered compounds that target FAS or CPT-1 to mice by single icv bolus and examined acute and prolonged effects on feeding and body weight. C75 decreased food intake rapidly and potently at all doses (1–56 nmol) and dose dependently inhibited intake on day 1. Dose-dependent weight loss on day 1 persisted through 4 days of postinjection monitoring. The FAS inhibitor cerulenin produced dose-dependent (560 nmol) hypophagia for 1 day, weight loss for 2 days, and weight regain to vehicle control by day 3. The CPT-1 inhibitor etomoxir (32, 320 nmol) did not alter overall day 1 feeding. However, etomoxir attenuated the hypophagia produced by C75, indicating that CPT-1 stimulation is important for C75's effect. A novel compound, C89b, was characterized in vitro as a selective stimulator of CPT-1 that does not affect fatty acid synthesis. C89b (100, 320 nmol) decreased feeding in mice for 3 days and produced persistent weight loss for 6 days without producing conditioned taste aversion. Similarly, intraperitoneal administration decreased feeding and body weight without producing conditioned taste aversion. These results suggest a role for brain CPT-1 in the regulation of energy balance and implicate CPT-1 stimulation as a pharmacological approach to weight loss.

central nervous system; fatty acid metabolism; energy intake; AMP-activated protein kinase

Intracellular mechanisms involved in the hypophagia and weight-loss responses to C75 and other molecules that alter fatty acid metabolic flux have been controversial. Of critical concern is the consequence of altered CPT-1 activity, particularly in brain, on feeding behavior and weight loss. C75 was originally thought to inhibit feeding by increasing the intracellular concentration of the FAS substrate malonyl-CoA, which inhibits CPT-1 (28). This would lead to elevated cytosolic concentrations of long-chain fatty acids and diacylglycerol, proposed to signal an energy surplus (42). However, the demonstration that C75 stimulated CPT-1 directly, and increased fatty acid oxidation (47), required the consideration of other mechanisms. C75 increased intracellular ATP in vitro (23) (25) (47), an outcome expected with increased beta-oxidation. An alternative or additional source of ATP might come from halting fatty acid synthesis (25).

In the present studies, we determined the relative contributions of brain FAS inhibition and CPT-1 stimulation to alterations in food intake and body weight. We used a pharmacological approach, employing established compounds as well as a novel CPT-1 stimulator, to reveal the effects of dynamic changes in metabolic flux.

	MATERIALS AND METHODS

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Animal preparation for food intake experiments. Male C57BL/6J mice (6- to 8-wk-old, Jackson Laboratories, Bar Harbor, ME) were housed individually (22 ± 2°C, 12:12-h light-dark cycle), weighed daily, and given ad libitum access to water and 1-g grain-based food pellets (FO173, BioServ). Mice were adapted to these conditions for 1 wk before undergoing cannulation of the lateral cerebroventricle.

Lateral cerebroventricle cannulas. Mice were anesthetized with ketamine-HCl (100 mg/kg), xylazine (20 mg/kg), and acepromazine maleate (3 mg/kg) given intraperitoneally (ip) and positioned in a stereotaxic instrument with incisor bar adjusted to achieve level-skull position. A hole drilled into the skull 0.6 mm caudal to bregma and 1.2 mm lateral to midline accommodated a 23-gauge stainless steel cannula, lowered to 2.2 mm below skull surface. Exposed skull surface was etched, coated with super-glue, and etched again. The cannula was secured using dental cement adhered to the etched surface. A 30-gauge stainless steel obturator maintained cannula patency. Mice received ip banamine (0.25 mg/100 g) for analgesia and penicillin (10,000 U) to prevent infection.

Icv injections were performed with a microliter syringe (Hamilton) attached to PE-10 tubing and a 30-gauge stainless steel injector, the tip of which extended 1.5 mm past the cannula into the lateral ventricle.

After 1 wk of postsurgical recovery, cannula placements were assessed by measuring food intake after icv neuropeptide Y (NPY; 0.25 nmol; American Peptide) vs. sterile 0.9% saline (2 µl) during a 1-h food access during the light. Only mice eating at least 0.5 g after NPY were used in experiments, which commenced 3–5 days after the cannula placement tests. When the behavioral studies concluded, cannulas were tested again in these mice. We analyzed data only from the mice with functional cannulas.

