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Am J Physiol Endocrinol Metab 283: E867-E879, 2002. First published July 9, 2002; doi:10.1152/ajpendo.00178.2002
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Vol. 283, Issue 5, E867-E879, November 2002

TRANSLATIONAL PHYSIOLOGY
Expression of FAS within hypothalamic neurons: a model for decreased food intake after C75 treatment

Eun-Kyoung Kim1,*, Ian Miller1,*, Leslie E. Landree1, Felice F. Borisy-Rudin1, Pierre Brown1, Tarik Tihan3, Craig A. Townsend6, Lee A. Witters7, Timothy H. Moran5, Francis P. Kuhajda3,4, and Gabriele V. Ronnett1,2

Departments of 1 Neuroscience, 2 Neurology, 3 Pathology, 4 Oncology, and 5 Psychiatry, The Johns Hopkins University School of Medicine, Baltimore 21205; 6 Department of Chemistry, The Johns Hopkins University, Baltimore, Maryland 21218; and 7 Endocrine-Metabolism Division, Departments of Medicine and Biochemistry, Dartmouth Medical School, Hanover, New Hampshire 03755


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

We previously demonstrated that C75, a specific and potent inhibitor of fatty acid synthase (FAS), reduced food intake and decreased body weight in mice. In the present study, we determined that these effects were not due to conditioned taste aversion. To investigate the mechanism of C75 action, we examined FAS brain expression. FAS was expressed in a number of brain regions, including arcuate and paraventricular nuclei (PVN) within regions that comprise the arcuate-PVN pathway in mouse and human. Although C75 and fasting significantly downregulated liver FAS, FAS levels remained high in hypothalamus, indicating that FAS levels were regulated differently in brain from those in liver. Double fluorescence in situ for FAS and neuropeptide Y (NPY) showed that FAS co-localized with NPY in neurons in the arcuate nucleus. NPY immnuoreactivity after C75 treatment was decreased in axon terminals that innervate the PVN and lateral hypothalamus. Collectively, these results demonstrate that FAS is present and active in neurons and suggests that C75 may alter food intake via interactions within the arcuate-PVN pathway mediated by NPY.

appetite; feeding; obesity


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

THE ROLE OF DIET IN HEALTH has become a world-wide epidemiological issue (32). Eating disorders, from malnutrition to obesity, have serious medical complications, including type 2 diabetes (37). Understanding the mechanisms that regulate feeding is critical to the development of therapeutic interventions. Specific central nervous system (CNS) pathways integrate peripheral and central signals to regulate food intake (58, 62, 73). For example, insulin and leptin are "adiposity" signals that act upon central pathways to influence food intake (6, 72). The circulating level of each of these hormones corresponds to the degree of body fat (5, 45). Both hormones enter the brain (5, 7), where they play an important role in regulating neuropeptides involved in feeding control.

We previously demonstrated (44) that a potent, selective inhibitor of fatty acid synthase (FAS), C75, was able to reduce food intake and induce a profound loss of body weight. FAS catalyzes long-chain fatty acid synthesis through the condensation of acetyl-CoA and malonyl-CoA in a complex seven-step reaction (Fig. 1). Malonyl-CoA is synthesized by acetyl-CoA carboxylase (ACC) and is degraded back to acetyl-CoA by malonyl-CoA decarboxylase (MCD). Although the role of FAS in energy homeostasis (fat synthesis) could explain some of the effects of C75, a mechanism to explain the role of FAS in feeding is unknown. FAS has been previously localized to cells with high lipid metabolism, to hormone-sensitive cells, to a subset of epithelial cells of the duodenum and stomach, and to some neurons, including basket cells (39). It has remained unclear whether FAS is also found in brain regions involved with the regulation of feeding, where C75 could have a direct effect on FAS activity to influence the expression or release of peptides that control food intake. However, the finding that the potent anorexic effect of C75 was reversed following central administration of neuropeptide Y (NPY) suggested that a neuronal system/circuit involving both FAS and NPY might exist (44).


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  		Fig. 1.   The fatty acid synthetic pathway. ACC, acetyl-CoA carboxylase; FAS, fatty acid synthase; MCD, malonyl-CoA decarboxylase.

NPY is one of several neuropeptides expressed within the arcuate-paraventricular hypothalamic pathway, an important region of the brain where signals such as insulin and leptin are integrated to influence food intake (6, 15, 58, 62, 72). NPY (42, 64) and agouti gene-related peptide (AGRP) (29, 48, 54) are orexigenic peptides in medial arcuate neurons that act to increase food intake. Proopiomelanocortin (POMC)-expressing neurons in the lateral arcuate synthesize alpha -melanocyte-stimulating hormone (28), an anorexigenic peptide that decreases food intake. Cocaine- and amphetamine-regulated transcript (CART) (34, 35) acts to reduce feeding (36, 40). NPY and AGRP (NPY/AGRP) are co-localized in neurons in the arcuate nucleus (10, 29), whereas POMC and CART (POMC/CART) are expressed in distinct neurons in the arcuate nucleus (22).

NPY/AGRP and POMC/CART neurons in the arcuate project to second-order neurons, including those in the paraventricular nucleus (PVN), perifornical area (PFA), and lateral hypothalamus (LHA) (21, 23-25). The second-order target neurons selectively express additional neuropeptides that can induce feeding [melanin-concentrating hormone (MCH) (53, 59) and orexins (19, 47, 56) in the LHA] or inhibit feeding [corticotropin-releasing hormone (CRH) (18, 46, 63, 68)], thyrotropin-releasing hormone (TRH) (33), and oxytocin (49) in the PVN]. Thus insulin/leptin activate or inhibit neurons (and pathways) in the arcuate nucleus (NPY/POMC) and influence how PVN, PFA, or LHA neurons respond to increase or decrease food intake.

