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APPETITE, OBESITY, DIGESTION, AND METABOLISM
Departments of 1Psychiatry and Behavioral Sciences, 3Neuroscience, and 4Pathology, Johns Hopkins University School of Medicine, Baltimore; and 2FASgen, Inc., Baltimore, Maryland
Submitted 17 January 2006 ; accepted in final form 13 February 2006
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONDISCLOSURESGRANTSREFERENCES |
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fatty acid synthase; carnitinepalmitoyl transferase-1; locomotor activity
Our recent work indicates that the mechanism by which C75 reduces food intake involves an increase in hypothalamic ATP with subsequent reduction of hypothalamic levels of the active, phosphorylated form of AMP-activated protein kinase (AMPK) (15). AMPK is a cellular fuel sensor that, when activated in response to stressors that reduce ATP, downregulates biosynthetic pathways that consume ATP and upregulates catabolic pathways that generate ATP (11). Recent work by Minokoshi et al. (23) has identified AMPK as a molecule with broad significance in the hypothalamic control of energy intake.
Decreases in food intake and body weight in response to central C75 have been reported, and intracellular mechanisms through which the actions of C75 are transduced have been proposed. However, how these effects are translated into changes in food intake and body weight have yet to be identified. In this work, we characterize behavioral responses to central C75, focusing on how C75 affects ongoing patterns of food intake, and investigate whether feeding changes can account for the C75-induced alterations in body weight. We then probe for potential brain neuropeptide mechanisms to explain the hypophagia and weight loss responses to C75. Our studies show that intracerebroventricular (ICV) C75 can reduce body weight more than can be accounted for by the hypophagia and that the weight loss is maintained for many days. Furthermore, we show that ICV C75 reduces food intake by decreasing meal frequency not meal size. The decreased appetitive drive coincides with a reduced expression of mRNA for agouti-related peptide (AgRP), implicating changes in melanocortin signaling as a key event downstream of C75’s direct actions in the brain.
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONDISCLOSURESGRANTSREFERENCES |
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Male Sprague-Dawley rats (200–250 g) obtained from Charles River (Kingston, NY) resided in 22 ± 2°C vivaria on a 12:12-h light-dark cycle. They were housed individually in hanging wire cages or in Plexiglas chambers equipped with pellet dispensers and nesting boxes and were weighed daily. Rats were adapted to these conditions for 1 wk before undergoing cannulation of the lateral cerebroventricle. All animal protocols were approved by the Johns Hopkins University Animal Care and Use Committee.
Chronic Lateral Cerebroventricle Cannulation
Rats were anesthetized with a mixture of xylazine (8.57 mg/kg) and ketamine HCl (57.14 mg/kg) administered intramuscularly. They were then positioned in a stereotaxic instrument with the incisor bar adjusted to achieve a flat-skull position. A hole drilled into the skull 1.0 mm caudal to bregma and 1.3 mm lateral to midline accommodated a 23-gauge stainless steel cannula aimed at the lateral ventricle and lowered to 5.0 mm below dura. The cannula was secured in place with dental cement adhered to stainless steel screws implanted in the skull. A 30-gauge stainless steel obturator inserted into the cannula maintained its patency. Rats received penicillin (60,000 units im) and banamine (1 mg/kg im) to prevent postoperative infection and pain.
ICV Injections
ICV injections were performed with a microliter syringe (Gilmont Instruments) attached to polyethylene tubing and a 30-gauge stainless steel injector, the tip of which extended 1.5 mm past the cannula into the lateral ventricle.
Tests for Cannula Placement and Patency
After rats recovered from surgeries for 1 wk, we assessed cannula placements by measuring water intake in response to ICV angiotensin II (ANG II; Sigma, St. Louis, MO). Rats deprived of water for 1 h were given ANG II (50 ng/5 µl) or sterile 0.9% saline vehicle via the ICV cannula, and allowed 30-min access to water from graduated tubes. Rats whose water intake after ANG II was at least 5 ml greater than intakes after saline were used in the experiments. When the behavioral studies were concluded, cannulas were tested again for patency. We analyzed data only from animals with functional cannulas at that time.