Compounds and vehicles for in vivo studies. For icv administrations, the combined FAS inhibitor and CPT-1 stimulator C75 [molecular weight (MW) = 254.15; 0–56 nmol] was given in 2 µl of 1x RPMI-1640 (no. 11879, Invitrogen). Other compounds required lipophilic vehicles, diluted as much as possible with aqueous solutions, to prevent long-lasting vehicle effects on food intake while maintaining the compounds in solution. The natural product FAS inhibitor cerulenin (MW = 223.3; 0–560 nmol) was given in 4 µl of 25% DMSO/75% RPMI. The CPT-1 inhibitor etomoxir (MW = 320.75; 0–320 nmol) was also given in 4 µl of 25% DMSO/75% RPMI. The selective CPT-1 stimulator C89b (MW = 322.2; 0–320 nmol) was given in 2 µl of 25% TEP/75% saline (TEP = 10% Tween-80, 10% ethanol, 80% polyethylene glycol). In other experiments, ip injections of compounds were given in 50 µl of 10% TEP/90% saline (testing the high icv doses ip), or 50 µl of 100% TEP (testing C89b at effective ip doses). FASgen provided C75 and C89b. Etomoxir was purchased from HPO Wolfe, Projekt-Entwicklung (Konstanz, Germany). Cerulenin was purchased from Sigma (St. Louis, MO).

Food intake and body weight experiments. For each feeding experiment, mice had ad libitum access to water and 22-h access to chow beginning at the onset of dark, permitting 2 h for preparations between trials. For icv experiments, mice received single injections of vehicles or compounds in half-log- and quarter-log-step doses dissolved in vehicles 30 min before lights out. We measured intakes of chow, corrected for spillage, at 0.5, 1, 2, 4, and 22 h on the first day, and 0- to 22-h intakes on subsequent days. Body weight was measured daily. Injections were given at one dose per week.

Conditioned taste aversion tests. Female BALB/c mice (6- to 8-wk-old, Jackson Laboratories, Bar Harbor, ME) or, in separate experiments, male C57BL/6J mice were trained for 2 wk to scheduled, daily, 2-h water access during the light. Trained mice were given a novel 0.15% saccharin solution to drink for the first 30 min of fluid access. The female mice were then injected ip with C89b (30 mg/kg, 93 µmol/kg) or 50 µl of 10% TEP vehicle. In different studies, male mice were injected icv with C89b (100 nmol) or 2 µl of 25% TEP vehicle. Injected mice were then given water for the remaining 90 min. The next day, mice were allowed to choose between water and 0.15% saccharin for 30 min. The data were expressed as % saccharin-preference {100 x [saccharin intake/(saccharin intake + water intake)]}.

MCF-7 cell line culture. MCF-7 cells were maintained in RPMI (no. 11875, Invitrogen) with 10% fetal bovine serum. Before assays, cells were incubated overnight at 37°C in 24-well plates at densities of 5 x 10⁴ cells/cm² for measuring acetate incorporation into fatty acids; 1 x 10⁶ cells/cm² for measuring CPT-1 activity; 2.5 x 10⁵ cells/cm² for measuring fatty acid oxidation.

Primary hypothalamic neuronal culture. E17 pups were harvested from euthanized, timed-pregnant Sprague-Dawley dams (Harlan, Indianapolis, IN). Hypothalami were removed and dissociated using a papain kit (Worthington Biochemical, Lakewood, NJ). Cells were plated on poly-D-lysine coated Corning Costar 24-well plates at 1 x 10⁶ cells/well in Neurobasal media (Gibco, no. 10888; Invitrogen) supplemented with B27 (2%), glutamine (2 mM), penicillin, and streptomycin. Cultures were grown in a sterile incubator (37°C, 95% O₂/5% CO₂) and maintained with 50% media changes on day 3 (with 1 µM cytosine arabinoside to inhibit glial proliferation) and day 6 (without cytosine arabinoside). Assays were performed on day 8. Drug treatments were performed with C89b resuspended in 100% DMSO or DMSO vehicle with a final well concentration of 0.1% or 0.25% DMSO.