The aim of this study was to determine whether FAS is active in brain and/or is expressed in neurons, and whether these neurons are part of known feeding pathways. We demonstrated that the effect of C75 on food intake was not due to the development of a conditioned taste aversion. We then examined whether FAS and enzymes involved in fatty acid synthesis were present in cells in the arcuate-PVN pathway. We also measured FAS expression and activity in brain and in liver to determine whether the regulation of FAS is similar in these two organs. Finally, having previously demonstrated that NPY could reverse the feeding-inhibitory effects of C75 (44), we examined whether C75 affected NPY levels/expression in the arcuate-PVN pathway.


    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Animal experimentation. All animal experimentation was done in accordance with guidelines on animal care and use as established by the Johns Hopkins University School of Medicine Institutional Animal Care and Use Committee. Male BALB/c mice (20-25 g) were obtained from Charles River Laboratories. The mice were housed in a controlled-light environment (lights on 0700-2100) and allowed ad libitum access to standard laboratory chow and water unless otherwise noted. In all experiments, mice were divided into three groups, fed, C75-treated, and fasted. Fed mice were intraperitoneally injected with 200 µl of RPMI medium. C75-treated mice were injected intraperitoneally with 30 mg/kg body wt C75 in 200 µl of RPMI (which caused an 11-15% weight reduction in mice over 24 h). Fasted mice were injected intraperitoneally with 200 µl of RPMI, and food was withdrawn from the cage; however, mice were allowed ad libitum access to water.

Conditioned taste aversion. Eighteen female C57/B6 mice served as the experimental subjects. Five days before testing, mice were placed on a schedule of having 2 h of daytime access to water. On the test day, mice were divided into three groups and were given access to 0.15% sodium saccharin rather than water for 30 min. Immediately after saccharin access, mice were injected intraperitoneally with 0.15 M LiCl (20 ml/kg body wt), C75 (15 mg/kg body wt) diluted in RPMI vehicle (20 ml/kg body wt), or RPMI vehicle. This dose of C75 had been shown by Loftus et al. (44) to reduce 24-h food intake by >90% in female mice. Twenty-four hours later, mice were given 2-h access to a two-bottle choice test of 0.15% saccharin vs. water. Intakes of both solutions were recorded, and data were expressed as saccharin preference (100 × saccharin intake/saccharin intake + water intake). Data were analyzed by one-way ANOVA and planned t-comparisons.

RNA in situ hybridization and localization. Antisense and sense digoxigenin (DIG)-labeled RNA probes were generated by in vitro transcription with the appropriate T7, T3, and SP6 RNA polymerases (Roche) from the plasmids pCMVSPORT6 (ACCalpha , GenBank no. AW215260), pBSmACCbeta (ACCbeta , AI596735), pT3T7FASm (FAS, AI390803), and pBSmMCDp (MCD, AI892746) (Image Consortium). The paraffin sagittal and coronal sections of brain were purchased from Novagen. All sections were matched for level by use of Bregma coordinates and neuroanatomical landmarks. Pretreatment and hybridization steps were performed with modifications as previously described (9). Sections (7 µm) were deparaffinated by heating at 65°C for 10 min, soaking in HemoDe solution, and washing in graded ethanol solutions to rehydrate the tissue. The sections were postfixed in Bouin's solution for 20 min. After treatment with 0.1 M HCl, the sections were permeabilized with 50 µg/ml proteinase K (Roche) at 37°C for 30 min and treated with 0.1 M TEA-0.5% acetic anhydride for 10 min. The tissue was dehydrated with a series of ethanol washes. DIG-labeled probes were diluted in hybridization solution (2× SSC, 10% dextran sulfate, 0.01% sheared salmon sperm DNA, 0.02% SDS, 50% formamide) and placed on a 95°C hot plate for 4 min. Hybridized slides were incubated in a humid chamber containing 2× SSC and 50% formamide at 65°C for 18 h. After hybridization, sections were washed in 2× SSC for 20 min three times at 55°C, 1× SSC for 15 min, and 0.2× SSC for 15 min at room temperature. For immunodetection, slides were blocked in fresh blocking buffer (50 mM Tris · HCl, pH 7.5, 150 mM NaCl, 10% FCS) and incubated with anti-DIG-Fab-AP conjugate (Roche) diluted to 1:1,000 for 1 h at room temperature. After a wash with buffer (100 mM Tris · HCl, pH 9.5, 100 mM NaCl, 50 mM MgCl2), the sections were developed with 4-nitroblue tetrazolium chloride (Roche) and x-phosphate/5-bromo-4-chloro-3-indolyl-phosphate (Roche) and 0.025% levamisole.

Double-fluorescent in situ hybridization was performed according to the Nonradioactive In situ Hybridization Application Manual (a Boehringer Mannheim publication). Pretreatment and hybridization steps followed the same in situ method. For FAS (FITC), the same DIG-labeled riboprobe was used. For NPY (Texas red), biotin-labeled riboprobe was generated from plasmid containing NPY gene (GenBank no. XM004941) with T7 RNA polymerase. After hybridization and washing using the same conditions as described, slides were incubated with TNB buffer (100 mM Tris · HCl, pH 7.5, 150 mM NaCl, and 0.5% blocking reagent) for 30 min at 37°C. For FITC detection, sheep FITC-conjugated anti-DIG antibody (1:50; Roche) was incubated in TNB buffer for 30 min at 37°C. For Texas red detection, streptavidin-Texas red (1:50; Amersham Pharmacia), rabbit anti-Texas red antibody (1:50; Molecular Probes), goat biotin-conjugated anti-rabbit IgG antibody (1:50; Santa Cruz Biotechnology), and streptavidin-Texas red (1:30) were incubated serially in TNB buffer at 37°C, each for 30 min.

Immunohistochemistry. Male BALB/c mice were anesthetized with 200 µl of 20 mg/ml pentobarbital sodium and perfused transcardially first with 4°C phosphate-buffered saline (PBS), pH 7.4, and then with 4°C filtered 4% paraformaldehyde in 0.15 M sodium phosphate buffer (PFA), pH 7.4. After perfusion, brains were removed and postfixed overnight in 4% PFA and cryoprotected by sequential overnights in 10 and 20% sucrose in PBS, pH 7.4. Brains were frozen by means of powdered dry ice and were sectioned into 40-µm coronal sections with a sliding microtome.