Drugs
FASgen provided the C75 (mol wt = 254.15). We injected rats with sterile RPMI-1640 1x medium containing 2 g/l glucose (11.1 mM) (Cambrex) as vehicle (RPMI, 5 µl), or half-log- and quarter-log-step doses of C75 (10, 32, and 56 µg; 39, 126, and 220 nmol) dissolved in RPMI in the lateral cerebroventricle 5 min before dark onset and food access in all experiments.
Experiments
Food intake and body weight. Ten cannulated rats in hanging wire cages had 22-h access to water and chow beginning at the onset of dark, which permitted 2 h at the end of the light for preparations between trials. Rats received a single ICV injection of RPMI or a dose range of C75 dissolved in RPMI. We measured daily intakes of chow, corrected for spillage, for at least 3 days after each injection and allowed at least 4 days for rats to return to preinjection body weights. C75 doses were given in the following order: 0, 32, 10, 56 µg. Data included in the analyses were from the seven rats with functional cannulas at the end of the study.
Locomotor activity. We moved 10 rats from hanging wire cages into open-field test chambers (Digiscan; Accuscan Instruments, Columbus, OH) to measure locomotor activity responses to ICV C75. The system consists of a 40-cm2 x 30-cm Plexiglas chamber surrounded by sensor panels that emit infrared beams across the open field. Each beam interruption is detected, and patterns of beam interruptions are analyzed to yield measures of locomotor activity. Activity counts were tallied and stored on file on a computer.
Rats remained in the monitors for 3 days with ad libitum access to food and water. The first day was considered a day of acclimation to the novel environment. On the next day, their activity was monitored in response to the ICV RPMI vehicle, and on the last day, their activity was monitored in response to 32 µg of C75. Injections were given just before onset of dark. Locomotor activity, as the number of beam interruptions in the horizontal plane, was monitored on all 3 days, in 11 bins of 2-h duration, for a total of 22 h each day, allowing 2 h for preparations between trials.
Meal patterns. In a separate group of rats, we examined patterns of food intake to further characterize the time course and ingestive behaviors underlying the hypophagic response to ICV C75. Six rats were acclimated to ad libitum access to water and 45-mg pellets in cages equipped with computer-monitored pellet dispensers (Med Associates, Georgia, VT) in which an infrared beam detects the removal of a single pellet from the feeding trough and triggers pellet replacement. The times of pellet removals were recorded to the nearest 10th of a second and stored on file on a computer. Files were formatted in Microsoft Excel 98 for subsequent analysis with TongueTwister (version 1.46a) on Macintosh computers (12).
C75 doses were given in the following order: 0, 10, 32, 56 µg. Data were analyzed for 3 days subsequent to drug or vehicle injections for latency to onset of the first meal of the day, as well as for pellet acquisitions, meal number, and meal size during (for each day): 0–24 h, 12–24 h light phase, 0–12 h dark phase, and 3- and 6-h segments of the dark phase. Meals consisted of at least five pellets with intervening intermeal intervals that lasted at least 10 min.
In situ hybridization for hypothalamic mRNA. Based on results from the food intake and meal pattern experiments, we analyzed C75-induced alterations in hypothalamic gene expression of feeding-related neuropeptides at two time points: 6 h after injection, when hypophagia commences, and 22 h postinjection, when the hypophagia is well established. For the 22-h time point, 18 rats in hanging wire cages were assigned to three groups: 1) RPMI injected, ad libitum fed (ADLIB; n = 6); 2) C75 injected (32 µg), ad libitum fed (C75; n = 6); and 3) RPMI injected, pair fed to the C75 group (PF; n = 6). Each PF rat was given the average C75 group chow intake plus its average amount spilled. PF rats received their allotment of food at lights out and ran out of food in a timeframe comparable with the delayed hypophagia that C75 produced in the meal pattern studies. Injections were given 5 min before onset of dark and chow access. At 22 h postinjection the rats were killed. In the other experiment, we killed six ADLIB and six C75-treated rats at 6 h after injections.
