Fat-depleted CLA-treated mice enter torpor after a short period of fasting

Appetite 42 (2004) 91–98 www.elsevier.com/locate/appet

Research Report

Fat-depleted CLA-treated mice enter torpor after a short period of fasting J.C. Bouthegourda, J.C. Martinb, D. Gripoisb, S. Roseaua, D. Tome´a, P.C. Evena,* a

UMR INRA/INA P-G 914, Physiologie de la Nutrition et du Comportement Alimentaire, Institut National de la Recherche Agronomique, 16, rue Claude Bernard, 75231 Paris Cedex 05, France b Laboratoire de Physiologie de la Nutrition, Baˆt. 447, Universite´ Paris-Sud, 91405 Orsay Cedex, France Received 22 April 2003; revised 9 July 2003; accepted 31 July 2003

Abstract Resting energy expenditure (Resting-EE), EE with treadmill exercise, and post-prandial thermogenesis were continuously monitored by indirect calorimetry during a 24 h recording session in control (CT) and CLA-treated (CLA) (1% CLA in the food, by weight) C57Bl/6 male mice. After 15 days of CLA treatment, the fat content of CLA mice had fallen to 20% of that in CT mice. CLA mice were able to face the energy challenge of exercise but used less lipid than CT mice. Resting-EE values fell during the post-exercise period. The thermogenic response to a calibrated test meal given 5 h after the run abolished the differences in EE and substrate oxidation between CT and CLA mice. However, 2.5 h after ingestion of the test meal onward, CT mice gradually increased their lipid oxidation to sustain resting-EE levels. In contrast, CLA mice did not increase their lipid oxidation and their resting-EE levels fell significantly until they entered into torpor. Blood leptin was low but similar in CT and CLA-treated mice suggesting that leptin is not critical to induce torpor. We suggest that the durable inhibition of lipid oxidation in fasting CLA mice was an adaptive behaviour devoted at sparing the residual adipose deposits. q 2003 Elsevier Ltd. All rights reserved. Keywords: Rapidly induced torpor; Indirect calorimetry; Conjugated linoleic acid; Activity; Leptin

Introduction The metabolic changes associated with torpor seem to differ depending on the stimulus inducing this state (Buck & Barnes, 2000; Geiser, Kortner, & Schmidt, 1998; Heldmaier et al., 1999; Snyder & Nestler, 1990; Song, Kortner, & Geiser, 1997). In species that spontaneously enter torpor, e.g. arctic hibernators or hamsters, entry into torpor seems to depend mainly on environmental factors such as light and temperature, and occurs before any energy deficit is observed (Buck & Barnes, 2000; Dark, Lewis, & Zucker, 1999; Geiser, Kortner, & Schmidt, 1998; Heldmaier et al., 1999; Snyder & Nestler, 1990; Song, Kortner, & Geiser, 1997). In contrast, mice do not enter torpor unless acute energy pressure, such as a reduction in temperature or a marked energy deficit is imposed (Gavrilova et al., 1999, 2000; Stamper & Dark, 1997; Webb et al., 1982). In general, the metabolic signal(s) for entry into torpor are as yet Abbreviations: CLA, conjugated linoleic acid; CT, control; EE, energy expenditure; Gox, glucose oxidation; Lox, lipid oxidation; TEF, thermal effect of food; TG, triacylglycerol. * Corresponding author. E-mail address: [email protected] (P.C. Even). 0195-6663/$ - see front matter q 2003 Elsevier Ltd. All rights reserved. doi:10.1016/j.appet.2003.07.003

unknown, although leptin has recently been proposed as a possible triggering factor because of its regulatory function on energy expenditure (Gavrilova et al., 1999). For example, rapidly induced torpor entry in ob/ob mice, which have massive triacylglycerol stores and low levels of circulating leptin, can be prevented by leptin replacement (Moitra et al., 1998). However, leptin infusion does not prevent transgenic A-ZIP/F-1 mice (genetically manipulated animals with no white fat tissue and low leptin levels (Gavrilova et al., 1999)) from responding rapidly and promptly entering torpor, thus suggesting that leptin is not the sole signal involved in the control of torpor (Moitra et al., 1998). For these reasons, the precise origin of torpor in rodent models is still not fully understood. A decrease in the respiratory quotient, indicating a reduced rate of carbohydrate oxidation, has been observed during the first hours of torpor. 2-Deoxy-D -glucose (which reduces glucose oxidation) or mercapto-acetate (which reduces free fatty acid oxidation) do not induce torpor in deer mice when injected alone, but can do so when injected together (Stamper & Dark, 1997). This suggests that torpor is not dependent on the availability of a specific substrate (i.e. glucose versus lipids) but rather requires an undifferentiated

