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Effects of Fluoxetine Administration on Neuropeptide Y and Orexins in Obese Zucker Rat Hypothalamus Arantza Gutie´rrez,* Gonzalo Saracı´bar,* Luis Casis,* Enrique Echevarrıá,* Vıćtor Manuel Rodrı´guez,† Maria Teresa Macarulla,† Luis C. Abecia,‡ and Marıá P. Portillo‡

Abstract GUTIE´ RREZ, ARANTZA, GONZALO SARACI´BAR, LUIS CASIS, ENRIQUE ECHEVARRIÁ, VIĆTOR MANUEL RODRI´GUEZ, MARIA TERESA MACARULLA, LUIS C. ABECIA, AND MARIÁ P. PORTILLO. Effects of fluoxetine administration on neuropeptide Y and orexins in obese Zucker rat hypothalamus. Obes Res. 2002;10: 532–540. Objective: The aim of this work was to study the potential involvement of neuropeptide Y (NPY) and orexins in the anorexigenic mechanism of fluoxetine in obese Zucker rats, assessing the effects of chronic fluoxetine treatment on NPY and orexin immunostaining in several hypothalamic regions. Research Methods and Procedures: Male obese Zucker (fa/fa) rats were administered fluoxetine (10 mg/kg intraperitoneally) daily for 2 weeks. The control group was administered 0.9% NaCl solution. Carcass composition was assessed using the official methods of the Association of Official Analytical Chemists. To test the potential thermogenic effect of fluoxetine administration, total body oxygen consumption was measured daily for 60 minutes before fluoxetine or saline injection and for 30 minutes after drug or saline injection. Hypothalamic arcuate and paraventricular nuclei, and the lateral hypothalamic area were immunostained for NPY, orexin A, and orexin B. Commercial kits were used for serum determinations. Results: Chronic fluoxetine administration in obese Zucker rats generated a reduction in body weight gain, food in-

Submitted for publication August 20, 2001. Accepted for publication in final form March 7, 2002. Departments of *Physiology, †Nutrition, and ‡Preventive Medicine and Public Health, Faculty of Pharmacy, University of the Basque Country, Paseo de la Universidad 7, 01006 Vitoria, Spain. Address correspondence to Dr. Enrique Echevarrıá, Department of Physiology, Faculty of Pharmacy, University of the Basque Country, Box 450, 01080 Vitoria, Spain. E-mail: [email protected] Copyright © 2002 NAASO

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take, adipocyte size, fat mass, and body protein. A decrease in NPY immunostaining in the paraventricular nucleus, without changes in the arcuate, was observed. However, no changes were observed in the number of neural cells immunostained for orexin A or orexin B in the lateral hypothalamic area. Discussion: Due to the hyperphagic effect of NPY in the paraventricular nucleus, these results suggest that NPY, but not orexins, could be involved in the anorexigenic effect of fluoxetine in obese Zucker rats. Key words: fluoxetine, Zucker rats, orexins, hypothalamus, neuropeptide Y

Introduction Maintenance of body weight is achieved by the balance between energy intake (food consumption) and expenditure. This energy homeostasis is ultimately governed by the brain, where a variety of afferent signals reflect the animal’s nutritional state and its external environment (1). The central site of integration is the hypothalamus (2– 4). The major appetite-modulating neurotransmitter is serotonin (5-HT), a potent anorexigenic agent, which is found in neurons projecting from the raphe nucleus to the hypothalamus (5). These 5-HT receptors are abundant in the hypothalamus, especially in the paraventricular (PVN) and arcuate (ARC) nuclei (6). Potential functional interactions among serotonin receptors and different neuromodulatory systems could be involved in the physiological regulation of appetite. Convincing evidence supporting important roles for certain hypothalamic neuropeptides in the central regulation of energy balance has been provided by molecular genetic studies in rodents (7). Neuropeptide Y (NPY), a 36-amino acid peptide, is a powerful feeding stimulant that is synthesized in the ARC nucleus of the hypothalamus and is found

Fluoxetine and NPY in Obese Zucker Rats, Gutie´rrez et al.

