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Endocrinology, doi:10.1210/en.2004-0039
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Endocrinology Vol. 145, No. 7 3197-3204
Copyright © 2004 by The Endocrine Society

Carnitine Palmitoyltransferase-1 (CPT-1) Activity Stimulation by Cerulenin via Sympathetic Nervous System Activation Overrides Cerulenin’s Peripheral Effect

Yong-Jun Jin, Song-Zhe Li, Zheng-Shan Zhao, Juan Ji An, Ryang Yeo Kim, Yoo Mee Kim, Ja-Hyun Baik and Sung-Kil Lim

Division of Endocrinology (Y.-J.J., S.-Z.L. Z.-S.Z., J.J.A., Y.M.K., S.-K.L.), Department of Internal Medicine, College of Medicine, and Brain Korea 21 Project for Medical Sciences (Y.-J.J., J.J.A., R.Y.K.), Yonsei University, Seoul 120-752, Korea; School of Life Sciences and Biotechnology (J.-H.B.), Korea University, Seoul 136-701, Korea; and Division of Endocrinology (Y.-J.J., J.J.A.), Department of Internal Medicine, Affiliated Hospital, Medical College, YanBian University, YanJi 133-000, People’s Republic of China

Address all correspondence and requests for reprints to: Sung-Kil Lim, 134 Shinchon-Dong, Seodaemoon-Gu, Seoul 120-752, Korea. E-mail: lsk{at}yumc.yonsei.ac.kr.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
To clarify the paradoxic effects of cerulenin, namely its in vitro inhibitory effects on fat catabolism and its in vivo reduction of fat mass, we studied the in vivo and in vitro effects of cerulenin on carnitine palmitoyltransferase-1 (CPT-1) activity, the rate-limiting enzyme of fatty acid oxidation. A single ip injection of cerulenin significantly reduced body weight and increased core temperature without significantly reducing food intake. In situ hybridization study revealed that a single injection of cerulenin did not affect the expression of orexigenic neuropeptide mRNA. Cerulenin’s effect on CPT-1 activity was biphasic in the liver and muscle: early suppression during the first 1 h and late stimulation in the 3–5 h after ip treatment. In vitro cerulenin treatment reduced CPT-1 activity, which was overcome by cotreating with catecholamine. Intracerebroventricular injection of cerulenin increased CPT-1 activity significantly in soleus muscle, and this effect was sustained for up to 3 h. Pretreatment with {alpha}-methyl-p-tyrosine inhibited the cerulenin-induced increase in core temperature and the late-phase stimulating effect of cerulenin on CPT-1 activity. In adrenalectomized mice, cerulenin also increased the activity. In vivo cerulenin treatment enhanced muscle CPT-1 activity in monosodium glutamate-treated arcuate nucleus lesioned mice but not in gold thioglucose-treated ventromedial hypothalamus lesioned mice. These findings suggest that cerulenin-induced late-phase stimulating effects on CPT-1 activity and energy expenditure is mediated by the activation of innervated sympathetic nervous system neurons through the firing of undefined neurons of the ventromedial hypothalamus, rather than the arcuate nucleus.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
EXCESS BODY WEIGHT is a major health problem in developed countries and is associated with an increased risk of type 2 diabetes, cardiovascular and cerebrovascular diseases, and increased mortality. The magnitude of this health problem and the limitations of successful weight-reduction therapy emphasize the need for different approaches to control body weight. Cerulenin, a new class of drug that inhibits fatty acid synthase (FAS), robustly and reversibly reduces body weight (1) and has brought hope to those needing an effective obesity management therapy.

Several trials have been undertaken to analyze the weight-reducing mechanisms of FAS inhibitors. A synthesized FAS inhibitor, C75 acts both centrally to reduce food intake and peripherally to increase fatty acid oxidation, thus leading to rapid and profound weight loss and loss of adipose mass (2). Treatment of C75 prevented fasting-induced up-regulation of hypothalamic agouti-related peptide (AgRP) and neuropeptide Y (NPY) mRNAs because well as down-regulation of cocaine- and amphetamine-related peptide and proopiomelanocortin mRNAs (3). In contrast to C75, a natural FAS inhibitor, cerulenin induced the preferential loss of adipose tissue, and this was not highly correlated with food intake. Furthermore, cerulenin, intriguingly stimulated heat production and metabolic activity in ad libitum-fed wild-type mice (4). Therefore, increased metabolic rate, rather than reduced food intake, has been considered to be the prime contributor to weight loss, especially with respect to the fat mass reduction induced by cerulenin.

