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Endocrinology, doi:10.1210/en.2007-0317
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Endocrinology Vol. 149, No. 2 615-625
Copyright © 2008 by The Endocrine Society

Lou/C Obesity-resistant Rat Exhibits Hyperactivity, Hypermetabolism, Alterations in White Adipose Tissue Cellularity, and Lipid Tissue Profiles

Christophe Soulage, Bader Zarrouki, Anisio Francesco Soares, Michel Lagarde and Alain Geloen

Université de Lyon 1, Lyon F-69003, France; Institut National de la Santé et de la Recherche Médicale, Unité 870, Institut Fédératif de Recherche 62, Hospices Civils de Lyon, and Institut National de la Recherche Agronomique, Unité Mixte de Recherche 1235, Lyon F-69008, France; and Institut National des Sciences Appliquées-Lyon, Régulations Métaboliques Nutrition et Diabètes, Villeurbanne F-6921, France

Address all correspondence and requests for reprints to: Dr. Christophe Soulage, U-870 Institut National de la Santé et de la Recherche Médicale, Unité 1235 Institut National de la Recherche Agronomique, Institut National des Sciences Appliquées-Lyon, Université Claude Bernard Lyon, Hospices Civils de Lyon, Régulations Métaboliques, Nutrition et Diabètes, Building Louis Pasteur (406), F-69621 Villeurbanne cedex, France. E-mail: Christophe.Soulage{at}insa-lyon.fr.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Lou/C obesity-resistant rat constitutes an original model to understand the phenomena of overweight and obesity. The aim of the present study was to identify metabolic causes for the outstanding leanness of Lou/C rat. To this end, the metabolic profiles (food intake, energy expenditure, and physical activity) and the cellular characteristics of white adipose tissue (lipogenesis, lipolysis, cellularity, and lipid composition) in 30-wk-old Lou/C rats were compared with age-matched Wistar rats. Lou/C rats exhibited a lower body weight (–45%), reduced adiposity (–80%), increased locomotor activity (+95%), and higher energy expenditure (+11%) than Wistar rats. Epididymal adipose tissue of Lou/C rat was twice lower than that of Wistar rat due to both a reduction in both adipocyte size (–25%) and number (three times). Basal lipolysis and sensitivity to noradrenaline were similar; however, the responsiveness to noradrenaline was lower in adipocytes from Lou/C compared with that from Wistar rats. Lipidomic analysis of plasma, adipose tissue, and liver revealed profound differences in lipid composition between the two strains. Of note, the desaturation indexes (ratio C16:1/C16:0 and C18:1/C18:0) were lower in Lou/C, indicating a blunted activity of {delta}-9-desaturase such as stearoyl-coenzyme A-desaturase-1. Increased physical activity, increased energy expenditure, and white adipose tissue cellularity are in good agreement with previous observations suggesting that a higher sympathetic tone in Lou/C could contribute to its lifelong leanness.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
OBESITY HAS BECOME the most common metabolic and nutritional disorder in industrial countries, and represents one of the most prevalent risk factors for the development of common chronic metabolic diseases such as atherosclerosis and diabetes. Most of the genetic studies in the field of human obesity have been focused on genes and polymorphisms associated with an obese phenotype. Considerably less attention has been paid to understand why certain people remain thin, and do not develop obesity, in the Western country obesogenic environment. In human populations, thinness has a strong genetic component, and inheritance of a thin body mass constitutes a strong protective factor that counteracts the development of obesity induced by environmental factors (1, 2). Many genetic manipulations and crossbreedings have created animal models of obesity providing an insight into the molecular mechanisms that affect energy balance and contribute to fat accretion. In contrast, few animal models are available to understand the leanness and the resistance to obesity. The present study was performed on a valuable model of obesity resistance, the Lou/C rat. The Lou/C rat, an inbred strain of Wistar origin is considered both as an obesity-resistant rat (3) and as a model of successful aging (4). Indeed, whereas the Wistar rats develop spontaneous obesity with increasing age (5), the Lou/C rats exhibit lighter body weight and lower adipose tissue accretion regardless of age (6). Interestingly, the Lou/C rat exhibits a longer life expectancy than most of the other laboratory rat strains (4), and develops fewer metabolic and neuroendocrine age-related disturbances (7). Several studies have been conducted to understand the endocrine determinants of low adiposity of the Lou/C rat (3, 7, 8). To date, however, the exact mechanisms responsible for the outstanding leanness of the Lou/C rats remain unknown. Surprisingly, no data are available in the literature concerning the characteristics of white adipose tissue (WAT) of Lou/C rat. The aims of the present study were to investigate thoroughly the energy balance, and to characterize the cellularity and the metabolic properties of the WAT in obesity- resistant Lou/C rat. To this end, 6-month-old Lou/C rats were compared with age-matched Wistar rats. Understanding the determinants of the outstanding leanness of Lou/C may yield important and complementary findings to the study of obesity models.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
Eleven-week-old male Wistar and Lou/C rats were purchased from Harlan (Gannat, France) and were raised in the Institut National des Sciences Appliquées animal house until use. Rats were kept at 24 ± 1 C on a 12-h light, 12-h dark cycle (light on from 0600–1800 h), with free access to food [3.2 kcal/g, 65% carbohydrates, 11% fat, 24% proteins (wt/wt), AO3, SAFE, Augy, France] and tap water. Body weights were measured weekly at the same time for up to 30 wk. All experiments were performed according to the guidelines laid down by the French Ministère de l’Agriculture and European Union Council Directive for the Care and Use of Laboratory Animals (No. 02889).

Food and water consumption
Rats were housed individually in metabolic cages for 1 wk, fed ad libitum with standard diet, and given free access to water. Food and water consumption were measured twice daily the last 5 d as the difference between the amount given and that removed from the cage. The spillage of food was considered in the calculation of the food intake.

