Effects of Chronic Administration of Sibutramine on Body Weight, Food

Exp. Anim. 49(4), 239Ð249, 2000

Effects of Chronic Administration of Sibutramine on Body Weight, Food Intake and Motor Activity in Neonatally Monosodium Glutamate-Treated Obese Female Rats: Relationship of Antiobesity Effect with Monoamines

Terutake NAKAGAWA1), Kiyoharu UKAI1), Tadashi OHYAMA1), Yutaka GOMITA2), and Hitoshi OKAMURA3)

1)Central Research Institute, Kaken Pharmaceutical Co. Ltd., 14 Shinomiya, Minamikawara-cho, Yamashina-ku, Kyoto 607-8042, 2)Department of Hospital Pharmacy, Okayama University Medical School, Shikada-cho 2Ð5Ð1, Okayama 700-8558, and 3)Department of Anatomy, Kobe University School of Medicine, 7Ð5Ð1 Chuohoku Kusunoki-cho, Kobe 650-0017, Japan

Abstract: When the hypothalamic ventromedial nucleus and arcuate nucleus were destroyed in rats by treatment with monosodium glutamate in the neonatal stage, increase in the Lee index (body weight 1/3/body length) and in retroperitoneal fat as well as decreases in spontaneous motor activity, food consumption and growth hormone secretion function associated with hypothalamic low body length obesity (monosodium glutamate- treated obesity; MSG-OB) were observed as these rats grew. Treatment with sibutramine at 3 and 10 mg/kg p.o. once a day continuously for 14 days improved these parameters, and the degree of improvement was dose related. The plasma lipid values in MSG-OB rats, which were the same as those in normal rats, were decreased by consecutive administration of sibutramine. Levels of hypothalamic monoamines (MAs) such as norepinephrine, 5-HT (serotonin) and dopamine and their metabolites DOPAC, HVA and 5- HIAA were decreased in MSG-OB rats, and further decrease in them, though slight, was observed with consecutive daily administration of sibutramine, probably as a result of the feedback attributable to an increase in MA in synapses caused by inhibition of MA uptake by sibutramine. These results suggest that sibutramine can activate the MA nervous system by MA uptake inhibition in regions of the brain such as the lateral hypothalamic area and the paraventricular nucleus, which control food intake and sympathetic nerve activity, and the nigrostriatal area related to the extrapyramidal motor system, and thereby exhibit anti-obesity effects in the MSG-OB rat. Key words: body weight, food intake, hypothalamic monoamine level, sibutramine, spontaneous motor activity

(Received 8 December 1999 / Accepted 5 April 2000) Address corresponding: T. Nakagawa, Central Research Institute, Kaken Pharmaceutical Co. Ltd., 14 Shinomiyaku, Minamikawara-cho, Kyoto 607-8042, Japan 240 T. NAKAGAWA, ET AL.