extensively throughout the central and peripheral nervous systems (8,9). It also reduces energy expenditure. Sakurai et al. (10) recently discovered and characterized orexins, a family of neuropeptides that stimulate food consumption. Orexin-positive neuronal cell bodies from the lateral hypothalamus project to several brain regions, where orexins may act as neuromodulators through their action on specific receptors, and mediate appetite and other behavioral functions in rodents. Anorexigenic effects have been described in rodents after chronic administration of fluoxetine, a specific serotonin reuptake inhibitor that enhances 5-HT action by blocking its reuptake at nerve endings (11,12). However, little is known about the effects of fluoxetine either on the NPY and orexin systems or on energy balance, in obese Zucker rats, a well-characterized animal obesity model, in which hypothalamic disfunctions, such as elevated content of NPY in arcuate, paraventricular, suprachiasmatic, and medial preoptic areas account for hyperphagia, decreased sympathetic activity, and increased lipogenic enzyme activities (13–16). This internal milieu promotes accretion of body fat (17). The aim of this work was to shed some light on the potential involvement of NPY and orexins in the anorexigenic mechanism of fluoxetine in obese Zucker rats, assessing the effects of chronic fluoxetine treatment on NPY and orexin immunostaining in several hypothalamic regions. Body weight and composition, food intake as well as changes in epididymal, perirenal, subcutaneous and brown interscapular adipose tissues, and serum parameters also are evaluated.

Research Methods and Procedures Animals and Experimental Design Male obese Zucker (fa/fa) rats were obtained from CRIFFA (Barcelona, Spain) and maintained in a temperature- (22 ⫾ 2 °C) and humidity- (50%) controlled room with 12 hour/12-hour light-dark cycle (lights on at 8:00 AM) and given free access to standard rat chow diet (pelleted) and water. They were housed individually in polycarbonate metabolic cages (Tecniplast, Gazzada, Italy). After 7 days of adaptation to these conditions, the rats (245 ⫾ 3 g) were randomly divided into two groups (n ⫽ 14/group). Body weight and food and water intakes were measured daily. Rats were carefully handled and all experiments were carried out in accordance with the local ethics board. Rats assigned to fluoxetine administration (fluoxetine hydrochloride; Lilly, Madrid, Spain) were subjected to daily intraperitoneal injections (10 mg/kg) for 2 weeks. The control group was given 0.9% NaCl solution in the same volume and duration as the fluoxetine-treated group. Twenty-four hours after the last treatment, six rats per group were anesthetized intraperitoneally with equithensin (2 mL/kg), an alcoholic solution of nembutal and chloral hydrate (Sigma, St. Louis,

MO) and perfused transcardially under deep anesthesia with saline plus 50 mM phosphate buffer (pH 7.4), followed by 4% paraformaldehyde (Sigma). The remaining eight rats in each group were killed after an overnight fast. Interscapular brown adipose tissue (IBAT) and white adipose tissue from different anatomical locations, subcutaneous (SC), perirenal (PR), and epididymal (EP), were dissected and weighed. Blood was collected and centrifuged, and serum was frozen and stored (⫺80 °C) until analysis. Adipocyte Isolation and Size Determination Isolated fat cells were obtained according to the Rodbell method (18) with minor modifications (19) by collagenase A digestion (1 mg/mL; 37 °C) (Boehringer Mannheim, Mannheim, Germany) in Krebs-Ringer-Bicarbonate (KRB) buffer, containing 3.5 g/100 mL of bovine serum albumin (BSA V) (Sigma) and 0.6 mmol/100 mL of glucose at pH 7.4 (KRBA). Adipocytes were filtered through nylon mesh and washed three times with the same incubation buffer (KRBA). Fat cell size was measured by optic microscopy (Olympus BX50, Tokyo, Japan), with the aid of a computerized image analysis system (Quantimet 500 MC; Leica Espan˜ a, Barcelona, Spain) and the mean diameter was calculated from the diameter of 200 cells. Body Composition After adipose tissue dissection, carcass composition was assessed using the official methods of the Association of Official Analytical Chemists (20). Frozen carcasses were minced in a grinder. Water content was determined gravimetrically, total protein by the Kjeldahl method, and fat by ether extraction in a Soxhlet apparatus. For total body fat calculation, carcass fat and adipose tissue weights were considered. Total-Body Oxygen Consumption To test the potential thermogenic effect of fluoxetine administration, total-body oxygen consumption was measured for 60 minutes before fluoxetine or saline injection (baseline value), and for 90 minutes after drug or saline injection, daily in an open circuit chamber system (Oxymax Monitoring System; Columbus Instruments, Columbus, OH). To avoid the influence of exploratory-type behavior, rats were transferred from their habitual cage to the calorimeter chamber 1 hour before starting measurements. Oxygen consumption was calculated as the product of air flow rate (1 liter/min) through the system multiplied by the oxygen concentration difference between chamber postexchange and room air, corrected for humidity (50%) and temperature (22 ⫾ 2 °C) changes. Results represent the mean value during the measuring period and are expressed as mmol O2/kg0.75 per hour. OBESITY RESEARCH Vol. 10 No. 6 June 2002