Actimetry
Locomotor activity was quantified using cages (0.30-m length x 0.20-m length x 0.17-m height; surface: 0.06 m2; volume: 0.01 m3) equipped with horizontal infrared beams (Imetronic, Pessac, France) placed 3 cm above the floor of each activity cage to detect horizontal locomotor activity. Beam interruptions were identified via an electrical interface, accumulated and recorded over 30-min intervals using a personal computer.

Energy expenditure measurement
Energy expenditure was measured by indirect calorimetry at 26 ± 1 C, the thermoneutrality for rats. Oxygen and carbon dioxide concentrations in downstream exhaust gas were successively measured in five different cages. To avoid errors resulting from sequential changes from one cage to another, common parts of the system were rinsed for 90 sec after which gas exchanges were measured for 40 sec. A computer-controlled system of three-way valves allowed sequential analysis of the five cages every 11 min. One cage was left vacant, and served as reference for measuring ambient O2 and CO2. Air samples were pumped at a constant flow rate, controlled within strict limits by a mass flowmeter (precision < 1%, Tylan, FM 380; Tylan General, San Diego, CA), and directed to a paramagnetic oxygen analyzer (range 0–100%, time delay < 3 sec; Klogor, Lannion, France) and an infrared carbon dioxide analyzer (range 0–1%, time delay < 3 sec, Gascard I; Edinburgh Sensors, Edinburgh Instruments Ltd., Livingston, UK) after being dried on a permapure system and calcium chloride, which were changed twice daily. The system was calibrated daily with pure nitrogen to set up the zero and with a standard gas mixture containing 20.5% O2 (accuracy 20.44–20.56%), 0.5% CO2 (accuracy 0.495–0.505%), and 79% nitrogen to set up the sensitivity. The measuring system was found to be accurate within ±1% by bleeding a known rate of CO2 and N2, as well as a known rate of O2 and N2. Analog signals from the analyzers and mass flowmeter were digitized with an interface card and stored in a desktop computer. After a period of acclimatization of 24 h, O2 and CO2 concentrations were measured continuously over 24 h. Thirty minutes were required to calibrate the system, clean the cages, change food and water, and weigh the animals. The total quantities of O2 consumed and CO2 produced in occupied cages minus O2 and CO2 concentrations in the empty reference cage, multiplied by the airflow through the cages yielded the respiratory gas exchange of animals. Energy expenditure was calculated according to the Depocas and Hart method (9). The 24-h energy expenditure records were divided into energy expended during quiescent (day) or active (night) phase of the animals. The respiratory quotient (RQ) was calculated as the ratio between CO2 production and O2 consumption. The body temperature was measured using a thermocouple thermometer (BAT-12; Harvard Apparatus, Les Ulis Cedex, France), the colonic temperature being taken as representative of body temperature. The colonic temperature was measured rectally at a depth of 3 cm. Five measurements were performed over a 30-min period, and the mean of these values was taken as the body temperature.

Blood sampling and tissue dissection
Rats were killed with CO2 between 0900 and 1000 h. Blood was rapidly withdrawn from puncture of the vena cava on heparinized tubes. Blood samples were centrifuged (1 min, 5000 x g), and plasma was collected and frozen in liquid nitrogen. Epididymal (eWAT), retroperitoneal (rWAT), inguinal (ingWAT) WATs, and interscapular brown adipose tissue (iBAT) were rapidly removed and cleaned. A portion of epididymal fat pad was immediately used for isolation of adipocytes, whereas the remaining tissues were snap frozen in liquid nitrogen and stored at –80 C until use.

Isolation of adipocytes
Adipocytes were isolated from 1- to 2-g epididymal fat pad by a modification of the Rodbell’s original procedure (10). Adipose tissues were weighed, minced, and digested in a 20-ml polystyrene vial containing 10 ml Krebs-Ringer bicarbonate (KRB) buffer, 25 mM HEPES, 6 mM glucose (pH 7.40) (KRB buffer), with 1.8 mg/ml collagenase (type II, C6885; Sigma, Saint Quentin Fallavier, France) and 1% of fatty acid free BSA (Sigma). The vial was shaken at 50 cycles/min at 37 C. The resulting cell suspension was filtered through a nylon mesh (250 µm) and washed three times with 5 ml KRB buffer 1% BSA and three times with KRB buffer containing 4% BSA. Adipocytes were then resuspended in KRB buffer 4% BSA, and a sample of the final cellular suspension was counted in a hemacytometer.

Adipose tissue cellularity
Cell size and number of adipocytes in epididymal adipose tissue were determined as described by Briquet-Laugier et al. (11). Briefly, microphotographs of isolated adipocytes were acquired from a light microscope equipped with a CCD camera, and 800-1000 cell diameters were measured using Axiocam software (Axiocam; Leica, Bron, France) (12, 13). The mean fat cell volume was calculated, and the fat cell number was calculated by dividing the tissue lipid content, estimated by lipid extraction using the method of Folch et al. (14), by the mean adipocyte weight (calculated by multiplying the mean adipocyte volume by the triacylglycerol density, namely 0.915).

In vitro lipolysis measurement
Aliquots of the cell suspension (75,000 cells) were distributed in 2-ml polystyrene vials containing KRB buffer 4% BSA with or without noradrenaline (-arterenol hydrochloride; Sigma) at various concentrations (final concentration ranging between 10–9 and 5 x 10–6 M). The final volume was 1 ml. Adipocytes were incubated with gentle shaking (50 cycles per minute) for 1 h. The reaction was stopped by placing vials on melting ice. The floating adipocytes were discarded by aspiration, and lipolysis was quantified by measuring the release of extracellular glycerol using a kit (Glycerin assay; Roche Molecular Biochemicals, Burgess Hill, UK). Each incubation was run in quadruplicate, and the results are the means of five to seven separate experiments performed on different days. Data are expressed as µmol glycerol release in 1 h/million cells and as percentage of the maximal response (10–6 M noradrenaline). Vmax and EC50 were calculated from the fitting of the dose-response curve of noradrenaline-stimulated lipolysis using JMP 5.1 software (Abacus Concepts, Berkeley, CA) for Macintosh. Vmax (responsiveness) represented the maximal stimulation of lipolysis. EC50 (an index of sensitivity) was defined as the concentration of noradrenaline inducing one half of the maximal response. The lipolytic activity of the isolated adipocytes was tested using 10–6 M isoproterenol (β-adrenergic agonist), 10–6 M BRL 37344 (β3-adrenergic agonist), and 5 x 10–5M IBMX (phosphodiesterase inhibitor). Antilipolytic responsiveness was explored on 10–6 M noradrenaline-stimulated lipolysis using 10–6 and 10–3 M clonidine ({alpha}2-adrenergic agonist).