Measurement of acetate incorporation. MCF-7 cells or hypothalamic neurons were pretreated with DMSO vehicle (0.25% final concentration) or C89b (10, 20, 40, 60, 80 µg/ml) for 15 min (neurons) or 4 h (MCF-7 cells) in conditioned media (RPMI or Neurobasal), then labeled with 100 µM [¹⁴C]acetate (PerkinElmer Life and Analytical Sciences, Wellesley, MA) for an additional 2 h, similar to previous protocols (39). C75 was a positive control to decrease fatty acid synthesis. Lipids were extracted with chloroform/methanol, dried under N₂, and counted by liquid scintillation.

Measurement of CPT-1 activity. CPT-1 activity was measured using digitonin permeabilization (45, 47). C89b (5, 10, 20, 40, 80 µg/ml) or vehicle was added to cultures in conditioned media (RPMI or Neurobasal). C75 was a positive control to increase CPT-1 activity. Medium was removed after 2 h (MCF-7 cells) or 1.5 h (neurons) and cells were washed with PBS. Cells were then incubated at 37°C with 700 µl of assay medium (50 mM imidazole, 70 mM KCL, 80 mM sucrose, 1 mM EGTA, 2 mM MgCl₂, 1 mM dithiothreitol, 1 mM KCN, 1 mM ATP, 0.1% fatty acid free bovine serum albumin, 70 µM palmitoyl-CoA, 0.25 µCi L-[methyl-¹⁴C]carnitine (GE Healthcare BioSciences, Piscataway, NJ) and 40 µg of digitonin, with or without 50 µM malonyl-CoA for positive control to decrease CPT-1 activity). After 6 min, the reaction was stopped with 500 µl of ice-cold 4 M perchloric acid. Cells were harvested, centrifuged (13,000 g, 5 min), washed with 500 µl of ice-cold 2 mM perchloric acid, and centrifuged again. The pellet was resuspended in 800 µl of distilled H₂O and extracted with 400 µl of butyl alcohol. The butyl alcohol phase, representing the acylcarnitine derivative, was measured by liquid scintillation.

Measurement of fatty acid oxidation. Fatty acid oxidation was measured as described previously (50) with modifications. MCF-7 cells were plated at 2.5 x 10⁵ cells/cm² in 24-well plates. Cells were treated in triplicate with C89b (0.6, 1.3, 3, 5, 10, 20, 40, 80 µg/ml) for 1.5 h in RPMI. C75 and etomoxir were positive controls to increase and decrease fatty acid oxidation. The media was changed to RPMI with compounds, plus carnitine (200 µM) and [¹⁴C]palmitate [100 uM, Moravek Biochemicals, Brea, CA; suspended in alpha-cyclodextran (10 mg/ml in 10 mM Tris)], and incubated an additional 0.5 h. The reaction was stopped with 2.6 N HClO₄ (50 µl/well). Well contents were transferred to tubes, hydrolyzed with 50 µl of 4 N KOH at 60°C for 1 h, and neutralized with H₂SO₄. Water-soluble products were extracted using chloroform/methanol and H₂O and quantified by liquid scintillation. We have found that measuring ¹⁴CO₂ yields negligible results in this cell system.

Cell viability assay. Hypothalamic neurons were treated with C89b or vehicle for 2 h. Cell viability was determined using calcein AM (Molecular Probes, Eugene, OR). Conversion of the cell permeant nonfluorescent calcein AM to intensely fluorescent calcein is catalyzed by intracellular esterase activity in live cells and is measured by detecting fluorescence at 485 nm/535 nm using a PerkinElmer Victor² 1420 plate reader.

