Increased Energy Expenditure Contributes More to the Body Weight-Reducing Effect of Rimonabant than Reduced Food Intake in Candy-Fed Wistar Rats

The CB1 receptor antagonist, rimonabant, affects the endocannabinoid system and causes a sustained reduction in body weight (BW) despite the transient nature of the reduction in food intake. Therefore, in a multiple-dose study, female candy-fed Wistar rats were treated with rimonabant (10 mg/kg) and matched with pair-fed rats to distinguish between hypophagic action and hypothesized effects on energy expenditure. Within the first week of treatment, rimonabant reduced BW nearly to levels of standard rat chow-fed rats. Evaluation of energy balance (energy expenditure measured by indirect calorimetry in relation to metabolizable energy intake calculated by bomb calorimetry) revealed that increased energy expenditure based on increased fat oxidation contributed more to sustained BW reduction than reduced food intake. A mere food reduction through pair feeding did not result in comparable effects because animals reduced their energy expenditure to save energy stores. Because fat oxidation measured by indirect calorimetry increased immediately after dosing in the postprandial state, the acute effect of rimonabant on lipolysis was investigated in postprandial male rats. Rimonabant elevated free fatty acids postprandially, demonstrating an inherent pharmacological activity of rimonabant to induce lipolysis and not secondarily postabsorptively due to reduced food intake. We conclude that the weight-reducing effect of rimonabant was due to continuously elevated energy expenditure based on increased fat oxidation driven by lipolysis from fat tissue as long as fat stores were elevated. When the amount of endogenous fat stores declined, rimonabant-induced increased energy expenditure was maintained by a re-increase in food intake.

ENERGY HOMEOSTASIS CONSISTS of the balance between energy intake and energy expenditure. The hypothalamus has been identified as the center of energy homeostasis with a detailed system of neurons, neuronal pathways, and neuronal connectivity mediating food intake and satiation. Peripheral and central signals to the hypothalamus are involved in the maintenance of energy homeostasis (1). Obesity can be explained by the result of an imbalance of energy intake and energy expenditure. It has become an epidemic not only in industrialized but also in developing countries (2–4).

Rimonabant (SR141716) is the first selective CB1 receptor antagonist (5) currently approved in Europe as a treatment for obesity. It is an effective adjunct to diet and exercise in overweight/obese patients with associated risk factors, such as type 2 diabetes and dyslipidemia. In four placebo-controlled, multinational, double-blind, clinical Rimonabant In Obesity studies, rimonabant has not only reduced body weight (BW) but additionally improved cardiometabolic risk factors such as waist circumference, glycosylated hemoglobin, high-density lipoprotein, and triglyceride in overweight/obese patients (6–9). Rimonabant was generally well tolerated. About 50% of the effects of rimonabant on dyslipidemia and glycemia parameters have been beyond that what would be expected by BW reduction alone (6, 9).

The antiobese effect of rimonabant is mainly ascribed in animals to its CB1 receptor antagonism in the hypothalamus (10, 11) and nucleus accumbens (12–14), resulting in reduced food intake (15–20), predominantly of palatable food (21–23). However, the reduction in food intake in laboratory animals was only transient (16–19, 24), although in the presence of sustained BW reduction. These results suggested that other factors than appetite are involved in the weight-reducing effect of CB1 receptor antagonists.

The CB1 receptor is an integral part of the endogenous endocannabinoid system in the central nervous system as well as peripherally. The peripheral endocannabinoid system is involved in the control of lipogenesis in the adipose tissue (25–27) and liver (28), as well as energy expenditure by the skeletal muscle (29). It becomes transiently activated after palatable food intake and is permanently overactive after chronic high-fat diets, which might contribute to excessive lipogenesis and reduced energy expenditure in obesity (25, 28, 30).

Because total caloric intake appears to be similar in wild-type and CB1 knockout mice on a high-fat diet (19, 28), the resistance of CB1-deficient mice to diet-induced obesity may be associated with increased energy expenditure. The ability of rimonabant to increase energy expenditure was also identified by gene expression profiles in lean and diet-induced obese mice, as well as in CB1 receptor-deficient mice. The decrease in BW and adipose tissue mass in obese mice produced by rimonabant was accompanied by a reversal of obesity induced changes in the expression of a wide range of genes involved in adipocyte differentiation, lipolysis, generation of futile cycles, and glycolysis (31). Despite this overwhelming evidence that rimonabant-induced reduction of BW may be associated with increased energy expenditure, there is only one study published using ob/ob mice describing increased oxygen consumption (VO₂) as a measure of increased energy expenditure (32).

