The Effects Of Rimonabant On Brown Adipose Tissue In Rat

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Abstract
The cannabinoid CB1 receptor antagonist rimonabant (SR 141716) produces a sustained decrease in body weight on a background of a transient reduction in food intake. An increase in energy expenditure has been implicated, possibly mediated via peripheral endocannabinoid system; however, the role of the central endocannabinoid system is unclear. The present study investigates this role. Rimonabant (10 mg/kg IP) was administered for 21 days to rats surgically implanted with biotelemetry devices to measure temperature in the interscapular brown adipose tissue (BAT). BAT temperature as a putative measure of thermogenesis in the BAT, physical activity, body weight, food intake, as well as changes in UCP1 messenger RNA (mRNA) and protein were measured. In addition, role of the CNS in mediating these actions of rimonabant was determined in rats where the BAT was sympathetically denervated. As expected, chronic administration of rimonabant significantly reduced body weight for the entire treatment period despite only a transient decrease in food intake. There was a profound increase in BAT temperature, particularly during the dark phase of each circadian cycle throughout the treatment period. A corresponding increase in uncoupling protein (UCP1) was also observed following chronic rimonabant treatment. The rimonabant-induced elevation in BAT temperature and decrease in body weight were significantly attenuated following denervation, indicating an involvement of the CNS. These findings suggest that the long-term weight loss associated with rimonabant treatment is due at least in part to an elevation in energy expenditure, represented here by elevated temperature recorded in the BAT, which is mediated primarily by the central endocannabinoid system.

Introduction
A well-established body of evidence has shown that the cannabinoid system is an important modulator of appetite. Administration of natural and endogenous cannabinoid ligands either systemically (1,2,3) or into discrete regions of the hypothalamus (4,5,6) stimulate appetite. Further, the levels of an endogenous cannabinoid ligand, 2-arachidonoylglycerol (2-AG), increase in the hypothalamus in normal animals during fasting, decrease during refeeding, and return to normal when rats are satiated (6). These observations point to an involvement of the CNS cannabinoid system in the normal regulation of energy balance. In obese rodent models, animals with increased food intake resulting from diet-induced obesity (DIO) (7) or leptin receptor mutation (8) show elevated levels of endocannabinoids within hypothalamic nuclei. Taken together, these types of studies provide strong evidence that activation of the endocannabinoid system impinges on energy balance and specifically produces overeating by widespread action in the hypothalamus and in elements of the mesolimbic circuitry associated with hedonistically based hyperphagia (9).

By extension, such observations also highlight the potential to use specific cannabinoid receptor antagonists to block appetite. Acute administration of the CB1 receptor antagonist SR 141716 (rimonabant) (10) reduces appetite in lean (11,12,13) and DIO (7) rodents. Chronic administration of rimonabant also produces a persistent reduction in body weight (11) despite the finding that tolerance develops to its anorectic effects within 5 days (11). This latter evidence supports a role for endogenous cannabinoids in regulating energy expenditure. Indeed, in experiments where food intake is controlled, DIO mice given SR 141716 lose more weight than their vehicle-treated counterparts (7). Furthermore, adult CB1 receptor knockout mice have a lean phenotype that is attributed more to "metabolic factors" than to the hypophagia that is pronounced in the immature knockout mice (14).

Studies examining the metabolic factors affected by cannabinoids have focused on the regulation of lipolysis (14). Treatment of DIO mice with SR 141716 changes the phenotype of obese adipocytes to that of lean mice (15). Importantly, these transcriptional changes are consistent with enhanced lipolysis and increased energy expenditure through futile cycles (15). The responses to CB1 receptor antagonists have however not been restricted to white adipose tissue. Although not well established, administration of another CB1 receptor antagonist, AM251, has been reported to elevate expression of uncoupling proteins in interscapular brown adipose tissue (BAT) suggesting a role for thermogenesis (16). These data are consistent with the microarray studies showing changes in the expression of genes associated with enhanced energy expenditure in BAT following rimonabant treatment (15).