Tissue processing and in situ hybridization. Brains were removed, frozen quickly in isopentane on dry ice, and stored at –80°C. They were cryosectioned into 14-µm slices in the coronal plane, thaw mounted onto electrostatically charged microscope slides, and air dried. Mounted slices were then fixed with 4% paraformaldehyde, dehydrated with ethanol, and stored at –80°C.
As described previously (2, 3), plasmids with cDNAs for neuropeptide Y (NPY), AgRP, proopiomelanocortin (POMC), and corticotropin-releasing factor (CRF) were linearized by recommended restriction enzymes. Antisense riboprobes were labeled with [35S]UTP (Amersham Pharmacia Biotech) by using in vitro transcription systems with appropriate polymerases according to the manufacturer’s protocols (Promega) and purified by Quick Spin Mini Columns (Roche Diagnotics) to yield specific radioactivity of 5 x 108 cpm/µg.
For in situ hybridization, frozen tissue sections were allowed to warm to room temperature, treated with acetic anhydride and ethanol, and incubated in hybridization buffer containing 50% formamide, 0.3 M NaCl, 10 mM Tris·HCl, pH 8.0, 1 mM EDTA, pH 8.0, 1x Denhardt’s solution (Eppendorf), 10% dextran sulfate, 10 mM dithiothrietol, 500 µg/ml yeast tRNA, and 107 cpm/ml of [35S]UTP-riboprobe at 55°C overnight. After hybridization, the sections were washed three times with 2x standard sodium citrate (SSC), treated with 20 µg/ml RNase A (Sigma) at 37°C for 30 min, then rinsed twice in 2x SSC at 55°C for 5 min and twice in 0.1x SSC at 55°C for 15 min. Slides were then dehydrated in gradient alcohols, air-dried, and exposed with BMR-2 film (Kodak) for 1–7 days.
Autoradiographic films were scanned (Epson), and the images were analyzed quantitatively with Scion Image (Version 1.62c, National Institutes of Health), by using autoradiographic 14C-labeled microscales (Amersham) as a standard. Data were averages obtained from three sections of the product of area and density of the region of interest. Data from C75 and PF rats in the 22-h experiment were normalized to ADLIB control average. Data from C75 rats in the 6-h experiment were normalized to the ADLIB vehicle control average. Final analyses included data from at least four rats per group (22-h experiment, n = 4 for C75 group CRF mRNA hybridization, all others n = 6).
Statistical Analysis
Food intake and body weight data for each day were analyzed by one-way repeated-measures ANOVA [F(dfbtwn treatments, dfresidual)]. Meal pattern data were analyzed by one-way repeated-measures ANOVA for each time segment. Data from in situ hybridization experiments were analyzed by one-way ANOVA [F(dfbtwn groups, dfresidual)]. Significant effects in the ANOVAs prompted post hoc tests for dose differences by Fisher’s least squares means. Locomotor activities for each 2-h bin were analyzed by Student’s paired t-test. Significance for all tests was assumed at P 
0.05.
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RESULTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONDISCLOSURESGRANTSREFERENCES |
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ICV C75 reduced chow intakes on day 1 [F(3,18) = 6.32, P = 0.004; Fig. 1A]. Although 10 µg of C75 did not reduce chow intake significantly compared with vehicle, the 32- and 56-µg doses produced significant and equal decreases of chow intake to 16.4 ± 1.7 and 15.8 ± 1.7 g, respectively (vs. vehicle: P = 0.004 and P = 0.002, respectively). Although reduced, chow intake on day 2 after C75 was not significantly different from intake after vehicle. On day 3, control levels of chow intake were fully restored. Chow intakes measured until day 4 after 32 µg of C75 and until day 5 after 56 µg did not differ from those after vehicle (data not shown).
