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

Pharmacological Stimulation of Brain Carnitinepalmitoyl-Transferase-1

Decreases Food Intake and Body Weight

Susan Aja1, Leslie E. Landree2, Amy M. Kleman2,

Susan M. Medghalchi3, Aravinda Vadlamudi3, Jill M. McFadden4,

Andrea Aplasca1, Jayson Hyun1, Erica Plummer1, Khadija Daniels1, Matthew Kemm1,

Craig A. Townsend4, Jagan N. Thupari5, Francis P. Kuhajda5, Timothy H. Moran1, and

Gabriele V. Ronnett2

Departments of Psychiatry and Behavioral Sciences1, Neuroscience2, and Pathology5

Johns Hopkins University School of Medicine

Baltimore, Maryland 21205

FASgen, Inc. 3

Baltimore, Maryland 21224

Department of Chemistry4, Johns Hopkins University

Baltimore, Maryland 21218

Running head: CNS CPT-1 and food intake

Address correspondence to: Susan Aja, Johns Hopkins School of Medicine, Department

of Psychiatry and Behavioral Sciences, 720 Rutland Avenue, Ross 618, Baltimore, MD

21205. Phone: (410) 955-2996, Fax: (410) 502-3769, e-mail: [email protected]

Page 1 of 41Articles in PresS. Am J Physiol Regul Integr Comp Physiol (December 5, 2007). doi:10.1152/ajpregu.00862.2006

Copyright © 2007 by the American Physiological Society.

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ABSTRACT

Inhibition of brain carnitinepalmitoyl-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 (i.c.v.). 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 i.c.v. bolus, and

examined acute and prolonged effects on feeding and body weight. C75 decreased food

intake rapidly and potently at all doses (1-56 ηmol), and dose dependently inhibited

intake on day 1. Dose dependent weight loss on day 1 persisted through four days of post-

injection monitoring. The FAS inhibitor cerulenin produced dose dependent (560 ηmol)

hypophagia for one day, weight loss for two days, and weight regain to vehicle control by

day 3. The CPT-1 inhibitor etomoxir (32, 320 ηmol) 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 inhibitor of CPT-1 that does not affect fatty acid synthesis. C89b (100,

320 ηmol) decreased feeding in mice for three days, and produced persistent weight loss

for six 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.

Keywords: Fatty acid metabolism, Energy intake, AMP-activated protein kinase

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INTRODUCTION

The rising prevalence of human obesity (36) and its resistance to diet and exercise

make the development of efficacious pharmacological approaches to weight management

a priority. Recently, manipulations of fatty acid metabolism yielded profound weight loss

in animal models. Administration of C75, a synthetic small molecule inhibitor of fatty

acid synthase (FAS) (23), decreased food intake and body weight in rodents (13) (18) (19)

(21) (28). In addition to inhibiting FAS, C75 increased the activity of carnitinepalmitoyl-

transferase-1 (CPT-1) (4) (25) (33) (47) (51), which transfers long-chain fatty acids into

mitochondria (30). By stimulating CPT-1, C75 increased the rate of fatty acid beta-

oxidation (25) (47) (46). In addition, C75 decreased feeding and body weight when

administered intracerebroventricularly (i.c.v.), implicating brain FAS and CPT-1 in

mediating the action of C75.

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).

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In the present studies, we sought to determine 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

Animal preparation for food intake experiments

Male C57BL6/J mice (6 - 8 weeks 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, NJ). Mice

were adapted to these conditions for one week 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 (i.p.), 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 i.p. banamine (0.25

mg/100g) for analgesia, and penicillin (10,000 U) to prevent infection.

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Intracebroventricular (i.c.v.) injections were performed with a microliter syringe

(Hamilton Co., NV) 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 one week of post-surgical recovery, cannula placements were assessed by

measuring food intake after i.c.v. neuropeptide Y (NPY, 0.25 ηmol; American Peptide

Co., CA) versus 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 i.c.v. administrations, the combined FAS inhibitor and CPT-1 stimulator C75

(MW = 254.15; 0 - 56 ηmol) was given in 2 µl of 1X RPMI-1640 (#12-167, Lonza,

Switzerland). Other compounds required lipophillic 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 ηmol) was given in 4 µl of 25% DMSO / 75% RPMI. The CPT-1

stimulator etomoxir (MW = 320.75; 0 - 320 ηmol) was also given in 4 µl of 25% DMSO /