Introduction increased, whereas blood glucose and free fatty acid concentrations increase. As a result, the fat and glyco- Obesity is defined as excessive accumulation of fat gen stored in the body as energy are oxidized and tissue (i.e., of somatic fat). When energy balance is decomposed, resulting in body weight decrease [14]. positive, that is, the number of calories contained in During development of sibutramine (N-1- (1-[4- ingested food greater than the energy released through chlorophenyl]-cyclobutyl)-3-methylbutyl-N, exercise, body temperature increase, etc., calories are N-dimethylamine hydrochloride monohydrate :BTS 54- stored in the body as fat, glycogen and protein, result- 524, ¨Meridia) as an antidepressant, it was found to ing in an increase in body weight [14]. Bray proposed have a body weight-reducing effect in clinical studies, “The Mona Lisa hypothesis in the time of leptin”, and and it has recently been developed as an anti-obesity indicated that inhibition of appetite and increased sym- drug [3]. In the mechanisms of centrally acting anorec- pathetic nervous system activity have anti-obesity tic drugs, monoamines (MAs) such as norepinephrine, effects [4]. Food intake is controlled through complex dopamine and 5-hydroxytryptamine (5-HT, serotonin) interactions of nervous systems in the brain which are play important roles [41]. related to appetite, exercise, reward and memory. Above The results of in vivo acute administration tests and all, various nuclei in the hypothalamus play central roles of in vitro tests have indicated the norepinephrine, 5- in controlling food intake. According to the so-called HT and dopamine reuptake-suppressing effects of dual center hypothesis, which attracted attention in the sibutramine [28]. past, the ventromedial nucleus (VMH), the satiety cen- It is of an interest to determine whether the food ter, and the lateral hypothalamic area (LH), the food intake suppression by acute administration of intake center, adjust appetite in the hypothalamus [14]. sibutramine to normal rats is attributable to effects of But recent studies on leptin, which is secreted from this agent mediated by the VMH and ARC or effects of peripheral fat cells and which suppresses food intake, this agent in other brain areas, e.g., the LH and PVN, and studies on the mechanism of action of neuropep- etc., and whether sibutramine acts on these hypotha- tide Y (NPY), reported that the paraventricular nucleus lamic nuclei to activate the sympathetic nervous system (PVN), perifornical hypothalamus (PFH), arcuate to increase energy expenditure [16, 19, 20, 44]. nucleus (ARC) and other brain areas are also involved Activation of the nigrostriatal dopaminergic system in food intake behavior [4]. is important because it increases the amount of motor Obesity results in rats and cats when the VMH is activity, thereby expending energy. In this regard, we electrically destroyed, but this method requires special have reported that sibutramine increased rat motor ac- devices and techniques and requires much time. On tivity due to activation of the dopaminergic system in the other hand, intraperitoneal administration of mono- the nigro-striatum [28]. Otherwise, there is also a re- sodium glutamate (MSG) to rats in the neonatal stage port that sibutramine does not have dopaminergic effects destroys the VMH and ARC in the hypothalamus, pro- [18]. It is still unclear whether continued administra- ducing neonatally MSG-treated obese (MSG-OB) rats tion of sibutramine maintains the increase in whose sympathetic nerve activity decreases with growth spontaneous motor activity, as observed after acute ad- [10, 34, 45, 46]. Hypothalamic obesity animal models ministration, which contributes to the anti-obesity effect may thus be easily prepared through destruction of the of sibutramine by energy expenditure. VMH by systemic administration of MSG. On the other Although the anti-obesity effect of sibutramine has hand, destruction of the lateral hypothalamus has ef- been clinically confirmed, few studies have examined fects opposite those of destruction of the ventromedial the anti-obesity effects of daily administration of hypothalamus, with reduction in food intake, decrease sibutramine on food intake or body weight (or Lee in- in body fat, and increase in sympathetic nerve system dex = body weight 1/3/body length) in animal models of activity [2]. obesity [9, 39, 40]. In the present study, sibutramine In the presence of anxiety and mental tension, the was chronically administered to normal and MSG-OB sympathetic nervous system is activated. Even when rats, and the relationship of behavioral pharmacologi- resting, secretion of norepinephrine and epinephrine is cal changes to hypothalamic MA levels was examined. ANTIOBESITY EFFECT OF SIBUTRAMINE 241