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Rats gained 4.4 ± 1.2 g by the end of day 1 after ICV injection of RPMI vehicle. Single ICV injections of C75 reduced body weight dose dependently on day 1 [F(3,18) = 10.637, P < 0.001; Fig. 1B]. Doses of 32 and 56 µg of C75 significantly reduced body weight, by 8.0 ± 3.8 g (P = 0.005 vs. vehicle) and 16.6 ± 5.2 g (P < 0.001 vs. vehicle), respectively. Although reductions in chow intake were equal after the 32 and 56 µg doses of C75 (Fig. 1A), body weight after 56 µg was significantly lower than after 32 µg (P = 0.041; Fig. 1B).
Although the principal weight losses with C75 occurred on day 1, reduced body weights were maintained over multiple days (Fig. 1B) [day 2: F(3,18) = 9.219, P < 0.01; day 3: F(3,18) = 7.697, P = 0.002; day 4: F(3,18) = 4.327, P = 0.018; day 5: F(3,18) = 6.521, P = 0.004]. Rates of weight gain or regain across doses over days 2-5 were comparable, such that doses of 32 and 56 µg resulted in significantly lower weights than vehicle consistently across days 2-5 (day 5: 32 µg, P = 0.002; 56 µg, P = 0.007). At day 5, the weight difference between the 56 µg dose and vehicle was 21.4 g, so a weight loss of 
5% was maintained for 5 days after a single ICV injection of C75.
Central C75 Does Not Alter Physical Activity
Rats exhibited essentially the same levels of activity in the horizontal plane, whether given vehicle or 32 µg of C75 ICV, during each of the 2-h bins of data collection on the day after injection (Fig. 2). Total counts of horizontal activity after C75 did not differ from those after vehicle during either the 0- to 12-h dark period [vehicle vs. C75: 42,500 ± 4,200 vs. 37,000 ± 4,600, t(18 df) = 0.887] or during the entire 0- to 22-h period of data collection [vehicle vs. C75: 54,100 ± 4,400 vs. 47,500 ± 4,900, t(18 df) = 1.002].
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Day 1 pellet intakes. ICV C75 reduced the acquisition of pellets on day 1 in the meal pattern experiment (Fig. 3, top) in a dose-response pattern consistent with that seen in the chow intake experiment (Fig. 1A). C75 did not produce significant feeding or pattern changes on days 2 or 3. C75 decreased the number of pellets acquired during the entire 0- to 24-h period of day 1 [F(3,15) = 4.237, P = 0.023], with significantly reduced and equal intakes after 32 and 56 µg (339 ± 63 and 325 ± 60 pellets, P = 0.031 and 0.022, respectively) compared with vehicle (528 ± 28 pellets).
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Delayed decrease in meal frequency. The C75-induced reduction in pellet intake appeared most clearly related to a decreased number of meals (Fig. 3, middle) rather than a reduction in meal size (Fig. 3, bottom). C75 decreased meal number during 0–24 h [F(3,15) = 3.63, P = 0.038], with significantly reduced and equal numbers of meals after the 32- and 56-µg doses (5.8 ± 1.4 and 6.0 ± 0.05 meals, P = 0.021 and 0.027, respectively) compared with vehicle (9.0 ± 1.3 meals). C75 did not decrease the number of meals during the first 0–6 h of dark, but did reduce meal number significantly during the 6- to 12-h period [F(3,15) = 4.479, P = 0.02], due to a reduction at the 56-µg doses of C75 (1.5 ± 0.04 vs. 3.3 ± 0.06 meals, P = 0.032).