Malonyl-CoA, in addition to its role as a substrate for FAS, is pivotally required for energy regulation through its reversible inhibition of carnitine palmitoyltransferase-1 (CPT-1) (5). During situations of excess energy, the increased malonyl-CoA-generated for fatty acid synthesis inhibits CPT-1 activity, preventing the oxidation of newly formed fatty acids bound for energy storage. However, during starvation, malonyl-CoA levels fall to permit the oxidation of fatty acids for energy production. Cerulenin, known as a FAS inhibitor at the cellular level, directly increases malonyl-CoA to inhibit CPT-1, the rate-limiting enzyme of fatty acid oxidation (6). Interestingly, in contrast to the in vitro inhibitory effects of cerulenin on fat catabolism, the in vivo treatment of cerulenin causes the opposite phenomenon, i.e. a selective reduction of fat mass. This phenomenon is referred to as the paradoxical effect of cerulenin.

Recently it was reported that FAS is also expressed in the hypothalamus, arcuate nucleus (ARC), ventromedial hypothalamus (VMH), and paraventricular nucleus (7). Moreover, considerable evidence has accumulated, suggesting the possibility that malonyl-CoA is one of the prime agents in the sensing of fuel supply in the brain and peripheral organs (8). In addition, the sympathetic nervous system (SNS) has been shown to play an important role in the regulation of both sides of the energy balance equation but especially in the regulation of energy expenditure. Studies based on treating endogenous catecholamines, involving norepinephrine (9, 10) and epinephrine (11, 12, 13) infusion have shown significant increases in energy expenditure, lipid oxidation, and lipolysis. Furthermore, reductions in sensitivity to certain levels of SNS activity and to lipid oxidation have also been observed in cases of obesity (14, 15, 16).

How could the paradoxical effects of cerulenin be explained, whereby a significant increase in the cellular levels of malonyl-CoA causes an opposite effect from that expected, namely lipid oxidation and a selective reduction in fat mass? In this study, we hypothesized that the modest and delayed central stimulating effects of cerulenin on CPT-1 activity and fatty acid oxidation through the SNS surpass the early peripheral inhibitory effects. To prove our hypothesis, we endeavored to clarify the catabolic effects of cerulenin on adipose mass by studying the following: 1) the effects of a single ip injection of cerulenin on food intake, body weight change, and core temperature; 2) the in vitro and in vivo effects of cerulenin on CPT-1 activity; 3) the role of the activation of the SNS for cerulenin-induced reversible fat catabolism, e.g. fatty acid oxidation; and 4) the central pathway for the cerulenin-induced activation of the SNS.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
Six-week-old C57BL/6J male mice were obtained from the Jackson Laboratory (Deahan Biolink Co., Chungbuk, Korea). Animals were maintained under a 12-h light, 12-h dark cycle at 23 C under institutional guidelines for the humane treatment of laboratory animals. All the experimental protocols were approved by the Animal Ethics Committee at the University of Yonsei, College of Medicine (Seoul, Korea). Mice were housed with free access to food and water. After a 1-wk acclimatization, animals were administered a single ip injection of cerulenin at 60 mg/kg body weight. Food intake and body weight changes were measured 24 h after the cerulenin treatment. Core temperatures were measured in fasted animals using a digital thermometer (Toshiba, Tokyo, Japan).

Drugs
Cerulenin, {alpha}-methyl-p-tyrosine (AMPT), monosodium glutamate (MSG) and gold thioglucose (GTG) were purchased from Sigma (St. Louis, MO). L-[methyl-14C]-carnitine was purchased from Amersham Pharmacia Biotech (Piscataway, NJ). Cerulenin was dissolved in RPMI 1640 medium and injected ip at 60 mg/kg of body weight (1) and intracerebroventrically (icv) at 10 µg/mouse. AMPT was dissolved in saline and injected ip at 300 mg/kg of body weight.

