The experimental protocol was approved by the French National Animal Care Committee. Male Wistar rats (Harlan Laboratories) weighed around 225 g (7 weeks) at their arrival in the laboratory. With the goal of keeping all the rats in the study and thus to avoid sacrificing many animals, the breeder was asked to select from its colony of 7-week-old rats the six lightest and the six heaviest in order to get rats with a large phenotypic heterogeneity. The composition of the diets is presented in Table 1. Animal facility conditions were a 12:12 Light-Dark cycle and 22°C.

This is outlined in Figure 1. The rats were weighed 2 days after arrival in the laboratory. After the selection performed by the breeder, the range of BWs of the rats was large (206–258 g) but the mean weights of the light and heavy rats not substantially different (219.8 ± 5.1 vs 233.5 ± 4.6, P = 0.07) with individuals overlapping between the two groups. After arrival and 5 days of adaptation to the animal facility, the 12 rats were split into three groups of 4 rats (the number of rats that could be studied simultaneously in the behavioral-metabolic device) and maintained under HCD in order to be studied in turn by group (1, 2, and 3 weeks after arrival in the laboratory). During a 5-day period each of the three groups was characterized for TEE, food intake (FI), and SPA during transition from HCD to HFD. After completion of the 5-days of calorimetric measurements, the rats were allowed to recover for 10 days under HCD, and then fed the HFD for 6 days to allow selection from BW gain between FS and FR rats. Body composition was analyzed from dissection and weighing of the main organs and tissues just after the end of the 6-day HFD period. These 12 rats could readily be separated into FS and FR groups, each of six rats with a highly significant difference in their BW gain response to the HFD. This suggested that despite initial small differences in BW, the selection performed by the breeder helped in the creation of a group with a large heterogeneity. There was however no correlation between the initial weight of the rats and their weight gain during the HFD period.

The goal was to obtain for each rat measures of FI pattern, SPA, TEE and respiratory quotient (RQ) during HCD, HFD and the transition between the two. Groups of four rats were housed at 18:00 in individual metabolic cages equipped with a weighed food cup (sensitivity better than 0.05 g) and an activity platform placed below (sensitivity better than 1 g). For gas analysis, the cages were multiplexed – all connected to the same gas analyzers. Thus VO₂ and VCO₂ were measured on each cage during 2 min every 10 min (2 min for each cage, plus 2 min on room air to correct values for room %O₂ and %CO₂). To reduce close to zero any energy expenditure for thermoregulation (non-shivering thermogenesis), temperature in the room was maintained at 25–26°C in order to maintain in the metabolic cage a temperature of 26–27°C.

Day 1 in the metabolic device was used for habituation. VO₂–VCO₂ and FI were measured during day 2 (HCD feeding) and during days 3–5 the rats were switched to the HFD. For each cage FI and SPA were measured in 5 s time bins on a separate computer. For analysis, data were pooled into 10 min bins and combined with the VO₂–VCO₂ data. Metabolic rate was computed from VO₂ and VCO₂ according to the Weir formula (Ferrannini, 1988; Even et al., 1994).

Rats were deeply anesthetized with an overdose of pentobarbital (40 mg/kg) then killed following exsanguination by blood sampling from the vena cava. The main tissues and organs (liver, spleen, kidneys, brain, heart, subcutaneous, retroperitoneal, inguinal, and mesenteric fat pads, skin) were dissected out, blotted dry, and weighed to the nearest 0.01 g.

Differences between groups (FR vs. FS and CR vs. CS) were assessed using Student’s t-test. A difference was considered statistically significant at P < 0.025 (Bonferroni correction) to take into account that the tests were repeated in the FR–FS and CR–CS study. Pearson product moment correlation analysis was also used to examine the relationship between the level of sensitivity to fat or carbohydrates and various metabolic and behavioral parameters that showed a trend toward significance between FR/FS or CR/CS rats.