Central C75 Decreases AgRP mRNA
At 22 h after injection and food access, rats pair fed (PF group) to the levels of food intake by hypophagic C75-injected rats [food intakes: ADLIB = 26.2 ± 2.1 g, C75 = 10.6 ± 1.6 g, PF = 9.1 ± 0.2 g; F(2,14) = 40.174, P < 0.001; PF vs. C75 not significant] had a significantly increased hybridization signal in the arcuate nucleus for AgRP mRNA compared with the ADLIB controls [F(2,13) = 6.123, P = 0.013; PF = 234 ± 32% of ADLIB, PF vs. ADLIB, P = 0.005] (Fig. 4A). In contrast, C75-injected rats, though hypophagic, had AgRP mRNA signal levels comparable to those of ADLIB (140 ± 30% of ADLIB, not significant) and significantly lower than those of PF (P = 0.034). Although reduced, the hybridization signal for NPY mRNA after C75 was not significantly different from the signal seen in ADLIB. Levels of hybridization to arcuate nucleus POMC mRNA and to paraventricular nucleus CRF mRNA did not differ among treatments.
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONDISCLOSURESGRANTSREFERENCES |
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The hypophagia produced by ICV C75 had a delayed onset. The 6- to 9-h period after injection was the earliest 3-h dark-phase bin to show a significant reduction in pellet acquisition. The hypophagic response might be delayed because C75 affects the de novo production of hypothalamic feeding-related neuropeptides. Consistent with data from mice (13), 1 day after ICV injection of C75 the rats, though hypophagic, did not exhibit the increase in arcuate nucleus expression of mRNA for AgRP seen in PF controls (Fig. 4A). Thus in opposition to gene expression changes due to an imposed food restriction, C75 decreases the expression of arcuate orexigenic neuropeptides (16). However, the question remained whether such C75 alteration of gene expression could drive the changes in feeding behavior. C75 decreases the level of expression of mRNA for AgRP at 6 h (Fig. 4B), just as the compound begins to reduce feeding. The delay in hypophagic response to C75 may include the time required for C75 to diffuse to critical sites in sufficient amounts. It is also possible that the biochemical or other changes occurring downstream of pharmacological fatty acid synthase inhibition and CPT-1 stimulation with C75 are subordinate to other physiological influences during the early dark phase.
In mice, C75 decreases the level of mRNA for NPY in the arcuate nucleus (5, 13, 15, 16, 29, 34). In these rat experiments, C75 tended to reduce levels of NPY mRNA. C75 prevents the expected hypophagia-induced increases in c-Fos in medial arcuate nucleus neurons that contain both NPY and AgRP (22). Results from recent experiments suggest a mechanism of action in which C75 increases hypothalamic ATP, reduces levels of gene expression for pAMPK, pCREB, and NPY, and reduces the levels of these proteins in mouse hypothalamus (15, 16). None of the experiments to date have examined the possibility that, in vivo, C75 alters synaptic release of neuropeptides or other neurotransmitters involved in food intake control, an action that could produce rapid changes in feeding behavior. Mice, unlike rats, reduce their food intake rapidly in response to ICV C75 (9, 15).
AgRP is an endogenous melanocortin antagonist at melanocortin-3 and -4 receptors (MC3R, MC4R) (8, 27, 35). The brain MC4R is most clearly involved in the control of food intake (14, 21). Recent reports suggest that AgRP (fragment 18-132) (Refs. 10, 26) and shorter fragments (6) also act as inverse agonists, keeping the MC4R in an inactive conformation and preventing their agonist-dependent activation and internalization (30). C75 decreased levels of AgRP mRNA at 6 h. We hypothesize that this should result in less AgRP signaling, so that MC4R previously held at the extracellular membrane by AgRP would be less likely to be occupied by AgRP. In such a case, neither POMC gene expression nor its processing to melanocortins would have to increase to achieve an increase in melanocortin signaling "tone," and to result in decreased food intake.