Adrenalectomy
Operations were carried out under ketamine (100 mg/kg; Korea United Pharm, Seoul, Korea) and xylazine (10 mg/kg; Bayer Korea Ltd., Seoul, Korea) anesthesia. Adrenalectomy was performed by bilateral flank incision. Control animals underwent sham operations in which the adrenal glands were grasped but not removed. At the time of adrenalectomy (ADX), mice were given dexamethasone (45 mg/kg body weight, ip) (17), and drinking water was replaced with saline (0.9% NaCl). CPT-1 activity assays were carried out 7–9 d after adrenalectomy.

Development of ARC and VMH lesion mice
To develop ARC lesions, pregnant C57BL/6J mice were housed singly before delivery. From d 1 to d 7, the offspring were injected sc with either MSG (2 mg/g body weight) or 0.9% NaCl daily (18). Animals were weaned at 21 d of age and then housed in groups of the same sex. All animals were allowed free access to water and standard rodent chow. Control and MSG-treated male mice were studied at 12 wk of age. To develop the VMH lesion model, 4-wk-old C57BL/6J male mice were single ip injected with GTG (0.5 mg/g body weight) or 0.9% NaCl (19). Animals were studied for CPT-1 activity 8 wk after injection when mice were killed, their brains removed, and frozen in liquid nitrogen. The brains were later sectioned through the hypothalamus using a cryostat, and the brain sections obtained were fixed with paraformaldehyde. Sections were stained with Cresyl violet and these stained sections were examined under a microscope to confirm the presence or absence of lesions in the ARC and VMH.

Measurement of CPT-1 activity in vitro
Primary hepatocytes were isolated from adult male C57BL/6J mice and prepared according to the Seglen method (20). CPT-1 was measured using digitonin-treated permeabilized cells (21). Primary hepatocytes 1 x 106 cells were plated in DMEM with 10% fetal bovine serum in six-well plates in triplicate. After overnight incubation, the drugs and vehicle controls were added as indicated. After 2 h, the medium was removed, and the cells were washed with PBS and then incubated with 700 µl of assay medium containing 50 mM imidazole, 70 mM KCl, 80 mM sucrose, 1 mM EGTA, 2 mM MgCl2, 1 mM dithiothreitol, 1 mM Kaliumcyanid, 1 mM ATP, 0.1% fatty acid-free BSA, 70 µM palmitoyl-CoA, 0.25 µCi L-[methyl-14C]-carnitine, and 40 µg digitonin. Cerulenin was added as specified for each experiment. After incubation for 6 min at 37 C, the reaction was stopped by the addition of 500 µl of ice-cold 4 M perchloric acid. Cells were then harvested and centrifuged at 13,000 x g for 5 min. The pellet was washed with 500 µl of ice-cold 2 mM perchloric acid and recentrifuged. The resulting pellet was resuspended in 800 µl of deionized H2O and extracted with 400 µl of butanol. 14C in the butanol phase was quantified; the results obtained represented the amount of acylcarnitine derivative.

Measurement of CPT-1 Activity in liver and muscle
Animals were killed by decapitation, and livers and soleus muscles were homogenized in 0.25 M sucrose medium containing 10 mM Tris-HCI (pH 7.4) and 1 mM EDTA, or in 0.15 M KCl medium containing 10 mM Tris-HCI (pH 7.4) and 1 mM EDTA, respectively. Mitochondria were isolated as described by Saggerson and Carpenter (22) and Saggerson (23) and finally resuspended in 0.3 M sucrose buffer. Mitochondrial protein was determined using the method of Lowry et al. (24). Enzyme activity was determined within 15 min of isolating mitochondria by measuring the incorporation of L-[methyl-14C]-carnitine into the n-butanol soluble product (25). Mitochondria (150–250 µg of protein, depending on the tissue) were preincubated at 25 C for 4 min in 1.0 ml of a mixture containing 150 mM sucrose, 60 mM KCl, 25 mM Tris-HCl (pH 7.4), 1 mM EDTA, 1 mM dithiothreitol, 40 µM palmitoyl-CoA, and 1.3 mg/ml albumin (fatty acid free). Reactions were started by adding 25 µl containing 1 µCi (0.4 µmol) of L-[methyl-14C]-carnitine and continued for up to 4 min. The reactions were then stopped by adding 1.0 ml of ice-cold 1 N HCl. Blank values were determined by replacing the mitochondria with an equal volume of resuspension buffer.

