Appetite Suppression and Weight Reduction by a Centrally Active Aminosterol Rexford S

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Appetite Suppression and Weight Reduction by a Centrally Active Aminosterol Rexford S. Ahima,1 Hiralben R. Patel,1 Nobuhiko Takahashi,1 Yong Qi,1 Stanley M. Hileman,2 and Michael A. Zasloff3

The rise in obesity and its complications has generated metabolite of cholesterol that was originally isolated from enormous interest in the regulation of feeding and body the dogfish shark (Squalus acanthias) liver during a weight. We show that a spermine metabolite of choles- search for naturally occurring antimicrobial compounds terol (MSI-1436) decreases body weight, specifically (4,5). MSI-1436 is structurally similar to squalamine (MSI- fat, by suppressing feeding and preventing the reduc- 1256) except for a spermine side-chain at C-3 on the tion in energy expenditure, hormonal changes, and pat- cholesterol A-ring (4,5). The bioactivity of MSI-1436 is also terns of neuropeptide expression normally associated dependent on a seven ␣-OH and sulfated moiety at C-25 with weight loss. MSI-1436 enters the brain after pe- (5). Unexpectedly, MSI-1436 was shown to inhibit feeding ripheral injection and is more potent when injected into and decrease body weight in a highly specific manner in the cerebral ventricle (intracerebroventricular [ICV]). Systemic or ICV MSI-1436 administration induced sim- normal and obese rodents (5). ilar patterns of Fos immunoreactivity in the brain, MSI-1436 is distributed to the brain and several periph- especially the paraventricular hypothalamic nucleus eral tissues (5). A single or intermittent treatment with (PVN). This brain region integrates neural signals from MSI-1436 results in a prolonged reduction in food intake hypothalamic and brain stem nuclei and regulates feed- and body weight and has been partly attributed to its long ing behavior, autonomic function, and neuroendocrine half-life (ϳ7 days in rodents) (5). However, it is unclear function. Microinjection of MSI-1436 into the PVN po- whether MSI-1436–induced weight loss is due to appetite tently suppressed feeding and reduced body weight for suppression alone. Moreover, although it had been sug- several days. Unlike caloric restriction, MSI-1436 gested that MSI-1436 is more potent when administered decreased mRNA levels of agouti-related peptide and into the cerebral ventricle, its targets in the central ner- neuropeptide Y in the hypothalamus. These findings indicate that MSI-1436 acts in the brain to regulate food vous system are unknown. The objective of this study was intake and energy expenditure, likely through suppres- to investigate the contributions of energy intake and sion of orexigenic hypothalamic pathways. Diabetes 51: expenditure to the sustained effect of MSI-1436 on body 2099–2104, 2002 weight and determine whether the biological activity of MSI-1436 in the brain is mediated by well-known hypothalamic neuronal pathways that mediate feeding behavior and energy balance. besity is highly prevalent in the U.S. and other developed countries and is increasing world- wide (1,2). This epidemic has serious public RESEARCH DESIGN AND METHODS health consequences because obesity is associ- Experiments were performed in accordance with guidelines and regulations O of the National Institutes of Health and Institutional Animal Care and Use ated with excess mortality and morbidity from type 2 Committee of the University of Pennsylvania. diabetes, cardiovascular disease, and other complications Determine the effect of MSI-1436 on food intake and energy expendi- (1,2). Diet and exercise remain the cornerstone of obesity ture. Male 12-week-old C57Bl/6J mice (Jackson Laboratories, Bar Harbor, management; however, it is likely that many patients will ME) were housed individually in a 12:12 h light-dark cycle (lights on at 0600; require drug treatment to reduce body weight and prevent temperature 