Notably, studies examining the effects of CB1 receptor antagonists on energy expenditure have assumed that effects are exerted via a peripheral mechanism (14) however there is no direct evidence to support this claim. On the other hand, CB1 receptors are associated with hypothalamic regions known to project to sympathetic outflows and have been localized specifically with neurochemically identified neurons that are "premotor" to sympathetic preganglionic neurons (14,17). On balance, it would appear therefore that despite intense interest in the efficacy of rimonabant in reducing body weight in the clinic and a focus on the mechanisms involved, there remains an uncertainty with regard to 1) the nature of the metabolic factors that may underpin the energy expenditure component of weight loss and 2) the extent to which these are mediated directly on the peripheral target tissues or via the CNS.

The present study addresses these two key issues basic to the understanding of the actions of rimonabant on body weight. First, a telemetric approach is used to directly measure changes in the BAT temperature in response to chronic peripheral administration of the CB1 receptor antagonist rimonabant. Second, in order to evaluate directly the extent to which rimonabants' effects on BAT temperature are mediated via central neural circuits, these experiments have also been conducted in rats where the BAT has been surgically isolated from its CNS innervation. It is hypothesized that chronic rimonabant treatment will change BAT temperature, an indirect measure of adaptive thermogenesis, in such a way as to contribute to the overall impact of the drug on body weight.

Materials and Methods
Animals, surgical, and histological procedures
Experimentally naive male Sprague-Dawley rats ~8 weeks old and weighing 200—300 g were used. Rats were housed individually in opaque polypropylene cages with stainless steel wire lids. Cages were lined with dust-free wood chips and were housed in a climate-controlled (20—23 °C) room maintained on a 12-h: 12-h reverse light—dark cycle (lights off at 0930 h). Experimental testing commenced at 0900 h, that is, 30 min before the onset of the dark cycle. Rats had ad libitum access to standard laboratory chow (GR2 rat and mouse cubes, Ridley AgriProducts, Pakenham, Victoria, Australia; Analysis: acid det. fiber = 10.2%, N.D.F. = 29.4%, protein = 22.4%, fat = 3.8%, crude fiber = 6.7%, digest. energy3 = 16.6 MJ/kg DM, av. CHO = 27.9%) and tap water in the cages.
This study was reviewed and approved by the Monash University School of Biomedical Sciences Animal Ethics Committee. All surgical procedures were performed under surgical anesthesia, induced and maintained by inhalation of isoflurane (Isorrane, Baxter Healthcare Pty Ltd, New South Wales, Australia), with depth of anesthesia assessed by loss of pedal withdrawal and corneal reflexes.