Materials and Methods dose of 100 µg/kg (0.1 ml/100 g) of GHRP was in- jected into the tail vein, and 15 min later the rats were Drugs decapitated and blood was collected. Immediately af- Sibutramine HCl and growth hormone (GH) secreta- ter blood collection, the hypothalamus and gogue (growth hormone releasing peptide retroperitoneal fat were extracted and weighed. The (GHRP:KP-102)) were synthesized at Kaken Pharma- blood samples were immediately centrifuged at 1,500 G ceutical Co. (Tokyo). Methamphetamine (Philopon¨) at 4°C for 15 min, and the plasma was stored at Ð20°C was purchased from Dainippon Pharmaceutical Co. or below until use. (Osaka). Other drugs were obtained from Sigma-RBI For measurement of hypothalamic MAs, each (St. Louis, MO, USA) . sample was homogenized in 0.06 M perchloric acid solution containing an internal standard, 3,4- Animals dihydroxybenzylamine (DHBA, 100 ng/ml), and Monosodium glutamate-treated obesity (MSG-OB) centrifuged with a high-speed refrigerated centrifuge rats were obtained by intraperitoneal administration of (4°C, 20,000 G, 20 min). The supernatant was fil- MSG to female neonates (born to female Sprague- tered, and MAs and their metabolites in the filtrate Dawley strain rats purchased from SLC Inc. (Shizuoka) were measured by means of HPLC with an electro- on Day 16 of gestation) at 2 mg/g body weight 5 times, chemical detector. i.e., 1, 3, 5, 7 and 9 days after birth. Physiological Plasma levels of total cholesterol, triglycerides and saline was administered in similar fashion 5 times in- high-density lipoprotein cholesterol (HDL-C) were traperitoneally to the control animals. The animals were quantified with a biochemical auto-analyzer (Fuji raised normally thereafter, and studied at the age of 10 Drychem 3030). Free fatty acid was measured by the weeks. The animals were kept through the experiments ACS-ACOD method (NEFA C Test, Wako), and GH on a 12-hr light-dark cycle at a room temperature of 21 was measured by radio-immunoassay. ± 1°C. Experiment 2: Effects of sibutramine and metham- Experiment 1: Effects on body weight, food intake, phetamine administered for 14 consecutive days on and levels of hypothalamic MAs and plasma GH and spontaneous motor activity rhythm of normal and MSG- lipid levels of normal and MSG-OB rats OB rats Three days before drug administration, rats in the Normal rats and MSG-OB rats were grouped by the neonatally physiological-saline-treated group (normal replication procedure mentioned above, and groups of group) underwent measurement of retroperitoneal fat 4 rats each were prepared. Body weight and food in- with a body-fat measuring apparatus for rats (Biotex, take were also measured by the methods described Kyoto) by the impedance method. On the basis of above. Spontaneous motor activity of the rats was moni- observed values, rats were distributed to a distilled wa- tored by the Rhythmer system with 16 channels for ter p.o. group, sibutramine 3 mg/kg p.o. group, and automatic simultaneous measurement of spontaneous sibutramine 10 mg/kg p.o. group by the replication motor activity, feeding and drinking (Biotex, Kyoto). method (block design); each group included 10 rats. Motor activity rhythm was depicted in a figure by MSG-OB rats were similarly divided into 3 groups, double-plotting for each rat with measurement of mo- each group with 10 rats. In each group, distilled water tor activity every 10 min. During the course of drug was given orally for 3 days before the start of drug administration for 14 days, 336 hourly data points were administration. collected for each rat. The data were subjected to Body weight and food consumption were measured rhythm analysis (lag 202 points (60%), 5-point moving at 1Ð2 p.m. every day. The drug was orally adminis- average, 0.027Ð0.25 frequency/hr) by the maximum tered at 4Ð5 p.m. daily for 14 days, from Day 0 through entropy method (MEM) to obtain cycle, amplitude, Day 13. Sixteen hours after the final administration, phase and mean motor activity parameters [30]. body-fat impedance, body weight (g) and body length (nasal-anal distance in cm) were measured, and the Lee Statistical analysis index (body weight 1/3/(body length)) was obtained. A Experiments were performed according to the MSG 242 T. NAKAGAWA, ET AL. treatmentásibutramine treatmentáreplicationáday design. but were steady thereafter until the end of administra- Time-course data for body weight, food intake and spon- tion (after 13 days of administration) (Fig. 1) [16, 28, taneous