Antilipolytic effect of insulin
Aliquots of the cell suspension (75,000 cells) were distributed in polystyrene vials containing KRB buffer 4% BSA with 10–6 M noradrenaline and with or without insulin at various concentrations (final concentrations: 25, 100, 500, and 1000 µU/ml). The final volume was 1 ml. Adipocytes were incubated with gentle shaking (50 cycles per minute) for 1 h. The reaction was stopped by placing vials on melting ice. The floating adipocytes were discarded by aspiration, and lipolysis was quantified by measuring the release of extracellular glycerol using a kit (Glycerin assay). Each incubation was run in quadruplicate, and the results are the means of five to seven separate experiments performed on different days.

In vivo lipogenesis
In vivo lipogenesis measurement was performed as described by Feingold and Grunfeld (15). Each rat was injected ip with 12 mCi/kg [3H] water (GE Healthcare, Orsay, France; original specific activity 50 mCi/ml diluted to 5 mCi/ml in saline) between 0900 and 1100 h. Exactly 1 h later, each animal was deeply anesthetized with pentobarbital (60 mg/kg ip), and 2 ml blood was withdrawn from heart puncture and used to measure the plasma [3H] water specific activity. Liver, epididymal, retroperitoneal, and inguinal white fat pads were rapidly removed, weighed, and frozen in liquid nitrogen. Total lipids were extracted with chloroform-ethanol (2:1 vol/vol), saponified with 5 N NaOH (80 C, 1 h), and the digitonin-precipitable sterols were isolated as described by Lowenstein et al. (16). Fatty acids were extracted from the same samples with 12 ml diethyl ether after acidification with 12 N H2SO4, followed by a second extraction with diethyl ether. The radioactivity was measured by liquid scintillation counting. The rates of fatty acid and cholesterol synthesis were calculated as micromoles of [3H] water incorporated into fatty acids or digitonin-precipitable sterols.

Plasma parameters
Triglycerides (Triglycerides PAP; BioMérieux, Marcy l’Etoile, France), nonesterified fatty acids (NEFAs) (NEFA-C; Wako, Neuss, Germany), and cholesterol (Cholesterol RTU; BioMérieux) were assayed using commercially available kits. Plasma glucose was assayed using the glucose oxidase method as previously described. Plasma insulin concentration was determined with a double antibody RIA (CIS Bio Intl., Gif sur Yvette, France) according to the manufacturer’s recommendations. All assays were performed at least in duplicate.

Fatty acid profiling
Plasma, liver, and rWAT were subjected to fatty acid profile analyses. Briefly, tissues were broken, and the total lipids were extracted using chloroform-ethanol (2:1 vol/vol) with heptadecanoic acid (C17:0) as an internal standard. Lipids were transmethylated using BF3 and methanol in a sealed vial under nitrogen at 100 C for 90 min. Fatty acid methyl esters obtained were extracted with isooctane and used for gas chromatography. Separation and quantification of fatty acid methyl esters were achieved with gas chromatography using a gas chromatograph (Agilent Technologies, Massy, France) with a silica column (60 m x 0.25 mm) and a flame-ionization detector.

RT-PCR analysis
The expression of some key genes related to lipid metabolism was measured using RT-PCR analysis. Briefly, total RNA was extracted using Sigma Tri Reagent. After RT of 1 mg total RNA using the OneStep QIAGEN kit (QIAGEN, Inc., Valencia, CA), PCR was performed on a Primus thermocycler (Nyxor, Paris, France). Semiquantitative RT-PCR analysis was performed for fatty acid synthetase (FAS), lipoprotein lipase (LPL), or stearoyl-coenzyme A-dehydrogenase-1 (SCD-1) using β-actin as a control to normalize gene expression. Primer sequences are shown in Table 1Go, and PCR conditions for LPL, FAS, and SCD-1 can be sent upon request.


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  		TABLE 1. Sequence of primers for semiquantitative PCR

 
Statistical analysis
The data are expressed as mean ± SEM. Statistical analyses were performed with StatView 4.5 software for Windows (Abacus Concepts) and JMP 5.1 (Abacus Concepts) for Macintosh. The body weights, food intake, locomotion, and energy expenditure values for the Wistar and Lou/C rats were compared using two-way ANOVA with one between-subject factor (strain) and one within-subject factor (time). When appropriate, individual comparisons were made with post hoc Fisher’s least significant difference test. Data concerning the energy expenditure are expressed in relation to the metabolic size as kg0.67 to standardize for differences in body weight (17) and were subjected to two-way ANOVA with one between-subject factor (strain) and one within-subject factor (time). For simple comparison between the two strains, Student’s t test was used. Lipogenesis data are expressed as mean ± SD and were compared using the Mann-Whitney U test. Adipocyte size distributions were compared using a Smirnov test. Differences were considered significant when P < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Lou/C rats exhibited a lower body weight and lower adiposity than Wistar rats
Lou/C rats and age-matched Wistar rats were raised for 20 wk in our animal house, and body weights were monitored weekly. The evolution of the body weights of Lou/C and Wistar rats is depicted in Fig. 1Go. At each age, the Lou/C rats exhibited a lower body weight than the Wistar rats. The ANOVA indicated a main effect of age (P < 0.001), a main effect of strain (P < 0.001), as well as an age vs. strain interaction (P < 0.001). At the time of euthanasia, the Lou/C rats exhibited a 45% (P < 0.0001) lower body weight than their Wistar counterparts. The daily weight gain (absolute or relative) was significantly lower in Lou/C than in Wistar rats (–55%; P < 0.005). Biometric data are shown in Table 2Go. The measurement of retroperitoneal fat tissue accretion is a direct indication of obesity in rats (5). A striking difference was observed in fat pad weights between Lou/C and Wistar rats. The amounts of iBAT (–60%; P = 0.0003), eWAT (–80%; P = 0.0001), rWAT (–85%; P = 0.0001), and ingWAT (–62%; P = 0.009) were significantly lower in Lou/C than in Wistar rats. The Lee index is a common marker of body mass in rats. According to the difference observed in WAT accretion, we noticed a lower Lee index in Lou/C compared with Wistar rats (–6%; P = 0.0002).