Statistical analyses and protocol review. Data are reported as means with SE. Data from in vivo experiments with icv injections of C75, cerulenin, C89b, or etomoxir were analyzed by one-way repeated-measures ANOVA. Data from the in vivo experiment with icv C75 and etomoxir were analyzed by two-way repeated-measures ANOVA. These ANOVAs, when they yielded significant overall effects, were followed by Fisher's least squared means tests for group comparisons. Data from the in vivo experiments with ip compounds were analyzed with paired two-tailed t-tests of compounds vs. vehicle control. Data from all in vitro concentration-response experiments with C89b were analyzed by one-way ANOVA, with Dunnett's test to compare treatments with control. Positive controls of C75 or malonyl-CoA were included to validate the assays and were compared with vehicle controls by unpaired one-tailed t-tests. For all tests, P 0.05 indicated significance. All experiments involving animals or their tissues were conducted according to guidelines on animal care and use established by the Johns Hopkins University institutional animal care and use committee.

	RESULTS

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Central nervous system (CNS) administration of C75 decreases food intake and body weight. We first established dose response curves and time courses for the effects of central C75 administration on body weight and ad libitum food intake in mice in order to compare C75's effects with responses to other compounds. C75 potently inhibited food intake and produced long-lasting weight reduction in mice (Fig. 1). C75 reduced food intake rapidly (Fig. 1A), consistent with other mouse studies (13, 21, 28). By 0.5 h, all doses of C75 (1–56 nmol) reduced chow intake to 30% of vehicle control (P < 0.01). At 4 h, all doses suppressed feeding to 65% of control (P < 0.05). By the end of day 1, the effect of C75 on feeding was dose dependent (Fig. 1B; 56 nmol, 65% of vehicle control, P = 0.001), as was the weight loss (Fig. 1C; +0.7 ± 0.5 g after vehicle; 10 nmol, –0.2 ± 0.3 g, P = 0.034; 56 nmol, –1.0 ± 0.2 g, P < 0.001). The feeding and weight changes following a single icv administration of C75 were remarkable because there was no rebound hyperphagia, and weight losses initiated on day 1 were sustained for many days. With this requisite characterization of mouse responses to C75, a combined FAS inhibitor and CPT-1 stimulator, we were positioned to examine the relative contributions of selective FAS inhibition or CPT-1 manipulation.

View larger version (21K): [in this window] [in a new window]   Fig. 1. The combined FAS inhibitor and carnitine palmitoyl-transferase-1 (CPT-1) stimulator C75 reduces food intake and body weight. A: single intracerebroventricular (icv) injections of C75 to mice reduced cumulative food intakes at very early time points during day 1. B: the C75-induced hypophagia was significant and dose dependent by the end of day 1. Subsequent days showed normal levels of food intake. C: weight loss after icv C75 was dose dependent on day 1. Weight losses were maintained relative to vehicle control on subsequent days. For all panels, n = 7. *P < 0.05 vs. 0-nmol control.

View larger version (20K): [in this window] [in a new window]   Fig. 2. Central nervous system (CNS) administration of the FAS inhibitor cerulenin reduces food intake and body weight. A: single icv injections of cerulenin to mice produced dose-dependent reductions in food intake at early time points, significant at 2 and 4 h. B: a high dose of cerulenin (560 nmol) reduced food intake on day 1, followed by slight hyperphagia on days 2–4. C: weight loss was significant on days 1 and 2 after this high dose of cerulenin but was regained by day 3. For all panels, n = 8. *P < 0.05 vs. 0-nmol control.

Etomoxir given icv increased food intake at 0.5 and 1 h (Fig. 3A), after which etomoxir had no effect on feeding during day 1 (Fig. 3, A and B). Mice lost weight on day 1 following treatment with 320 nmol of etomoxir (–1.0 ± 0.4 g, P = 0.025) (Fig. 3C). This was followed by both persistent hyperphagia (Fig. 3B) and significant weight gain over the next 6 days (Fig. 3C) that might be compensatory responses. Rat studies have either reported decreased weight after acute icv administration of CPT-1 inhibitors (41) or no weight change despite hypophagia after chronic icv infusion (8).

View larger version (22K): [in this window] [in a new window]   Fig. 3. Effects of CNS administration of the CPT-1 inhibitor etomoxir in mice. A: a single injection of etomoxir could increase food intake at very early time points on day 1. B: there was no overall change in food intake on day 1 or 2 after etomoxir, although subsequent days 3–7 showed hyperphagia. C: although a high dose of etomoxir did not affect cumulative food intake on day 1 after injection, it did reduce body weight. Subsequent days showed a return to, then surpassing of preinjection body weight. For all panels, n = 11. *P < 0.05 vs. 0-nmol control.