75% RPMI. The selective CPT-1 stimulator C89b (MW = 322.2; 0 - 320 ηmol) was given

in 2 µl of 25% TEP / 75% saline (TEP = 10% Tween-80, 10% ethanol, 80% polyethylene

glycol). In other experiments, i.p. injections of compounds were given in 50 µl of 10%

TEP / 90% saline (testing the high i.c.v. doses i.p.), or 50 µl of 100% TEP (testing C89b

at effective i.p. doses). FASgen, Inc. provided C75 and C89b. Etomoxir was purchased

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from H. P. O. Wolfe, Projekt-Entwicklung (Konstanz, Germany). Cerulenin was

purchased from Sigma (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

i.c.v. experiments, mice received single injections of vehicles or compounds in half-log-

and quarter-log-step doses dissolved in vehicles 30-min prior to lights-out. We measured

intakes of chow, corrected for spillage, at 0.5, 1, 2, 4, and 22 h on the first day, and 0-22 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 – 8 weeks old, Jackson Laboratories, Bar Harbor, ME)

or, in separate experiments, male C57Bl6/J mice, were trained for two weeks 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 i.p. with C89b (30 mg/kg) or 50 µl of 10% TEP vehicle. In different

studies, male mice were injected i.c.v. with C89b (100 nmol) or 2 ul 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

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MCF-7 cells were maintained in RPMI with 10% fetal bovine serum. Prior to

assays, cells were incubated overnight at 37 ºC 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 Corporation, 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, #10888; Invitrogen, CA) supplemented with B27 (2 %), glutamine (2 mM),

penicillin and streptomycin. Cultures were grown in a sterile incubator (37 ºC, 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

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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 ºC with 700 µl 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-CoAfor 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 x g, 5 min), washed with 500 µl ice-cold

2 mM perchloric acid, and centrifuged again. The pellet was resuspended in 800 µl dH2O

and extracted with 400 µl 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)

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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 ºC 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 hours. Cell

viability was determined using calcein AM (Molecular Probes, Eugene, OR). Conversion

of the cell permeant non-fluorescent 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 averages with standard error of the mean. Data from in vivo

experiments with i.c.v. injections of C75, cerulenin, C89b, or etomoxir were analyzed by

One-Way Repeated Measures ANOVA. Data from the in vivo experiment with i.c.v. 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 experiment with i.p.

compounds were analyzed with paired two-tailed t-tests of compounds versus 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.

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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 AND DISCUSSION

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 ηmol) reduced chow intake to about 30% of vehicle control (P <

0.01). At 4 h, all doses suppressed feeding to about 65% of control (P < 0.05). By the end

of day 1, the effect of C75 on feeding was dose dependent (Fig. 1B; 56 ηmol, 65% of

vehicle control, P = 0.001), as was the weight loss (Fig. 1C; +0.7 ± 0.5 g after vehicle; 10

ηmol, -0.2 ± 0.3 g, P = 0.034; 56 ηmol, -1.0 ± 0.2 g, P < 0.001). The feeding and weight

changes following a single i.c.v. 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.

Selective inhibition of FAS in the CNS decreases food intake and body weight

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FAS inhibition by the natural product cerulenin (38) has been shown previously to

reduce food intake and body weight, but prior work has not provided complete

characterization. Weight loss was robust after high doses of systemic cerulenin (20) (28)

(29) but feeding reductions did not reach statistical significance (20) (29). Food intake

and body weight changes have not been reported following central administration of the

FAS inhibitor cerulenin (28). Cerulenin decreased mouse food intake significantly and

dose dependently by 2 h (Fig. 2A; 320 ηmol, 38% of vehicle control, P = 0.015; 560

ηmol, 10% of control, P < 0.001). Cerulenin at the highest dose of 560 ηmol produced

hypophagia lasting one day (Fig. 2B; 60% of vehicle control, P = 0.005). Weight loss one

day after this high dose was greater (-2.4 ± 0.5 g) than the feeding reduction (-1.4 ± 0.5 g;

paired two-tailed t-test, t = 12.72, 7 d.f., P < 0.0001), suggesting potential effects on

energy expenditure. Following the initial reductions in feeding and body weight on day 1

with 560 ηmol of cerulenin, food intake was slightly increased on days 2-4, and the lost

weight was regained by day 3 (Fig. 2C). These results showed that FAS inhibition in the

brain can decrease both food intake and body weight. However, although cerulenin is at

least as potent as C75 at inhibiting FAS (11) (40), the dose of cerulenin required for

reducing food intake was considerably higher than that of C75. Because cerulenin lacks

the CPT-1 stimulating action of C75, the pattern of results suggests an important role for

CPT-1 stimulation in the effects of C75.