Drugs
Rimonabant (5-(p-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-N-piperidinopyrazole-3-carboxamide, Sequoia Research Products (Pangbourne, UK)) was mixed with a few drops of Tween 80 (polyoxtethylene sorbitan monooleate, ICN Biomedicals, Seven Hills, New South Wales, Australia). Physiological saline was then added, and solution was stirred and sonicated. The final vehicle solution contained 15 l Tween 80/2 ml saline. Rimonabant was administered intraperitoneally (IP) at a dose of 10 mg/kg in a volume of 1 ml/kg at the same time every day for 21 days.
Implantation of telemetry probes
To implant the temperature sensitive TA10TA-F40 biotelemetry probes (DSI Systems, St Paul, MN) a 2-cm long incision was made in the flank region and a pouch formed by blunt dissection to accommodate the radio transmitter. A second incision was made between the scapulae and the BAT exposed by dissecting the overlying fascia. Further blunt dissection was used to form a subcutaneous channel between the two openings. The thermistor tip of the biotelemetry cannula was guided though this channel and sutured to muscle to maintain its position between the left and right lobes of the BAT. The flank wound was closed with Michel clips and the interscapular incision with silk sutures. The position of the thermistor tip in relation to the BAT was confirmed postmortem.
Denervation of BAT
The BAT was bilaterally denervated by exposing and identifying the nerve bundles entering each of the left and right pads. In most cases, four nerve bundles were found to project into the fat pads, with smaller branches sometimes running alongside blood vessels entering the fat pad. Each of the branches was cut and the blood vessels carefully stripped, leaving the vascular supply intact. For the sham-operated rats, the BAT and nerves were identified and exposed but not transected.
Analgesia
Following each surgical procedure, lignocaine HCL (2% Lignomav; MAVLAB Australia) was administered subcutaneously (s.c.) in the area surrounding the incisions. Meloxicam (Metacam 1.0 mg/kg s.c.; Boehringer Ingelheim Products) was administered during the surgery, and again 8—12 h later to manage postoperative pain and inflammation. Animals were closely monitored and allowed to recover for at least 7 days before testing.
BAT temperature measurement
BAT temperature was measured using biotelemetry probes (DSI Systems, St Paul, MN), with transmitted signals received by RPC-1 receiving plates mounted under the cages. Each cage was positioned at least 32 cm apart to eliminate any interference (DSI Systems, St Paul, MN). The data were relayed to a data-exchange matrix (DSI Systems, St Paul, MN) connected to an IBM desktop computer. Data were converted into a temperature reading through a set of precalibrated functions and recorded using a Dataquest ART 2.2 Gold acquisition system (DSI Systems, St Paul, MN). BAT temperature was recorded in this way at 3-min intervals for 24-h periods on alternate days for 21 days.
Physical activity
Locomotor activity was assessed using the same biotelemetry probes. Activity was expressed in ambulatory units and represented the displacement or distance traveled by the animal. Activity was measured once every 10 s and a value recorded every min. Similar to BAT recording, activity counts were obtained at 3 min interval for 24 h periods on alternate days for 21 days.
Experimental regimens
Allowing at least 7 days to recover from implantation of biotelemetry devices into the BAT, recordings of BAT temperature, physical activity, food intake, and body weight were commenced and continued for 21 days. On the first day, baseline parameters were recorded over a 24-h period following IP injection of vehicle (Tween 80 in saline) 30 min before the onset of the dark cycle. Subsequently, either rimonabant (10 mg/kg; n = 7) or vehicle (n = 7) were injected daily as described above. Food consumption was measured both at 2 h and 24 h following injections. Body weight was measured every 24 h generally just before the drug or vehicle administration. Recordings were made from each rat every second day and groups were divided such that recordings were made from half of the drug- and vehicle-treated groups each day.
Following the 21 days of measurement, half (n = 7) of the total group of rats underwent bilateral surgical denervation of the BAT and the other half (n = 7) were sham operated. Rats comprising the denervated and sham groups were derived equally from those previously exposed to either rimonabant or vehicle. After 3 days recovery from surgery, both groups of rats were exposed to rimonabant daily for 8 days with each rat having recordings of BAT temperature and body weight on every second day as outlined above.

Another cohort of rats (N = 10) was given rimonbant daily for 21 days and killed by brief CO2 exposure and the intrascapular BAT removed and snap-frozen in liquid nitrogen and stored at -80 °C for UCP1 protein and mRNA quantification.

Histological verification of denervation
Following completion of the experimental protocol, rats were deeply anesthetized and perfused transcardially with aldehydes, their BAT was removed and placed in 4% paraformaldehyde at 4 °C for 24 h. The BAT was then transferred to 15% sucrose in a phosphate buffer (PB, 0.1 M, pH 7.3) solution at 4 °C for 24 h, and finally placed in 30% sucrose solution in PB at 4 °C for 48 h. The BAT was histologically sectioned at 20 m using a cryostat and sections mounted directly onto gelatin coated slides and stored at -80 °C.
Slides were thawed and sections demarcated with a PAP pen and given three 10-min washes in immunobuffer (IB, 10 mmol/l Tris, 0.05% thimerosal, 10 mmol/l sodium phosphate buffer, pH 7.4 and 0.3% Triton X-100). Sections were given a 30 min incubation in 10% normal horse serum in IB followed by a further incubation for 24 h at 4 °C in mouse anti-rat dopamine hydoxylase (DH) antibody (1:1000; Chemicon Int, Temecula, CA) diluted in IB containing 10% normal horse serum. Slides were then exposed to three 10-min washes in IB and incubated for 60 min in Cy-3 conjugated donkey anti-mouse IgG (Jackson ImmunoResearch, West Grove, PA) diluted 1:200. After another three 10-min washes with IB, slides were allowed to dry and coverslipped with buffered glycerol and sealed.