22°C) and allowed normal laboratory diet and water ad libitum. complications (3). Here, we describe the anti-obesity Synthetic MSI-1436 and squalamine (MSI-1256) were provided by Genaera (Plymouth Meeting, PA). Our preliminary studies confirmed that squalamine action of a novel aminosterol. MSI-1436 is a spermine did not affect food intake or body weight (5). In contrast, intraperitoneal (IP) injection of MSI-1436 during the light or dark cycle reduced food intake and body weight in a dose-dependent manner (data not shown). We selected a 1 From the Division of Endocrinology, Diabetes and Metabolism, University of lower dose of MSI-1436 that did not suppress drinking as previously described Pennsylvania School of Medicine, Philadelphia, Pennsylvania; the 2Depart- (5). MSI-1436 (5 mg/kg IP ϫ three doses) was administered at 0900–1000 at ment of Physiology and Pharmacology, Robert C. Byrd Health Sciences ϭ ϭ Center, West Virginia University, Morgantown, West Virginia; and the 3George- 3-day intervals to a group of mice (n 6). Control mice (n 6) were treated ␮ ϭ town University School of Medicine, Washington, DC. with vehicle (100 l endotoxin-free H2O IP). A third group of mice (n 6) was Address correspondence and reprint requests to Rexford S. Ahima, Univer- pair-fed to the daily intake of MSI-1436 mice. Indirect calorimetry was sity of Pennsylvania School of Medicine, Division of Endocrinology, Diabetes performed after the final treatment. The mice were acclimatized to the test and Metabolism, 764 Clinical Research Bldg., 415 Curie Blvd., Philadelphia, PA cage for 2 days, and energy expenditure was measured at 15-min intervals for 19104. E-mail: [email protected]. 24 h on the third day (Oxymax Equalflow System; Columbus Instruments, Received for publication 13 February 2002 and accepted in revised form 3 Columbus, OH) (6). The following settings were used per cycle: air flow 500 April 2002. ml/min, sample flow 400 ml/min, settle time 120 s, measuring time 60 s, AGRP, agouti-related peptide; BAT, brown adipose tissue; CART, cocaine- ϭ and amphetamine-regulated transcript; MCR, melanocortin receptor; NEFA, temperature 22°C, respiratory exchange ratio (RER) volume of carbon nonesterified fatty acid; NPY, neuropeptide Y; POMC, pro-opiomelanocortin; dioxide generated (VCO2) divided by oxygen consumption (VO2). Heat (kcal/ PVN, paraventricular hypothalamic nucleus; RER, respiratory exchange ratio; h) ϭ 3.815 ϩ 1.232 ϫ RER. Total and ambulatory activity were measured UCP, uncoupling protein. simultaneously with photodetectors (Optovarimex System; Columbus Instru-

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ments). Rectal temperature was measured with a thermistor (Physitemp histologically, and only data from conﬁrmed PVN injection (n ϭ 4/group) were Instruments, Clifton, NJ). included in the analysis.

The mice were killed by CO2 inhalation, and blood was obtained by cardiac Analyze the effect of MSI-1436 on hypothalamic neuropeptide expres- puncture. Glucose and triglycerides (Sigma, St. Louis, MO) and nonesterified sion. Male 12-week-old C57Bl/6J mice were treated with three injections of ␮ fatty acids (NEFAs) (Wako Chemicals, Richmond, VA) were measured with MSI-1436 (5 mg/kg IP) or vehicle (100 l endotoxin-free H2O) at 3-day intervals. enzyme assays. Plasma insulin, leptin, corticosterone, and thyroxine were A third group was pair-fed to MSI-1436 treatment. Three days after the last measured by radioimmunoassay as previously described (7,8). Uncoupling injection, the mice were killed, hypothalami were dissected, and quantitative protein (UCP)-1 mRNA was measured in brown adipose tissue (BAT) by RT-PCR analysis was performed using specific primers for agouti-related peptide Northern blot analysis using a cDNA probe (provided by Mitch Lazar, (AGRP), neuropeptide Y (NPY), pro-opiomelanocortin (POMC), cocaine- and University of Pennsylvania). The rest of the carcass was dried to a constant amphetamine-regulated