In situ hybridization
Mouse brain cryostat sections (10 µm) were prepared and hybridized with specific antisense riboprobes. These antisense and sense RNA probes were labeled with [35S]CTP (>800 Ci/mmol; Amersham) in the standard conditions using T3, T7, or SP6 polymerase (Promega, Madison WI). In situ hybridization was performed as previously described (26). Dried brain sections were first exposed to XAR-OMAT film (Kodak, Rochester, NY) for autoradiography and then dipped in a Kodak NTB2 nuclear emulsion. Slides were exposed at 4 C for 24 h or 1 wk, developed in Kodak D19, fixed with Kodak Unifix, and counterstained with toluidine blue. The specificity of the in situ hybridization results was confirmed by using sense strand riboprobes, which showed no detectable signals. The dark-field images obtained were processed using an Imaging Technology image digitalizer to quantify the in situ hybridizations. The digitalized images were then analyzed on a MCID image-analysis system (Imaging Research Inc., St. Catherines, Ontario, Canada). Autoradiograms were analyzed in parallel using a digital scanning densitometer (Luminescent image analyzer, LAS-1000 CH, Fuji, Tokyo, Japan), operating using the image acquisition and analysis program, TINA on BAS 2500 (Fuji). AgRP and NPY mRNA were measured in the ARC, and melanin-concentrating hormone (MCH) was measured at the lateral hypothalamus.

Statistical analysis
All values are reported as means ± SEM. Statistical significance was determined by using the Student’s t test and one-way ANOVA followed by Tukey’s post hoc test, using GraphPad Software (San Diego, CA).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Effects of cerulenin on food intake, body weight change, and core temperature
To investigate the physiological consequences of the in vivo inhibition of fatty acid synthesis on global fat metabolism, we administered cerulenin (60 mg/kg body weight) to mice by a single ip injection. Twenty-four hours later food intake and body weight were measured. As previously described, cerulenin produced a significant reduction in body weight; it also tended to reduce food intake but not significantly (Fig. 1Go, A and B). Fasted mice treated with cerulenin lost 15% more weight than the fasted control mice (Fig. 1CGo).



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  		FIG. 1. Effects of cerulenin (Cer) on food intake, body weight change, and core temperature. Cerulenin was single ip injected at 60 mg/kg body weight. After 24 h, food intake (A) and body weight changes were measured in fed (B) and fasted (C) mice. Core temperatures were monitored over the 24-h period after cerulenin treatment in fasted mice (D). Data are expressed as mean ± SEM. *, P < 0.05 vs. control (n = 12).

 
To investigate cerulenin’s effects on energy expenditure, we measured the core temperature of fasted mice after cerulenin or RPMI 1640 treatment. The core temperatures of the RPMI 1640-administered control mice were found to have fallen dramatically throughout a 24-h fasting period (Fig. 1DGo). In contrast, a single administration of cerulenin (60 mg/kg body weight) significantly prevented this reduction in core temperature at 3 and 5 h after treatment. Tyrosine hydroxylase (TH) is a rate-limiting enzyme of catecholamine synthesis. To test whether the effects of cerulenin on the core temperatures of fasted mice were mediated through the activation of SNS, mice were pretreated with AMPT, an inhibitor of TH. As shown in Fig. 1DGo, the cerulenin-induced elevation of core temperature was blocked in the AMPT-pretreated mice.

Effects of cerulenin on the expression of hypothalamic orexigenic neuropeptide mRNA in mice
To investigate whether cerulenin alters the levels of neuropeptides known to regulate food intake, the mRNA expressions of AgRP, NPY, and MCH were assessed by in situ hybridization. Ad libitum-fed mice were ip injected with either RPMI 1640 or cerulenin and then killed 3 h after injection. A previous study reported that cerulenin treatment did not reduce hypothalamic AgRP or NPY mRNA and did not elevate proopiomelanocortin mRNA when compared with the control group in wild-type mice (4). In the present study, cerulenin treatment did not reduce hypothalamic orexigenic neuropeptide, AgRP, NPY, or MCH mRNA (Fig. 2Go).