CPT-1 stimulation is involved in hypophagic responses to i.c.v. C75

Having established that CNS FAS inhibition could decrease feeding and body

weight, we next assessed the effects of altering CPT-1 activity on these parameters.

Numerous studies have shown that inhibition of fatty acid oxidation, and direct inhibition

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of CPT-1 in the periphery increase food intake (27). Effects on body weight are typically

not discussed, but studies that have examined body weight show no significant effect (10)

(17). In contrast, CPT-1 inhibition in the brain as shown by others decreased feeding (8)

(34) and body weight (41). Investigations into the role of CPT-1 in energy balance have

been limited by a lack of selective CPT-1 stimulators. To assess the role of CPT-1

activity in the regulation of food intake and body weight, we first antagonized the CPT-1

stimulating action of C75, using the well characterized CPT-1 inhibitor etomoxir (44).

Etomoxir given i.c.v. increased food intake at 0.5 h and 1 h (Fig. 3A), after which

etomoxir had no effect on feeding during day 1 (Fig. 3A, 3B). Mice lost weight on day 1

following treatment with 320 ηmol 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 i.c.v. administration of CPT-1 inhibitors (41), or no

weight change despite hypophagia after chronic i.c.v. infusion (8).

To counteract C75’s CPT-1 stimulating action, and thereby determine whether

CPT-1 stimulation contributes to C75’s hypophagic effect, we pretreated mice with the

highest dose of etomoxir (320 ηmol, i.c.v.) before administering C75 (32 ηmol, i.c.v.)

(Fig. 4). As expected, C75 alone suppressed feeding at 0.5, 1, 2, and 4 h compared with

double-vehicle control (P < 0.01) (Fig. 4A). However, after combined treatment with

etomoxir and C75, food intakes at 1, 2, and 4 h were not significantly lower than those

after given etomoxir alone (Fig. 4A). Cumulative intakes on day 1 further demonstrated

etomoxir’s ability to prevent the full hypophagic effect of C75 (C75 vs. vehicle, P =

0.049; ETO vs. C75+ETO, no significant difference) (Fig. 4B). Body weight reduction on

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day 1 after C75 was not different from that seen after C75 with an etomoxir pretreatment

(Fig. 4C). Beyond day 3 after i.c.v. injection, the mice given an etomoxir pretreatment

with C75 became compromised and most were euthanized (Data shown in Fig. 4 are from

mice that were healthy until day 3, n = 4 or 5.). This longer-term effect of C75 plus

etomoxir was unexpected, given that FAS inhibition alone (which we tried to

approximate with C75 + etomoxir) with cerulenin is well-tolerated, and that neither i.c.v.

C75 nor i.c.v. etomoxir had untoward effects on mice. The data did suggest that part of

C75’s feeding inhibitory action depended on its ability to stimulate CPT-1. Blockade of

that action reduced C75’s feeding inhibitory capacity. In the next experiment, we sought

to more fully explore the effect of CPT-1 stimulation on feeding and body weight using a

novel, selective CPT-1 stimulator.

C89b, a novel CPT-1 stimulator

We used compound C89b (26) to stimulate CPT-1. We first characterized the FAS

inhibitory and and CPT-1 stimulatory actions of C89b using MCF-7 breast cancer cells.

C89b did not affect the level of fatty acid synthesis as determined by 14C-acetate

incorporation into fatty acids (Fig. 5A, P = 0.0635, Dunnett’s tests all P > 0.05). C89b did

increase CPT-1 activity in digitonin permeabilized MCF-7 cells, as indicated by a

concentration dependent increase in radiolabeled palmitoyl-carnitine (Fig. 5B, P =

0.0275), with 20 µg/ml (62.1 µM) significantly increasing CPT-1 activity (144% control,

P < 0.05). Consistent with stimulation of CPT-1, C89b increased beta-oxidation of fatty

acids in MCF-7 cells in a concentration dependent manner (Fig. 5C, P < 0.0001). Beta-

oxidation with C89b was greater than vehicle control at concentrations of 1.3 µg/ml (4.0

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µM) or higher (P < 0.01), reaching maximal levels at 10 µg/ml (31 µM, 177% of control).