motor activity were tested by analysis of 33]. The decreases in body weight and the Lee index variance (ANOVA) for repeated measurements. Other in the normal and MSG-OB rats were dose-dependent, data were tested by two-way ANOVA of MSG but body length was independent of the dose (Fig. 1, treatmentásibutramine treatmentáreplication. Thereafter, Table 1). Food intake in the MSG-OB group was sig- 2 × 3 point parallel line bioassay, i.e., MSG treatment nificantly less than in the normal group (Fig. 2). effect, sibutramine treatment effects (linearity and qua- Administration of sibutramine at 3 and 10 mg/kg p.o. dratic curve) and effects of their interaction (linear decreased food intake by both normal and MSG-OB non-parallelism and quadratic non-parallelism), was per- rats (Fig. 2). The amount of retroperitoneal fat in the formed [7]. MSG-OB group rats was significantly greater than that When either one of the two non-parallelism tests indi- in the normal group rats. Sibutramine at 3 and 10 mg/ cating interaction was significant, interaction was judged kg p.o. decreased retroperitoneal fat dose-dependently to be present and expressed as “I” for p<0.05. Then in both normal and MSG-OB rats (Table 1). Imped- multiple comparison testing of the corresponding control ance values obtained with the body fat measuring group and sibutramine treatment at each dose was per- apparatus were closely correlated with the amount of formed by Dunnett’s method. The finding of significant retroperitoneal fat (r=0.93, p<0.01). difference between the saline treatment control and the No significant differences between the MSG-OB group MSG treatment control was expressed as “#” for p<0.05 and the normal group were found in plasma levels of or “##” for p<0.01 (two-tailed test). When neither of total cholesterol, triglyceride, high-density lipoprotein the two interactions was significant, multiple compari- cholesterol or free fatty acid. Sibutramine at 3 and 10 son testing with the crossed control group, which places mg/kg p.o. dose-dependently and significantly lowered the saline treatment control group and MSG treatment plasma levels of total cholesterol, triglyceride, high-den- control group together, and crossed sibutramine treat- sity lipoprotein cholesterol and free fatty acid in both ment group at each dose was performed by Dunnett’s normal rats and MSG-OB rats (Table 2). method. Significances of p<0.05 and p<0.01 were ex- GH secretion into plasma induced by loading of 100 pressed as “$” and “$$” for the repeated measurement µg/kg i.v. of GHRP was significantly less in the MSG- design, and by “*” and “**” for others. Significances OB group than in the normal group (Table 2). of p<0.05 and p<0.01 were expressed as “#” and “##”, Sibutramine at 3 and 10 mg/kg p.o. did not affect GH respectively, in comparisons of the crossed saline treat- secretion into plasma induced by GHRP loading in nor- ment group and crossed MSG treatment group. mal rats, but significantly accelerated GH secretion into plasma in MSG-OB rats (Table 2). Results Hypothalamic levels of norepinephrine, dopamine, 5- HT and their metabolites, dihydroxyphenylacetic acid Experiment 1: Effects of sibutramine administered (DOPAC), homovanillic acid (HVA) and 5- for 14 consecutive days on body weight, food intake, hydroxyindole-3-acetic acid (5-HIAA), were hypothalamic MAs, and plasma GH and lipid levels of significantly lower in the MSG-OB group than in the normal and MSG-OB rats normal group, but the turnover rates of DOPAC/dopam- Compared with the control physiological saline group, ine and 5-HIAA/5-HT were similar in between these rats in the neonatally MSG 2 mg/g i.p.-treated group two groups (Table 3). Sibutramine at 3 and 10 mg/kg exhibited lower body weight, shorter body length, higher p.o. significantly lowered the norepinephrine level in Lee index, and more retroperitoneal fat, and thus dem- both normal rats and MSG-OB rats (Table 3). onstrated successful preparation of a growth-suppressed Sibutramine at 3 and 10 mg/kg p.o. tended to lower the obese rat model (Fig. 1, Table 1). During daily admin- dopamine level but affected neither the levels of its istration of sibutramine at 3 and 10 mg/kg p.o., which metabolites, DOPAC and HVA, nor the turnover rate inhibited MA reuptake in vivo, body weights of normal of DOPAC/dopamine, in either normal rats or MSG- and MSG-OB rats decreased daily for the first 4 days OB rats (Table 3). Sibutramine at 3 and 10 mg/kg p.o. ANTIOBESITY EFFECT OF SIBUTRAMINE 243