Here, we describe that rimonabant increased energy expenditure inherently. We suggest that rimonabant-induced increase in energy expenditure was more important for the sustained reduction of BW, than the reduction of food intake, which was only transient in this study.

Materials and Methods

Animals

All experimental procedures were conducted in accordance with the German Animal Protection Law, as well as according to international animal welfare legislation and rules. Female and male Wistar rats (HsdCpb:WU; Harlan Winkelman GmbH, Borchen, Germany) were used for these studies. A total of 64 female Wistar rats weighing 250 g at the beginning was used for the multiple-dose study. An additional 16 male Wistar rats were used for the single-dose study on lipolysis and glycogenolysis. When not mentioned otherwise, they were housed two per cage in an environmentally controlled room with a 12-h light, 12-h dark circle (light on at 0600 h), and ad libitum access to food and water. Standard rat chow (17.7 kJ/g, R/M-H, V1535; Ssniff, Soest, Germany) and a self-made candy diet (21.3 kJ/g) were used during the studies.

Candy diet

The candy diet was made of several candy bars in a ratio of 1:1:1:1 (Mars, Snickers, Twix, and Bounty; Masterfoods, Viersen, Germany). The candy bars were frozen in dry ice, pulverized with a commercial available cutter (Kutter HTC; Alexanderwerk AG, Remscheid, Germany), and pressed into pellets. The candy diet (21.3 kJ/g) contained crude protein, sugar (mono-/disaccharides), starch, and crude fat of 5.6, 42.3, 11.3, and 24%, compared with the standard rat chow of 18.9, 4.7, 30.7, and 3.5%, respectively (values represent percentage of weight; food analysis done by LUFA, Kiel, Germany). The high-fat and high-carbohydrate (CH) candy diet was used to induce a phenotype similar to the human syndrome of obesity and dyslipidemia.

Experimental design

Two main studies were performed: 1) a multiple-dose study in candy-fed female Wistar rats, which had been on the diet 6 wk before the start of the long-term treatment with rimonabant for an additional 6 wk; and 2) a single-dose study for the acute effects of CB1 blockade in normal-fed postprandial male Wistar rats for the investigation of direct effects of rimonabant on metabolic blood and hepatic biomarkers. Rimonabant was synthesized by the chemical department of Sanofi-Aventis, and was administered orally at the dose of 10 mg/kg by gavage in a vehicle consisting of 0.5% hydroxyethylcellulose and 0.2% Tween 80 at a volume of 5 ml/kg. The rats in all control groups received vehicle only.

Multiple-dose study

Animals were separated into four weight-matched groups, each with n = 16 animals. Group 1 served as a control group and was fed ad libitum with standard rat chow (control chow) throughout the experiment of 12 wk. The animals of the three other groups (groups 2–4) also had access to the standard rat chow but additionally to the candy diet (ad libitum for all three groups during the whole study period, except for the pair-fed control group during the last 6 wk). After the initial period of 6 wk with the candy diet, the treatment period started. Group 2 served as the candy diet control group (control candy diet) and was fed standard rat chow plus candy diet ad libitum. Rats in group 3 were treated with rimonabant (10 mg/kg; rimonabant candy diet). It was administered orally once daily to rats in the postprandial state, which had free access to food (standard rat chow and candy diet) and water during the preceding night (dark phase), at the beginning of the light phase (between 0800 and 0900 h). At 1700 h, rats in group 4 were fed delayed with the amount of standard rat chow and candy diet the rimonabant-treated rats consumed the previous day (pair-fed candy diet). They had free access to water. All rats that did not get rimonabant were treated with the vehicle (0.5% hydroxyethylcellulose/0.2% Tween 80) orally.

Part of each group (n = 8) was used for the in vivo measurement of energy expenditure (indirect calorimetry) and energy consumption (metabolizable energy intake: energy intake minus fecal caloric loss measured by bomb calorimetry). Energy expenditure as well as metabolizable energy intake were measured at the beginning (treatment d 1, treatment d 2, and treatment d 3) for the rimonabant group and the pair-fed candy diet group. The two control groups (control chow, control candy diet) were measured subsequently a week later. In addition, energy expenditure and metabolizable energy intake were determined at the end of the treatment period (treatment wk 5 = study wk 11). At this time point, half of the animals in each group were measured in the first part of the week, and the other half was measured in the second part of the week.