View larger version (17K): [in this window] [in a new window]   Fig. 4. Pretreatment with the CPT-1 inhibitor etomoxir (ETO) prevents the full expression of hypophagia after C75. A: at 30 min, C75 reduced food intake relative to vehicle control and reduced food intake when given after ETO pretreatment, compared with ETO alone. At 1, 2, and 4 h, C75 still reduced food intake compared with vehicle control. However, C75 did not significantly reduce food intake when given after ETO, compared with ETO alone. B: ETO's ability to attenuate C75-induced hypophagia remained evident by the end of day 1. C: decreases in body weight after C75 with ETO pretreatment did not differ from those after C75 alone (n = 4). For A and B, n = 5. *P < 0.05 comparing C75 vs. double-vehicle control and comparing etomoxir + C75 vs. etomoxir alone.

View larger version (19K): [in this window] [in a new window]   Fig. 5. In vitro characterization of fatty acid metabolism after C89b, a novel CPT-1 stimulator. A: C89b across a broad concentration range did not alter levels of fatty acid synthesis in MCF-7 cells, as measured by uptake of 14C-acetate into fatty acids. B: C89b increases the activity of CPT-1, as indicated by a concentration-dependent increase in 14C-carnitine-palmitate in assays on digitonin permeabilized MCF-7 cells. C: C89b increases beta-oxidation of 14C-palmitate in MCF-7 cells, as measured by radiolabeled water soluble product. D: as in MCF-7 cells, C89b did not significantly alter uptake of 14C-acetate into fatty acids in hypothalamic neuronal cultures. E: C89b increases the activity of CPT-1 in a concentration-dependent manner in hypothalamic neuronal cultures. In A and B, n = 3 per condition. In C and D, n = 6 per condition. In E, C75 and malonyl-CoA, n = 12; concentrations: 0 µg/ml, n = 11; 5–40 µg/ml, n = 8; 80 µg/ml, n = 4. Molar equivalents of the concentrations (µg/ml: µM) for C75 = 20: 78.7, 40: 157.4; for C89b = 0.6: 1.9, 1.3: 4.0, 2.5: 7.8, 3: 9.3, 5: 15.5, 10: 31.0, 20: 62.1, 40: 124.2, 60: 186.2, 80: 248.3, 160: 496.6, 320: 993.1. *P < 0.05 vs. 0 µg/ml control (Dunnett's test). #P < 0.05 vs. 0 µg/ml control (unpaired one-tailed t-test).

Stimulation of CPT-1 in the CNS with C89b produces persistent hypophagia and weight loss. C89b administered to mice icv was 100 times less potent than C75 to suppress feeding, despite the compounds' similar potencies to stimulate CPT-1 in vitro (Fig. 5, B, C, and E). By the end of day 1, the higher doses of C89b (100 and 320 nmol) decreased food intake to 45% of vehicle control (P < 0.001) (Fig. 6B). Hypophagia persisted through day 2 after these doses (73% of control, P < 0.05) and day 3 after the high dose (84% of control, P = 0.007) (Fig. 6B). Thus hypophagia after C89b lasted longer than that seen after combined FAS inhibition and CPT-1 stimulation with C75. Unlike the FAS inhibitor cerulenin (Fig. 2B), the CPT-1 stimulator C89b did not produce rebound hyperphagia. Consistent with C89b's ability to reduce feeding, both 100 and 320 nmol caused weight loss on day 1 compared with control (vehicle, gain of +0.3 ± 0.0 g; 100 nmol, –1.6 ± 0.5 g, P = 0.002; 320 nmol, –1.4 ± 0.6 g, P = 0.004) (Fig. 6C). Compared with control, these weight losses were maintained for at least 6 days after a single bolus injection of C89b (Fig. 6C). The decrease in food intake after icv C89b was unlikely to be caused by malaise, because C89b at 100 nmol failed to produce a conditioned taste aversion to saccharin in a two-bottle test (Fig. 7).