The estimated EC50 was 3 µg/ml (9.3 µM, 139% of control).

We further examined the effects of C89b on fatty acid metabolism in primary

cultures of rat hypothalamic neurons, because the hypothalamus has well known

importance for controlling food intake and whole body energy balance. Results from

biochemical assays of cultured hypothalamic neurons were similar to those of MCF-7

cells. Unlike C75, C89b did not decrease acetate incorporation into fatty acids (Fig. 5D, P

= 0.5843). However, C89b did increase CPT-1 activity in cultured neurons in a

concentration dependent manner (Fig. 5E, P = 0.0173, with maximal response at 20

µg/ml (62.1 µM, 150% of control, P < 0.05), without adversely affecting the health of the

cultures, as determined by calcein fluorescence in live cells (P = 0.5603, data not shown).

The data show concordance of C89b effects in two different cell types, indicating that the

pharmacological mechanism of C89b action is to stimulate CPT-1 activity without

affecting fatty acid biosynthesis.

Stimulation of CPT-1 in the CNS with C89b produces persistent hypophagia and

weight loss

C89b administered to mice i.c.v. was about 100 times less potent than C75 to

suppress feeding, despite the compounds’ similar potencies to stimulate CPT-1 in vitro

(Fig. 5B, 5C, 5E). By the end of day 1, the higher doses of C89b (100 and 320 ηmol)

decreased food intake to about 45% of vehicle control (P < 0.001) (Fig. 6B). Hypophagia

persisted through day 2 after these doses (about 73% of control, P < .05), and day 3 after

the high dose (84% of control, P = 0.007) (Fig. 6B). Thus, hypophagia after C89b lasted

longer that that seen after combined FAS inhibition and CPT-1 stimulation with C75.

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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

ηmol caused weight loss on day 1, compared with control (vehicle, gain of +0.3 ± 0.0 g;

100 ηmol, -1.6 ± 0.5 g, P = 0.002; 320 ηmol, -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 i.c.v C89b was

unlikely to be caused by malaise, because C89b at 100 ηmol failed to produce a

conditioned taste aversion to saccharin in a two-bottle test (Fig. 7).

Systemic administration of C89b and other compounds

Doses of compounds given i.c.v. did not elicit their effects on feeding and body

weight by leaking to and acting at peripheral tissues. The highest i.c.v. doses were

injected intraperitoneally (i.p.), and had no effects on cumulative chow intakes at 2, 4,

and 22 h (Fig. 8A), or on body weight (Fig. 8B). C89b given peripherally, at much higher

doses, decreased body weight (Fig. 9A). Further study showed that C89b given i.p. (30

mg/kg, 93 µmol/kg) produced hypophagia (Fig. 9B) and weight loss (Fig. 9C) without

eliciting conditioned taste aversion (Fig. 9D). This indicates that C89b given systemically

would not decrease feeding by causing sickness behavior.

General Discussion

Central FAS inhibition and CPT-1 stimulation decreased food intake and body

weight in a rodent model. The FAS inhibitor cerulenin reduced feeding but led to rebound

hyperphagia and regain of lost weight in the time interval examined. By comparison, the

selective CPT-1 stimulator C89b produced longer lasting hypophagia and persistent

weight loss. C89b administered either in the CNS or systemically also reduced feeding

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and body weight without producing sickness behavior. Together, these data suggest CPT-

1 stimulation as a potential approach to weight loss and management.

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 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 if C89b,

like C75, will produce long-lasting changes in gene expression for hypothalamic

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neuropeptides (1) (21) and for peripheral enzymes and proteins (48) (6) (7) 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 i.c.v. 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 ((8)

vs. (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 i.c.v. 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 i.c.v. etomoxir in mice prior to the present

study. It is possible that mice had increased energy expenditure after i.c.v. etomoxir. We

have given C75 i.c.v to rats and 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 i.c.v. 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

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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/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 versus 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

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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/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 to 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

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on AMP/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/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 if 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.

ACKNOWLEDGEMENTS

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Ms. Erica Plummer and Ms. Khadija Daniels participated in these experiments

through their enrollment in the Research Practicum (Baltimore Polytechnic, Baltimore,

MD).