transcript (CART), melanin-concentrating hormone, and ␤ weight at 60°C to determine water content and digested in ethanol-KOH, and -actin (14). For corticotropin-releasing hormone, the following primers were Ј Ј Ј fat (triglyceride) content was measured with a colorimetric assay (Sigma) (8). used: upstream, 5 -gcatcctgagagaagtccctctg-3 ; downstream, 5 -aagttagccgcagcgt- Ј We determined whether MSI-1436 could prevent the hyperphagia normally ggtc-3 , corresponding to nucleotides 437–459 and 1476–1495 (15). The signal was associated with fasting. Male 12-week-old C57Bl/6J mice were fasted for 48 h measured by Phosphoimager (Molecular Dynamics, Sunnyvale, CA), and densi- and received a single injection of MSI-1436 (5 mg/kg IP) or vehicle (n ϭ ties corresponding to mRNA expression of various peptides were normalized to ␤ 6/group) after the fast. They were housed in individual cages and allowed ad -actin (14). Data analysis. The effects of MSI-1436 on food intake, body weight, and other libitum access to normal diet and water. Food intake and body weight were parameters were compared by ANOVA. Pairwise differences between groups measured daily. Feeding frequency and duration were monitored using were determined using Fisher’s protected least significant differences test; infrared detectors (Vitalview System; Mini Mitter, Bend, OR). The data on P Ͻ 0.05 was considered significant. feeding frequency and duration were collated in 1-h bins and analyzed using Actiview software (Mini Mitter). Determine the dose response to intracerebroventricular versus sys- RESULTS temic MSI-1436 treatment. Male Sprague-Dawley rats (250–300 g) were obtained from Taconic Farms (Germantown, NY), housed in a 12:12 h MSI-1436 decreases body weight by inhibiting food light-dark cycle (lights on at 0600; temperature 22°C), and allowed normal intake and increasing energy expenditure. To ascer- laboratory food and water ad libitum. The animals were anesthetized with tain whether weight reduction by MSI-1436 was mediated sodium pentobarbital, and a 22-gauge stainless steel guide cannula with entirely by inhibition of feeding, the daily intake of a group obturator (Plastics One, Roanoke, VA) was implanted unilaterally in the of pair-fed mice was matched with that of MSI-1436– lateral cerebral ventricle using the following coordinates: 0.8 mm posterior to bregma, 1.5 mm lateral to the midline, and 4 mm below the skull. The treated mice. Cumulative food intake was decreased by intracerebroventricular (ICV) cannula was attached to the cranium with ϳ20% in MSI-1436–treated and pair-fed mice (Fig. 1A); stainless steel screws and dental cement. Cannula placement was verified with however, body weight (Fig. 1B) and body fat (Fig. 1C) the drinking response to ICV angiotensin II injection and histologically after were significantly lower in MSI-1436–treated mice than in the experiment. Only data from rats with correctly positioned cannulas were pair-fed mice, indicating that inhibition of food intake included in the analysis. The animals were housed individually after surgery and handled daily to alone could not account for the weight loss in MSI-1436– habituate, and two sham injections were performed at 3-day intervals. treated mice. Oxygen consumption (VO2) (Fig. 1D) and Experiments were performed after restoration of body weight (ϳ1 week). heat (data not shown) were maintained at high levels in MSI-1436 (2, 10, or 30 ␮g) or vehicle (5 ␮l endotoxin-free H O) was 2 MSI-1436–treated mice despite weight reduction. RER, an administered ICV over 1 min to unanesthetized rats (n ϭ 6) with an injector protruding 1 mm below the guide cannula into the cerebral ventricle. Other index of metabolic fuel use, was not different between rats (n ϭ 6/group) received a single intraperitoneal injection of MSI-1436 (5 MSI-1436–treated (0.8 Ϯ 0.01) and vehicle-treated (0.84 Ϯ ␮ mg/kg) or vehicle (200 l endotoxin-free H2O). The animals were presented 0.02) mice. In