Diaminobenzidine (DAB) immunohistochemistry was performed as previously described (13). Briefly, sections were incubated in blocking solution (5% nonfat dry milk, 2% normal serum, and 0.2% Triton X-100 in PBS) for 1 h at room temperature. They were then incubated overnight in primary antibody diluted in blocking solution at 4°C. Rabbit polyclonal antibody JH-3593 against mouse FAS 2 peptide (CLADLGLDSLMSAPVRQTLERE) was diluted at 1:2,000. To control for specificity, antiserum against FAS 2 peptide was preabsorbed for 24 h with FAS 2 peptide, which successfully blocked immunostaining (data not shown). To reduce background, JH-3593 was purified using a protein A column. After overnight incubation in primary antibody, sections were washed 3 × 10 min in PBS and then incubated in biotinylated secondary antibody (1:200) in 2% normal serum in PBS for 30 min. Sections were washed 3 × 10 min in PBS and incubated in avidin-biotin (Vector) solution for 30 min. They were washed 2 × 10 min in PBS and transferred to TN buffer (50 mM Tris · HCl, pH 7.4, 100 mM NaCl) for 10 min. Sections were developed using DAB (0.5 mg DAB/ml and 0.01% H2O2 in TN buffer).

For NPY projection experiments, immunostaining was repeated twice with three groups of two mice each for a total of 12 mice, fed (n = 4), C75 (n = 4), and fasted (n = 4). Mice were anesthetized and subsequently perfused 24 h posttreatment, and the brains were processed for immunohistochemistry as described. Sections were matched using Bregma coordinates -0.90 mm for the PVN, -1.64 mm for the arcuate, and -1.40 mm for the LHA. Rabbit polyclonal antibody against NPY (17) was diluted 1:20,000 and used in DAB immunohistochemistry as described. Mice in each group of six were processed exactly at the same time using the same reagents and developing time.

Immunohistochemistry of human brain sections. Autopsied brains from two males and two females (aged 40-65 yr) were selected for immunolocalization studies. All patients had died of causes other than CNS-related disorders and had no evidence of ischemia or hypoxia in comprehensive postmortem neuropathological evaluation. A thick coronal slice caudal to the mammillary bodies and rostral to the optic chiasm was removed, and 0.2-cm parallel sections were obtained from the hypothalamic region. The sections were fixed in 10% buffered formalin and embedded in paraffin. Ten serial sections (5 µm thick) were obtained and were stained with hematoxylin histochemical stain as well as Bielschowsky silver stain. Anti-human monoclonal antibody against FAS was previously characterized (27) and was used at 1:3,000 dilution. Antigen was detected using the LSAB2 system from DAKO with a DAKO Autostainer. All dilutions and incubations were kept constant, and all slides were stained in a single run with appropriate positive and negative controls. Immunostaining was visualized using 3-amino-9-ethylcarbazole (AEC), and sections were counterstained with hematoxylin. Similarly treated paraffin sections from breast and colonic adenocarcinomas, known to express high FAS activity, were used as positive controls. The sections were mapped on an overlayer grid to include the staining pattern of all patients, and the common staining pattern was mapped onto a coronal section of the hypothalamic region.

RNA preparation and Northern blot analysis. Hypothalamus and liver were harvested for RNA preparation, and total RNA was purified using TRIzol reagent (GIBCO-BRL). Hypothalamus was dissected using as landmarks the optic chiasm rostrally and the mammillary bodies caudally. Tissue was harvested to a depth of 2 mm. Northern blot analysis using 15 µg of total RNA was performed as previously described (4). RNA was separated on a 0.8% agarose, MOPS-formaldehyde gel and capillary transferred to Hybond N+ membrane (Amersham Pharmacia Biotech). RNA was then UV cross-linked using a Stratalinker (Stratagene) and prehybridized for 1 h at 42°C in Ultrahyb solution (Ambion). The blot was then hybridized with random primed 32P-labeled DNA probes (106 cpm/ml hybridization buffer) made by Amersham Pharmacia labeling kit for FAS (AI390803, 184-2545 nt), NPY (XM004941, 80-490 nt), CRH (AA189886, 1-546 nt), TRH (AA208912, 1-759 nt), and oxytocin (AA197698, 1-433 nt). The blot was washed three times at room temperature each for 10 min in 2× SSC and 0.1% SDS and three times each for 20 min at 65°C in 0.1× SSC and 0.1% SDS. As a loading control, the probe for GAPDH gene was used at the same blot. Signals were quantified using an image analyzer (Molecular Dynamics) and Imagequant software.

Western blot analysis. The dissected hypothalamus and liver tissues were homogenized in lysis buffer (50 mM Tris · HCl, pH 7.5, 50 mM NaF, 5 mM sodium pyrophosphate, 0.25 M sucrose, 1 mM EDTA, 1 mM EGTA, 1 mM DTT, 0.1 mM PMSF, and 0.5% SDS). The extracts were harvested by centrifugation at 12,000 rpm for 15 min at 4°C. Protein was measured with a Bradford assay kit (Bio-Rad) with bovine serum albumin as a standard. Sixty micrograms of protein per sample were run on 4-15% SDS-polyacrylamide gradient gel. The transferred membrane was blocked in TBST solution (10 mM Tris · HCl, pH 7.4, 150 mM NaCl and 0.1% Tween 20) containing 5% skim milk for 1 h at room temperature. Slides were incubated with sheep anti-FAS antibody (26) at 1:1,000 dilution in TBST solution containing 1% skim milk at 4°C overnight, and peroxidase-conjugated rabbit anti-sheep secondary antibody was used at 1:5,000 dilution in the same TBST solution for 1 h. After the blot was stripped, monoclonal anti-actin antibody, JLA29 (43) was used for a loading control. The signal was visualized by enhanced chemiluminescence (Pierce).