GRANTS

This work was funded by grants from the National Institutes of Health:

DK068054 to S.A., DK064000 to G.V.R., CA091634 to J.M.M., CA087850 to F.P.K.,

and DK019302 to T.H.M.

DISCLOSURES

FASgen, Inc. provided C75 and C89b for the experiments. Under a licensing agreement

between FASgen, Inc. and the Johns Hopkins University, LEL, JNT, CAT, FPK, and

GVR are entitled to a share of royalties received by the University on sales of products

described in this article. FPK and CAT own FASgen stock. GVR and TH.M 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.

FIGURE LEGENDS

FIG. 1. The combined FAS inhibitor and CPT-1 stimulator C75 reduces food intake and

body weight. (A) Single i.c.v. 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 i.c.v. C75 was dose-dependent on day 1. Weight losses were

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maintained relative to vehicle control on subsequent days. For all panels, n = 7, * P <

0.05 versus 0 ηmol control.

FIG. 2. CNS administration of the FAS inhibitor cerulenin reduces food intake and body

weight. (A) Single i.c.v. 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 ηmol) 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 versus 0 ηmol

control.

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 days 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, pre-injection body weight. For all

panels, n = 11, * P < 0.05 versus 0 ηmol control.

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 etomoxir pretreatment,

compared with etomoxir alone. At 1, 2, and 4 h, C75 still reduced food intake compared

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to vehicle control. However, C75 did not significantly reduce food intake when given

after etomoxir, compared with etomoxir alone. (B) Etomoxir’s ability to attenuate C75-

induced hypophagia remained evident by the end of day 1. For panels A and B, n = 5, * P

< 0.05 comparing C75 versus double-vehicle control, and comparing etomoxir + C75

versus etomoxir alone. (C) Decreases in body weight after C75 with etomoxir

pretreatment did not differ from those after C75 alone (n = 4).

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 carnitinepalmitoyl-transferase-1 (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 panels (A) and (B), n = 3 per condition. In panels (C)

and (D), n = 6 per condition. In panel (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 for C75 (ug/ml : uM) 20 : 78.7 uM, 40 : 157.4 uM; for C89b (ug/ml : uM)

0.6 : 1.9 uM, 1.3 : 4.0 uM, 2.5 : 7.8 uM, 3 : 9.3 uM, 5 : 15.5 uM, 10 : 31.0 uM, 20 : 62.1

uM, 40 : 124.2 uM, 60 : 186.2 uM, 80 : 248.3 uM, 160 : 496.6 uM, 320 : 993.1 uM. For

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all panels, * P < 0.05 versus 0 µg/ml control, Dunnett’s test; # P < 0.05 versus 0 µg/ml

control, unpaired one-tailed t-test.

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 i.c.v.

administration. (B) A single i.c.v. injection of C89b to mice at a dose of either 100 or 320

ηmol 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 versus 0 ηmol control.

FIG. 7. CNS administration of the CPT-1 stimulator C89b does not produce conditioned

taste aversion. Male C57BL/6J mice injected i.c.v with C89b at 100 ηmol did not exhibit

conditioned taste aversion to a novel saccharin solution in a two-bottle test (vehicle

controls, n = 7; C89b, n = 6).

FIG. 8. Systemic administrations of compounds at the highest concentrations from i.c.v.

experiments do not produce major effects on food intake and body weight. (A) Single i.p.

injections of compounds at the highest doses from i.c.v. experiments (C75 (56 ηmol),

cerulenin (560 ηmol), C89b (320 ηmol), and etomoxir (320 ηmol)) had no effect on

cumulative chow intakes at 2, 4, and 22 h versus vehicle control (50 µl of 10% TEP, 90%

saline vehicle; TEP = 80% polyethylene glycol, 10% ethanol, 10% Tween-80). (B) These

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doses of compounds, given i.p., did not significantly alter body weight, compared with

vehicle control.

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 - 8 weeks

old) on ad libitum chow and water were injected i.p. with either C89b (15, 30, 60 mg/kg)

or vehicle (50 µl of TEP; TEP = 80% polyethylene glycol, 10% ethanol, 10% Tween-80)

(n = 3 per group). All i.p. 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, i.p. This dose (A) decreased food intake for the day and (B) produced significant

weight loss. (D) In a separate group of mice, C89b at this i.p. dose did not produce

conditioned taste aversion to a novel saccharin solution (vehicle controls, n = 8; C89b, n

= 7).

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