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but marked energy deficit. On the other hand, in A-ZIP/F-1 mice, rapid entry into torpor is observed in response to fasting, and may compensate for the inability of these mice to release free fatty acids as substrates to maintain their basal metabolic rate. The objective of the present study was to investigate whether torpor was dependent on the inability of mice to use lipids as energy substrates. For this purpose, a reduction in fatty acid availability was achieved by dietary treatment with conjugated linoleic acids (CLAs). In mice, CLAs promote almost complete fat depletion that can be compared to that observed in A-ZIP/F1 mice (Park et al., 1997; Tsuboyama-Kasaoka et al., 2000; West et al., 1998). Our results showed that CLA-treated fat-depleted mice entered torpor after a few hours of fasting, while untreated mice sustained resting-EE levels by relying to a considerable extent upon lipid oxidation. Because we observed that no more than several hours before torpor these same mice were able to greatly increase their energy expenditure and fat oxidation in response to exercise, we suggest that torpor did not directly result from lipid scarcity but more probably occurred in response to an adaptive but still unknown physiological signal generated to reduce energy expenditure and preserve lipid stores.

Materials and methods Animals and diets. Forty-five C57Bl/6 male mice (six weeks old) were housed three per cage in a controlled environment, with a constant temperature (24 8C ^ 1), and an artificial light – dark cycle (dark from 18:00 to 06:00). After 10 days of adaptation to the laboratory conditions, they were divided into two groups: one was fed a standard synthetic diet, the other received the same regimen augmented with 1% by weight (3% by energy) of a CLA free fatty acids mixture kindly donated by Loders Croklaan (Wormerveer, Netherlands). Food cups were refilled every day at 18:00. The standard diet was composed as follows (ingredient, g/kg): corn starch: 480; saccharose: 150; ‘vitamin-free’ casein: 210; rapeseed oil: 50; cellulose: 50; mineral mix: 35; vitamin mix: 10. The fatty acid content and isomer composition of the CLA mixture were determined by GC/MS on methyl-triazoline dione (MTAD) derivatives and GC analysis of fatty acid methyl esters (FAME). The lipid content was made up of 78.8% CLA isomers, 4.26% palmitic acid, 1.48% stearic acid, 12.48% oleic acid, 0.57% cis-vaccenic acid, 0.10% trans-9, cis-12, 0.17% cis-9, trans12, 2.16% linoleic acid. The composition of CLA isomers as determined by capillary GC was 48.54% cis-9, trans-11; 49.62% trans-10, cis-12; 0.80% cis-10, cis-12 1.00m cis-9, cis-11; 0.05% trans-9, trans-11 and trans-10, trans-12. Thirty mice were designed to fulfil a 22 h measurement of energy expenditure including a treadmill exercise. Fifteen other mice were designed to be sacrificed just after the run for a body composition evaluation.

Measurements of energy expenditure components Thirty mice (five groups of three for each condition) were placed for 22 h in a metabolic cage connected to an opencircuit, indirect calorimetry system with its airflow adjusted to 0.5 l/min. The temperature in the metabolic cage (24 ^ 1 8C) was stable and controlled throughout the experiment. Because of the low metabolic rate of such small animals, respiratory exchange measurements had to be performed on groups of three animals. Oxygen consumption and carbon dioxide production were recorded at 10 s intervals using a computer-assisted data acquisition program (Even, Perrier, Aucouturier, & Nicolaı¨dis, 1991). Throughout the daytime, spontaneous activity was also recorded quantitatively by means of piezo-electric strain gauges (sensitivity 0.1 g) located beneath the metabolic cage. Computer-assisted processing of respiratory exchanges and spontaneous activity signals made it possible to compute that part of the total metabolic rate devoted to fuelling the energy cost of activity (Bouthegourd et al., 2002; Even, Mokhtarian, & Pele´, 1994; Even et al., 1991). Thus, by continuously extracting the energy expended with activity, it was possible to compute the resting metabolism of these free-moving mice (Bouthegourd et al., 2002; Even, Mokhtarian, & Pele´, 1994) (Fig. 1). At 18:00 on the day before the calorimetric investigation, the mice received ø 50% of their spontaneous 24 h food intake (2.5 g of dry food per mouse) so that their last meal was eaten no later than the middle of the night. Experimental protocol The respiratory exchanges measurements started with an adaptive period to the metabolic cage environment during 2 h between 10:00 and 12:00. Between 12:00 and 13:00, mice were exercised on a treadmill, while respiratory exchanges measurements where continued. Then they returned for 5 h in the metabolic cage with no access to food. At 18:00 a calibrated test meal was introduced in the cage so that mice can be re-fed for the measurement of the thermic effect of feeding. The experiment then lasted during the whole following night. Mice were removed from the metabolic cage the next morning. Next sections describe the key periods of this experimental protocol. Those key periods are: post-absorptive period (1), exercise, postexercise period (2), thermic effect of feeding (TEF), postTEF period (3), fasting period (4). See Fig. 1. Post-absorptive energy expenditure (1) At 10:00, the mice were housed in the metabolic cage with free access to water but no food. Post-absorptive resting-EE levels were measured between 11:00 and 12:00.