contrast, VO2 was lower in pair-fed mice with a preweighed amount of pellet food, and food consumption and body (Fig. 1D). RER was significantly lower in pair-fed mice weight were measured daily. The effect of MSI-1436 on conditioned taste (0.77 Ϯ 0.02) than in vehicle-treated mice (0.84 Ϯ 0.02) aversion was investigated in other rats using the two-bottle paradigm (9). The Ͻ animals (n ϭ 4 per group) were treated with a single injection of MSI-1436 (30 (P 0.05). Body temperature, UCP-1 mRNA expression in ␮g ICV), MSI-1436 (10 mg/kg IP), vehicle (ICV or IP), or LiCl (100 mg/kg). BAT, and locomotor activity (beam breaks) were not Saccharin preference ratio was determined as the volume of 0.1% saccharin affected by MSI-1436 (data not shown). divided by water consumed in 24 h (9). Serum triglycerides, insulin, and leptin were reduced in Determine the effect of MSI-1436 on Fos immunoreactivity and response to paraventricular hypothalamic nucleus injection. Expression both MSI-1436–treated and pair-fed mice (Table 1). NEFAs of the immediate-early gene c-fos has been used to map neuronal pathways for and the NEFA-to-triglyceride ratio were twofold greater in feeding regulation (10–13). Fos immunostaining was performed on coronal pair-fed mice than in MSI-1436–treated mice, consistent brain sections from pathogen-free male Sprague-Dawley rats (Taconic Farms) with increased lipolysis (Table 1). Thyroxine was de- as previously described (10). The animals were treated with MSI-1436 (5 ␮ ␮ ␮ creased and corticosterone was increased in pair-fed mice mg/kg in 200 l endotoxin-free H2OIPor20 gin5 l endotoxin-free H2O ICV; n ϭ 4/group). Control rats (n ϭ 4) were treated with vehicle (IP or ICV). The but remained normal after MSI-1436 treatment (Table 1). animals were anesthetized with sodium pentobarbital 2 h later (1000–1200) Feeding frequency and food consumption increased rap- and perfused transcardially with PBS followed by 10% neutral-buffered idly in vehicle-treated fasted mice, leading to restoration formalin. Brains were cryoprotected in 20% sucrose/PBS, and free-floating of body weight within 48 h (Fig. 2A–C). In contrast, coronal sections (40 ␮m) were cut on a sliding microtome and processed for Fos immunoreactivity (10). The sections were examined with a Nikon E600 MSI-1436 blunted the postfast hyperphagia and weight microscope equipped with a digital camera and image analysis system (SPOT gain (Fig. 2A–C). The duration of feeding bouts was not Diagnostic Instruments, Sterling Heights, MI). Fos-positive cells/region/hemi- significantly different between MSI-1436 treatment and sphere with distinct nuclear staining were counted by an observer blinded to pair-feeding (data not shown). the experimental protocol using software provided by the manufacturer The potency of MSI-1436 is greater after central (Phase 3 Imaging, Glenmills, PA). Based on the induction of strong Fos-immunoreactivity in the paraventricu- administration than after peripheral treatment. Cere- lar hypothalamic nucleus (PVN) (see RESULTS), we determined whether bral ventricular injection of MSI-1436 resulted in a dose- injection of MSI-1436 into this hypothalamic region would recapitulate the related decrease in food intake and body weight (Fig. 3A response to ICV administration. A 26-gauge stainless steel cannula with and B). Water intake was reduced slightly by the highest obturator (Plastics One) was implanted into the PVN in rats as described (13). ␮ ␮ ␮ ICV dose (30 g MSI-1436) but was not statistically signif- One week later, MSI-1436 (0.5 or 1 g) or vehicle (0.5 lH2O) was microinjected into the PVN. The animals were housed singly, and food intake icant (33.7 Ϯ 2.6 vs. 30 Ϯ 4.6 ml/24 h; P ϭ 0.21). The and body weight were measured daily. The injection site was verified effective ICV dose of MSI-1436 was Ͻ1,000 of the periph-