Western blot and qRT-PCR
Total protein extraction was performed on BAT by homogenization in extraction buffer (50 mmol/l Tris-HCl pH 7.4, 1% SDS, protease inhibitor–Roche Complete Mini tablet), gently shaking for 1 h at 4 °C, and then by centrifugation for 10 min, 10,000g, 4 °C. The supernatant were collected and the pellet discarded. Protein concentrations were determined using the Quantichrom Protein Assay Kit (Bioassay Systems, Hayward, CA). A total of 10 g of protein were loaded onto a 4—20% SDS polyacrylamide gel (NuSep, Frenchs Forest, NSW, Australia) and transferred onto a nitrocellulose membrane (Schleicher and Schuell, Bartell Instruments, Australia). Adequate transfer was ensured by staining the membrane with 2% Ponceau S. The membrane was blocked with 5% non-fat dry milk powder in Tris-buffered saline/1% Tween 20 (Sigma, St Louis, MO) for 1 h and incubated overnight at 4 °C with the primary antibody for UCP1 (UCP11-A, Alpha Diagnostics International, San Antonio, TX; Rabbit polyclonal, diluted 1:1000) in 5% BSA in Tris-buffered saline/Tween 20 solution. Following secondary antibody incubation using anti-rabbit HRP (diluted 1:2000, gift from Prof. Iain Clark, Monash University) protein expression signals were visualized by chemiluminescence using the LumiGlo kit (Cell Signalling Technologies, Danvers, MA) on Amersham Hyperfilm ECL (GE Healthcare Ltd, Buckinghamshire, UK). Relative densities of protein bands were assessed using a densitometer and SynGene Gene Tools analysis software (SynGene Laboratories, Frederick, MD).
Total RNA was extracted from the BAT tissue using RNeasy micro kits (Qiagen, Melbourne, Australia) and treated with DNase to remove any residual genomic DNA. One microgram of each sample was reverse transcribed to cDNA using Quantitect Reverse Transcriptase Kit (Qiagen, Melbourne, Australia). Real-time PCR was performed on an EP- Gradient-S Real Time PCR machine (Eppendorf, Germany). Relative levels of mRNA expression for UCP1 (Applied Biosystems, Foster City, CA; assay code number Rn00562126_M1) were analyzed. A comparative Ct (cycle of threshold fluorescence) method was used with -actin gene (Applied Biosystems, Foster City, CA; part number 4352340E) as an endogenous reference to calculate the relative expression levels in each sample (Livak & Schmittgen 2001). Briefly, Ct values for the -actin gene were subtracted from the Ct values of the gene of interest to give a Ct value. The Ct value of the calibrator (the mean Ct value of the saline infused group) was then subtracted from each individual sample to give a Ct value. This number was then inserted into the formula 2-Ct to give the expression level relative to the calibrator.

Statistical analysis
The BAT temperature (°C), physical activity measurement, and amount of food consumed (g) were used as dependent variables and analyzed separately. Data for the baseline BAT temperature, activity count, food consumed, and body weight were analyzed using paired t-tests or repeated measures ANOVAs.
Data for the BAT temperature and physical activity were collapsed into 1-h time intervals. The first hour was further subdivided into 15 min and 30 min intervals. This was done to determine whether there was an observable elevation in thermogenesis immediately following rimonabant treatment as acute effects of the drug on food intake appear during this time. These data were further averaged for each time interval, for each rat in each treatment group, for the duration of the treatment period to give 24 h variation in BAT temperature. Time was treated as a factor. Repeated measures (treatment by time) ANOVAs with time being the repeated measure was conducted on each dependent variable. Similarly, body weight for each day for each group was averaged and analyzed using a repeated measures (treatment by day) ANOVA with day as the repeated factor. Where significant main effects were found, pairwise comparisons were conducted using Bonferroni adjustments for multiple comparisons. Mauchly's W was computed to check for violations of the sphericity assumptions. When Mauchly's W test was significant, the Greenhouse—Geisser correction was applied. For western-blot and qRT-PCR, t-test was performed to determine if the gene and protein expression of the various groups were significantly differentially expressed.