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Fluoxetine and NPY in Obese Zucker Rats, Gutie´ rrez et al.

NPY and Orexin Hypothalamus Immunostaining The brains were removed, cut into smaller pieces, and immersed in the same fixative medium overnight. They were stored for 2 days in 0.1 M phosphate buffer containing 30% sucrose at 4 °C. Fifty micrometer sections were cut using a cryostatic microtome (Cryocut 3.000; Leica Espan˜ a) with a stereotaxic atlas guide and immunostained for NPY, orexin A, and orexin B with polyclonal antisera raised in rabbits. The antigens were detected by the avidin-peroxidase technique, using 3,3⬘-diaminobenzidine as chromogen (Sigma). After reduction of endogenous peroxidases with 1% hydrogen peroxide (Sigma) and blocking of nonspecific background staining with 5% normal goat serum (Sigma), the sections were incubated with the following immunoreagents: 1-primary antisera: rabbit anti orexin A, rabbit anti orexin B, and rabbit anti-NPY (Chemicon International Inc., Temecula, CA), commercially obtained polyclonal antibodies raised in rabbits (dilution 1:1,000); 2-goat anti-rabbit immunoglobulin: goat anti-rabbit biotinylated (Chemicon International Inc.), dilution 1:200; 3-avidin-peroxidase complex: strept ABC complex HRP (Dako A/S, Glostrup, Denmark), dilution 1:300; 4-chromogen: 3,3⬘-diaminobenzidine (Sigma), 0.3 mg/mL in 0.2 M Tris HCl buffer containing 0.03% hydrogen peroxide. Each step was followed by an appropriate wash per triplicate in phosphate buffer saline, and 0.3% Triton X-100 (Sigma) was used. Sections were carefully extended, dehydrated, and mounted (DPX mountant for histology; Fluka Chemie AG, Buchs, Switzerland), examined with an Olympus BX50F optic microscope (Olympus Optical Co. Ltd.). For NPY immunostaining, arcuate and paraventricular nuclei were selected. In the case of orexins A and B, only the lateral hypothalamic area was studied. It must be pointed out that the intensity of chromogen deposition on each stained neural structure (cell body or fiber) is not necessarily proportional to the antigen quantity that it contains. Thus, in this work, only the change in the numbers of NPY-immunostained neural fibers or cell bodies in both selected brain regions has been considered to suggest a variation in regional forebrain NPY expression in fluoxetine-treated rats compared with the controls. A hypothetical fluoxetine-induced decrease in the number of these stained fibers, which could be easily observed through optic microscopy in a selected brain region, will reflect a regional decrease in NPY levels, suggesting a possible change in the activity of NPY circuits. However, the possible lack of changes in other regions will require computerized image analysis (Quantimet 500 MC; Leica Espan˜ a S. A.) to be confirmed, due to the fact that NPY levels are not exactly the same as the number of NPY-immunostained neural fibers. The pattern of NPY immunostaining in most of the hypothalamic regions shows a high density of stained neural fibers, which are very difficult to count. Consequently, regional optical density (or gray level) was measured, with 534