Body weight and composition data are shown in Table 2. The six FS rats were selected according to their larger daily BW gain during the period of HFD. Accordingly, BW gain during HF feeding was 44% larger in the FS than in the FR rats but no differences in body adiposity were observed between the two groups at the end of the study. However, within each group and therefore amongst all 12 rats combined, there was a great variation in adiposity. This allowed a second classification according to adiposity into five leaner rats (mean adiposity 10.68 ± 1.07%) and seven fattier rats (mean adiposity 15.12 ± 0.49%). We refer to the leaner rats as CR and to the fattier as CS since the adiposity differences were accumulated in rats fed under HCD during most of their life. In addition, because the CR and CS rats were approximately evenly distributed (five vs seven) into the FS and FR groups, we assumed that the 6-days of HFD affected similarly the body fat gain during this period in the CR and CS rats. Despite these large differences in body fat and body adiposity, final BWs of the CS and CR rats were not significantly different. Of the 31-g difference in BW, 20 g was accounted for by body fat (+53% in the CS rats) while only 10 g was accounted for by LBM (+3.7% in the CS rats). BW gain during HF feeding was also very similar in CR and CS rats.

The meal pattern under HCD in rats classified as FS was characterized by significantly more numerous meals compared with FR during HCD but the difference decreased during HFD (Table 3). In parallel, inter-meal interval (IMI) and meal size tended to be lower which explains the absence of a significantly higher overall CI. No differences were observed between FR and FS rats for the components of SPA (Table 4). TEE was similar in FS and FR rats under HCD, but decreased progressively in FS rats during HFD and became significantly lower than in FR rats after 3 days (Table 5). When expressed relative to HCD, TEE during HFD was significantly more decreased in FS rats after 2 and 3 days of HFD. In parallel, RQ tended to decrease more in FR rats, indicating a better adaptation to the increased fat content of the diet, and became significantly lower in FR than FS rats after 3 days of HFD. In summary, FS rats can be distinguished from FR rats from their larger meal number under HCD, and a larger decrease in TEE and maintenance of a higher RQ in response to HFD.

None of the parameters of FI showed significant differences between CR and CS rats during HCD as well as during HFD. In both groups, HFD increased CI. CS rats tended to spend more time active than CR rats during HCD (+23%, P = 0.054; Table 4), but this tendency disappeared completely during HFD. In contrast, during HCD CS rats developed bursts of SPA that were of significantly lower intensity (−29%, P = 0.004,) than those recorded in the CR rats. This characteristic vanished progressively under HFD. No differences were observed between the two groups in TEE or RQ throughout the 4-days (Table 5). In summary, CS rats can be characterized primarily from the lower intensity of their bursts of SPA relative to CR rats during HCD.

According to the differences observed in the FS/FR and CS/CR rats, we re-analyzed the data starting from the potential predictive parameters to test if these parameters could indeed be good predictive factors.

It is noteworthy that the FS rats did not gain more weight (therefore most probably not more fat) than the FR groups under HCD. Diets high in fat and consequently of higher energy density encourage food consumption (Poppitt, 1995), and accordingly in this study CI was larger during HFD than during HCD for all rats. However, FS rats did not eat significantly more food (g) than FR rats under either HFD or HCD. In contrast, FS rats ate significantly more meals under both diets but had smaller meals during the two first days of HFD. However, the reduction in meal size was only transient and vanished after 2–3 days of HFD. The fact that meal size was not larger in FS rats tends to contradict the idea that FS rats eat larger meals. However, such observations are usually made on rats that are already overweight after high fat feeding (Farley et al., 2003) which may confound pre-existing differences in meal pattern with current obesity, metabolic, and endocrine adaptation to HFD.