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FIG. 1. Effect of MSI-1436 on food intake and energy expenditure. Male 12-week-old C57Bl/6J mice were treated with MSI-1436 (5 mg/kg IP at -3-day intervals ؋ three doses) or vehicle (endo

toxin-free H2O). Another group of mice were pair-fed (PF) to the MSI-1436–treated group (Table 1). A: Food intake; B: body weight; C: body fat (carcass analysis); D: resting oxygen ؍ ؎ consumption (VO2). Data are means SE; n 6; *P < 0.05 vs. vehicle, ␦P < 0.05 vs. MSI-1436. eral dose (Fig. 3A and B). In all cases, body weight was (Fig. 4C) and body weight (Fig. 4D). As with ICV treat- restored several days after MSI-1436 treatment (Fig. 3B). ment, the response to a single PVN injection persisted for MSI-1436 treatment did not cause taste aversion, as evi- several days (Fig. 4C and D). denced by a normal saccharin preference ratio of 0.8 in MSI-1436 inhibits the expression of orexigenic hypo- vehicle, ICV, and IP MSI-1436–treated rats. In contrast, thalamic neuropeptides. The expression of hypotha- saccharin preference ratio was markedly suppressed to lamic neuropeptides was analyzed after MS-1436 0.38 (P Ͻ 0.001) after LiCl administration, consistent with treatment or pair-feeding (caloric restriction) (Fig. 5). aversion behavior. MSI-1436 decreased NPY mRNA expression in the hypo- MSI-1436 induces Fos immunoreactivity in the PVN thalamus by 10% (P ϭ 0.1) and AGRP mRNA by 60% (P Ͻ and reduces food intake and body weight after direct 0.001) (Fig. 5). In contrast, NPY mRNA increased by 12% injection into the PVN. The distribution of potential (P ϭ 0.08) and AGRP mRNA by 27% (P Ͻ 0.01) in pair-fed MSI-1436 targets in the brain was evaluated using Fos mice (Fig. 5). The expression of POMC, corticotropin- immunohistochemistry. A robust induction of Fos-immu- releasing hormone, CART, and melanin-concentrating hor- noreactive cells was detected in the PVN after central or mone was not affected by MSI-1436 or caloric restriction peripheral MSI-1436 administration (Fig. 4A and B). Fewer (Fig. 5). numbers of Fos-positive cells were observed in other forebrain regions involved in ingestive behavior, energy DISCUSSION balance, and glucose homeostasis, i.e., arcuate nucleus, MSI-1436 and other aminosterols that affect feeding were perifornical region, zona incerta, lateral hypothalamic discovered serendipitously during a search for antimicro- area, dorsolateral and ventromedial hypothalamic nuclei, bial compounds in the dogfish shark (4,5). Initial studies and central amygdala (Fig. 4B). Injection of MSI-1436 into showed that MSI-1436 decreased body weight over long the PVN caused a dose-dependent reduction in food intake periods. However, there was a lack of understanding as to whether the sustained reduction in body weight was due TABLE 1 solely to appetite suppression. Our studies provide evi- Effects of MSI-1436 versus pair-feeding on hormone levels dence in support of a specific suppression of feeding as Vehicle MSI-1436 Pair-fed well as increased energy expenditure after MSI-1436 treatment. Importantly, MSI-1436 reduced body weight (specif- Glucose (mg/dl) 128 Ϯ 18 102 Ϯ 12 98 Ϯ 7 Ϯ Ϯ Ϯ ically fat) without provoking the well-known response to Triglycerides (mg/dl) 138 12 90 13* 102 14* caloric depletion (16,17). In the physiological model of NEFAs (mEq/l) 0.55 Ϯ 0.1 0.48 Ϯ 0.08 1.6 Ϯ 0.05*† Insulin (ng/ml) 0.9 Ϯ 0.1 0.2 Ϯ 0.1* 0.31 Ϯ 0.06* energy homeostasis, the amount of energy stored in adi- Leptin (ng/ml) 3.4 Ϯ 0.2 1.6 Ϯ 0.08* 1.8 Ϯ 0.2* pose tissue reflects the balance between energy intake and Corticosterone (ng/ml) 63 Ϯ 988Ϯ 15 134 Ϯ 21*† expenditure (16). A decrease in energy stores from fasting Thyroxine (␮g/dl) 4.8 Ϯ 0.8 4.5 Ϯ 0.2 3.2 Ϯ 0.1*† triggers various responses, e.g., reduced energy expenditure and overfeeding in an attempt to restore body weight Data are means Ϯ SE. n ϭ 6. Male 12-week-old C57B1/6J mice were treated with MSI-1436 (three doses of 5 mg/kg IP every 3 days) or (16). Other typical responses to food deprivation include ␮ decreased thyroid hormone, increased glucocorticoids, vehicle (100 l endotoxin-free H2O). A group of mice was pair-fed to MSI-1436 treatment. *P Ͻ 0.05 vs. vehicle; †P Ͻ 0.05 vs. MSI-1436. and increased lipolysis (evidenced by an increased NEFA-