the aid of a black and white computerized image analysis system, to indirectly valerate the possible fluoxetine-induced regional changes in the number of NPY-stained neural fibers in both studied regions (hypothalamic arcuate and paraventricular nuclei), with respect to the controls. In this procedure, the term optical density is analogous to gray level in every region studied. Thus, every immunostained brain slice, obtained from experimental animals, was observed through optic microscopy and a black and white video signal was generated, sent to an interface in a computerized image analyzer (doted with an specific software), and digitized. Each point or pixel in this digitized black and white image is automatically transformed to a number from 0 (black) to 255 (white). Gray level in each pixel is measured and the median value of gray level in the selected region, which is previously delimited with an optic pencil (with the aid of a stereotaxic atlas), is expressed as arbitrary units (gray level or optical density). Because background staining could be slightly different in every brain slice analyzed, gray level values were referred as increments with respect to a control region that shows a lack of NPY immunostaining, such as the corpus callosum. In both selected regions, this procedure implies an elevated number of measurements. Thus, to take into account the hypothetical variations in NPY immunostaining through different rostrocaudal levels of a brain region, such as for instance the paraventricular nucleus, at least six different brain slices (that is six different rostrocaudal levels) were analyzed. Moreover, this procedure was repeated in six experimental animals and their respective controls. Median values were obtained (mean ⫾ SEM). In the case of orexin immunostaining, scattered and intensely stained cell bodies can be easily observed and counted in the lateral hypothalamus. Consequently, counts were made in the selected hypothalamic regions with the aid of a black and white computerized image analysis system. Median values were obtained (mean ⫾ SEM) and the results were referred as the numbers of positively immunostained cells in 1 mm2 of nervous tissue, in treated and control animals. Serum Parameters Commercial kits were used for serum determinations. Glucose, triglycerides, and free fatty acids were measured spectrophotometrically (TCD-Glucose, Triglyceride GPOPAP and Free Fatty Acids Half Micro test; Boehringer Mannheim GmbH, Mannheim, Germany), leptin and insulin by ELISA (Linco Research, St. Charles, MO; Rat Insulin ELISA; DRG Instruments, Mannheim, Germany) and thyroxine (T4) and triiodothyronine (T3) by RIA (T4 and T3 Radioimmunoassay kits; Farmos Diagnostica, Espoo, Finland).


Table 1. Final body weight, body weight gain, cumulative food and water intakes, adipose tissue weights, and body composition of control and treated rats Characteristic Final body weight (g) Body weight change (g) Cumulative food intake (g) Total water intake (mL) Adipose tissue weights (g) interscapular brown perirrenal epididymal subcutaneous Body composition total fat (g) total fat (%) protein (g) water (g) ash (g) fat mass per fat-free mass

Control (n ⴝ 8)

Fluoxetine (n ⴝ 8) p value

304 ⫾ 7

243 ⫾ 5

⬍0.001

51 ⫾ 4

⫺5 ⫾ 3

⬍0.001

284 ⫾ 12

161 ⫾ 11

⬍0.001

225 ⫾ 18

111 ⫾ 15

⬍0.001

0.78 ⫾ 0.07 0.61 ⫾ 0.05 ⬍0.05 3.3 ⫾ 0.3 2.5 ⫾ 0.1 ⬍0.05 4.5 ⫾ 0.3 3.4 ⫾ 0.2 ⬍0.01 34.2 ⫾ 1.8 23.9 ⫾ 1.1 ⬍0.001 138.1 ⫾ 8.1 87.8 ⫾ 4.4 45.5 ⫾ 2.9 34.7 ⫾ 1.8 40.1 ⫾ 1.1 36.4 ⫾ 0.8 143.1 ⫾ 6.5 110.8 ⫾ 2.1 9.8 ⫾ 0.7 7.8 ⫾ 0.7

⬍0.001 ⬍0.01 ⬍0.05 ⬍0.001 NS

0.86 ⫾ 0.12 0.57 ⫾ 0.03 ⬍0.05

Values are presented as mean ⫾ SEM. NS, not significant.