Activity of brain and liver FAS. To compare brain and liver fatty acid synthesis activity from fed, C75-treated, and fasted mice (3 mice/group, repeated twice; total n = 6), hypothalamus and liver tissue were harvested and ex vivo labeled using [14C]acetate. Lipids were extracted by the Folch method, and the organic phase was dried under N2 gas as described previously (52). The amount of labeled lipid was quantified by scintillation counting and expressed as counts per minute per milligram of protein. We normalized these values to relative activity and expressed them as a percentage of the fed control.

Microscopy. Images were visualized using an Axiocam HRc (Carl Zeiss) digital camera mounted directly on Axiophot microscope (Carl Zeiss). Fluorescent images were taken using a mercury arc lamp (Ushio). An arc lamp power supply (Carl Zeiss) and fluorescent filters that corresponded to the emission and absorption peaks for the riboprobes and antibodies were used. Images were acquired using Improvision Openlab software and analyzed using Adobe Photoshop on a Macintosh G4.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Conditioned taste aversion testing. Although we demonstrated that C75 reduced food intake and caused significant weight loss (44), the effect of C75 on feeding could have been due to illness or malaise. To assess this possibility, we examined whether C75 administration produced a conditioned taste aversion response (Fig. 2). Analyses of saccharin preference indicated a significant treatment effect, F(2,15) = 8.198, P < 0.004. Planned t-comparisons showed that LiCl administration resulted in a significant decrease in saccharin preference (26.6%) relative to both vehicle (70.1%) and C75 (61.1%). C75 administration had no effect on saccharin preference relative to vehicle control. Thus C75 does not appear to reduce food intake secondarily to the production of illness or malaise.


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  		Fig. 2.   Saccharin preference in mice after pairing of saccharin ingestion with vehicle, LiCl, or the FAS inhibitor C75. LiCl significantly reduced saccharin preference relative to vehicle control, whereas C75 administration did not. Data were statistically analyzed by planned t-comparisons. ** P < 0.01 vs. vehicle control.

Distribution of FAS, ACCalpha , ACCbeta , and MCD in brain. Previous studies (39) suggested that, although FAS is expressed in human brain, it is expressed predominantly in nonneuronal cells. To further elucidate the expression patterns of FAS and associated enzymes in the brain, in situ hybridization was performed on mouse sagittal brain sections by use of DIG-labeled antisense riboprobes for FAS, ACCalpha , ACCbeta , and MCD (Fig. 3). FAS message was expressed in olfactory bulb, cortex, hippocampus, cerebellum, brain stem nuclei, hypothalamus, and choroid plexus (Fig. 3A). With higher magnification, the cellular mRNA distribution of FAS was examined (Fig. 3, B-F). FAS was expressed in cells of the occipital cortex in a distribution consistent with a neuronal localization (Fig. 3B). In cerebellum, message was expressed in Purkinje cells and in the granule cell layer (Fig. 3C). FAS was seen in the frontal cortex in outer cortical layers (Fig. 3D), similar to that seen in occipital cortex. In olfactory bulb (Fig. 3E), FAS was expressed in cells in the olfactory nerve layer, the periglomerular layer, the mitral cell layer, and in the granule cell layer. Expression was also observed in the basal forebrain (Fig. 3F). FAS was also expressed in hypothalamic nuclei, which were further visualized in coronal section (Fig. 3K). Hybridization using a DIG-labeled sense probe was negative (Fig. 3G).


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  		Fig. 3.   In situ hybridization of FAS, ACCalpha , ACCbeta , and MCD in mouse brain. Paraffin sagittal sections were hybridized with the corresponding digoxigenin (DIG)-labeled antisense riboprobes (A-J), as described in MATERIALS AND METHODS. Message was detected for FAS (A-F and K), ACCalpha (H and L), ACCbeta (I and M), and MCD (J and N). High-magnification photomicrographs show the distribution of FAS within specific subdivisions of the cells of the occipital cortex (B), cerebellum (C), frontal cortex (D), olfactory bulb (E), and basal forebrain (F). Hybridization using a DIG-labeled sense FAS probe was used as a negative control (G). In situ hybridizations for all 4 enzymes (K-N) were performed with hypothalamic coronal sections (symbol I in A). ARC, arcuate nucleus; VMH, ventromedial hypothalamus; DMH, dorsomedial hypothalamus.

DIG-labeled antisense riboprobes for three additional enzymes that generate (ACC) and degrade (MCD) malonyl-CoA, the substrate required by FAS, were also used to determine whether this biosynthetic pathway was present in the brain (Fig. 3, H-J). Their expression patterns were very similar to the pattern of FAS (Fig. 3, H-J). Mammals have two isoforms of ACC, ACCalpha and ACCbeta , which are encoded by different genes and distributed in distinct tissue (1, 2, 8). Whereas ACCalpha is expressed mainly in liver and adipose tissue, ACCbeta is highly expressed in heart and muscle. Interestingly, these two isoforms showed similar expression patterns in brain (Fig. 3, H and I). As with the sense FAS riboprobe (Fig. 3G), the other sense riboprobes corresponding to each protein did not show any significant signals (data not shown). Coronal sections were used to examine the expression of the message for these enzymes in the arcuate (Fig. 3, K-N). All four enzymes were also expressed in ventromedial (VMH) and dorsomedial hypothalamus (DMH) in addition to the arcuate nucleus.