That FS rats eat more meals than FR rats when fed a HCD has been reported previously in genetically selected FR and FS rats (Ricci and Levin, 2003; Cottone et al., 2007). Interestingly, these genetically selected rats seem similar to the outbred rats of this study since they were as lean as or even leaner than their FR counterparts when maintained under HCD. Meal fractioning has been proposed as a means to oppose obesity in humans (Cohn et al., 1965; Nicklas et al., 2001; Parks and McCrory, 2005) which contradicts the present observation that meal fractioning is associated with an increased sensitivity to obesity. In fact, meal fractioning does not seem to influence the predisposition to gain weight or fat. In FS rats, meal fractioning was higher under HCD when FS rats did not gain more weight or fat than FR rats, and was maintained under HFD when FS rats gained more weight. Therefore, meal fractioning is probably not responsible per se for the higher sensitivity to HF feeding in FS rats. Rather it may reveal some defect in the mechanisms controlling FI. For example, among potential defective signal(s), leptin production, or central sensitivity to leptin may be postulated. Indeed, central administration of leptin has been shown to reduce meal frequency in rats (Zorrilla et al., 2005) and to favor post-meal satiety (Montague et al., 1997; Chapelot et al., 2000; Westerterp-Plantenga et al., 2001). A defect in leptin sensitivity has also been reported in genetically selected FS rats (Levin et al., 2004; Clegg et al., 2005; Irani et al., 2007). Thus, measurement of meal number under HCD seems to be a criterion to easily and non-invasively separate FR and FS rats in a colony of young, non-obese unselected outbred rats, bearing in mind however that while there is indeed a good correlation, there is also overlap of individual values.

The fact that hyperphagia is a characteristic of FS rats is widely accepted and was even suggested to be a faithful predictor of sensitivity to obesity (Dourmashkin et al., 2006). Hyperphagia as the critical stimulus to increase BW gain in FS rats fed a HFD has also been suggested by the observation that FS rats have no increased inherent capacity for dietary fat retention (Commerford et al., 2001). However, in virtually every animal model of obesity in which hyperphagia is a characteristic feature, increased fat deposition has been shown to be independent of the increase in FI (Friedman, 1990, 1998). Therefore, if hyperphagia cannot be ruled out as a component of the sensitivity to HFD, at least for some of the FS rats, the present results confirm that increased FI is only one among several components responsible for increased weight gain of FS subjects under HFD.

Spontaneous physical activity is considered an important component involved in the resistance to HFD (Levine et al., 1999), but analysis of the various components of activity in FS and FR rats in this study did not reveal any significant difference, neither during HCD nor during HFD. In line with this absence of differences, genetically selected FS rats have not been shown to be less active than FR rats under HCD but to become less active only after overweight develops following long-term HFD (29 days; Novak et al., 2006). Thus, following the lack of a short-term effect of the HFD on SPA observed here, it is probable that the decrease in SPA after long-term HFD accompanies or results from, but is not responsible for, the gain in weight and the development of metabolic disturbances that progress during HFD.

Analysis of TEE and RQ suggests that the multiple pre-existing abnormalities reported in FS rats do not significantly affect these components of energy expenditure under HCD, a result that can be considered as normal since FS and FR rats maintained under the HCD ended the study with the same BW, LBM, carcass mass, fat mass, and adiposity. In contrast, during transition to the HF diet, TEE decreased more in FS rats while RQ tended to decrease less so that after 3 days of HFD RQ was higher in FS rats. These responses are obviously able to promote fat storage under HFD by saving energy and reducing fat oxidation. Therefore, studying the transition from HCD to HFD was able to reveal metabolic defects that were not visible under HCD. This suggests that processes specifically related to partitioning of dietary lipids between storage and oxidation are affected in FS rats during HFD but not HCD. Many potential mechanisms have been suggested in the literature, but one possible mechanism that we consider worth further investigation may be a defective lipid oxidation located specifically in muscles which progressively leads to fat accumulation in muscle and insulin resistance if HFD is continued (Galgani et al., 2008). To our knowledge, there has been no direct demonstration that this phenomenon pre-exists in still lean FS rats, but it has been reported that a significantly lower proportion of type I muscle fibers in the medial head of the gastrocnemius muscle of FS rats already before HFD may play a role in determining susceptibility to dietary obesity (Abou Mrad et al., 1992). Thus, changes in TEE and RQ during the first days of adaptation to HFD are potential parameters to discriminate between FR and FS rats.