Results
The rats (N = 14) were randomly assigned to two equal groups, rimonabant or vehicle (Tween 80 in saline) such that the body weights between the two groups were evenly matched. These groups did not show any difference in baseline values of BAT temperature, physical activity, and food intake.

Effects of rimonabant on food intake and body weight
When rimonabant was administered IP to rats daily just before the beginning of the dark phase, (10 mg/kg/day for 21 days) food intake was significantly reduced in the 24 h following treatment (F(1,12) = 15.01; P < 0.01) (Figure 1). This effect lasted for approximately the first half of the 21 day exposure to the drug (Figure 1a). Thereafter, the food consumed in each 24 h period was indistinguishable in the rimonabant- and vehicle-treated groups. Analysis of the food consumed in the 2 h immediately following administration of rimonabant showed essentially the same time course of reduced intake over the treatment period with a return to levels of food intake seen in vehicle-treated rats after ~12 days (F(1,12) = 12.17; P < 0.01) (Figure 1b). Comparison of 2 and 24 h food intake measurements indicated that most of the observed daily reduction in feeding occurred in the first 2 h, a feature that is borne out by examination of the last 22 h of daily food consumption where there is no difference between control and treated groups (Figure 1c).

Despite the return of food intake to control levels midway through the treatment protocol, body weight gain in the rimonabant-treated group remained low compared to vehicle-treated rats (F(1,12) = 7.90; P < 0.05) (Figure 2). The reduction in body weight gain was greatest during the first 10 days (treatment by time interaction (F(4,48) = 3.33; P < 0.01) (see Figure 2, part A). In the 11 days following the return of food intake to control levels (see Figure 1a and Figure 2, part B), the reduction in body weight gain in the rimonabant-treated group persisted for the remainder of the treatment period (treatment by time interaction (F(4,48) = 2.82; P < 0.05) (see Figure 2, part B).

Effect of rimonabant on BAT temperature
During the same 21 days of exposure of rats to rimonabant and vehicle, BAT temperature was measured with an indwelling thermister positioned between the lobes of the BAT. Vehicle-treated rats displayed the well-described circadian shift in BAT temperature with an elevation throughout the dark phase that was immediately reduced at the beginning of the light phase (Figure 3). When rimonabant was administered, there was a profound elevation of temperature (1.5—2.0 °C) measured in the BAT that differed significantly from controls during the entire dark phase (F(1,12) = 32.05; P < 0.001) (Figure 3). This elevation of temperature tended to persist into the light phase when compared to controls, however the latter difference did not reach significance (i.e., P = 0.08).

Locomotor activity was assessed throughout the testing period in all rats using biotelemetry devices (see methods) that measured point to point displacement. While there was a distinct diurnal rhythm in activity this was unaffected by treatment with rimonabant (Figure 4).

Effect of denervation of the BAT on the BAT temperature stimulating effect of rimonabant
Following the 21 days of rimonabant treatment outlined above, rats (n = 14) were randomly allocated to two equal groups, one underwent surgical denervation of the nerves directed to the BAT and the other was sham operated (see methods).
Administration of rimonabant to the denervated group resulted in an amelioration of the elevation of BAT temperature seen following the exposure of the drug to sham-operated rats. Rimonabant treatment of the denervated animals produced a temperature response that was statistically indistinguishable from the vehicle-treated controls but was significantly reduced from the effect of rimonabant on sham-operated rats (F(1,18) = 11.87; P < 0.001) (Figure 5). As might be expected, the effect of rimonabant on BAT temperature in the sham-operated group was indistinguishable from intact rats treated with the drug (see Figure 3).