To confirm these patterns of mRNA expression, immunohistochemistry was performed (Fig. 4). Mouse sagittal sections were stained with an anti-mouse FAS antibody (Fig. 4A). The distribution of FAS-positive cells was similar to that seen for FAS mRNA expression by in situ hybridization. Correspondingly, the immunohistochemistry data for ACCalpha , ACCbeta , and MCD showed the same expression patterns compared with in situ hybridization (data not shown). FAS immunoreactivity was clearly seen within the arcuate nucleus (Fig. 4B) and the PVN (Fig. 4C). These results indicated that the enzymes involved in the regulation of fatty acid synthesis are widely distributed in brain and are present in the hypothalamus in a number of nuclei, including the arcuate and the PVN. Thus FAS was expressed in the appropriate nuclei to influence peptides involved in feeding behavior.


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  		Fig. 4.   Immunohistochemical localization of FAS in mouse paraventricular (PVN) and arcuate nuclei. A: floating sagittal sections were prepared and immunostained with anti-FAS antibody. Hypothalamic floating slices containing the arcuate nucleus (B) and PVN (C) were further examined. FAS immunoreactivity was seen throughout the arcuate nucleus, median eminence, and PVN.

FAS immunohistochemistry in human hypothalamus. To determine whether FAS was expressed in human hypothalamic neurons, immunohistochemistry for FAS was performed using paraffin sections of human hypothalamus and anti-human FAS antibody (Fig. 5). Sections were prepared from patients who died of causes other than CNS-related disorders; these sections displayed no evidence of ischemia or hypoxia at the time of comprehensive postmortem neuropathological evaluation. Immunostaining was visualized by AEC, and sections were counterstained with hematoxylin.


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  		Fig. 5.   Immunohistochemical localization of FAS in human hypothalamus. Paraffin sections were obtained from human postmortem brains and processed as described in MATERIALS AND METHODS. Immunostaining was visualized with 3-amino-9-ethylcarbazole, and sections were counterstained with hematoxylin. A: line drawing of human hypothalamus depicting the relative density of immunostaining for FAS in these regions (stippling); boxes depict the region of hypothalamus visualized in B, C, and D. Pv, periventricular region; A, arcuate nucleus; f, fornix. B: light micrograph of immunostaining of paraventricular region (Pav). C: light micrograph of immunostaining of VMH region (Vm). D: light micrograph of immunostaining of arcuate nucleus.

The distribution of the staining among the hypothalamic nuclei is summarized in Fig. 5A. The relative density of immunoreactive cells is represented by dots on the right side of the line drawing. FAS immunoreactivity was localized to human hypothalamus in a punctate somatic expression pattern (Fig. 5, B-D) similar to that seen in mouse brain (Fig. 4). Sections from all cases demonstrated a highly reproducible staining pattern, predominantly highlighting the neuronal perikaria. Endothelial and glial cells showed no demonstrable immunoreactivity. The majority of the neurons in the arcuate, the ventromedial nuclei, and the periventricular zone showed strong immunoreactivity for FAS (Fig. 5, B-D). In addition, there were scattered positive neurons within the dorsomedial nuclei. Neurons without significant staining could be distinguished even in the regions where the staining was the strongest. The staining pattern did not differ to any significant extent among the patients. Thus FAS immunoreactivity was detected in neurons in a number of brain regions in human, including hypothalamic sites that are involved in the regulation of feeding behavior.

Comparison of FAS levels in hypothalamus and liver from fed, C75-treated, and fasted animals. Fatty acid synthesis occurs during periods of energy surplus. Concomitantly, FAS expression is downregulated in liver during starvation (50). To investigate the regulation of hypothalamic FAS expression, we compared FAS mRNA levels in hypothalamic and liver tissues in fed, C75-treated, and fasted mice (Fig. 6A). One group of adult mice (n = 3) was maintained on a normal diet and fed ad libitum, a second group was treated with C75 (30 mg/kg body wt), whereas a third group was denied access to food for 24 h before tissue harvesting. As reported by Paulauskis and Sul (50), a single 8.2-kb mRNA for FAS was detected in hypothalamus and liver (Fig. 6A). Mouse hypothalamus harvested from the fed controls expressed significant levels of FAS message, similar to those in liver.


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  		Fig. 6.   Regulation of FAS expression in the hypothalamus (left) and liver (right) from fed, C75-treated, and fasted mice. A: hypothalamus and liver were harvested for RNA and protein extract as described in MATERIALS AND METHODS. Northern blot analysis was performed for FAS mRNA levels in hypothalamus and liver. Relative amounts of RNAs were normalized to the amount of control GAPDH signal (data not shown) and expressed as % relative to the mRNA level of control fed mice (in graph). Graphs represent the averages ± SE from quantification of blots from 3 mice each. B: FAS Western analysis was performed with hypothalamic and liver tissue from 2 mice each in 3 groups. C: FAS activity was measured using hypothalamic or liver tissue from 6 mice each per group. Levels of [14C]acetate incorporation into fatty acids were measured from tissue lysates. Average values from each tissue were reported as cpm/mg of total protein ± SE. We normalized these values to relative activity and expressed them as % of control. Data were statistically analyzed by the t-test. * P < 0.05 and ** P < 0.01 vs. fed group.

The regulation of hypothalamic FAS in response to C75 treatment or starvation differed from that of liver FAS. Hypothalamic FAS mRNA levels were not significantly reduced by either fasting or C75 administration. FAS mRNA levels remained at 83.1 ± 0.69 and 72.8 ± 0.98% of control in fasted and C75-treated mice, respectively, compared with fed control mice (Fig. 6A). In contrast, C75 treatment or fasting for 24 h resulted in significant reductions of liver FAS mRNA expression to 14.47 ± 0.40 and 14.58 ± 0.38%, respectively, of control fed animals (Fig. 6A). Treatment of animals on an every-other-day regimen with C75 (10 mg/kg body wt) for 2 wk had no effect on FAS mRNA level in the hypothalamus (data not shown). Western blot analysis revealed that FAS protein levels followed mRNA levels in the hypothalamus and liver (Fig. 6B). The correlation of mRNA message and protein levels is consistent with reported results that FAS expression is regulated at the transcriptional level (50). These results indicated that hypothalamic FAS levels were not altered by feeding status or C75 administration and suggested that transcriptional control of hypothalamic FAS message was different from that in liver.