At the end of the multiple-dose study, rats were fasted for 4 h. Blood samples were collected from the tip of the tail for determination of glucose, as well as retro-orbitally for the determination of serum parameters [triglycerides, free fatty acids (FFAs)] in terminal isoflurane anesthesia. All blood samples were placed in serum gel tubes and were centrifuged at 4 C at 5000 rpm. After laparotomy the liver was removed, immediately freeze clamped in liquid nitrogen, and stored in −80 C for subsequent analysis of liver glycogen.

Single-dose study

Rimonabant (10 mg/kg) was administered to rats in a postprandial state, which had free access to standard rat chow and water during the preceding night (dark phase), at the beginning of the light phase (between 0800 and 0900 h). Metabolic blood and tissue parameters up to 6 h after administration were measured. After 2 and 5 h after administration, blood was collected from the tip of the tail for determination of glucose, as well as during short-term isoflurane anesthesia retro-orbitally for determination of FFAs and triglycerides. One hour later (6 h after administration), rats were anesthetized with isoflurane, laparotomized, and then killed by terminal blood collection from the aorta. The liver was removed, and specimens (about 1 g) were freeze clamped in liquid nitrogen and stored at −80 C until liver glycogen content was determined. Although food intake is minimal during the light phase according to the physiological behavior of rats, food was withdrawn for 6 h after dosing in this study to monitor primary acute effects of rimonabant on intermediary metabolism and to exclude any secondary acute effects of rimonabant on metabolic parameters due to reduced food consumption.

BW and food consumption

During the multiple-dose study, the BW and food intake (standard rat chow and candy diet) of all animals were monitored twice weekly in the initial period (first 6 wk of the study) and daily in the treatment period (last 6 wk of the study). Food intake was expressed as daily consumption in gram per animal separately for the standard rat chow and candy diet.

Indirect calorimetry

The setting consisted of 17 cages, of which 16 cages were used for the individual housing of the animals during the study, and one cage served as a reference cage for corrections of O₂ and CO₂ measurements. All rats were accustomed to the special calorimetry cages, equipped with commercial available grid panels, at least 24 h before the start of the measurements. O₂ consumption and CO₂ production were measured every 16 min/cage for 1 min (gas analyzers: Magnos 16 and Uras 14; ABB, Frankfurt/Main, Germany) and recorded by a computer. Values were expressed as the means of liter/d of VCO₂ and VO₂, respectively. For interpretation of substrate use, the respiratory quotient (RQ) was calculated as the quotient of VCO₂/VO₂ (33). Total energy expenditure (TEE) was calculated according to the formula:

$$TEE(kJ)\ {=}\ 16.17\ \mathrm{VO}_{2}(\mathrm{liter})\ {+}\ 5.03\ \mathrm{VCO}_{2}(\mathrm{liter})\ {-}\ 5.98\ \mathrm{N(g)}$$

where nitrogen loss was set constantly to 0.2 g/d and expressed as TEE per metabolic body mass (kg^0.75) per day (34). Fat oxidation and CH oxidation were calculated according to Ferrannini et al. (35) and modified (36) by applying the following formulas:

$$Fat\ oxidation(g)\ {=}\ 1.72\ \mathrm{VO}_{2}(\mathrm{liter})\ {-}\ 1.72\ \mathrm{VCO}_{2}(\mathrm{liter})\ {-}\ 1.96\ \mathrm{N(g)}$$

and

$$CH\ oxidation(g)\ {=}\ {-}2.97\ \mathrm{VO}_{2}(\mathrm{liter})\ {+}\ 4.17\ \mathrm{VCO}_{2}(\mathrm{liter})\ {-}\ 2.44\ \mathrm{N(g)}$$

and expressed as g/d.

Bomb calorimetry

BW, food and water consumption, as well as feces production were measured simultaneously to measurements of indirect calorimetry as described previously (37). Food consumption was corrected by segregating the uneaten or scattered food from feces. Samples of standard rat chow, candy diet, and feces (∼1 g) were dried at 60 C for up to 7 d, homogenized in a grinder, and squeezed to a pill for determination of energy content in an oxygen bomb calorimeter (model 6300; Parr Instruments, Frankfurt, Germany). Energy intake was determined as the product of food consumed and the caloric value of the food. To obtain metabolizable energy intake, the energy content of feces and urine [2% of energy intake (38)] was subtracted from energy intake. The energy loss, defined as fecal caloric loss, was calculated from the feces produced per day and the energy content of feces. The use of food energy was calculated as food assimilation efficiency indicating the relation of metabolizable energy intake and energy intake.