View larger version (25K): [in this window] [in a new window]   Fig. 6. The CPT-1 stimulator C89b produces persistent hypophagia and weight loss. A: C89b begins to produce significant reductions in food intake in mice by 4 h after icv administration. B: a single icv injection of C89b to mice at a dose of either 100 or 320 nmol reduced cumulative food intake on day 1. The hypophagia remained significant for 2–3 days. C: weight losses elicited by C89b on day 1 were very long lasting, remaining significant compared with vehicle control even at 6 days after a single bolus administration. For all panels, n = 7. *P < 0.05 vs. 0-nmol control.

View larger version (9K): [in this window] [in a new window]   Fig. 7. CNS administration of the CPT-1 stimulator C89b does not produce conditioned taste aversion. Male C57BL/6J mice injected icv with C89b at 100 nmol did not exhibit conditioned taste aversion to a novel saccharin solution in a two-bottle test (vehicle controls, n = 7; C89b, n = 6).

View larger version (16K): [in this window] [in a new window]   Fig. 8. Systemic administrations of compounds at the highest concentrations from icv experiments do not produce major effects on food intake and body weight. A: single ip injections of compounds at the highest doses from icv experiments [C75 (56 nmol), cerulenin (560 nmol), C89b (320 nmol), and etomoxir (320 nmol)] had no effect on cumulative chow intakes at 2, 4, and 22 h vs. vehicle control (50 µl of 10% TEP, 90% saline vehicle; TEP = 80% polyethylene glycol, 10% ethanol, 10% Tween-80). B: these doses of compounds, given ip, did not significantly alter body weight compared with vehicle control.

View larger version (9K): [in this window] [in a new window]   Fig. 9. Systemic administration of the CPT-1 stimulator C89b reduces food intake and body weight without producing sickness behavior. A: female BALB/c mice (6- to 8-wk-old) on ad libitum chow and water were injected ip with either C89b (15, 30, 60 mg/kg; 46.5, 93, 186, µmol/kg) or vehicle (50 µl of TEP; TEP = 80% polyethylene glycol, 10% ethanol, 10% Tween-80) (n = 3 per group). All ip doses of C89b reduced body weight comparably compared with vehicle control (unpaired Student's t-tests vs. vehicle: 15 mg/kg, P = 0.075; 30 mg/kg, P = 0.056; 60 mg/kg, P = 0.009). Male C57BL/6J mice were given C89b at 30 mg/kg ip. This dose decreased food intake for the day (B) and produced significant weight loss (C). D: in a separate group of mice, C89b at this ip dose did not produce conditioned taste aversion to a novel saccharin solution (vehicle controls, n = 8; C89b, n = 7).

	DISCUSSION

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In addition, these experiments indicate that C75's mechanism of action involves both FAS and CPT-1. First, cerulenin and C89b decrease feeding, but the response to C75 is more rapid. Second, significant hypophagia is produced by lower doses of C75 than either cerulenin or C89b. This is striking, given that the degree of inhibition of fatty acid synthesis with cerulenin is comparable to or greater than that obtained with C75 (11, 40) and that C75 and C89b exhibit similar potencies for stimulating CPT-1 (Fig. 5). It might be the case that FAS inhibition and CPT-1 stimulation synergize to have a supra-additive effect on acute feeding behavior. Finally, longer term effects of C75 on food intake, weight loss, and weight regain are intermediate between patterns obtained using cerulenin and C89b. These studies did not assess whether the compounds have differing half-lives or pharmacokinetics in CNS that could contribute to their different temporal patterns of effect.

There has been controversy concerning C75's effect on CPT-1 activity. The FAS-inhibiting action of C75 could lead to CPT-1 inhibition, as cerulenin increases malonyl-CoA, inhibits CPT-1, and reduces beta-oxidation (40). It has even been proposed that C75, in a C75-CoA form, inhibits CPT-1 (2). Despite these possibilities, studies show that the net effect of C75 on CPT-1 is to increase its activity (4, 25, 33, 47, 51). Our result, that etomoxir attenuated C75-induced hypophagia, is consistent with the latter hypothesis. The CPT-1-stimulating action of C75 might be particularly important to set up the long-term effect of weight loss and maintenance. Further study will show whether C89b, like C75, will produce long-lasting changes in gene expression for hypothalamic neuropeptides (1, 21) and for peripheral enzymes and proteins (6, 7, 48) involved in energy balance.