Figure 1
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  		FIG. 1. Body weights of male Wistar ({square}) and Lou/C (bullet) rats with increasing age. Data are expressed as mean ± SEM. At each age, Lou/C rats exhibit a significant lower body weight than age-matched Wistar (P < 0.005, two-way ANOVA, Lou/C vs. Wistar; n = 10 in each strain).

 

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  		TABLE 2. Biometric data and adipose tissue weights of 30-wk-old Wistar and Lou/C rats involved in the study

 
Lou/C rats exhibited a particular energy balance and a higher physical activity
To investigate whether changes in energy balance could account for the lower adiposity observed in Lou/C rats, metabolic rate, locomotor activity, and food intake were measured. The daily energy expenditure, corrected per metabolic body mass and expressed as J/min·kg0.67, is depicted in Table 3Go. The Lou/C rats exhibited higher energy expenditure than their Wistar counterparts during both the quiescent (day, +13%; P < 0.05) and the active phase (night, +9%; P < 0.05) of the animals. No difference in RQ was noticed between the two strains. The colonic temperature, taken as representative of body temperature, did not differ between the two strains. The daily cumulative locomotor activity of Lou/C and Wistar rat is depicted in Fig. 2Go. Locomotor activity, estimated by interrupted infrared beams, was significantly enhanced in the Lou/C rat. The number of movements observed during the active (light off) and quiescent phase (light on) was increased respectively by 87 (P < 0.005) and 102% (P < 0.005) in Lou/C compared with Wistar rats. The food intake of Lou/C and Wistar rat is depicted in Table 4Go. The daily food intake was higher in Lou/C rats than in their Wistar counterparts (+17%; P < 0.005). A striking difference was noticed in the amount of food ingested during the active phase (+24%; P < 0.01), whereas no difference was detected in the amount of food ingested during the quiescent phase. No difference was observed in the daily partition (quiescent vs. active phase) of the food intake. Metabolic efficiency was calculated as the total body mass gain divided by the total amount of food eaten by the same animal. Metabolic efficiency was 6.58 ± 0.57% in Wistar and 4.81 ± 0.24% in Lou/C rats (P < 0.05). In good agreement, the relative water intake was also higher in the Lou/C strain than in the Wistar strain (+45%; P < 0.01).


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  		TABLE 3. Daily energy expenditure in 30-wk-old Wistar and Lou/C rats

 

Figure 2
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  		FIG. 2. Daily locomotor activity in 30-wk-old Wistar and Lou/C rats. Interval measurements were taken every 30 min for 24 h as described in Materials and Methods. The data represent the number of times the light beams were broken due to animal movements. The data are expressed as mean ± SEM (n = 8 in each group). Note that locomotor activity was higher in Lou/C than in Wistar rats (P < 0.005, two-way ANOVA, Lou/C vs. Wistar).

 

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  		TABLE 4. Food and water intake in 30-wk-old Wistar and Lou/C rats

 
Plasma parameters
Blood metabolic parameters for Wistar and Lou/C rats are shown in Table 5Go. As previously described, the plasma glucose concentration, insulin, and NEFA were similar in both strains. However, triglyceride concentration was lowest in Lou/C, whereas cholesterol concentration was found to be higher in Lou/C than in Wistar rats.


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  		TABLE 5. Plasma parameters in 30-wk-old Wistar and Lou/C rats

 
Lou/C rats presented some alterations in WAT cellularity
A 5-fold difference was observed in the weight of epididymal fat pad between Lou/C and Wistar rats. To investigate whether changes in WAT accretion were due to a reduction in the number of adipocytes, a decrease in triglyceride accumulation in adipocytes, or both, we performed a cytological analysis of eWAT. The cellular characteristics of eWAT in Lou/C and Wistar rats are shown in Fig. 3Go. The frequency distributions of adipocyte diameters were biphasic in Wistar as well as in Lou/C rats. The mean adipocyte diameters were 129.0 ± 9.1 µm in Wistar and 102.3 ± 4.39 µm in Lou/C rat (P < 0.01). The number of adipocytes per epididymal fat pad were 7.10 ± 0.78 and 2.30 ± 0.46 million in Wistar and Lou/C rat, respectively. Thus, the reduction in WAT accretion in Lou/C results from both a hypotrophia (i.e. a reduction in the volume of adipocytes) and to a hypoplasia (i.e. a reduction in the number of white adipocytes).


Figure 3
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  		FIG. 3. A, Frequency distribution of adipocyte diameters in 30-wk-old Wistar and Lou/C rat epididymal fat pad. B, Number of adipocytes in epididymal white fat pad of Wistar and Lou/C rats. For both strains, individual measurements were performed on 800–1000 isolated adipocytes. Results are expressed as mean ± SEM from five rats in each strain. Note that adipocyte size distributions were different between the two strains (P < 0.001, Smirnov test, Lou/C vs. Wistar). *, P < 0.05; **, P < 0.01, significant difference between the two strains estimated using Student’s t test.