Figure 1: Mean values of total-body oxygen consumption over 60 minutes (baseline) and 30 minutes (60 to 90 minutes) after fluoxetine administration in treated obese Zucker rats.

in PR white adipose tissue and 41.8 ⫾ 4.4 ␮m vs. 55.7 ⫾ 6.0 ␮m in EP white adipose tissue. Data concerning body composition showed that the reduction in body-weight gain induced by fluoxetine treatment was accounted for by both body fat and lean body mass (Table 1). Total fat mass was reduced in 36.4%. When the fat mass/fat free mass ratio, an important marker of obesity, was calculated, a significant reduction (34%) was observed in fluoxetine-treated rats. Total-Body Oxygen Consumption Total-body oxygen consumption (VO2) was measured before and after the administration of fluoxetine. No thermogenic effect of the drug was found because VO2 values were not significantly different. This situation was maintained during the 14 days of experimental treatment (Figure 1). The measurements performed in control rats demonstrated that the intraperitoneal injection of saline did not modify body oxygen consumption.

Statistical Analysis Data were expressed as mean ⫾ SEM. Statistical analysis was performed using SPSS 8.0 (SPSS Inc., Chicago, IL). The Mann-Whitney U test was used for comparisons between both groups. P values ⬍ 0.05 were considered statistically significant.

Results Body Weight, Food and Water Intakes, Adipose Tissue Weights, Adipocyte Size, and Body Composition Fluoxetine administration significantly decreased food intake (⫺43.3%), body weight (⫺20.1%), and water intake (⫺50.7%). The weights of all the measured adipose depots were lower in treated rats than in controls (Table 1). In accordance with this, adipocyte sizes were smaller in treated rats than in controls: 43.9 ⫾ 1.9 ␮m vs. 75.8 ⫾ 6.7 ␮m in SC white adipose tissue, 43.2 ⫾ 5.3 ␮m vs. 56.5 ⫾ 6.0 ␮m

Figure 2: Variations in neuropeptide Y (NPY) immunostaining in the selected hypothalamic regions, expressed as median values of integrated gray level (mean ⫾ SEM), in control and fluoxetinetreated obese Zucker rats. The key for the representation is as follows: ARC, arcuate nucleus; PAV, paraventricular nucleus. *p ⬍ 0.05.


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Figure 4: Variations in the number of neural cells immunostained for orexin A and orexin B in the lateral hypothalamic area (LHA), expressed as cells per square millimeter, in controls and fluoxetine-treated obese Zucker rats.

Discussion

Figure 3: Light micrographs showing neuropeptide Y (NPY) immunostaining in controls (A) and fluoxetine-treated obese Zucker rats (B). Abundant stained neural structures can be seen in the hypothalamic paraventricular nucleus. Magnification, 10⫻.

NPY and Orexin Hypothalamic Immunostaining Fluoxetine administration in obese Zucker rats generated a significant decrease in NPY immunostaining in the paraventricular nucleus, without changes in the arcuate nucleus (Figures 2 and 3). However, lack of changes in the number of neural cells immunostained for orexin A and orexin B was observed in the lateral hypothalamic area after fluoxetine treatment, with respect to controls (Figures 4–6). Serum Parameters Fluoxetine treatment induced significant reductions in glucose, triglycerides, and T3 and T4 levels. Leptin was 25% reduced; nevertheless, the difference between both experimental groups did not reach statistical significance (Table 2). 536


This study has shown that fluoxetine (10 mg/kg per day), a selective serotonin-reuptake inhibitor widely used as antidepressant drug, reduced body weight gain in obese Zucker rats. Although this effects has been proven in lean rodents (21,22), there is scarce information concerning fluoxetine action on body weight and energy balance in genetically obese models. In the results reported here, the sustained satiety-inducing effect of the drug during the treatment period is involved in body weight reduction. By analyzing body composition, it was observed that reduced body weight was accounted for mainly by fat stores, with little reduction in body protein (6.6%). Concerning white adipose depots, the subcutaneous fat pad seems to be the most sensitive to treatment: it was reduced by 30.2%, whereas epididymal and perirenal depots were reduced by 25.3% and 23.3%, respectively. A similar situation was found in previous studies performed in our laboratory, where obese Zucker rats were submitted to a 16% energy restriction without pharmacological treatment (23). This higher sensitivity of subcutaneous depot cannot be explained in terms of lipolysis because it has been demonstrated that lipolytic capacity of the subcutaneous depot is weaker than that of deeper depots, such as perirenal (24,25). Higher diminution of lipid deposition in this depot, with respect to visceral tissues, is more likely. To shed light onto the mechanisms underlying changes induced by fluoxetine treatment, NPY and orexin immunostaining in several hypothalamic areas was performed. The potential interactions between 5-HT and NPY neurons in the control of feeding are particularly interesting because there seem to be close anatomical links between the NPY and 5-HT neurons, especially in the PVN (26) and ARC nucleus (27). In obese Zucker rats, hyperactivity of the NPY arcuate– paraventricular projection, and reduced hypothalamic density of Y5 NPY receptors, have been described (28,29). This work showed a reduced NPY immunostaining that is a