The hypophagia and weight loss after CPT-1 stimulation in the CNS seem in conflict with other studies that report that inhibiting central CPT-1 activity decreases food intake (34) (41) and body weight (41) and that icv administration of oleic acid decreases food intake (35). In our mouse studies, the CPT-1 inhibitor etomoxir did not decrease feeding, but it transiently reduced body weight. In rats, hypophagia has been reported after central CPT-1 inhibition, but weight loss has not always been observed (Ref. 8 vs. Ref. 41). Species differences and differences in experimental designs, including times of outcome measurements, might account for some discrepancies in the manifestation of effects of CPT-1 inhibitors on energy balance. Studies thus far, including the present work, have examined changes in feeding and weight after icv injections, not directly in the hypothalamus, so there is potential response from direct actions at multiple brain sites. In the few studies that discuss body weight after systemic CPT-1 inhibition, etomoxir produced no significant weight change in rats (10) or mice (17). Changes in body weight have not been investigated after icv etomoxir in mice before the present study. It is possible that mice had increased energy expenditure after icv etomoxir. We have given C75 icv to rats and have seen decreases in body weight that seemed greater than could be accounted for by the hypophagia (1). Our recent studies have confirmed the additional weight loss using pair-fed controls, and icv C75 increased oxygen consumption and fat oxidation (2). Another group has shown similar effects in mice, with evidence for increased sympathetic neural output and elevated fat oxidation in skeletal muscle (6, 7). Changes in overall metabolism and energy expenditure after different manipulations of CNS fatty acid metabolism should continue to be addressed.

One current hypothesis contends that the cytosolic concentration of long-chain fatty acids serves as a gauge of nutritional status, with increased long-chain fatty acids providing a "signal of plenty" and leading to decreased feeding and body weight (16, 24, 35, 41). Another more generalized hypothesis is that the altered hypothalamic neuronal AMPK activity provides the impetus for changes in overall energy balance (3, 21, 22, 26, 31, 32), with decreased AMP-to-ATP ratio and/or decreased AMPK activity leading to reductions in feeding and body weight. Regarding how CNS fatty acid metabolism might be involved in the regulation of energy balance, a critical issue for both hypotheses may be involvement of CPT-1 activity. CNS administration of a CPT-1 stimulator decreased food intake and body weight in mice. Conversely, CPT-1 inhibition would be expected to decrease neuronal ATP, increase AMPK activity, and thereby increase food intake. Indeed, mice showed transient hyperphagia after etomoxir. Further investigation of differing mechanisms of action with CPT-1 stimulation vs. inhibition in CNS and hypothalamus are warranted.

These data do not support the current long-chain fatty acid-sensing hypothesis for regulating organism energy balance (16, 24, 35, 41). CPT-1 stimulation and inhibition should have opposite effects on cytosolic long-chain fatty-acyl-CoA levels. According to the fatty acid-sensing hypothesis, CPT-1 inhibition should inhibit feeding by increasing cytosolic fatty-acyl-CoA levels. Instead, the first response of mice to etomoxir was to increase food intake (Fig. 3A). Furthermore, the fatty acid-sensing hypothesis would predict that CPT-1 stimulation would increase food intake due to a drop in cytosolic fatty-acyl-CoA levels as fatty acids are transferred into the mitochondria. Instead, mice decreased their food intake substantially in response to the CPT-1 stimulator C89b.