 
Metabolic potentialities of WAT from Lou/C rat
Figure 4AGo shows the dose-response effect of noradrenaline on the lipolysis of adipocytes isolated from epididymal adipose deposits in Wistar and Lou/C rats with a higher response of the former. When results are expressed as the percentage of the maximal stimulation for noradrenaline (i.e. 5 x 10–5 M) (Fig. 4BGo), no significant difference was observed between the two strains. Pharmacodynamic parameters of the noradrenaline-stimulated lipolysis (V0, VMAX, and EC50) were calculated from the fitting of the dose-response curve (Fig. 4AGo), as described in Materials and Methods. V0 represents the basal level of lipolysis in the absence of noradrenaline stimulation, VMAX (responsiveness) the maximal stimulation of lipolysis with noradrenaline. EC50, a common index of sensitivity, is defined as the concentration of noradrenaline inducing one half of the maximal response. No difference was observed in basal lipolysis or in noradrenaline sensitivity indicated by similar EC50 values for noradrenaline. However, the responsiveness (VMAX) to noradrenaline was significantly lower in adipocytes isolated from Lou/C WAT than in those isolated from Wistar rat WAT (Table 6Go). The lipolytic activity of the isolated adipocytes was tested using the β-adrenergic agonist isoproterenol (10–6 M) and the highly selective β3-adrenergic agonist BRL 37344 (10–6 M). The lipolytic potencies of isoproterenol were similar in the two strains, whereas agonist BRL 37344 induced a stronger stimulation of lipolysis in Lou/C rats. The use of the phosphodiesterase inhibitor IBMX (5 x 10–5 M) resulted in both strains in a submaximal stimulation of lipolysis (Fig. 5Go). Antilipolytic responsiveness was explored on 10–6 M noradrenaline-stimulated adipocytes using the {alpha}2-adrenergic agonist clonidine (Fig. 6Go). The 10–6 M clonidine failed to show any antilipolytic activity in either strain under our experimental conditions. However, 10–3 M clonidine resulted in a 30% inhibition of noradrenaline-stimulated lipolysis in Lou/C, whereas it remained unaltered in Wistar rat. Therefore, the efficiency of {alpha}2-adrenergic agonist was greater in Lou/C than Wistar rats. Insulin induced a dose-dependent inhibition of the noradrenaline-stimulated lipolysis, but no difference was observed between the two strains (Fig. 7Go). Together, these results indicate that apart from a greater {alpha}2-adrenergic efficiency in Lou/C rats, there is no major modification in the lipolytic potencies of isolated adipocytes from Lou/C or Wistar rats.


Figure 4
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  		FIG. 4. Dose-response curves of noradrenaline-stimulated lipolysis of adipocytes isolated from epididymal adipose deposits of 30-wk-old Wistar and Lou/C rats. The dose response-curves are expressed in µmol glycerol released from 106 cells in 1 h (A) or as the percentage of maximal stimulation by 5 x 10–6 M noradrenaline (B). In each group, values are mean ± SEM of five experiments performed in quadruplicate. *, Significant difference between Wistar and Lou/C rats estimated using two-way ANOVA (P < 0.05). Ba, Baseline.

 

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  		TABLE 6. Basal and noradrenaline stimulated lipolysis in isolated epididymal adipocytes of 30-wk-old Wistar and Lou/C rats

 

Figure 5
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  		FIG. 5. Effect of various agonists on lipolytic response of isolated adipocytes from 30-wk-old Wistar and Lou/C rats. The maximal lipolytic effect (100%) corresponds to the response induced by 10–6 M noradrenaline. The data are the mean ± SEM from five to eight animals per group. *, Difference between Wistar and Lou/C rats estimated using Student’s t test (P < 0.01). BRL, BRL 37344 (β3 agonist); IBMX, 3-isobutyl-1-methylxanthine (phosphodiesterase inhibitor); ISO, isoproterenol (β agonist).

 

Figure 6
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  		FIG. 6. Antilipolytic effect of clonidine ({alpha}2-adrenoceptor agonist) on noradrenaline-stimulated lipolysis in adipocytes isolated from epididymal adipose deposits of 30-wk-old Wistar and Lou/C rats. In each group, values are mean ± SEM of five to eight experiments performed in quadruplicate. The maximal lipolytic effect (100%) corresponds to the response induced by 10–6 M noradrenaline. a, Difference from the maximal stimulation (P < 0.05). b, Difference between 10–6 and 10–3 M clonidine estimated using two-way ANOVA (Lou/C vs. Wistar) (P < 0.05).

 

Figure 7
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  		FIG. 7. Antilipolytic effect of insulin on noradrenaline-stimulated lipolysis in adipocytes isolated from epididymal adipose deposits of 30-wk-old Wistar and Lou/C rats. The dose-response curves are expressed as the percentage of maximal stimulation by 10–6 M noradrenaline. In each group, values are mean ± SEM of six experiments performed in quadruplicate. No significant difference was found between Wistar and Lou/C rats. £, Significant difference from the maximal stimulation estimated using two-way ANOVA (Lou/C vs. Wistar) (P < 0.05).

 
In rat, white adipose cells produce a significant amount of lipids rather than importing them all from the liver. Therefore, a reduced lipogenesis in WAT could contribute to the modest fat accretion noticed in Lou/C rat. Thus, we performed an in vivo lipid biosynthesis experiment with [3H] water as a tracer. Wistar and Lou/C rats were given a dose of [3H] water, and 1 h later, liver and several white adipose fat pads were excised, and the content of [3H]-labeled fatty acids was measured in liver and adipose tissue, whereas the content of [3H]-labeled digitonin-precipitable sterols was assayed in the liver. No difference was noticed in fatty acid biosynthesis rate between the two strains (Fig. 8AGo). However, a 1.6-fold increase in sterol synthesis (P < 0.05) was observed in the liver of Lou/C rats compared with their Wistar counterparts (Fig. 8BGo).