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  		FIG. 2. Effects of cerulenin (Cer) on the mRNA levels of the neuropeptides, AgRP (A), NPY (B), and MCH (C). Mice were treated ip with either RPMI 1640 or cerulenin and killed 3 h later. Data are expressed as percentages of the corresponding mean RPMI 1640-injected ad libitum-fed levels ± SEM (n = 4).

 
Effects of cerulenin on CPT-1 activity in vivo and in vitro
A prior study found that cerulenin preferentially reduced fat mass (4). To investigate cerulenin’s effects on fat catabolism, i.e. fatty acid oxidation, we measured the activity of CPT-1, the rate-limiting enzyme of fatty acid ß-oxidation, in the liver and soleus muscle. Mice were ip injected with cerulenin at 60 mg/kg body weight, and CPT-1 activities were measured at 0, 0.5, 1, 3, and 5 h after cerulenin treatment. In the liver, CPT-1 activity was found to be significantly reduced 0.5 h after treatment but recovered 1 h after treatment (Fig. 3AGo). More interestingly, CPT-1 activities were significantly elevated 3 and 5 h after treatment (by 16 and 9% vs. baseline, respectively). The effect of cerulenin on muscular CPT-1 activity was similar to its affect on the liver (Fig. 3BGo). Based on these results, we assessed CPT-1 activity 3 h after cerulenin treatment in all other experiments.



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  		FIG. 3. Effects of cerulenin (Cer) on CPT-1 activity in liver and soleus muscle in vivo. Mice were treated with RPMI 1640 or cerulenin at 60 mg/kg body weight At 0, 0.5, 1, 3, and 5 h after treatment, CPT-1 activities were measured in liver (A) and soleus muscle (B). *, P < 0.05 and **, P < 0.01 vs. 0 h (n = 8)

 
To determine how cerulenin initially reduced and then elevated CPT-1 activity, the mice were icv injected with 10 µg cerulenin, and the CPT-1 activity of soleus muscles were measured at 0, 0.5, 1, 3, and 5 h after cerulenin treatment. CPT-1 activity was found to be significantly elevated at 1 h, and this was maintained 3 h after treatment (Fig. 4Go), which supports the notion that the late stimulating effect of cerulenin on CPT-1 activity occurred via the central nervous system. Then we examined the direct effects of cerulenin on CPT-1 activity in primarily incubated hepatocytes. Cerulenin treatment was found to reduce CPT-1 activity in primary hepatocytes dose dependently (Fig. 5AGo). At 40 µg/ml, cerulenin reduced hepatocytes CPT-1 activity by up to 50% vs. the control. Therefore, we speculated that the initial reduction in CPT-1 activity might have been mediated through the direct effects of the malonyl-CoA elevation induced by FAS inhibition and that the delayed elevation of CPT-1 activity might have been mediated through another mechanism, an indirect central effect of cerulenin via the central nervous system, which leads to the biphasic response of CPT-1 activity by cerulenin.



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  		FIG. 4. Effects of icv administered cerulenin (Cer) on muscular CPT-1 activity. Mice were icv administered 10 µg cerulenin. At 0, 0.5, 1, 3, and 5 h after the injection, CPT-1 activities were measured in the soleus muscle. *, P < 0.05 and **, P < 0.01 vs. 0 h (n = 8).

 


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  		FIG. 5. Effects of cerulenin (Cer) and catecholamines (E and NE) on CPT-1 activity in primary cultured hepatocytes in vitro. Primary cultured hepatocytes were treated with cerulenin at 10, 20, 30, and 40 µg/ml, and CPT-1 activities were determined (A). Hepatocytes were treated with epinephrine or norepinephrine at 0.1 and 1 µg/ml in the presence (C) and absence (B) of an inhibitory concentration of cerulenin (10 µg/ml). *, P < 0.05, **, P < 0.01 and ***, P < 0.001 vs. control. ##, P < 0.01 vs. Cer10.