Fig. 1. Effects of chronic systemic administration of sibutramine on the body weight of normal and MSG-OB female rats. Normal rats: control, sibutramine 3 mg/kg and sibutramine 10 mg/kg. MSG-OB rats: control, sibutramine 3 mg/kg and sibutramine 10 mg/kg. Each value is the mean ± SD for 10 rats. ##: p<0.01 compared with the saline-treated crossed group, since interaction was not significant on ANOVA. $$: p<0.01 compared with the crossed control group, since interac- tion was not significant on ANOVA.

Table 1. Effects of chronic systemic administration of sibutramine on body weight, body length, Lee index and retro- peritoneal fat weight in normal and MSG-OB female rats

Neonatal Drugs Dose Body Nasal-anal Lee Retroperitoneal Treatment (mg/kg, p.o) Weight (g) Length (cm) index Fat Weight (g)

Saline-treated Control 253 ± 14 21.5 ± 0.363 0.294 ± 0.004 9.951 ± 3.950 Sibutramine 3 248 ± 12 21.3 ± 0.262 0.294 ± 0.004 7.863 ± 2.974 Sibutramine 10 234 ± 7.5* 21.4 ± 0.386 0.288 ± 0.005** 3.711 ± 1.694** MSG-treated Control 229 ± 29# 19.8 ± 0.911# 0.309 ± 0.008# 16.82 ± 6.209# Sibutramine 3 218 ± 24 19.5 ± 0.774 0.308 ± 0.012 14.71 ± 6.057 Sibutramine 10 203 ± 25* 19.8 ± 0.675 0.296 ± 0.009** 11.26 ± 4.296**

Each value is the mean ± SD for 10 rats. The Lee index is (body weight (g)) 1/3/(nasal-anal length (cm)). #: p<0.05 compared with the neonatally saline-treated group, since interaction was not significant on ANOVA. *: p<0.05, **: p<0.01 compared with the crossed-control group, since interaction was not significant on ANOVA.

tended to lower the 5-HT level, lowered the level of its phetamine administered for 14 consecutive days on metabolite, 5-HIAA, and decreased the turnover rate of spontaneous motor activity rhythm of normal and MSG- 5-HIAA/5-HT in both normal rats and MSG-OB rats OB rats (Table 3). Sibutramine at 3 and 10 mg/kg p.o. decreased body Experiment 2: Effects of sibutramine and metham- weight and food consumption in the MSG-OB group, 244 T. NAKAGAWA, ET AL.

Fig. 2. Effects of chronic systemic administration of sibutramine on food intake by normal and MSG-OB female rats. Each value is the mean ± SD for 10 rats. #: p<0.05 compared with the saline-treated group. **: p<0.01 compared with each control group.

Table 2. Effects of chronic systemic administration of sibutramine on total cholesterol, triglyceride, HDL-cholesterol and free fatty acid levels, and GHRP-loaded GH secretion in plasma of normal and MSG-OB female rats

Neonatal Drugs Dose GH@ T-Cho TG HDL-Cho NEFA treatment (mg/kg, p.o.) (log ng/ml) (mg/dl) (mg/dl) (mg/dl)(mEq/l)

Saline-treated Control 2.077 ± 0.643 89 ± 13 120 ± 72 54 ± 12 0.638 ± 0.226 Sibutramine 3 1.801 ± 0.602 82 ± 11 100 ± 28* 45 ± 5** 0.406 ± 0.160** Sibutramine 10 1.980 ± 0.450 66 ± 10** 76 ± 12** 39 ± 6** 0.419 ± 0.132** MSG-treated Control 1.539 ± 0.245I 85 ± 14 161 ± 90 48 ± 11 0.561 ± 0.121 Sibutramine 3 1.883 ± 0.500† 82 ± 12 109 ± 45* 46 ± 21** 0.513 ± 0.119** Sibutramine 10 1.867 ± 0.633† 68 ± 10** 92 ± 41** 38 ± 7** 0.425 ± 0.091**

Each value is the mean ± SD for 10 rats. @: loaded with GHRP 100 µg/kg, i.v.. GH: growth hormone, T-Cho: total cholesterol, TG: triglyceride, HDL-Cho: high-density lipoprotein cholesterol, NEFA: free fatty acid. *: p<0.05, **p<0.01 compared with the crossed-control group, since interaction was not significant on ANOVA. I: p<0.05 compared with the neonatally saline-treated group, since interaction was significant on ANOVA. †: p<0.05 different from MSG-control. similar to the decreases shown in Table 1 and Fig. 1. No significant difference between the normal and MSG- Methamphetamine at 10 mg/kg p.o. decreased body OB control groups in this group was found in either the weight and food consumption in the MSG-OB group to diurnal motor activity rhythm or phase (Table 4). the same degree as did sibutramine 10 mg/kg p.o. (data Sibutramine at 3 mg/kg p.o. increased the mean spon- not shown). The mean spontaneous motor activity in taneous motor activity in the MSG-OB group, but no the MSG-OB group was significantly less than that in changes were found in either the diurnal motor activity the normal group for 14 consecutive days, and the mean rhythm or phase in this group (Table 4). Sibutramine amplitude of the activity rhythm in the MSG-OB group at 10 mg/kg p.o. and methamphetamine at 10 mg/kg tended to decrease, although not to a significant extent. p.o. increased both mean spontaneous motor activity ANTIOBESITY EFFECT OF SIBUTRAMINE 245

Table 3. Effects of chronic systemic administration of sibutramine on hypothalamic norepinephrine, DOPAC, dopamine, DOPAC/ dopamine, 5-HIAA, 5-HT, 5-HIAA/5-HT and HVA levels in normal and MSG-OB female rats

Neonatal Dose Drug NE DOPAC DA DOPAC/DA 5-HIAA 5-HT 5-HIAA/5-HT HVA treatment (mg/kg, p.o.)