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FIG. 3. Dose-effect of ICV versus IP MSI-1436 treatment. Male Sprague-Dawley rats were treated with a single dose of MSI-1436 (5 ␮ mg/kg IP) or vehicle (200 l endotoxin-free H2O IP), or ICV MSI-1436 ␮ ␮ (10 or 30 g) or vehicle (5 l endotoxin-free H2O). Food intake (A) .4 ؍ and body weight (B) were measured daily. Data are means ؎ SE; n

to-triglyceride ratio and reduced RER). These responses, which are mediated at least in part by reduced leptin level, are designed to defend body weight and lean mass by reducing energy expenditure and stimulating feeding (16,17). The counter-regulatory mechanisms may be re- sponsible for the failure to sustain weight loss during forced caloric restriction (16). MSI-1436 was effective in reducing food intake and body weight in ad libitum–fed animals for several days and also prevented the hyperphagia and rapid weight gain normally associated with fasting, indicating that its pharmacological effects can override the physiological tendency to gain weight. The ability of MSI-1436 to maintain high-energy expenditure despite weight loss is not likely to be mediated through UCP-1 or locomotor activity because these factors were not altered. Based on a previous study (5) and our own study (data not shown) showing that MSI-1436 reaches the brain after peripheral injection, we compared the dose-effect of ICV treatment versus systemic MSI-1436 treatment. Adminis- tration of MSI-1436 ICV was Ͼ1,000 times more potent FIG. 2. MSI-1436 prevents postfast hyperphagia and weight gain. C57Bl/6J mice (12 weeks old) were fasted for 48 h, treated with than intraperitoneal treatment. Central or peripheral MSI- MSI-1436 (5 mg/kg IP) or vehicle after the fast, and allowed normal 1436 treatment did not cause taste aversion, thus provid- food and water ad libitum thereafter. Food intake (A), feeding frequency (B), and body weight (C) were monitored. Data are means ؎ ing evidence against visceral illness as a cause of the -P < 0.05 vs. vehicle. reduced food intake and body weight. We mapped poten* ;6 ؍ SE; n

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FIG. 4. Fos immunostaining was performed on coronal brain sections from pathogen-free male Sprague-Dawley rats treated with MSI- 1436 (5 mg/kg IP or 20 ␮g ICV). Control rats

were treated with endotoxin-free H2O (200 ␮lIPor5␮l ICV). A: Photomicrographs comparing Fos immunoreactivity in the PVN after treatment with ICV vehicle or MSI- ␮m. B: Analysis of 250 ؍ Scale bar .1436 Fos-positive cells/region/hemisphere. The ICV and IP vehicle (not shown) produced similar background levels of Fos immunore- ,Arc .4 ؍ activity. Data are means ؎ SE; n arcuate nucleus; CeA, central amygdala; LH, lateral hypothalamic area; NTS, nucleus trac- tus solitarius; VMN, ventromedial hypothalamic nucleus. The response to PVN injection of MSI-436 (0.5 vs. 1 ␮g) on food intake (C) and body weight (D) was analyzed in male ;Sprague-Dawley rats. Data are means ؎ SE -There was a significant dose-depen .4 ؍ n dent reduction in food intake and body weight. tial targets of MSI-1436 in the brain using Fos immuno- would produce an effect similar to that of ICV or periph- staining. The induction of Fos immunoreactivity in the eral MSI-1436 injection. Administration of one-thirtieth the PVN is consistent with neuronal activation and might ICV dose of MSI-1436 into the PVN resulted in a sustained explain the effects of MSI-1436 on feeding and energy reduction in food intake and body weight, suggesting that expenditure because this hypothalamic region mediates the PVN is a specific target of MSI-1436. feeding behavior and autonomic function (10,16,17). Fos The PVN receives neuronal input from the arcuate immunoreactivity in the