 
To determine whether the delayed stimulating effects of cerulenin on CPT-1 activity might be mediated through the activation of the SNS, we applied the adrenergic agonists, epinephrine and norepinephrine, to primary incubated hepatocytes. Epinephrine significantly enhanced CPT-1 activity at a 1 µg/ml, and norepinephrine also significantly enhanced CPT-1 activity at 0.1 and 1 µg/ml (Fig. 5BGo), whereas cerulenin inhibited CPT-1 activity (by 20% of the control level at 10 µg/ml). When catecholamines were applied with cerulenin to the incubation buffer, cerulenin did not inhibit CPT-1 activity. Even at high norepinephrine concentrations (1 µg/ml), CPT-1 activity remained higher than that of the control. (Fig. 5CGo). To examine whether increased CPT-1 activity by cerulenin is mediated via the activation of the SNS, we pretreated mice with AMPT before cerulenin treatment. Pretreatment with AMPT attenuated cerulenin’s stimulating effects on CPT-1 activity in liver and muscle (Fig. 6Go, A and B).



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  		FIG. 6. Effects of cerulenin (Cer) on CPT-1 activity in AMPT-pretreated and ADX mice. Cerulenin was administered as described above to either AMPT-pretreated or ADX mice. Three hours after cerulenin treatment, CPT-1 activity was determined in liver (A and C) and soleus muscle (B and D). *, P < 0.05 and **, P < 0.01 vs. control; #, P < 0.05 and ##, P < 0.01 vs. ADX (n = 8–12).

 
Catecholamines are known to be secreted from two main sources: the sympathetic nerves and adrenal glands. To determine whether the adrenal production of catecholamines is involved in the regulation of the CPT-1 activity in cerulenin-treated mice, we analyzed mice in which the adrenal medulla, the main source of circulating epinephrine, had been surgically removed. The CPT-1 activity of the liver was significantly reduced in ADX mice, whereas the CPT-1 activity of muscle was not significantly different in ADX and sham-operated mice (Fig. 6Go, C and D). Treatment with cerulenin significantly stimulated CPT-1 activity in ADX mice, and muscular CPT-1 activity reached similar levels to those of non-ADX cerulenin-treated mice.

To further study the central pathways required for SNS activation as mediated by cerulenin, we treated newborn mouse pups with MSG, which predominantly damages the ARC nucleus structures (27, 28). Baseline CPT-1 activity in the muscles of 8-wk-old MSG-treated mice was not significantly different from those of saline-treated mice. Moreover, an ip injection of cerulenin in MSG-treated mice significantly elevated muscular CPT-1 activity as in control mice. In MSG-treated mice, liver CPT-1 activity was found to be reduced to 25% of that of the control, and cerulenin treatment enhanced the activity (Fig. 7Go, A and B). This indicates that MSG-sensitive neurons were not responsible for the activation of the SNS. Immunohistochemical studies showed that MSG treatment did not damage the VMH neurons or other neurons outside the hypothalamus (data not shown). To study the roles of VMH neurons in the mediation of the cerulenin-induced activation of the SNS, we treated 4-wk-old mice with GTG, a compound that destroys glucose-sensitive neurons in the VMH (29). These mice were treated with cerulenin, and liver and muscular CPT-1 activities were analyzed at 8 wk after GTG treatment. GTG treatment was found to significantly reduce CPT-1 activity; however, cerulenin failed to stimulate CPT-1 activity (Fig. 7Go, C and D).



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  		FIG. 7. Effects of cerulenin (Cer) on CPT-1 activities in saline-treated nonobese and MSG- and GTG-treated obese mouse models. MSG- and GTG-induced obese mice were treated with cerulenin. Three hours after cerulenin treatment, liver (A and C) and soleus muscle (B and D) CPT-1 activities were measured. *, P < 0.05 and ***, P < 0.001 vs. saline; #, P < 0.05 vs. MSG (n = 10–12).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Recently there has been considerable interest in the role of fatty acid oxidation in the development of obesity and insulin resistance (30, 31, 32, 33). One aspect of insulin resistance has been explained by the abnormal accumulation of triglycerides in nonclassic fat-storing tissues, such as muscle and pancreatic ß-cells. Because fatty acid flux into muscle is similar in obese and lean subjects, it has been speculated that triglyceride accumulation in the skeletal muscle of obese subjects might be the result of reduced fatty acid oxidation (30). Accordingly, many studies have focused on the potential links between changes in fatty acid oxidation and the development of obesity.