Saline-treated Control 1143 ± 227 104 ± 26 558 ± 124 0.187 ± 0.032 309 ± 80 581 ± 105 0.541 ± 0.160 37 ± 11 Sibutramine 3 930 ± 185* 127 ± 38 536 ± 134 0.244 ± 0.073 275 ± 59 547 ± 95 0.523 ± 0.174 39 ± 10 Sibutramine 10 927 ± 142** 117 ± 49 525 ± 236+ 0.246 ± 0.115 239 ± 58** 548 ± 79+ 0.437 ± 0.103* 38 ± 11 MSG-treated Control 843 ± 224# 78 ± 21# 420 ± 97# 0.191 ± 0.055 265 ± 22# 475 ± 81# 0.574 ± 0.113 31 ± 9# Sibutramine 3 680 ± 227* 63 ± 21 352 ± 103 0.178 ± 0.027 222 ± 28 437 ± 54 0.512 ± 0.069 30 ± 7 Sibutramine 10 624 ± 164** 87 ± 22 332 ± 139+ 0.215 ± 0.081 207 ± 39** 429 ± 85+ 0.506 ± 0.165* 29 ± 11

Each value is the mean ± SD (ng/g wet weight of tissue) for 10 rats. #: p<0.05 compared with saline-treated and the MSG-treated crossed group, since interaction was not significant on ANOVA. +: p<0.1, *: p<0.05, **: p<0.01 compared with the crossed-control group, since interaction was not significant on ANOVA.

Fig. 3. Representative double plot of effects of sibutramine and methamphetamine on spontaneous motor activity rhythm of normal and MSG-OB female rats. Control (distilled water), administration of sibutramine 10 mg/kg p.o. and administration of methamphet- amine 10 mg/kg p.o. for 14 days to MSG-OB rats.

Table 4. Effects of chronic systemic administration of sibutramine and methamphetamine on spontaneous motor activity rhythms of normal and MSG-OB female rats

Neonatal Drugs Dose Period Amplitude Phase Mean activity treatment (mg/kg, p.o.) (hr) (logarithm) (hr) (logarithm/day)

Saline-treated Control 23.58 ± 0.27 0.661 ± 0.257 2.61 ± 1.03 5.25 ± 0.99 Sibutramine 10 23.44 ± 0.06 0.985 ± 0.402* 3.22 ± 0.26 6.16 ± 0.37** MSG-treated Control 24.01 ± 0.62 0.506 ± 0.346 0.63 ± 3.45 4.40 ± 0.28## Sibutramine 3 23.71 ± 0.52 0.645 ± 0.285 1.37 ± 1.80 5.66 ± 0.41** Sibutramine 10 23.67 ± 0.33 1.089 ± 0.236* 0.97 ± 3.49 5.33 ± 0.20** Methamphetamine 10 23.94 ± 0.21 1.070 ± 0.184* Ð1.72 ± 4.08 5.46 ± 0.39**