Analytical procedures of blood parameters

Blood metabolic parameters were determined enzymatically using commercially available kits on a Hitachi 912 (Hitachi Ltd., Tokyo, Japan) for glucose (Gluco-quant Glucose/HK kit; Roche, Mannheim, Germany), FFAs (NEFAC kit; Wako Chemicals, Neuss, Germany), and triglycerides (GPD-PAP; Roche). Standard procedures were used to determine hepatic glycogen (amyloglucosidase digestion followed by glucose analysis) (39).

Statistical analysis

Data are presented as means ± sem. Depending on the homogeneity of variances (Levène test), significant differences were tested by a one-way ANOVA, followed by a post hoc Newman-Keuls multiple comparison test or by a Kruskal-Wallis analysis. When testing for differences between different time points, a two-way ANOVA (for repeated measures) followed by a post hoc Newman-Keuls multiple comparison test was used. For all statistical calculations, the software package Everstat V5 (Sanofi-Synthelabo based on SAS 8; SAS Institute Inc., Cary, NC) was used. A P < 0.05 was considered statistically significant.

Results

The availability of the candy diet in addition to the standard rat chow ad libitum resulted in an increased BW in female Wistar rats, reaching a plateau after 4 wk on the candy diet, which was about 10% above that of the group receiving standard rat chow solely (Fig. 1A). The rats obviously preferred the candy diet because immediately the consumption of standard rat chow decreased to about 30% compared with the group receiving standard rat chow solely (Fig. 1, C and D). Administration of rimonabant to rats, which had free access to the candy diet and standard rat chow, caused a decrease of BW during the first week of treatment. The level of reduced BW nearly reached the level of the control chow group, which was maintained during the whole treatment period (Fig. 1A and Table 1). Rimonabant caused a transient reduction of food consumption, which was more pronounced and longer lasting for the candy diet, than for the standard rat chow. After 2-wk treatment, there was a slightly higher food consumption of the standard rat chow and still a slightly reduced consumption of the candy diet for the rimonabant-treated group (Fig. 1, C and D). The change of BW during the treatment period is shown in Fig. 1B for the groups receiving the candy diet: control candy diet, rimonabant candy diet, and pair-fed candy diet. BW gain of the control candy diet group showed a slight increase during the second half of the study, whereas rimonabant-treated animals lost weight during the first week of treatment, which then continuously paralleled that of the control candy diet group, but on a much lower level. In contrast, BW development of the pair-fed candy diet group was very close to that of the rimonabant candy diet group for the first 3 wk of the treatment period, but later on started to differ significantly.

Fig. 1

A, BW development of control chow rats (standard rat chow ▿) and control candy diet rats (standard rat chow plus candy diet ○). B, BW change of control candy diet-fed rats (○), rimonabant-treated candy diet-fed rats (•), as well as pair-fed rats (pair-fed to rimonabant with standard rat chow plus candy diet ▪). Food intake of standard rat chow (C) and candy diet (D) in control chow Wistar rats (▿) and control candy diet rats (○), as well as in rimonabant-treated candy diet rats (•). Rimonabant was administered orally. Values are mean ± sem (n = 16). *, P < 0.05 vs. control chow. #, P < 0.05 vs. control candy diet. §, P < 0.05 vs. rimonabant candy diet. Statistical significance for food intake is not shown.

Table 1

Body weight change, energy consumption (measured by bomb calorimetry), and expenditure (measured by indirect calorimetry) of Wistar rats on standard rat chow, on the candy diet, as well as of candy diet fed rats treated with rimonabant and pair-fed control

Treatment	Standard rat chow (17.7 kJ/g)			Candy diet (21.3 kJ/g) + standard rat chow (17.7 kJ/g)
	Control			Control			Rimonabant				Pair fed
	d 0	d 1	wk 5	d 0	d 1	wk 5	d 0	d 1	wk 5	d 0	d 1	wk 5
BW (d 0) gain (d 1–44) (g)	276 ± 8.1	−1.3 ± 3.4	+5.3 ± 1.8	300 ± 13.0	+0.4 ± 3.6	+16.7 ± 4.2	301 ± 13.0	−13.1 ± 1.7ac	−8.8 ± 3.6ac	284 ± 7.6	−11.5 ± 1.8ac	−1.3 ± 1.5ab
Energy intake (kJ per rat per 24 h)		257.2 ± 26.1	281.5 ± 17.7		226.5 ± 26.8	300.6 ± 16.8		52.5 ± 2.9ac	267.7 ± 18.6		39.6 ± 7.3ac	208.6 ± 31.0a
Emet (kJ per rat per 24 h)		202.2 ± 24.4	214.3 ± 17.0		194.1 ± 23.4	263.0 ± 14.2		37.1 ± 2.1ac	231.1 ± 16.5		25.4 ± 5.5ac	176.7 ± 30.0a
Food assimilation efficiency (%)		76.7 ± 3.1	75.7 ± 2.0		85.5 ± 1.2c	87.6 ± 1.1c		71.1 ± 4.8a	86.3 ± 1.5c		63.8 ± 5.3a	82.3 ± 2.9c
TEE (kJ per rat per 24 h)		181.0 ± 2.4	182.0 ± 3.7		197.2 ± 3.4c	199.9 ± 2.9c		250.9 ± 15.0ac	212.0 ± 6.2c		182.6 ± 5.3ab	182.3 ± 7.1ab
Energy balance ratio Emet × TEE−1		1.11 ± 0.13	1.18 ± 0.08		0.99 ± 0.12	1.32 ± 0.07		0.15 ± 0.01ac	1.10 ± 0.09		0.14 ± 0.03ac	0.96 ± 0.15a