FAS enzyme activity assays were also performed at 24 h to correlate FAS protein and message expression with activity (Fig. 6C). Twenty-four hours after a single dose of C75, we determined that C75 had been cleared from the brain (44) and therefore could not inhibit FAS activity directly. Thus FAS activity at 24 h should reflect the level of FAS protein expression. FAS activity was measured by determining the amount of [14C]acetate incorporated into lipid. Substantial activity was found in control hypothalamus compared with liver, suggesting that there is indeed a high level of fatty acid biosynthesis in the brain. In contrast to liver, in which FAS activity was reduced with C75 treatment and starvation to ~55 and 60% of control, respectively, hypothalamic FAS activity was not decreased. Collectively, the results obtained by Western and Northern blot analyses and enzyme activity assays indicate that FAS is not downregulated by starvation or C75 treatment.

NPY-FAS co-localization. To further investigate the mechanism by which C75 treatment resulted in a decrease in NPY message expression in the hypothalamus (44), we determined whether NPY and FAS were localized within the same neurons within the hypothalamus, specifically the arcuate nucleus (Fig. 7A). FAS message was detected by immunofluorescence in cells of the arcuate nucleus, consistent with results obtained using DIG-labeled antisense probes (Fig. 3F) and DAB immunohistochemistry (Fig. 4B). NPY message was detected in arcuate nucleus (Fig. 7B). Overlay of the two images revealed that FAS and NPY were co-expressed in the same neurons (Fig. 7C). NPY was expressed in a subset of neurons expressing FAS. These results indicate that FAS is positioned to influence NPY expression in a cell-autonomous manner.


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  		Fig. 7.   Double-fluorescence in situ hybridization of FAS and neuropeptide Y (NPY) in arcuate nucleus. Paraffin coronal sections of mouse brain containing arcuate nucleus were hybridized with FITC and Texas red-conjugated DIG-labeled riboprobes for FAS (A) and NPY (B), respectively. Overlay of the images in A and B revealed co-localization of the FAS and NPY (C).

Changes in NPY mRNA level and its projections to the hypothalamus following C75 treatment. We (44) previously demonstrated that C75 blocked the normal increase in hypothalamic NPY mRNA levels induced by decreased food intake and that the effect of C75 on feeding was reversed by central infusion of NPY. Consistent with this result, C75 inhibited NPY transcription and the fasting-induced increase in NPY mRNA levels in the hypothalamus (Fig. 8A). Compared with fed controls, the level of NPY mRNA in C75-treated mice was decreased to 58.97 ± 1.52%, and in fasted mice NPY was upregulated to 156.6 ± 4.79%. These changes in NPY mRNA levels were different from other published data (60), which showed no change and an 85% increase in NPY mRNA in C75-treated and fasted mice, respectively. Although FAS mRNA levels in the hypothalamus 24 h after C75 treatment were not changed compared with fasted mice (Fig. 6A), C75 treatment decreased NPY message compared with controls in our case (Fig. 8A).


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  		Fig. 8.   Effect of C75 on NPY expression in arcuate, projection to PVN, and second-order molecule expression from fed control, C75-treated, and fasted mice. A: NPY mRNA levels in hypothalamus of 3 groups (3 mice each) were determined by Northern blot analysis. B: at the levels of the arcuate nucleus (1, 2, 3), PVN (4, 5, 6), and lateral hypothalamus (LHA; 7, 8 9), the changes in NPY immunoreactivity were detected in fed (1, 4, 7), C75 treated (2, 5, 8) or fasted (3, 6, 9) animals. C: mRNA levels of CRH, oxytocin, and TRH in hypothalamus from fed (open bar), C75-treated (filled bar), and fasted (gray bar) mice (3 mice each) were analyzed in the same blot. All graphs show the quantification of Northern results by normalization and analysis as means ± SE, the same as in Fig. 6A. Data were analyzed statistically with the t-test. * P < 0.05 and ** P < 0.01 vs. fed group.

Many hypothalamic areas, including the PVN, PFA, and LHA, are richly supplied by axons from the NPY/AGRP neurons in the arcuate nucleus (21, 23). Because hypothalamic NPY mRNA expression was affected by C75, we assessed whether C75 affected the level and distribution of NPY peptide in the arcuate nucleus, PVN, and LHA (Fig. 8B). Within the arcuate nucleus, there was no obvious change in NPY immnuoreactivity seen in either cell somas or axons following C75 treatment (Fig. 8B, 2), compared with controls (fed animals; Fig. 8B, 1). Fasted animals did display increased immunoreactivity within the arcuate (Fig. 8B, 3). Control animals demonstrated prominent NPY-immunoreactive axons in the PVN (Fig. 8B, 4). At 24 h post-C75 treatment, NPY-immunoreactivity in the PVN was reduced (Fig. 8B, 5). The reduced NPY immunostaining appeared to be due to decreased NPY levels within the axons and not simply a reduction in total axon density, since neurites were observed at a similar density in the control sections by using Nomarski optics, albeit with weaker staining. In contrast, NPY immunoreactivity was increased in the PVN in fasted animals (Fig. 8B, 6). NPY immunoreactivity was also seen in the LHA in control-fed animals (Fig. 8B, 7). C75 decreased the immunostaining of NPY projections in the LHA (Fig. 8B, 8), whereas fasting increased immunoreactivity in these projections as expected (Fig. 8B, 9). Taken together, these results indicate that C75 resulted in decreased NPY levels within specific regions of the hypothalamus, which could be, in part, responsible for the reduction in food intake.