Consistent with the data shown in Figure 2, sham-operated animals given rimonabant showed a significant reduction in body weight gain when compared to vehicle-treated animals for the entire duration of the drug exposure (F(1,12) = 5.59; P < 0.05) (Figure 6). Interestingly, when denervated animals were injected with rimonabant there was a significant attenuation of the body weight reducing effect of the drug compared to its impact on sham-operated controls (F(1,12) = 26.45; P < 0.001) (Figure 6). However, the body weight gain achieved by the denervated animals treated with rimonabant was still significantly lower compared to the vehicle-treated group during days 6 and 8 of the treatment regimen.

In order to confirm the completeness of the denervation, BAT from the denervated and sham-operated groups were exposed to antisera raised against dopamine hydroxlase, a marker of noradrenergic (sympathetic) inputs to the BAT. In the sections examined, no immunoreactive fibers could be detected in the denervated tissue whereas these fibers were abundant in the sham-operated BAT (Figure 7).

Effect of rimonabant on the expression of UCP1 mRNA and protein
In order to determine whether changes in UCP1 mRNA and protein could be detected in BAT following rimonabant treatment, rats (N = 10) were randomly assigned to two equal groups and administered either vehicle or rimonabant for 21 days. Using western blot analysis, a significant elevation in UCP1 protein in rats given long-term rimonabant was observed (Figure 8), however, no such elevation was seen in the mRNA for UCP1 in the BAT of rimonabant-treated animals.

Discussion
The major findings of the present study are that rimonabant delivered intraperitoneally produces a reduction in body weight commensurate with a transient reduction of food intake, which occurs only in the first half of the chronic treatment, and a persistent elevation of BAT temperature. Importantly, the impact of rimonabant on BAT temperature is predominantly mediated via the CNS as demonstrated by the elimination of the effect following the denervation of the BAT. It appears that physical activity does not play a role in the reduction of body weight associated with treatment with the CB1 receptor antagonist.

That rimonabant induces an immediate and sustained decrease in body weight despite only a transient decrease in food intake is in agreement with pervious studies (11,14,15,18,19). These observations have consistently implicated energy expenditure as a mechanism underlying the anti-obesity effect of rimonabant. More direct evidence in support of this concept arises from studies showing that rimonabant given chronically elevates basal oxygen consumption (18) and increases the expression of genes favoring energy-expenditure (15). The results presented here provide the first measurement of elevated BAT temperature arising from chronic administration of rimonabant. In these and many other experiments, the dissipation of heat from the BAT resulting from the UCP1-mediated uncoupling of oxidative phosphorylation is seen as a reliable measure of the extent of thermogenesis in that organ. (for review, see ref. 20). In data derived from other experiments in our laboratory (Stefanidis, Verty & Oldfield, unpublished data), we have shown that BAT temperature, as a measure of thermogenesis, varies in the same direction as, but is temporally independent of, shifts in core temperature. It is likely that the same relationship between the two measures exists in the present experiments. We also show for the first time that chronic rimonabant treatment is associated with an elevation in the protein rather than the message for the UCP1 gene indicative of a long-term activation.

The question arises as to the mechanism by which rimonabant reduces body weight through an effect on energy expenditure. There is a current view that supports the idea that cannabinoids and their receptor antagonists directly modulate the activity of CB1 receptors located in peripheral tissues such as BAT (14,15,21). An alternate possibility is that the effects of rimonabant are mediated through the CNS, particularly in view of the well defined central neural pathway modulating the sympathetic input to BAT (17,22). In this respect, the mRNA for CB1-R has been localized in neurons expressing orexin-A, melanin concentrating hormone (MCH) and cocaine and amphetamine regulated transcript (CART) (14). Each of these peptides has been directly demonstrated to be key peptidergic components of the descending, synaptically linked projections to BAT (16). Furthermore, we have recently shown that rimonabant-induced elevation of Fos occurs in neurons expressing the peptides CART, orexin, MCH, and alpha-melanocyte stimulating hormone (-MSH) (23). Each of these observations, and the clear indication from the present data of a major central component to the control of BAT temperature by rimonabant, point to this pathway as a conduit for the actions of endocannabinoids on energy balance.