Values are mean ± sem (n = 4–8). E_met, Metabolizable energy intake.

^a

P < 0.05 vs. control candy diet.

^b

P < 0.05 vs. rimonabant.

^c

P < 0.05 vs. control chow.

Energy expenditure showed a clear circadian rhythm, with higher energy expenditure throughout the nocturnal phase. Obviously, there was no difference in TEE, calculated per metabolic BW, between the control chow group and control candy diet group (Fig. 2A), although the animals of the control chow group had a higher CH oxidation rate (Fig. 2D), whereas the animals of the control candy diet group had a higher fat oxidation rate (Fig. 2C). This was represented by the RQ, shown in Fig. 2B. The control animals had a RQ near 1, indicating almost pure combustion of CHs. The control candy diet group had a RQ near 0.9, indicating a rather mixed oxidation with increased percentage of fat oxidation.

Fig. 2

Effects of rimonabant (•) after single-oral administration (treatment d 1; between 0800 and 0900 h) as well as pair feeding (▪; pair feeding at about 1700 h) on TEE (A), RQ (B), fat [fat oxidation (FO)] (C), and CH oxidation (CHO) (D) in candy diet-fed Wistar rats. Respective data are also shown for control rats on standard rat chow only (▿) and plus candy diet (○). Values are mean ± sem (n = 7–8).

First administration of rimonabant to rats in the postprandial state immediately led to an increase in TEE (Fig. 2A) based on a strongly increased fat oxidation (Fig. 2C), even in their inactive period during the day. This was concurrent with a decrease in CH oxidation (Fig. 2D). The shift to higher fat oxidation rate is shown in Fig. 2B, with a RQ close to 0.7. This effect of rimonabant lasted over the whole treatment period (measured on treatment d 1, 2, and 3, and in treatment wk 5; Figs. 2 and 3), whereas during treatment wk 5, the effect was slightly diminished. In contrast, only pair feeding did not show comparable effects. At the first treatment day, pair-fed animals behaved like the control candy diet rats, until they totally consumed their limited food (placed at 1700 h). They then strongly reduced their energy expenditure, even during the nocturnal activity period, to save their energy stores. This reduction of energy expenditure was maintained over the whole study period (Figs. 2 and 3).

Fig. 3

Effects of rimonabant (•) after multiple-oral administration (treatment d 2 and 3, as well as after 5 wk; administration between 0800 and 0900 h) as well as pair feeding (▪; pair feeding at about 1700 h) on TEE (column A), RQ (column B), in candy diet-fed Wistar rats. Respective data are also shown for control rats on standard rat chow only (▿) and plus candy diet (○). Values are mean ± sem (n = 7–8).

The bomb calorimetric experiments (Table 1) showed alterations in energy intake and metabolizable energy intake during the investigated days (treatment d 1, 2, and 3, and in treatment wk 5). Effects observed at d 1 for rimonabant were maintained during the next 2 d; Table 1 showed data for d 1 and for a day in wk 5 only. Interestingly, the energy intake in kJ did not differ between the control chow group and control candy diet group throughout the study, although the caloric value of the candy diet (21.3 kJ/g) was higher than that of standard rat chow (17.7 kJ/g). This was caused by decreased food consumption of candy diet-fed rats, which was accompanied by a significantly decreased fecal caloric loss, resulting in enhanced food assimilation efficiency.