Changes in cytosolic fatty acid levels could play a role in the regulation of energy balance through downstream alterations in AMP-to-ATP ratio and AMPK activity. One possibility is that elevated cytosolic long-chain fatty acids would inhibit acetyl-CoA carboxylase allosterically (14), conserving ATP. Other possible mechanisms include changes in conductance through ion channels (15). In some tissues, long-chain fatty acids have been shown to regulate the conductance or probability of open-state ion channels, including the Na⁺-K⁺-ATPase (37) and the ATP-sensitive K⁺ channel (K⁺_ATP) (5). Interestingly, K⁺_ATP also responds to changes in AMPK activity (43). In a heterogeneous tissue such as hypothalamus, effects are likely to be complex. Oleic acid has been shown to inhibit, or excite, distinct populations of neurons in the arcuate nucleus (49).

Interestingly, both the CPT-1 inhibitor etomoxir and the FAS inhibitor cerulenin produced weight loss on the first day after administration in excess of that accounted for by the level of food intake. If etomoxir increases cytosolic long-chain fatty acids, they could then allosterically inhibit acetyl-CoA carboxylase, decreasing flux through the fatty acid synthetic pathway. Compared with etomoxir and cerulenin, a single injection of the CPT-1 stimulator C89b elicited long-lasting hypophagia, with no apparent compensation for the lost calories.

Measuring multiple parameters at multiple time points is needed to clarify how manipulations of CNS fatty acid metabolism are translated into changes in organism energy balance. Altered hypothalamic AMPK activity has been identified as a leading contender for regulating organism energy balance (31) and is important for C75's effects on AMP-to-ATP ratio to be translated into changes in hypothalamic gene expression and altered feeding behavior (21). We are mindful that AMPK activity can be altered by signals other than changes in AMP-to-ATP ratio, including changes in redox state, which could be affected by altering fatty acid metabolism (FAS requires NADPH). The present studies were not designed to determine whether altered fatty acid metabolism in the hypothalamus is involved in physiological controls of organism energy balance, but the issue is an important subject of current research. Recent data by Gao et al. indicate that leptin's ability to decrease food intake and body weight are mediated by an increase in metabolic flux through the fatty acid synthetic pathway, downstream of leptin-induced decrease in hypothalamic AMPK activity (12).

CPT-1 stimulation poses certain advantages over CPT-1 inhibition as a potential treatment strategy for obesity. First, systemic administrations of CPT-1 inhibitors are known to increase food intake (27) and do not appear to produce weight loss (10, 17). Second, although CPT-1 inhibitors lower plasma glucose in the short term (9), prolonged treatment might increase intramyocellular lipids in skeletal muscle and increase insulin resistance (10). Thus, if CPT-1 inhibition is a potential obesity treatment, it would have to be administered to bypass peripheral tissues and function exclusively in the CNS. In contrast, C89b administered systemically to mice produced feeding reduction and weight loss without behavioral signs of toxicity. If CPT-1 stimulation is shown to be safe, effective, and feasible, it may have utility for obesity treatment and warrants further clarification of the underlying mechanisms.

	GRANTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION GRANTS DISCLOSURES REFERENCES

 
This work was funded by grants from the National Institutes of Health: DK-068054 to S. Aja, DK-064000 to G. V. Ronnett, CA-091634 to J. M. McFadden, CA-087850 to F. P. Kuhajda, and DK-019302 to T. H. Moran.

	DISCLOSURES

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION GRANTS DISCLOSURES REFERENCES

 
FASgen provided C75 and C89b for the experiments. Under a licensing agreement between FASgen and the Johns Hopkins University, L. E. Landree, J. N. Thupari, C. A. Townsend, F. P. Kuhajda, and G. V. Ronnett are entitled to a share of royalties received by the University on sales of products described in this article. F. P. Kuhajda and C. A. Townsend own FASgen stock. G. V. Ronnett and T. H. Moran have an interest in FASgen stock that is subject to restrictions under University policy. The Johns Hopkins University manages the terms of these agreements in accordance with its policies on conflict of interest.

	ACKNOWLEDGMENTS

 
E. Plummer and K. Daniels were enrolled in the Research Practicum (Baltimore Polytechnic, Baltimore, MD).

	FOOTNOTES

 

Address for reprint requests and other correspondence: S. Aja, Johns Hopkins School of Medicine, Dept. of Psychiatry and Behavioral Sciences, 720 Rutland Ave., Ross 618, Baltimore, MD 21205 (e-mail: saja1{at}jhmi.edu)

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.

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