At the level of individual meals, feeding reductions can occur via decreased meal size or decreased meal frequency. The melanocortin feeding system has not been extensively tested for effects on meal pattern. One report shows that ICV administration of the MC-3/4-R agonist melanotan-II decreases food intake in rats by decreasing meal duration and size selectively (1). However, other data have demonstrated a decrease in meal number as well (25). Central C75 reduced pellet acquisitions in the meal pattern experiment during 0–24 h, specifically during 6–12 h. The 0- to 24-h reduction in pellet intake was clearly characterized by a reduction in the number of meals during 0–24 h. The reduced meal number occurred specifically during 6–12 h, with 56 µg of C75. Although C75 did not produce a significant reduction in meal size, subtle reductions in meal size may contribute to the overall feeding reduction. However, our data are more consistent with the notion that C75 acts in the brain primarily to reduce the drive to initiate and reinitiate energy intake, rather than to truncate meals once they have begun.
Although hypophagia was a major contributor to weight loss in rats given C75 centrally, additional factors cannot be discounted. Although 32 and 56 µg of C75 produced the same level of hypophagia on day 1, the 56-µg dose produced the greater weight loss. Furthermore, the 56-µg dose reduced food intake by 7 g, yet produced 22 g of weight loss relative to vehicle control. The present experiments do not examine hydration, overall metabolic measures, or carcass composition. They do show that ICV C75 did not alter the level of physical activity. This is consistent with other data showing that C75 alters brain c-Fos activation only in hypothalamic and hindbrain sites involved in the control of feeding and involuntary energy expenditure (9, 22) without altered c-Fos in cortex, cerebellum, or other brain regions (22). Thus, although C75 has been termed a "nonspecific neuronal activator" in some in vitro studies (31), this seems unlikely in vivo. It has been suggested that C75 decreases feeding and body weight secondary to illness (7, 28, 31). Relevant to the present study, ICV C75 does not produce conditioned taste aversion in rats (7).
Until very recently, effects of central C75 administration on peripheral tissue metabolism had not yet been explored. Cha et al. (4) report that ICV C75 increases whole body fatty acid oxidation in mice in vivo and in mouse skeletal muscle in vitro at an early 2-h time point. Muscle is a large organ that primarily uses fatty acids for fuel, so it seems reasonable that increasing fatty acid oxidation in skeletal muscle could be one way that central C75 reduces body weight. Notably, intrahypothalamic injection of leptin also increases fatty acid oxidation in skeletal muscle (24). Leptin is also one of several anorexigenic treatments recently shown to decrease hypothalamic AMPK activity (23), as does C75 (15).
The feeding and weight changes after C75 are unusual in that there is no rebound hyperphagia and that the weight loss initiated on day 1 is sustained for many days, without return to preinjection baseline body weight (Fig. 1). The present study did not include measures of hypothalamic mRNA beyond a 22-h time point. It would be interesting to know whether AgRP mRNA remains decreased in C75 treated rats for several days or whether other events downstream of altered melanocortin signaling are affected at later time points.
Perspectives: C75, AMPK, and AgRP/Melanocortin Signaling
Our data suggest that ICV C75 decreases the drive to initiate energy intake. The sequential timing of C75’s reduction of AgRP gene expression and the hypophagia suggest that altered melanocortin signaling is important for the hypophagia. A recent report has demonstrated that the increased AgRP in response to a low-energy state requires the activity of AMPK (19). Conversely, decreased hypothalamic AMPK activity decreases mRNA for AgRP and reduces food intake and body weight (23). Thus the decreased appetitive drive with ICV C75 may derive from its ability to increase hypothalamic ATP and decrease the activity of AMPK (15). This in turn may reduce AgRP gene expression and AgRP blockade of MC4Rs to increase melanocortin signaling "tone."
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DISCLOSURES |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONDISCLOSURESGRANTSREFERENCES |
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GRANTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONDISCLOSURESGRANTSREFERENCES |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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