Comparing rimonabant-treated rats with control candy diet rats showed a strong decrease of energy intake during the first days of treatment, paralleled by lowered fecal caloric loss and reduced metabolizable energy intake. Although food assimilation efficiency strongly decreased on the first day of treatment, it recovered on the following days. The strong reduction of food and energy intake concurrent with continuously elevated fat metabolism, indicated by significantly elevated TEE, led to a pronounced loss of BW. This BW reduction was the result of an imbalance between metabolizable energy intake and energy expenditure. Although both control groups showed ratios varying between 0.87 and 1.25, it was at 0.15 on the first day of rimonabant treatment. The quotient slightly increased to 0.45 during the following days of the first week of treatment (data not shown for treatment d 2 and 3). The pair-fed rats necessarily showed similar reduced energy intake values but significantly decreased their energy expenditure, which indicated an adaptive metabolic process to save their energy stores. However, their energy balance during the first days of treatment remained negative (less than one), which also led to a BW decrease.

The continuative treatment with rimonabant over 5 wk revealed a recovery of energy intake and metabolizable energy intake compared with control candy diet-fed rats. However, even after 5-wk treatment, TEE was elevated compared with the pair-fed candy diet group avoiding a regain of BW. In Figs. 2 and 3, TEE is expressed per metabolic body mass and, therefore, allows direct comparisons between study groups, regardless of different BWs of the groups, which appeared during the study. TEE in Table 1 is expressed per animal in absolute terms because this is essential for an energy balance calculation, which was the intention of this table. Therefore, TEE at treatment wk 5 for rimonabant-treated rats was not significantly elevated in absolute terms compared with TEE of the much heavier candy diet-fed control group (BW of candy diet-fed control rats: 316 g vs. rimonabant-treated rats: 292 g). Despite this lower BW (24 g: 8% below that of the candy diet-fed controls), TEE of the rimonabant-treated rats (still numerically higher, but not numerically significantly different) represents an elevated value relative to their lower BW compared with the candy diet-fed control rats.

After 5-wk treatment, the energy balance of control groups (control chow and control candy diet) was still slightly elevated (more than one), which indicated a higher metabolizable energy intake in relation to energetic demands (standard rat chow-fed group 1.18 ± 0.08; candy diet-fed group 1.32 ± 0.07). The energy balance of the chronically rimonabant-treated and pair-fed group stabilized on a lower level, resulting in moderate alterations of BW (rimonabant at 1.1 ± 0.09, pair fed at 0.96 ± 0.15) (Table 1, wk 5).

The metabolic plasma parameters were obtained after a short fast of 4 h at the end of the multiple-dose study after 12 wk on the candy diet. Obviously, the high-fat, high-CH candy diet led to a significant increase of FFA, triglyceride, and blood glucose levels compared with the control chow group (Fig. 4, A–C). Rimonabant administration to candy diet-fed rats resulted in significantly decreased FFA and triglyceride levels, according to the strong decrease in their BW. Pair feeding led to a similar decrease of triglycerides, whereas FFA levels were only slightly decreased in comparison to candy diet-fed control rats and were not significant. Blood glucose levels were significantly decreased by pair feeding compared with candy diet control rats. This might be the result of the longer period without food for these rats because they totally took up their limited amount of food at the beginning of the dark phase. Liver glycogen levels were not changed in any group (Fig. 4D).

Fig. 4

FFA (A), triglyceride (B), blood glucose (C), and liver glycogen content (D) in control chow rats, control candy diet rats, rimonabant-treated candy diet rats, as well as pair-fed rats at the end of the treatment period of the multiple-dose study. Rimonabant was administered orally. Values are mean ± sem (n = 7–8). *, P < 0.05 vs. control chow. #, P < 0.05 vs. control candy diet. §, P < 0.05 vs. rimonabant.

For the investigation of the acute inherent effects on metabolic parameters, a single-dose study in normal-fed Wistar rats was performed. These rats had free access to standard rat chow and water during the preceding night (postprandial state and similar to the rats on d 1 of the multiple-dose study). To exclude any secondary effects of reduced food uptake that might occur even at the beginning of the light phase on metabolic blood and tissue parameters, food was removed during the 6-h study period. Administration of rimonabant to these rats caused an increase in FFAs, indicating increased lipolysis from fat tissue (Fig. 5A). Despite elevated FFA levels, serum triglyceride levels did not increase during the study period (Fig. 5B). After 2 and 5 h after administration, blood glucose was slightly elevated, but not significantly (Fig. 5C), despite increased glycogenolysis, measured as a reduction of liver glycogen down to about 40% of that of the control group 6 h after administration (Fig. 5D). In summary, these findings demonstrate an acute primary effect of rimonabant on induction of: 1) lipolysis from fat tissue (increased FFAs); and 2) glycogenolysis from the liver (reduced hepatic glycogen), without any increases in serum triglyceride levels or meaningful increases of blood glucose. These findings were clearly not secondary due to reduced food intake because food was removed during the 6-h study period for the control and treatment groups.