Malonyl-CoA presents a prime means for sensing the fuel supply in the brain and peripheral organs (8). Malonyl-CoA also affects fatty acid oxidation by inhibiting CPT-1, which controls fatty acid entry into mitochondria for oxidation. Cerulenin (2,3-epoxy-4-oxo-6-dodecadienoylamide), an antifungal antibiotic found in culture of Cephalosporium caerulens, is a strong inhibitor of fatty acid synthesis; it blocks the condensation of acetyl and malonyl-CoA (34). A synthetic FAS inhibitor, C75, acted centrally to reduce food intake by altering in the levels of hypothalamic NPY and other hypothalamic peptides (3). In contrast to C75, a single injection of cerulenin did not reduce food intake significantly in this study, and our in situ hybridization study reconfirmed that a single injection of cerulenin does not affect the expressions of hypothalamic AgRP or NPY mRNA. MCH is also a strong orexigenic neuropeptide. However, no alteration in the expression of MCH was observed after treating cerulenin in the present study. We cannot explain this discrepancy between C75 and cerulenin on food intake. However, in previous studies (1, 4), cerulenin was also found to cause a preferential loss of adipose tissue, but this was not found to be significantly correlated with an altered food intake. Nevertheless, in the present study, without a significant reduction in food intake, a single injection of cerulenin significantly reduced body weight in ad libitum-fed mice. Furthermore, even in a fasted state, cerulenin reduced body weight by 15% vs. the control. The core temperatures of control mice given RPMI 1640 was found to drop dramatically during the 24-h period after food deprivation (Fig. 1DGo), which is consistent with the normal reduction of energy use observed after fasting (35). However, a single administration of cerulenin significantly prevented this core temperature reduction at 3 and 5 h after injection. Taken together, these results indicate that cerulenin induces weight loss by increasing energy expenditure rather than by reducing food intake.

Cerulenin as a natural FAS inhibitor induces the preferential reduction of adipose mass (36), and fatty liver (1) in the setting of cellular increased malonyl-CoA levels. The synthetic FAS inhibitor, C75, paradoxically stimulated CPT-1 activity and fatty acid oxidation by binding to CPT-1 (2). The difference of these two FAS inhibitors was partially explained by the presence of dicarbonyl groups and the amphipathic nature, which are required for direct contact with CPT-1, whereas cerulenin contains only a single dicarbonyl group in the cyclized and not in the amphipathic form (37, 38, 39). Previous in vitro study has revealed that cerulenin has no significant effect on CPT-1 activity (6); however, in the present study, CPT-1 activity was inhibited dose dependently by cerulenin. Even though cerulenin does not bind directly to CPT-1, it is a natural FAS inhibitor, which increases cellular malonyl-CoA, and, therefore, it should decrease CPT-1 activity indirectly. This discrepancy might be explained by the cerulenin dosage because only a single dose of 10 µg/ml was tested in the study of Thupari et al. (6). The most striking observation of this study was that a single ip injection of cerulenin produced a biphasic response, whereby the early-phase suppression of CPT-1 activity was followed by the late-phase stimulation. The biphasic response of CPT-1 activity to cerulenin led us to speculate that this delayed effect might be due to the indirect central effect of cerulenin, and here we found that the CPT-1 activity was peaked at 1 h after icv injection of cerulenin and that this was sustained for up to 3 h (Fig. 4Go). Taken together, these findings suggest that the central stimulating effect of cerulenin override the early peripheral inhibition of CPT-1 activity and that the modest and delayed central effect of cerulenin might be the major contributor to energy expenditure and the preferential reduction of adipose mass.