PVN neurons produce several neuropeptides; among these, CRH (41), TRH (69), and oxytocin (49) have been implicated in the regulation of food intake and body weight. To investigate the effect of the reduction of NPY in the projections from the arcuate on CRH, TRH, and oxytocin levels, we performed Northern blot analysis on hypothalamic tissue with cDNA probes to these peptides (Fig. 8C). If these PVN neurons are second-order effectors located downstream of NPY projections, the levels of the peptides made by these neurons might be altered by the C75-induced changes in NPY levels. Among these anorexigenic peptide candidates, CRH was upregulated to 179.3 ± 7.52% in C75-treated mice and downregulated to 57.33 ± 1.92% in fasted mice. Oxytocin expression was also significantly increased by 144.8 ± 7.44% in C75-treated mice, whereas its expression did not change (95.3 ± 0.64%) in fasted mice. TRH did not change significantly in either C75-treated (98.37 ± 0.29%) or fasted mice (102.8 ± 0.95%). These results show that C75 treatment resulted in alteration of CRH and oxytocin gene expression, presumably by relieving the inhibitory effect of NPY on these neurons (30).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

In this study, we investigated the CNS expression and regulation of FAS to provide a framework for elucidation of the mechanism(s) by which C75 may mediate its anorectic and weight loss effects. The effects of C75 on food intake are unlikely secondary to illness or malaise, since doses of C75 that resulted in greater than a 90% suppression of 24-h food intake (44) did not induce a conditioned taste aversion. The ability of a compound to reduce food intake is not sufficient evidence to conclude that the involved pathways and signaling systems play a role in energy regulation. Anorexia can be induced by a variety of states, including motor impairments, behavioral disorganization, or aversive states. The conditioned taste aversion paradigm employed here provides an assessment of C75's ability to produce an aversive state arising from such things as visceral illness or malaise. Although previous work has shown that the dose of C75 employed in this assessment produces up to a 90% decrease in food intake (44), this dose failed to support the development of a conditioned taste aversion. C75-treated mice showed a postexposure preference for saccharin that was similar in magnitude to that of vehicle-treated mice and significantly greater than that seen in LiCl-treated mice. This result suggests that the anorectic actions of C75 are not secondary to a learned aversion to the diet or secondary to malaise. Such findings provide support for the view that the anorectic actions of C75 may be mediated through systems normally involved in feeding control.

A central mechanism of action for C75 is also supported by the localization of FAS to neurons in the hypothalamus. FAS was previously localized to hormone-sensitive cells, proliferating fetal tissues, and cells with high lipid metabolism and was detected in the CNS only in scattered cortical neurons and basket cells in the cerebellum (39). We demonstrate that FAS is present and enzymatically active in the brain, where FAS is expressed along with other enzymes involved in fatty acid synthesis, namely ACCalpha , ACCbeta , and MCD. Using in situ hybridization and immunohistochemistry, we show that FAS is expressed in a number of brain regions, including cortex, olfactory bulb, cerebellum, brain stem nuclei, and hypothalamus. FAS was detected in some axonal tracts, such as the olfactory nerve layer in the olfactory bulb, but the most prominent expression was in neurons. FAS is localized to neurons in hypothalamic pathways known to regulate food intake, including the arcuate and paraventricular nuclei. FAS has a similar localization in human hypothalamus. The variability in expression pattern often obtained with immunohistochemistry may explain the differences between our results and those of previous studies, as immunostaining is influenced by tissue preparation and antibody reactivity. In contrast to liver FAS, the levels of hypothalamic FAS message and protein are not downregulated by fasting or C75 treatment. These findings suggest that C75 may act on hypothalamic pathways to mediate its effects and position FAS to have a potential role in modulating these pathways.

The direct co-localization of FAS and NPY to neurons in the medial hypothalamus provides a mechanism whereby C75 can influence NPY production. To directly address a role for NPY in the ability of C75 to inhibit feeding, immunohistochemistry was performed to determine whether C75 treatment affected NPY levels in the arcuate and other regions of the hypothalamus. The reduction in NPY immunoreactivity in axons innervating the PVN and LHA with C75 treatment suggests that C75 may affect projecting NPY neurons in the arcuate nucleus by inhibiting FAS within the same neuron. This suggestion can be supported by our results that C75 inhibition of FAS in NPY-expressing arcuate neurons reduced NPY synthesis primarily at the mRNA level, decreasing NPY peptide levels in axon terminals that innervate the PVN and LHA.

The decrease in NPY immunoreactivity in these projections was correlated with an increase in mRNA levels of putative anorexigenic effectors such as CRH and oxytocin. Alternatively, NPY transport along axon terminals may have been affected if FAS is involved in vesicular packaging and transport to synapses, but this is unlikely, as NPY immunoreactivity did not increase in cell bodies within the arcuate nucleus. In the brain, NPY is produced in many cell populations (14, 16), and it has been reported that some of the NPY fibers in the PVN originate in brain stem nuclei (11, 12, 57). Even though we cannot exclude the possibility that NPY levels in brain stem nuclei are affected, contributing to the change in NPY immunoreactivity in the PVN, a significant contribution to the decrease of NPY immunostaining in the PVN may be attributed to changes in the arcuate nucleus, as C75 reduced NPY mRNA levels in the arcuate.

These results suggest that the actions of C75 on FAS/NPY-containing neurons in the medial arcuate nucleus could contribute substantially to C75-induced reductions in food intake. It is unlikely that FAS itself can regulate the NPY expression directly. Rather, inhibition of FAS activity by C75 may result in changes in substrate flux, such as increasing malonyl-CoA levels (44) to affect the cellular perception of energy status. Although it is known that arcuate NPY mRNA expression is activated by reduced leptin (71), an effect of C75 on leptin signaling appears unlikely, as the effects of C75 are independent of leptin (44).

Although CNS pathways play a role in controlling energy homeostasis, the roles of FAS and endogenous fatty acid synthesis in the brain are unknown. Endogenous synthesis might be presumed to be unnecessary, as fatty acids are transported into the brain. It is highly unlikely that brain FAS functions to produce palmitate for energy storage, because neurons preferentially utilize glucose and ketone bodies as energy sources, whereas astrocytes use fatty acids (20). Endogenous palmitate synthesis may serve several functions that could vary in different brain regions. The demand for palmitate for structural needs in some brain regions may require local production. Fatty acids may be produced for myelination, axonal transport, or vesicular packaging and release. Furthermore, FAS may serve different functions during development and in the adult brain.