Classification of CR and CS rats according to their body fat content at the end of the study showed that despite large differences in body fat, CS rats were only slightly, not significantly heavier than the CR ones, and had similar LBM and carcass mass. This may explain why this kind of rat had not been clearly distinguished, except, to our knowledge, in one publication (Dourmashkin et al., 2005) in which overfeeding and BW gain was boosted by a high content of saccharose in the diet which was not the case here. In addition we did not find in this study (but the number of rats was small) that CS rats were more sensitive to HFD than CR ones.

Contrary to what was observed between FR and FS rats, no differences were observed between CR and CS rats in any of the components of meal pattern under either HCD or HFD. In contrast, differences were observed at the level of SPA. CR rats developed bursts of SPA of higher intensity than the CS ones. The difference was particularly strong and significant during HCD, which is when the rats were fed the diet for which they exhibited a different sensitivity for fat deposition. The bursts of SPA correspond to the intensity of the mechanical work developed when the rats are active in contrast to the mean SPA that is the mean mechanical work developed during 24 h which includes resting periods. This component cannot be quantified with the usual red-beams device but requires a method able to quantitatively measure the forces that develop on the floor of the cage by force transducers located beneath the cage. We have extensive experience with this system and have been able to control that the intensity of the activity signal tightly correlates to the energy expended in relation to muscular work (Even et al., 1994).

In humans, a reduced SPA is considered a trait of individuals sensitive to obesity and cardiovascular problems under HFD (Moore, 2000) and the increasingly sedentary habits associated with modern lifestyles are suggested to play an important role in the development of obesity (Prentice and Jebb, 1995). However, the influence on adiposity of the type of activity in which people engage during their daily life, or the way they perform standardized activities (walking, stair-climbing,) has not been systematically investigated. Here, we observed that the intensity of the bursts of SPA was very significantly lower in CS rats during HCD. This result suggests that rather than, or in conjunction with the absolute amount of activity, the type of activity, and specifically its intensity or its briskness may be involved in the resistance/sensitivity to fat deposition under HCD, possibly by influencing the control of energy metabolism and the aerobic/glycolytic capacities in muscles. For example, it was recently shown that rats with greater aerobic capacities were more active and also more resistant to HFD-induced obesity and responded differently to i.c.v. injections of orexin-A (Novak et al., 2010). Thus, activity and aerobic capacities in muscle are both important components of the response to HFD and we bring here preliminary data suggesting that this may well also be the case in rats that exhibit different sensitivities to HCD. On practical grounds, the amplitude of the differences in the bursts of activity between CS and CR rats makes this component of activity a potentially robust parameter to separate the two groups. It is in fact the only parameter for which we observed no overlap between CR and CS rats. This observation deserves further attention to confirm this phenomenon and further analyze how it is connected with the increased adiposity that develops in CS rats, in particular to what extent muscle metabolism is involved.

Measurements of TEE did not reveal differences between CR and CS rats. Comparison of CI with TEE showed that energy balance (EB) was not significantly different in the two groups under HC as well as under HF feeding. The increase in EB during HF feeding was mainly the result of an increase in CI while EE was unchanged, the amplitude of this phenomenon being comparable in the two groups. The fact that TEE does not increase (or only marginally so) after HF feeding has been reported in various studies (Dallosso and James, 1984; Schutz et al., 1989). However, it is interesting to note that despite not being significantly different, EB was steadily higher in CS than in CR rats. The difference was the largest (25%) under HC feeding. This was confirmed by differences between RQ values that were also larger in CS rats. The fact the EB and RQ values were not significantly different may be disappointing, but one must keep in mind that obesity develops very progressively as a result a very tiny daily differences that are necessarily very difficult to reveal by direct measurement of energy expenditure. Differences in RQ have also been reported under HF feeding between FS and FR (Chang et al., 1990), and the authors suggested that the capacity to adjust nutrient oxidation to nutrient intake was a major mechanism underlying the sensitivity to HF feeding. This is obviously a potential mechanism, and it seems logical that in the CS rats of the present study, differences in RQ may be larger under HCD than under HFD.