Figure 8
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  		FIG. 8. A, In vivo synthesis of fatty acids in WAT pads of 30-wk-old Wistar and Lou/C rats. B, Hepatic synthesis of fatty acids and sterols in 30-wk-old Wistar and Lou/C rats. Each value represents the mean ± SD of four animals of each strain. [3H] water was injected ip, and 1 h later, liver and several white fat pads were removed for the measurement of their content of [3H]-labeled fatty acids and/or digitonin-precipitable sterols. *, Difference between Wistar and Lou/C rats estimated using Mann-Whitney U test (P < 0.05).

 
Lou/C rats exhibited profound changes in tissue lipid composition
We investigated changes in lipid metabolism between Lou/C and Wistar rats using a comprehensive lipid profiling. Table 7Go represents the differences observed within entire fatty acid families in plasma (an index of lipid exchange), liver (the site of lipid biosynthesis), and WAT (the main site of lipid storage) in Lou/C and Wistar rats. The most striking changes are a higher proportion of saturated fatty acids in plasma, liver, and WAT from Lou/C rats with a reciprocal decrease in monounsaturated fatty acid (MUFA) proportion, except in WAT. In plasma and WAT, a decrease in n-3 without significant alteration in n-6 fatty acids was observed, whereas in plasma, the n-3 decrease was associated with an increase in n-6 fatty acids leading to an altered n-6/n-3 ratio. The "heat map" presented in Fig. 9Go summarizes the differences observed in individual fatty acid composition of total lipids extracted from plasma, WAT, and liver of Lou/C and Wistar rats. In plasma from Lou/C rats, the general trend was a decrease in the content of several classes of fatty acids. Indeed, we observed significant decreases in 16:1n-7 (palmitoleic), 18:1n-7 (vaccenic), 18:1n-9 (oleic), 18:3n-3 ({alpha}-linolenic), 20:5n-3 (eicosapentaenoic), 18:2n-6 (linoleic), 20:2n-6, and 20:3n-6 (dihomo-{gamma}-linolenic), whereas increases were observed for downstream members of n-6 fatty acids, i.e. 20:4n-6 (arachidonic), 22:4n-6, and 22:5n-6. Large alterations of lipid composition were also observed in the liver from Lou/C rats. We noticed a decrease of 16:1n-7, 18:1n-7, 16:1n-9 (hexadecenoic), 18:3n-3, 18:2n-6, 20:2n-6, and 20:3n-6 and an increase of 18:0 (stearic), 20:5n-3, 22:5n-3, 20:4n-6, and 22:4n-6. In contrast to what was observed in plasma and liver, fewer changes occurred in the Lou/C WAT. The most striking change was an increase in saturated fatty acid proportions, whereas few alterations were observed in other classes of fatty acids (i.e. decrease in 18:1n-7 and 18:3n-3). The desaturation indexes were calculated for the C16 and C18 fatty acids. Interestingly, the ratio of the MUFAs to saturated fatty acids was significantly lower in tissues from Lou/C than in tissue from Wistar rats (Fig. 10Go).


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  		TABLE 7. Fatty acid composition of total lipids extracted from plasma, liver, or rWAT of 30-wk-old Wistar and Lou/C rats

 

Figure 9
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  		FIG. 9. Analysis of fatty acid composition. "Heat map" showing individual fatty acid proportion in total lipid extracts from plasma, liver, and rWAT of 30-wk-old Lou/C rats. The fatty acid composition was determined using gas chromatography coupled to a flame-ionization detector. Quantitative data were used to calculate the percent increase (green) or decrease (yellow to red) in Lou/C compared with Wistar rats (n = 7 in each group). Significant differences were analyzed using the Student’s t test with color denoting P < 0.05. Differences not reaching P < 0.05 are shown in black. Statistical significance: a, P < 0.05, b, P < 0.01, and c, P < 0.005.

 

Figure 10
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  		FIG. 10. Lipid desaturation indexes in plasma, liver, and rWAT from 30-wk-old Wistar and Lou/C rats. Saturated and MUFAs in plasma, liver, and rWAT were measured by gas chromatography coupled with a flame-ionization detector. Index of desaturation corresponds to the ratio between the proportions of MUFAs and saturated fatty acids. Data are expressed as mean ± SEM from seven in each group. *, P < 0.05; **, P < 0.005, difference between Wistar and Lou/C rats estimated using the Student’s t test. C16:0, Palmitic acid; C16:1n-7, palmitoleic acid; C18:0, stearic acid; C18:1n-9, oleic acid.

 
Analysis of gene expression
We measured the expression of some key genes related to lipid metabolism. Semiquantitative RT-PCR analysis was performed for FAS, LPL, or SCD-1. Using β-actin as an internal standard, the ratios to β-actin were compared in epididymal fat pad and liver in both strains. In the liver no difference was noticed for FAS or SCD-1. A lower LPL expression (P < 0.05) was observed in eWAT from Lou/C rats (Fig. 11Go), whereas no change was observed in SCD-1 expression.