Figure 5: Light micrographs showing orexin A immunostaining in controls (A) and fluoxetine-treated obese Zucker rats (B). Abundant stained neural cells can be seen in the lateral hypothalamic area. Magnification, 4⫻.

Figure 6: Light micrographs showing orexin B immunostaining in controls (A) and fluoxetine-treated obese Zucker rats (B). Abundant stained neural cells can be seen in the lateral hypothalamic area. Magnification, 10⫻.

significant decrease in positively stained neural cells and fibers in PVN after fluoxetine treatment. In contrast, no differences were found between both experimental groups in the ARC nucleus. These results are in accordance with those published by Dryden et al. (30) after a shorter treatment (7 days) with fluoxetine, using the same dose (10 mg/kg per day). They reported reduced NPY concentrations in the PVN, without changes in ARC, no changes in hypothalamic NPY mRNA levels and a decrease in NPY axonal transport and liberation from ARC to PVN. Accordingly, the results here reported suggest that the ARC-PVN NPY projection is involved in the anorexigenic effect of fluoxetine in obese Zucker rats. Indeed, the previous work by Dryden et al. (30) has been essential to design the experiments and to understand the results of this study. It is interesting to emphasize that despite the accordance between this study and the study of Dryden et al. (30) in terms of food intake and hypothalamic NPY changes, several important differences were found. Dryden et al. (30) could not demonstrate an effect of fluoxetine on body

weight or on plasma glucose concentration. In contrast, these data show significant reductions in these parameters in fluoxetine-treated rats compared with the controls. A potential explanation for this discordance is the age and maturation stage of the rats. Whereas Dryden et al. (30) used mature rats (initial body weight: ⬃400 g), this study was performed in young growing rats (initial body weight: 245 g), which are known to be in a dynamic state of fat accumulation (23). In contrast, it has been demonstrated that, besides hyperphagia, the high activity of lipogenic enzymes contributes strongly to fat accumulation in obese Zucker rats (31,32). Moreover, these activities are higher in young than in mature and old rats (32). These features lead to a more evident effect of energy intake reduction on fat accumulation and body weight in young rats. Due to the fact that body fat decrease leads to improved insulin sensitivity, the observed discordance in plasma glucose levels could be related to the above mentioned differences in body weight reduction. By comparing these two studies, it can be sugOBESITY RESEARCH Vol. 10 No. 6 June 2002

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Table 2. Serum parameters of control and treated rats Parameter Glucose (mg/dL) Triglycerides (mg/dL) Free fatty acids (mM) Insulin (pM) T3 (nM) T4 (nM) Leptin (ng/mL)

Control (n ⴝ 8)

Fluoxetine (n ⴝ 8) p value

130 ⫾ 6 111 ⫾ 5 ⬍0.05 346 ⫾ 53 175 ⫾ 12 ⬍0.01 0.96 ⫾ 0.07 0.93 ⫾ 0.03 NS 620 ⫾ 60 621 ⫾ 52 NS 1.50 ⫾ 0.31 0.36 ⫾ 0.03 ⬍0.01 80 ⫾ 7 51 ⫾ 5 ⬍0.001 40 ⫾ 8 30 ⫾ 4 NS

Values are presented as mean ⫾ SEM. NS, not significant.