Fig. 5

Acute effects of rimonabant (black bars) after single-oral administration on metabolic blood parameters of lipid and glucose metabolism compared with control (white bars) in postprandial normal-fed Wistar rats: FFA (A), triglyceride (B), blood glucose (C), and liver glycogen content (D). Values are mean ± sem (n = 8). *, P < 0.05 vs. control.

Discussion

Female Wistar rats were chosen for this study because they reached a plateau in their physiological BW development, in contrast to male rats, which progressively gained weight, particularly on a high caloric diet (20, 24). In female rats the candy diet caused a BW plateau, which was about 10% above that of the control rats receiving standard rat chow only (40). After 6-wk feeding, both control groups (control chow and control candy diet) dissipated nearly exactly the same amount of total energy (TEE) calculated for their metabolic body mass (41) but demonstrated different RQ values. According to the higher fat content in their candy diet, these rats had a lower RQ and demonstrated an increased calculated fat oxidation, in the presence of a reduced CH oxidation. Despite the different caloric values of candy diet (21.3 kJ/g) and standard rat chow (17.7 kJ/g), total energy intake was similar in both control groups. This was provoked by a lower amount of food intake per day. The calculated higher food assimilation efficiency indicated an adaptation of feeding behavior for energy homeostasis. Therefore, the BW gain resulted from higher food assimilation efficiencies on the one hand and probably also from reduced energy costs of lipid synthesis associated with high-fat diets (42) in the presence of an overactivated peripheral endocannabinoid system favoring lipogenesis (25, 26). Although the nutritional quality of the candy diet compared with standard chow with respect to essential nutrient intake (e.g. protein, micronutrients) was lower, the simultaneous offer of standard chow and candy diet to the candy diet-fed rats avoided any malnutrition because their lean body mass was not affected as well because no clinical symptoms or hints in clinical chemistry for any malnutrition or undersupply occurred (data not shown).

The well-reported pharmacological effects of rimonabant on BW and food intake were confirmed by this study (16–20, 24). Rimonabant did not only reduce the BW gain as it has been reported from genetic rat models of obesity (16, 20, 24) but clearly reduced the BW, similar to the diet-induced mouse studies (18, 19), approaching the BW level of the normal-fed controls. In agreement with other reported studies, BW reduction was maintained during the whole study period, although the reduction of food intake was only transient (16–20, 24). A more pronounced reduction in palatable food has also been reported earlier (21–23). In our study rimonabant obviously caused a more prolonged inhibitory effect on the consumption of palatable food (candy diet) compared with standard rat chow.

The explanation for the discrepancy between sustained BW reduction on the one hand and a transient reduction of food intake on the other hand was elucidated to be an increased energy expenditure, which has already been postulated (16, 31), indirectly demonstrated (24), and measured in ob/ob mice for up to 180 min after acute treatment and after 7-d treatment (32). In a 14-d study, Doyon et al. (24) showed that in obese rats, energy intake (∼−20%) and energy gain (∼−50%) significantly decreased with rimonabant treatment, whereas energy expenditure calculated by comprehensive energy balance profile based on whole body composition analysis remained stable, whereas a decrease would have been expected. They suggested that rimonabant exerted stimulating effects on thermogenesis consequently leading to a reduction in fat and, thus, weight gain. Whereas in the present study, the detailed analysis of energy balance obviously proved that rimonabant-treated rats dissipated more energy coming from endogenous fat stores during the first days of treatment, resulting in a pronounced reduction of BW. The increased energy expenditure based on increased fat oxidation started immediately after the first administration. Because these rats had free access to their food during the preceding night (dark phase), they were in a postprandial state. According to the physiological food intake behavior of rodents, which appeared predominantly during the dark phase, the immediate increase of fat oxidation after the first administration of rimonabant at the beginning of the light phase could not be the result of starvation-induced lipolysis with subsequent increased fat oxidation. A rimonabant-induced increase of energy expenditure was maintained over the whole study period, although to a lesser extent at the end of the study.

With respect to energy homeostasis, one could speculate that the transience of the reduction of food intake during the treatment with rimonabant appeared in relation to the decreasing endogenous fat stores. When the amount of endogenous fat stores declined, rimonabant-induced increased energy expenditure was maintained by a re-increase in food intake. This speculation can be supported by two studies, in which it was reported that the period of reduced food intake was significantly shorter in lean rats compared with obese ones (20, 24). A similar observation on energy homeostasis has been reported for leptin substitution to leptin-deficient ob/ob mice (43).