Figure 11
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  		FIG. 11. Gene expression in liver and rWAT. FAS (A), SCD-1 (B), and LPL (C) expression in 30-wk-old Wistar and Lou/C rats (n = 5 per group) were measured using semiquantitative RT-PCR. Quantification of RT-PCR was performed by scanning densitometry using β-actin as a control to normalize gene expression. **, Difference between Wistar and Lou/C rats estimated using the Student’s t test (P < 0.01). AU, Arbitrary units.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The Lou/C rat has been described as a model of obesity resistance (3). To gain insight into the mechanisms of the outstanding leanness of Lou/C rats, we performed a detailed comparison with age-matched Wistar rats with particular focus on energy balance and cellular characteristics of the WAT. To date, no data are available in the literature regarding the structural and metabolic features of WAT in Lou/C rats. In good agreement with previous reports (3, 7), we found that Lou/C rats exhibited a reduced and stable adiposity during aging, as indicated by a low Lee adiposity index. The weights of adipose tissue pads, visceral fat pads (i.e. retroperitoneal or epididymal), as well as sc fat pads (i.e. inguinal), whose growths are correlated with the accretion of body lipid (5), are strikingly lower in Lou/C compared with Wistar rats. Indeed, 7-, 5-, and 3-fold differences were noticed in retroperitoneal, epididymal, and inguinal fat pad weights, respectively. Cellularity measurements of epididymal fat pad indicate that the lower adiposity was due both to a hypotrophia (i.e. a decreased adipocyte size) and a hypoplasia (i.e. a reduction in the number of adipocytes). This finding is in good agreement with previous observations made on sympathetic activity in WAT from Lou/C rats. Indeed, we have previously reported that noradrenaline content and biosynthesis were higher in WAT of Lou/C compared with Wistar rats (18), suggesting either a higher density of sympathetic fibers or a greater sympathetic activity. A higher sympathetic activity in WAT could lead to enhanced lipolysis and inhibition of WAT development (19, 20, 21, 22, 23). We observed a striking change in LPL expression in WAT of Lou/C rats (6-fold lower than in Wistar). In WAT, catecholamines, through β-adrenoceptors, can decrease LPL gene transcription (24). LPL is responsible for the mobilization of lipids from plasma lipoproteins and is, therefore, a key enzyme for triglyceride accumulation in WAT. Thus, a decreased LPL expression in WAT could result in a lower uptake of lipids in the WAT and participate in the modest WAT accretion observed in Lou/C rats.

Energy accumulation, i.e. increased adiposity, results from the strict difference between energy intake and energy expenditure. Therefore, we thoroughly compared the metabolic profiles of Lou/C and Wistar rats through the measurements of caloric intake, energy expenditure, and physical activity. Some discrepancies exist in the literature regarding the caloric intake of Lou/C rats. Several authors reported that Lou/C rats exhibit a spontaneous 40% reduction in caloric intake and, therefore, presented Lou/C strain as a spontaneous model of caloric restriction (3, 8, 25, 26). Other investigators did not observe any reduction in food intake when compared with obesity prone strains Wistar or Fischer F344 rats (7, 27, 28). These discrepancies result mainly from differences in the expression of caloric intake, namely whether these results were normalized for body mass or metabolic mass (kg 0.67). In the present study, we observed a slightly higher food intake in Lou/C compared with age-matched Wistar rats when results were normalized for the body mass. Lou/C rats exhibited a 11% increase in daily energy expenditure compared with Wistar rats due to increases in energy expenditure in both quiescent (+13%) and active (+10%) phases. The RQ was very similar in the two strains excluding a large difference in metabolic fuel utilization, and indicated the oxidation of both carbohydrates and lipids as energy substrates. Lou/C rats showed a decrease in feed efficiency, as estimated by the ratio of total body mass gain divided by total amount of food consumed by the same animal. This ratio is significantly lower in Lou/C rats, suggesting the occurrence of futile cycles in the metabolic pathways. Locomotor activity was significantly increased in Lou/C compared with Wistar rats and may contribute to some extent to a higher energy dissipation (29). Thus, the lower adiposity observed in Lou/C rats could be partly explained by a higher locomotor activity associated with a higher energy expenditure. However, during the quiescent phase, the metabolic rate was higher in Lou/C than in Wistar rats, suggesting that the higher daily energy expenditure cannot totally be explained by an increased physical activity. We previously reported that Lou/C rats exhibited a selective increase in noradrenaline content and catecholamine biosynthesis activity in iBAT (7, 18), the main site of facultative thermogenesis in rodents. Obese rats, such as Zucker fatty rats, exhibit a blunted sympathetic activity in BAT that results in its reduced thermogenic function and, in this way, contributes to the development of obesity (30, 31, 32). We may hypothesize that a higher sympathetic tone in Lou/C rats, resulting in an enhanced thermogenic function, could account for the higher metabolic rate observed in this strain. An alternative hypothesis is that the reduced fat deposition decreases the body isolation, increases heat dissipation, and increases heat loss. In search of additional factors to explain the modest fat deposition in Lou/C rats, we investigated the metabolic potentialities of their white adipocytes. Indeed, a reduced adiposity could result from an increased lipolytic activity, a lower lipogenic activity, or both. To test these hypotheses, we measured the lipolysis on isolated adipocytes, whereas lipogenesis was measured in vivo using tritiated water. No difference was noticed in lipid biosynthesis activity (lipogenesis) measured through the incorporation of in [3H] water in fatty acids in liver or WAT. However, in good agreement with the higher plasma cholesterol concentrations, a significant increase in hepatic sterologenesis was observed in Lou/C rats. The lipolysis, i.e. the capacity to hydrolyze triglycerides and release glycerol, was measured in isolated epididymal adipocytes, in the basal state, and after stimulation with noradrenaline. The resulting lipolysis revealed neither changes in the basal production rate of glycerol, nor in noradrenaline sensitivity as indicated by similar EC50 values between the two strains. However, we noticed a strong decrease in noradrenaline responsiveness in adipocytes from Lou/C rats as indicated by the blunted VMAX value. This decreased responsiveness without decreased sensitivity could result from a smaller size of Lou/C adipocytes and, thus, a reduced number of noradrenaline binding sites. Indeed, when results are expressed as percentage of the VMAX, no difference was noted. Thus, an increased lipid mobilization could not explain the difference observed in adiposity between the two strains. Lafontan and Berlan (33) reported that the bigger the adipocytes are, the more they possess antilipolytic {alpha}2-adrenoceptors. Thus, with an intact sensitivity to noradrenaline, the small size of adipocytes from Lou/C rats could result from a decreased number of {alpha}2-adrenoceptors. Surprisingly, the antilipolytic {alpha}2-adrenergic responsiveness was enhanced in Lou/C rats because clonidine produced a significant inhibition of noradrenaline-stimulated lipolysis. The use of the highly selective β3-adrenergic agonist BRL 37344 led to a higher lipolytic activity in Lou/C adipocytes than in Wistar ones, suggesting a higher density and/or higher affinity of β3-adrenergic receptors on Lou/C adipocytes. In the present study, several adipose depots have been dissected out, however, for practical reasons, it has not been possible to perform the lipolysis measurements on each depot. Because lipolysis measurements were restricted to epididymal fat pad, we cannot exclude that the regional differences exist between adipose deposits.