It is clear from our current experiments that the surgical section of nerve branches leading to the BAT results in an attenuation of the rimonabant-induced elevation of BAT temperature to levels that are not statistically different from vehicle; however there is a tendency particularly during the dark phase for temperatures to remain slightly elevated. If this is the case, the possibility remains that in addition to the substantial central control indicated by the denervation, there is a residual effect of rimonabant that is attributable to a peripheral action perhaps directly on CB1 receptors in the BAT. Alternatively, it is conceivable that any remaining slight elevation of temperature is due to surviving small sympathetic branches possibly running with blood vessels to the BAT. The latter seems less likely given our evidence that sympathetic nerve terminals are not detectable in the BAT after denervation. It should be recognized however that it is not possible to state with certainty that no terminals remained.

Similar to the reduction of the BAT temperature in denervated animals given rimonabant, there was also an attenuation of the reduction of body weight gain of these animals when compared to a sham-operated rimonabant-treated group. However, the body weight gain in the denervated rats was still significantly lower compared to vehicle controls (see Figure 6). These data are not surprising considering that the BAT is not the only organ in the body responsible for energy expenditure (24). Our data are consistent with the possibility that other organs in the body that express CB1, are still actively responding to the energy expending effect of rimonabant (18,25).

The finding that chronic (21 day) application of rimonabant produced a decrease in 2 h food intake for only the first 10 days after which energy intake returned to baseline is in good agreement with previous reports showing tolerance to the anorectic effects of rimonabant treatment in lean (11), ob/ob mice (18), and diet-induced rats (19). Unlike the study by Liu et al. (18) where suppression of food intake was seen 5 days after commencement of rimonabant treatment, the onset of hypophagia detected in the present study was immediate and sustained and signs of tolerance were seen only on the 8th day of rimonabant treatment. The mechanisms by which rimonabant produces a short term anorectic effect is not well-defined.

The observed effect of rimonabant on food intake could be due to an interaction between the hypothalamic endocannabinoid system and other mediators of food intake. Indeed, the hyperphagia induced by a melanocortin receptor specific antagonist is significantly attenuated by rimonabant, (26) consistent with an involvement of endocannabinoids in melanocortin receptor inhibition. In addition to modulating the activity of anorexigenic mediators of food intake, rimonabant also decreased food intake induced by the application of morphine into the hypothalamic paraventricular nucleus (27). Furthermore, rimonabant reduces energy intake in food-deprived NPY-deficient mice (8) and, AM251, an analogue of rimonabant, inhibits expression of NPY mRNA in rat hypothalamic explants (28). Other clues as to the nature of the interaction between rimonabant and hypothalamic circuits mediating energy balance arise from co-localization studies, outlined above, that demonstrate co-expression of CB1 R mRNA and a range of hypothalamic peptides including orexin A, MCH and CART (14). Collectively, these data indicate the potential for an interaction between endocannabinoids and neurons regulating food intake in the lateral hypothalamus. The neural circuitry underlying the complementary effect of rimonabant on energy expenditure however is less well defined.

Whilst further mechanistic studies are required, it is our conclusion that energy expenditure, and thermogenesis in particular, may represent key components of the actions of rimonabant. Furthermore, we conclude that much of this effect is mediated via the CNS. The latter has important implications for the informed use of this anti obesity therapy, although caution needs to be exercised in the translation of data and the doses used in the present experiments to the human situation. However, much of the current debate related to the efficacy of rimonabant centers on its possible side effects on mood and other centrally derived depressive disorders (24,25). In this context, it is imperative to have a clear understanding of the effects of the drug that are mediated via peripheral vs. central mechanisms.

Source, Graphs and Figures: Obesity - The Effects of Rimonabant on Brown Adipose Tissue in Rat: Implications for Energy Expenditure
 
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