The autonomic nervous system is involved in processes whereby the brain controls energy expenditure. In an attempt to identify the central pathway responsible for the biphasic response of fatty oxidation and increased energy expenditure to cerulenin, we performed the following: 1) tested the in vitro effects of catecholamine, epinephrine, and norepinephrine on CPT-1 activity in the presence and absence of cerulenin; 2) pretreated mice with AMPT, an inhibitor of TH that is the rate-limiting enzyme of catecholamine synthesis; and 3) removed the adrenal glands before treating with cerulenin. As we had expected, cerulenin-induced inhibition of CPT-1 activity was overcome by cotreating with epinephrine or norepinephrine. Moreover, pretreatment with AMPT inhibited the cerulenin-induced increase in core temperature and the late-phase stimulating effect of cerulenin on CPT-1 activity in both liver and muscle. In ADX mice, cerulenin treatment enhanced the CPT-1 activity in both the liver and muscle. These findings suggest that a major portion of the cerulenin-induced late-phase stimulation of CPT-1 activity occurred through the activation of innervated SNS neurons. The cerulenin-induced delayed activation of CPT-1 through the SNS might be also partially explained by the recent observation that leptin induced the delayed activation of {alpha}2-AMP-activated protein kinase through the SNS because the activation of {alpha}2-AMP-activated protein kinase reduces ACC activity, which results in the stimulation of CPT-1 activity (40).

FAS is also expressed in the hypothalamus: ARC, VMH, and paraventricular nucleus (7). Although fasting and treatment with a FAS inhibitor significantly down-regulated FAS activity in the liver, FAS mRNA remained high in the hypothalamus, indicating that FAS is regulated differently in the brain (7). VMH neurons were found to mediate leptin-induced increases in catecholamine secretion (41), and a chemical lesion reduced the activity of the SNS (42). To further elucidate the central pathways involved in the cerulenin-mediated activation of the SNS, we used well-established MSG-treated (ARC lesion) (27, 43) and GTG-treated (VMH lesion) (29, 43) mouse models. Mice treated with MSG or GTG showed a significant increase in body weight, compared with saline-treated mice (26.2 ± 0.6 g vs. 29.5 ± 0.4 g, P < 0.05; 32.3 ± 0.8 g vs. 36.8 ± 0.5 g, P < 0.05, respectively). Cerulenin treatment enhanced CPT-1 activity in the liver and muscle of MSG-treated mice; however, it did not enhance CPT-1 activity in GTG-treated mice. In addition, initial hepatic CPT-1 activity decreased in MSG-treated mice. Previous studies (44, 45, 46, 47) have shown that MSG-treated mice became significantly obese and had higher levels of serum blood glucose, total cholesterol, and cholinesterase. Furthermore, compared with control mice, the MSG-treated mice revealed high levels of triglycerides in the liver and even showed evidence of fatty liver. By this pathologic change, MSG treatment might contribute to this unexpected initial suppression of CPT-1 activity. Taken together, our results suggest that enhanced CPT-1 activity by cerulenin is mediated through VMH activation, rather than through ARC nucleus activation, to activate the SNS. However, because both GTG and MSG may influence several populations of neurons and because both altered basal CPT-1 activity in this study, any interpretation concerning the ability of cerulenin to alter CPT-1 activity based on these animal models needs to be treated with caution. Further study is necessary to define the neuronal target of cerulenin in the VMH.

In conclusion, our findings provide some insight into the paradoxical effect of a FAS inhibitor, cerulenin, which was found to induce a biphasic response on CPT-1 activity by acting directly on the peripheral and indirectly in the central hypothalamic-SNS; moreover, we found that the central stimulating effect of cerulenin surpasses its peripheral inhibitory effect.


   Acknowledgments
 
We thank Dr. John Roberts for English language revision.


   Footnotes
 
This work was supported by research grants from the Korea Ministry of Health and Welfare (03-PJ1-PG1-CH05-0005) and by the Brain Korea 21 Project for Medical Sciences, Yonsei University.

Abbreviations: ADX, Adrenalectomy; AgRP, agouti-related protein; AMPT, {alpha}-methyl-p-tyrosine; ARC, arcuate nucleus; CPT-1, carnitine palmitoyltransferase-1; FAS, fatty acid synthase; GTG, gold thioglucose; icv, intracerebroventrically; MCH, melanin-concentrating hormone; MSG, monosodium glutamate; NPY, neuropeptide Y; SNS, sympathetic nervous system; TH, tyrosine hydroxylase; VMH, ventromedial hypothalamus.

Received January 14, 2004.

Accepted for publication March 17, 2004.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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