Each value is the mean ± SD for 4 rats. ##: p<0.01 compared with the control group treated with saline neonatally. *: P<0.05, **: P<0.01 compared with each control. 246 T. NAKAGAWA, ET AL. and movement (amplitude) in the active phase, but af- tors [5, 17, 26]. The decreases in MA and its metabolite fected neither the diurnal rhythm nor the phase in either concentrations in the tissue of MSG-OB rats appear to the normal or the MSG-OB group (Fig. 3, Table 4). be attributable to the destruction of the VMH and ARC in the hypothalamus. Slight decreases in the concentra- Discussion tions of norepinephrine, 5-HT and dopamine and its metabolites in hypothalamic tissue observed after chronic In the present study we found that 12-week-old MSG- administration of sibutramine to normal and MSG-OB OB female rats neonatally treated with MSG exhibited rats were assumed to be due to the MA uptake inhibition obesity with reduced body weight and body length and by sibutramine in the hypothalamic areas which had not increased Lee index, compared with control normal rats been destroyed, which promoted function of the MA neonatally treated with saline. An increase in retro- nervous system by increasing MA levels in synapses. peritoneal white adipose tissue weight was also found Consecutive daily administration of sibutramine at 3 in MSG-OB rats. Food intake by MSG-OB rats during and 10 mg/kg p.o. for 14 days induced body weight loss the 14-day period from 10 through 12 weeks of age and improved the Lee index in normal and MSG-OB was smaller than that by saline-treated rats. Hypotha- female rats. According to Bray, food intake and sympa- lamic levels of norepinephrine, dopamine and 5-HT, thetic nerve activity play important roles in obesity. and their metabolites, DOPAC, HVA and 5-HIAA, in These activities are controlled by various nuclei in the the MSG-OB group were lower than in the normal hypothalamus [4]. Food intake was dose-dependently group. These observations are consistent with the re- suppressed by systemic administration of a dopamine sults of many studies [21, 31, 34Ð36, 46]. release drug, amantadine, or by microinjection of this Centrally-acting antiobesity drugs now clinically used drug into the LH. Furthermore, chronic administration can be classified into serotonergic drugs, such as of amantadine decreased the body weight of obese rats fluoxetine and fenfluramine, and adrenergic drugs, such administered sulpiride, a dopamine antagonist [1]. Sys- as mazindol, and these drugs are thought to exert anti- temic administration of fluoextine, which specifically obesity effects in the hypothalamus [41]. Acute or suppresses 5-HT uptake, not only suppressed food in- chronic administration of MA uptake inhibitors in- take but also increased the extracellular 5-HT creases MA in the synapse by inhibiting MA uptake. concentration in the PVN [37]. Leibowitz reported that As a result, metabolic turnover of MA is decreased by microinjection of dopamine and norepinephrine into the reduction and feedback of MA receptors, with a de- LH strongly suppressed food intake, but that food intake crease in tissue concentrations of MA and its was increased by injection of these agents into the PVN metabolites. Methamphetamine, which releases MA, [23Ð25]. This increase in food intake caused by norepi- and pargyline, which suppresses MA oxidase, change nephrine was antagonized by microinjection of 5-HT and MA and MA metabolite levels several-fold [18, 28]. fluoxetine into the PVN [13, 42]. It was reported that On the other hand, the changes caused by MA uptake microinjection of neuropeptide Y into the PVN increased inhibitors in the MA level in cerebral tissue were statis- food intake, but did not affect food intake when admin- tically almost insignificant [17, 22, 24, 26, 28]. Wortley istered into the LH and VMH [8]. et al. and we have already reported that sibutramine Sakaguchi and Bray recorded the firing rate of sym- strongly inhibited reuptake of 5-HT, norepinephrine and pathetic nerves innervating interscapular brown adipose dopamine in mice in vivo, and caused large increases in tissue (BAT) after microinjection of 5-HT or norepi- extracellular 5-HT and norepinephrine levels and a small nephrine into the VMH or PVN [38]. They found that increase in the dopamine level, as measured by these drugs produced marked dose-dependent increases microdialysis, in the hypothalamus and striatum of rats in the firing rate of sympathetic nerves. It has been [28, 33, 44]. Inhibitory effects of sibutramine on this reported that in VMH-lesioned MSG-OB rats, lipolysis MA reuptake have also been confirmed by in in vitro and thermogenesis are decreased in association with studies [5]. It was reported that sibutramine signifi- weakened sympathetic activity by lesions in the VMH; β α cantly reduced adrenaline 1- and 2- receptors and these changes are considered to be reasons for obesity tended to decrease the number of dopamine D2-recep- in MSG-OB rats despite decreased food intake [10, 45]. ANTIOBESITY EFFECT OF SIBUTRAMINE 247