The BW of rimonabant-treated candy diet-fed rats decreased nearly to that level of standard rat chow-fed animals, although food intake increased in rimonabant-treated rats during the study for standard rat chow as well as the candy diet. In addition, there was a tendency of a more preferred uptake of standard rat chow compared with candy diet, which due to the different energy densities of the chow and diet, might contribute to the persistent BW reduction of the rimonabant-treated rats. Obviously, these rats were in energy balance at a reduced BW level at the end of the study, indicating that the weight loss caused by rimonabant reflected the need to achieve a new “carbohydrate” and “fat” balance (44) in response to the metabolic effects of the drug.

The difference between rimonabant-treated candy diet rats and pair-fed candy diet rats, which consumed the same amount of food, could be obviously seen in their BW development. The rats of the pair-fed candy diet group started to gain more weight at the end of the study compared with the rimonabant-treated group. Again, the detailed analysis of energy balance demonstrated that the pair-fed animals reduced their energy expenditure after they had consumed their limited amount of food to save their energy stores (45). This was in clear contrast to the TEE profile of the rimonabant-treated rats. Their TEE was always above that of both control groups and even more above that of the pair-fed rats.

The immediate increase of FFAs in the single-dose study using rats in the postprandial state demonstrated an inherent pharmacological effect of rimonabant to induce lipolysis in vivo. The concomitant decrease of serum triglycerides after 5 h in the presence of increased lipolysis was consistent with an increased combustion of fatty acids by increased fat oxidation, as demonstrated by the indirect calorimetric data of the multiple-dose study. In addition to increased lipolysis, liver glycogen levels were significantly reduced 6 h after single administration to postprandial rats. These acute effects in postprandial rats demonstrated an inherent pharmacological characteristic of the compound and not a secondary one due to reduced food intake and subsequent starvation-like postabsorptive changes in intermediary metabolism.

In contrast to the acute findings of increased lipolysis and glycogenolysis produced by rimonabant, long-term treatment resulted in reduced FFA levels reflecting the decreased fat mass, and normalized hepatic glycogen levels. It seems reasonable that increased fat oxidation did not appear in the adipocytes themselves, but in the skeletal muscle and liver. Therefore, increased lipolysis must precede the fat oxidation in muscle and liver, and this resulted subsequently in the reduction of body fat and BW. Mobilization of adipocytic fat stores initially resulted in increased serum FFAs, but later on, FFA levels decreased when total fat mass was reduced.

The fine-tuned effects as demonstrated in this rat study of increased fat oxidation on the one hand, which has already been postulated earlier (16, 31), and increased lipolysis on the other hand revealed a very coordinated cluster of pharmacological effects of rimonabant. Fat oxidation is a major contributor of energy expenditure and, thus, an integral part of energy homeostasis. The hypothalamus is known as the center of energy homeostasis controlling peripheral metabolism by the autonomic nervous system and neuroendocrine signals (46), and the role of the endocannabinoid system in the hypothalamus is well described (30). Therefore, inhibition of food intake as well as increased energy expenditure caused by rimonabant as shown in this study could be ascribed to its interference with the hypothalamic endocannabinoid system.

A putative peripheral mechanism of rimonabant-induced fat oxidation could be linked to adiponectin. Rimonabant elevated adiponectin mRNA expression in adipose tissue of Zucker fatty rats and in cultured adipocytes in vitro (16). Increased adiponectin levels were also seen in human studies (6). Although chronic effects of adiponectin in obesity are much more complex (47), it has been hypothesized in the context of the endocannabinoid system and fat oxidation that the increase in adiponectin levels and subsequent activation of AMP-activated protein kinase were causally involved in the increased fat oxidation caused by rimonabant (48). The relative contribution of central vs. peripheral actions of rimonabant to increase fat oxidation remains to be established.

Regardless of the mechanisms involved, we demonstrated here that increased energy expenditure caused by rimonabant contributed more to the sustained BW-reducing effect and probably also to the improvement of metabolic risk factors, like the reduction of serum triglyceride levels, than reduced food intake.

Acknowledgments

Disclosure Statement: A.W.H., R.E., G.H., and W.K. are employed by and hold shares of Sanofi-Aventis, which manufactures and markets among others also pharmaceuticals related to the treatment of diabetes. S.K. was previously employed by Sanofi-Aventis.

Abbreviations

 
• BW

  Body weight

 
• CH

  carbohydrate

 
• FFA

  free fatty acid

 
• RQ

  respiratory quotient

 
• TEE

  total energy expenditure

 
• VCO₂

  volume of carbon dioxide production

 
• VO₂

  volume of oxygen consumption