We used a comprehensive lipid profiling to characterize the lipid metabolism in liver and WAT of obese-resistant Lou/C rats. This lipidomic analysis of tissues and plasma from Lou/C and Wistar rats reveals profound differences in lipid metabolism between the two strains. Fatty acid composition in Lou/C (plasma, WAT, and liver) rats is characterized by an increased content of saturated fatty acids and a decreased content in monosaturated fatty acids. The serum fatty acid composition has predicted the risk of diabetes, cardiovascular diseases, (34) and metabolic syndrome (35, 36). Indeed, the fatty acid composition in individuals with insulin resistance is characterized by high concentrations of polyunsaturated fatty acids (18:2n-6) and a proportionally higher level of palmitoleic (16:1n-7) and di-homo-{gamma}-linolenic (20:3n-6) acids (36). In plasma Lou/C rats, 18:2n-6 was found decreased compared with Wistar rat, which is consistent with the increase observed in man with metabolic syndrome since Lou/C rat showed an improved insulin sensitivity (7). Plasma concentrations of 16:1n-7 and 20:3n-6 have been found decreased in Lou/C compared with Wistar rats, which can be observed in insulin-resistant patients, suggesting that these fatty acids do not account for insulin sensitivity in Lou/C rats. Other findings have suggested a physiological and pathological role of fatty acid composition on protein handling that is proposed to be relevant to insulin sensitivity (37). Indeed, acyl-coenzyme A synthase-1 ligates NEFA to coenzyme A (CoA) and directs fatty acids primarily into triglyceride synthetic pathways (38). Its expression was associated negatively with adipose tissue 16:0 and positively with NEFA 16:1 (37). Adipose tissue 16:0 content was positively related to insulin sensitivity (homeostatic model assessment index). Fatty acid transport protein expression was correlated with adipose tissue 20:4 n-6, which in turn positively correlated with insulin sensitivity (homeostatic model assessment index). Although these data obtained in Lou/C rats are in good agreement with results from human beings, they do not allow the identification of causal relationships; nevertheless, they suggest a link between fatty acids, ACS1, and fatty acid transport protein expression levels and insulin resistance. Genetic differences in fatty acid desaturases might predispose to obesity. SCDs convert some saturated fatty acids into MUFAs and represent the rate-limiting step in MUFA biosynthesis in vivo. The natural substrates of these enzymes are palmitoyl-CoA (16:0) and stearoyl-CoA (18.0), which are converted into palmitoleoyl-CoA (16:1n-7) and oleoyl-CoA (18:1n-9), respectively. Although several isoforms of SCD exist in rodents, only SCD-1 is expressed in liver and WAT. There is now accumulating evidence that SCD-1 plays a crucial role in lipid metabolism and body weight control (39, 40). Indeed, mice with SCD-1 inactivation, either using knockout mutation or RNA interference, exhibited a hypermetabolism, hyperactivity, reduced adiposity, and resistance to diet-induced obesity (41, 42, 43, 44). Interestingly, Lou/C rats share several metabolic characteristics with SCD-1-deficient animals, namely hypermetabolism, increased locomotor activity, reduced adiposity, and resistance to obesity. In good agreement, the lipid profiling reveals that in plasma and liver from Lou/C rats, there were lower MUFAs than Wistar rats, and that C16 and C18 desaturation indexes were significantly blunted, suggesting a defect in the SCD pathways. This would imply that the particular metabolism and outstanding leanness of Lou/C rats might result from an inherited deficiency in SCD-1. To explore this hypothesis, we measured the relative expression of the SCD-1 gene in WAT and liver of both strains. Surprisingly, we failed to find any difference in the level of SCD-1 expression in liver or WAT of Lou/C compared with Wistar rats. This finding excludes a genetic deficiency in SCD-1 in the Lou/C strain. However, the changes in C18 and C16 desaturation index reported previously clearly indicate a defect in SCD-1 activity. These differences could result from different levels of SCD-1 enzymatic activity and regulations. However, these points need further exploration.

Despite extensive literature, the present study constitutes the first description of cellularity and metabolic properties of WAT in Lou/C obesity-resistant rats. The outstanding leanness and the modest fat accretion observed in the Lou/C strain might result from systemic differences between the two strains rather than from white adipocyte metabolic characteristics. The increased physical activity and higher energy expenditure observed in Lou/C rats can partly explain the lower adiposity observed in these rats. Whether differences in sympathetic activity in Lou/C tissues involved in energy regulation could account for the leanness of Lou/C rats remains to be explored.


   Footnotes
 
This work was supported by Institut National de la Santé et de la Recherche Médicale, l’Institut Multidisciplinaire de Biochimie des Lipides, and Région Rhône-Alpes. A.F.S. was a recipient Capes Brazil (No. 0159 026). B.Z. was supported by a grant from the French "Ministère de l’Education Nationale, de la Recherche et de la Technologie."

Disclosure Statement: The authors have nothing to declare.

First Published Online November 15, 2007

Abbreviations: CoA, Coenzyme A; eWAT, epididymal white adipose tissue; FAS, fatty acid synthetase; iBAT, interscapular brown adipose tissue; ingWAT, inguinal white adipose tissue; KRB, Krebs-Ringer bicarbonate; LPL, lipoprotein lipase; MUFA, monounsaturated fatty acid; NEFA, nonesterified fatty acid; RQ, respiratory quotient; rWAT, retroperitoneal white adipose tissue; SCD-1, stearoyl-coenzyme A-dehydrogenase-1; WAT, white adipose tissue.

Received March 7, 2007.

Accepted for publication November 5, 2007.


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 Introduction
 Materials and Methods
 Results
 Discussion
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