It was reported that microinjection of neuropeptide losses via stimulation of energy expenditure. Y into the PVN suppressed sympathetic nerve activity It was reported that systemic administration of in BAT, but that injections of this agent into the LH sibutramine antagonized the food intake increase in- and VMH did not do so [12]. Nevertheless, the results duced by administration of neuropeptide Y into the PVN of experiments have not always reached consistent con- [15]. This also suggested the possibility of antagonism clusions, since food intake and sympathetic nerve by sibutramine of the suppression of the sympathetic activity are controlled through various mechanisms in- nervous system activity in BAT induced by microinjec- cluding MAs such as 5-HT, dopamine and tion of neuropeptide Y into the PVN [12]. In fact, β norepinephrine as well as peptides. The mechanism of sibutramine, similarly to 3 adrenoreceptor agonists, control of various nuclei in the hypothalamus therefore enhances oxygen uptake, glucose utilization and ther- requires further investigation. mogenesis in BAT [6]. Studies of microinjection of Edwards et al. reported that chronic systemic admin- sibutramine into hypothalamic areas and electrophysi- istration of 5-HT, which does not cross the blood-brain ological determination of the sympathetic nervous barrier, and must therefore act peripherally, induced system activity in BAT should be performed in order to body weight losses in male and female rats, but that a elucidate further the elevation of sympathetic nerve ac- food intake-suppressing effect of 5-HT was found in tivity caused by sibutramine in future. male rats but not female rats [11]. For this reason, we Corresponding to previous report, the results of this used female rats in the present study but a marked de- experiment indicated a decrease in spontaneous motor crease in food consumption caused by the administration activity by MSG-OB rats [34]. Daily administration of of sibutramine was observed for both normal and MSG- sibutramine at 10 mg/kg p.o. or of methamphetamine OB female rats. This suggests that peripheral and central (MA releaser) at 10 mg/kg p.o. for 14 consecutive days effects of some factor other than 5-HT are involved in increased the amplitude of the diurnal motor activity the suppression of food intake due to sibutramine. Simi- rhythm and the mean motor activity of normal and lar findings of suppression of food intake by sibutramine MSG-OB rats. These findings suggest that sibutramine have been observed for obese rats fed a high-fat diet, enhanced motor activity in the active phase rather than genetically obese Zucker rats, obese-diabetic ob/ob mice in the resting phase. Many studies have indicated the and normal rats [9, 20, 39, 40]. participation of MA in the motor activity rhythm. Meth- The monoaminergic effects of sibutramine also ex- amphetamine increases spontaneous motor activity via plain the finding that suppression of food consumption release of dopamine from the corpus striatum [33]. We by sibutramine is antagonized by pretreatment with reported that sibutramine has dopaminergic effects, β blockers of 5-HT2A/2C receptors, 1 adrenoreceptors, and since it increased the extracellular dopamine level in α particularly 1 adrenoreceptors [19]. Interestingly, Jack- the rat corpus striatum as measured by a microdialysis son et al. reported that low-dose administration of either method, induced ipsilateral circling behavior in rats in the selective 5-HT reuptake inhibitor fluoxetine or the which the corpus striatum had been lesioned unilater- selective norepinephrine reuptake inhibitor nisoxetin did ally by means of 6-hydroxydopamine, and antagonized not suppress food intake, but that combined administra- reserpine-induced suppression of conditioned avoidance tion of the two noticeably suppressed food intake [20]. behavior [19, 28, 32, 33]. The food intake-suppressing effect of sibutramine there- These findings suggest that the reversal by sibutramine fore appears to be due to a synergistic effect of of the decrease in rat motor activity caused by neonatal noradorenergic and serotonergic systems resulting from MSG treatment is due to dopamine reuptake inhibition norepinephrine and 5-HT reuptake inhibition. Toler- in the striatum in addition to inhibition of MA reuptake ance did not occur in food intake suppression since in the hypothalamus. Tolerance to sibutramine did not interaction of sibutramine x day on ANOVA for food occur in the increase in spontaneous motor activity, since intake was not significant. We believe that the food the interaction of sibutramine x day on ANOVA for mo- intake-suppressing effect of sibutramine mediated via tor activity was not significant. We believe that the norepinephrine, 5-HT and dopamine reuptake inhibi- increase in spontaneous motor activity caused by tion continued for 14 days and caused body weight sibutramine via MA reuptake inhibition in rat striatum 248 T. NAKAGAWA, ET AL. and hypothalamus continued for 14 days and caused body Psychopharmacol. Biol. Psychiatry. 12: 575Ð584. weight losses via an increase in energy expenditure. 6. Connoley, I.P., Liu, Y.L., Frost, I., Reckless, I.P., Heal, D.J., and Stock, M.J. 1999. 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