gested that the effectiveness of fluoxetine treatment in obese Zucker rats depends on the stage of obesity development. The effects of fluoxetine on NPY immunostaining could also explain other features observed in treated rats, such as reduced water consumption. In this context, Stanley et al. (33) found an increase in drinking intake when NPY was injected in PVN of rats. Nevertheless, in this work, the opposite situation occurs: fluoxetine-treated rats showed both a reduced NPY immunostain in the PVN and a decreased water intake. Injection into the PVN has been described as reducing sympathetic activity in brown adipose tissue, consequently decreasing thermogenesis (34,35). It has also been described that acute administration of 5-HT as well as fenfluramine, a serotonin reuptake inhibitor, induces an increase in metabolic rate of ⬃21% in lean rats (36). Taken together, these reports may suggest a potential involvement of increased thermogenesis in the fluoxetine-induced weight reduction in obese Zucker rats. Accordingly, in this work, the reduced body weight observed in fluoxetine-treated rats could not only be a consequence of food intake decrease, but also of increased IBAT sympathetic activity caused by an impaired ARC-PVN NPY projection. To test this hypothesis, body oxygen consumption was measured before and after fluoxetine administration, showing no changes. These results suggest that a hypothetical action of fluoxetine on thermogenesis is not underlying body weight reduction in obese rats. It could be supposed that fluoxetine induced changes in NPY immunostaining are not big enough to stimulate thermogenesis. The high levels of NPY in obese Zucker rats are also involved in the high de novo lipogenesis observed in adipose tissue from these rats (16). Zajevski et al. (17) found that chronic NPY intracerebroventricular injection in lean Zucker rats induces an increase in the activity of acetyl-CoA-carbox538


ylase, the rate-limiting enzyme of fatty acid biosynthesis, and lipoprotein-lipase, the enzyme that allows adipose tissue the uptake of circulating triacylglycerols. Taking all these features into account, it seems likely that the reduction in PVN NPY immunostaining observed in fluoxetine-treated rats could lead to a reduced activity of the enzymes responsible for fat accumulation in adipose tissue and, consequently, to the reduction in fat depot sizes observed in these rats. The lateral area of the hypothalamus has also been implicated in the regulation of feeding behavior and energy homeostasis. It has been demonstrated that orexin nerve terminals project from the lateral hypothalamus to NPY neurons in the ARC nucleus, suggesting a functional link between NPY and orexins (37). Moreover, it has been reported that orexins, administered directly in the lateral ventricle of rats, stimulate food consumption (10). Finally, it has been suggested that NPY is involved in orexin induced feeding behavior (38,39). Consequently, the possibility that orexins could have a physiological role in fluoxetine regulation of food intake should not be discarded. The results reported here show no changes in the numbers of neural cells immunostained for orexin A and orexin B in the lateral hypothalamic area after fluoxetine treatment, suggesting a lack of orexin involvement in fluoxetine orexigenic action. Nevertheless, it should be considered that orexin immunostaining does not entirely describe the activity of orexin system, due to the fact that variations in orexin receptor numbers, receptor affinity, peptide release, peptide degradation, or even intracellular transduction could alter the function of orexin neural circuits. Insulin resistance is a well-known metabolic characteristic of Zucker rats (16,31). In this work, fluoxetinetreated rats showed significantly decreased concentrations of glucose without changes in insulin levels, suggesting improved insulin sensitivity. Due to the fact that body weight reduction per se, and more particularly adiposity reduction, produces this effect (40), it is not clear whether the observed amelioration of insulin resistance in treated rats is a direct effect of fluoxetine or a subsequent effect of body weight decrease. Finally, thyroid hormones were significantly reduced in treated rats. This effect might represent an attempt to compensate for the reduced energy intake showed by these rats, to reduce its impact on body-weight gain. In conclusion, chronic fluoxetine administration in fa/fa Zucker rats leads to a decrease in body weight, due to a reduced food intake mediated by NPY in ARC-PVN projection. However, a lack of orexin involvement in fluoxetine orexigenic action could be suggested. Additional studies focusing on the possible involvement of other hypothalamic neuropeptides in fluoxetine anorexigenic mechanism are needed.


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