PVN has been shown to be nucleus, other hypothalamic regions, and the brainstem regulated by a variety of factors, such as leptin, urocortin, (16,17). The orexigenic neuropeptides NPY and AGRP are corticotropin-releasing factor, and AGRP (10–13). We ob- produced by the same neurons in the arcuate nucleus and served fewer Fos-positive cells in other hypothalamic and increase body weight by stimulating appetite and increas- forebrain regions implicated in energy homeostasis. How- ing energy expenditure (16,17). Conversely, ␣-melanocyte- ever, activation of Fos within the PVN does not necessarily stimulating hormone (␣-MSH) is produced by POMC prove that MSI-1436 acts at this site. Thus, we determined neurons in the arcuate nucleus and decreases body weight whether direct administration of MSI-1436 into the PVN by inhibiting appetite and increasing expenditure (16,17). The cellular action of ␣-MSH is mediated through antago- nism of AGRP at melanocortin receptor-3 (MCR3) and MCR4. Hypothalamic neuronal circuits expressing these neuropeptides respond to leptin, insulin, glucocorticoids, and other endogenous factors (16,17) and can be pharma- cologically influenced by other molecules such as ciliary neurotropic factor (CNTF) and the fatty acid synthase inhibitor C75 (18–21). MSI-1436 significantly inhibited AGRP mRNA and, to a lesser extent, NPY mRNA expression in the hypothalamus. This response was distinct from caloric deprivation, in which AGRP and NPY mRNA expression increased, consistent with their roles as stimula- tors of feeding (16,17). We speculate that the central action of MSI-1436 is mediated at least in part through inhibition of AGRP/NPY projection to the PVN (16,17). The suppression of AGRP may explain the prolonged inhibition of feeding by MSI- 1436 because AGRP has the unique ability to regulate food FIG. 5. Effects of MSI-1436 vs. pair-feeding (PF) on mRNA expression intake and body weight for several days after a single ,CRH .4 ؍ of hypothalamic neuropeptides. Data are means ؎ SE; n corticotropin-releasing hormone; MCH, melanin-concentrating hor- central injection (21). The ability of MSI-1436 to pro- mone. foundly inhibit appetite while increasing energy expendi-

DIABETES, VOL. 51, JULY 2002 2103 CHOLESTEROL METABOLITE DECREASES WEIGHT ture for long periods distinguishes it from other weight- circadian rhythm of leptin by feeding: implications for energy homeostasis reducing compounds. In case the pharmacology of MSI- and neuroendocrine function. J Clin Invest 101:1020–1027, 1998 9. Wang C, Mullet MA, Glass MJ, Billington CJ, Levine AS, Kotz CM: Feeding 1436 is shown to be similar in humans, these properties inhibition by urocortin in the rat hypothalamic paraventricular nucleus. could prove to be advantageous by counteracting the Am J Physiol Regul Integr Comp Physiol 280:R473–R480, 2001 homeostatic metabolic responses that resist weight loss. 10. Elias CF, Kelly JF, Lee CE, Ahima RS, Drucker DJ, Saper CB, Elmquist JK: Chemical characterization of leptin-activated neurons in the rat brain. ACKNOWLEDGMENTS J Comp Neurol 423:261–281, 2000 11. Wang L, Martinez V, Vale W, Tache Y: Fos induction in selective hypotha- This work was supported by National Institutes of Health lamic neuroendocrine and medullary nuclei by intravenous injection of Grant P30 DK50306. urocortin and corticotropin-releasing factor in rats. Brain Res 855:47–57, The authors thank Genaera Corporation (Plymouth 2000 Meeting, PA) for providing MSI-1436 and squalamine, 12. Hagan MM, Benoit SC, Rushing PA, Pritchard LM, Woods SC, Seeley RJ: Qinglan Jiang for initial studies, Mitchell A. Lazar for Immediate and prolonged patterns of Agouti-related peptide-(83–132)- induced c-Fos activation in hypothalamic and extrahypothalamic sites. helpful discussions, and the Mouse Phenotyping, Physiol- Endocrinology 142:1050–1056, 2001 ogy & Metabolism and Radioimmunoassay Cores of the 13. 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