The finding that hypothalamic FAS levels are not altered by food intake further supports the hypothesis that FAS does not function merely in energy storage in the brain. It has been previously reported that lipogenic enzymes are regulated in a tissue-specific manner (31). Within the liver, expression of the FAS gene is regulated primarily at the transcriptional level and is responsive to feeding status, nutrients, and hormones such as glucose, insulin, glucagons, glucocorticoids, and thyroid hormone (51, 61, 65). Here, we show that FAS mRNA, protein levels, and enzyme activity remained high in brain after starvation or C75 treatment compared with liver, demonstrating that brain FAS is regulated differently than liver FAS. Endogenous FAS activity that is maintained even when systemic energy sources are low may ensure the continued production of palmitate for membrane and vesicle production. Our data do not demonstrate a normal physiological role for FAS in the response to food intake, merely that inhibition of FAS results in decreased NPY expression in the hypothalamus. This pathway may be fortuitously manipulated by C75 to signal a change in the neuronal perception of energy availability, mimicking a fed state.

Flux through the fatty acid synthetic pathway is indeed monitored in other tissues. In muscle, malonyl-CoA levels regulate carnitine palmitoyltransferase I (CPT I), the rate-limiting enzyme for entry of long-chain acyl-CoAs into the mitochondria for fatty acid oxidation (66). During conditions of energy excess, malonyl-CoA is generated for fatty acid synthesis, and it inhibits CPT I, preventing the oxidation of newly synthesized fatty acids destined for storage. The opposite occurs during starvation, when malonyl-CoA levels fall, favoring fatty acid oxidation. FAS, although a complex multifunctional enzyme, is not the regulated step in fatty acid synthesis. Rather, ACC is negatively regulated by long-chain fatty acyl-CoAs, and citrate is an allosteric activator of ACC (55). It is possible that monitoring of the fatty acid synthetic pathway occurs in neurons to sense energy status. The maintenance of hypothalamic FAS levels independent of diet indicates that FAS activity might be responsive to fluctuations in substrate availability (acetyl-CoA), which reflects energy status. Whether FAS has a physiological role in feeding remains to be determined. At present, our data suggest that blockade of FAS synthesis and buildup of malonyl-CoA may affect food intake.

Our recent data indicate that C75 acts not only centrally to reduce food intake but also peripherally to increase fatty acid oxidation by increasing CPT I activity (67). This action of C75 explained a paradox that we observed. C75 reduced adipose tissue and fatty liver despite high levels of malonyl-CoA, which should block the oxidation of fats by inhibiting CPT I. C75-treated diet-induced obese (DIO) mice displayed greater weight loss and increased production of energy through increased fatty acid oxidation compared with pair-fed controls. Etomoxir, an inhibitor of CPT I, reversed the C75-induced increased energy expenditure seen in DIO mice, confirming that C75 was likely affecting CPT I. Thus, C75 acts both centrally to reduce food intake and peripherally to increase fatty acid oxidation, leading to rapid and profound weight loss, while inducing loss of adipose mass and resolution of fatty liver.

Several issues concerning the long-term use of C75 remain to be addressed. Here, we focused on the effect of C75 on hypothalamic NPY levels 24 h after a single treatment. It will be important to examine neuropeptide levels after chronic dosing of C75 to ascertain C75's potential therapeutic utility. According to a recent report (38), tolerance to C75 was induced more quickly in lean mice than in ob/ob or DIO mice by use of a daily dosing regimen. Tolerance to C75 could reflect downregulation of FAS with extended C75 treatment; however, our data do not support this conclusion. The tolerance to C75 observed by Kumar et al. (38) could be due to the daily dosing regimen or could reflect a decreased effect of C75 in lean animals that do not have as much fat to serve as a substrate for CPT I- mediated oxidation. Our studies using an every-other-day dosing regimen (67) indicate that mice do not develop tachyphylaxis to C75 treatment. Animals treated for 2 wk continued to display reduced food intake and weight loss.

Understanding the regulation of food intake and body weight is crucial in designing rational therapies for a spectrum of diseases, from malnutrition to obesity. Our studies suggest that FAS may serve as a target for pharmacological manipulation of feeding. C75 also has peripheral effects that result in weight loss. The effect of C75 on other aspects of metabolism needs to be determined, as the hypothalamus not only regulates food intake but is central in the regulation of thermogenesis, endocrine responses, and circadian systems (3, 70, 74), to name but a few.


    ACKNOWLEDGEMENTS

This work was supported by National Institutes of Health Grants DC-02979 to G. V. Ronnett, CA-87850 to F. P. Kuhajda, DK-19302 to T. H. Moran, DK-35712 to L. A. Witters, and National Institute of Neurological Disorders and Stroke Grant F-32 to L. E. Landree.


    FOOTNOTES

* These authors contributed equally to this work.

Funding for the study described in this article was provided by FASgen, LLC. Under a licensing agreement between FASgen and the Johns Hopkins University, G. V. Ronnett, F. P. Kuhajda, and C. A. Townsend 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, and G. V. Ronnett has an interest in, FASgen stock, which is subject to certain restrictions under University policy. The Johns Hopkins University, in accordance with its conflict of interest policies, is managing the terms of this arrangement.

Address for reprint requests and other correspondence: G. V. Ronnett, Dept. of Neuroscience, 1006B Preclinical Teaching Bldg., Johns Hopkins Univ. School of Medicine, 725 North Wolfe St., Baltimore, MD 21205 (E-mail: gronnett{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.

July 9, 2002;10.1152/ajpendo.00178.2002

Received 29 April 2002; accepted in final form 26 June 2002.


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Am J Physiol Endocrinol Metab 283(5):E867-E879
0193-1849/02 $5.00 Copyright © 2002 the American Physiological Society


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