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Fig. 1. Changes in energy expenditure, glucose and lipid oxidation during calorimetric measurements observed in groups of three mice (n ¼ 5 per group before the meal, n ¼ 5 in control and 4 in CLA after the meal). Key periods are represented on the graph as described in Section 2.2. The arrow at 02:00 indicates the point at which no further activity could be recorded in CLA mice.

Exercise energy expenditure Between 12:00 and 13:00, the mice were exercised on a metabolic treadmill (10% slope) at a moderate intensity (exercise period). The treadmill was placed beside the cage and linked to the same open-circuit indirect calorimetry system. After a 2-minute warm-up period, the speed of the treadmill was adjusted to 12 m·min21 (50 – 60% of

the VO2max of the mice). The moderate intensity of the run was chosen to create a workload during which lipids would participate for about 50% in the energy requirements of the run (Bouthegourd et al., 2002; Brooks & Mercier, 1994). The three animals were placed in the same treadmill line and exercised together. The airflow through the treadmill was adjusted to 1 l/min to take into account the increased oxygen consumption during the run. Respiratory exchanges during

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exercise were recorded as described above. Energy expenditure was analysed during the last 50 min of the run, after the mice had adopted a regular pace. Post-exercise energy expenditure (2) After this exercise, the mice were taken back to the metabolic cage and allowed to rest for 5 h. Post-exercise resting-EE levels were measured between 16:00 and 18:00, i.e. 3 – 5 h after the end of exercise.

described (Even, Rolland, Roseau, Bouthegourd, & Tome´, 2001). The liver triaglycerol (TG) content was assayed on liver portions (250 mg) ground with a turrax in 4 ml isopropanol. The extract was then heated for 90 min at 60 8C in teflon screw-capped tubes and precipitated protein was pelleted by low-speed centrifugation. The protein pellet was reextracted the same way and the isopropanol phases were pooled. Triacylglycerol in the isopropanol extract was assessed using an enzymatic kit (Bio-Me´rieux, MarcyL’e´toile, France).

Thermic effect of feeding Statistics At 18:00, a food cup containing a calibrated test meal (1.5 g of the usual dry diet given in three 0.5 g portions) was placed in the cage. The TEF was computed as the excess energy expenditure arising from the increase in resting metabolism induced by the meal (18:00 –21:00).

The results are presented as means ^ SEM. Comparisons were made using a bilateral t-test for unpaired samples. Differences were considered significant for P , 0:05:

Post-TEF (3) and fasting metabolism (4)

Results

Post-TEF resting energy expenditure was measured over a 3-hour period after the end of TEF (21:00– 00:00). The fasting metabolism was measured for the last 3 h of the study (04:00 – 07:00). At this time, CLA-treated mice were in a torpid state.

Body weight and body composition

Blood sampling procedures Fifteen mice not used for respiratory measurements were deeply anaesthetized with Pentobarbital (4.8 mg/kg) (Sanofi Sante´, France) immediately after the treadmill run. Blood samples (200 ml) were drawn by cardiac puncture < 2 min after anaesthesia. The plasma was immediately separated by centrifugation (4 8C, 20 min, 3000g) and stored at 2 80 8C for the further determination of glycaemia, insulin and leptin levels. Glycaemia was measured using the glucose oxidase method with the Glucose Biochem kit (Chimie Clinique Biochem, Rungis, France). Plasma levels of insulin and leptin were determined by radio-immuno assay (sensitive rat insulin RIA kit, Linco St Charles, NO, USA, Leptin multispecies RIA kit, Linco, St Charles, NO, USA). The intra and inter specific assays coefficient were, respectively, 4.75 and 7.4% for the insulin kit and 3.2 and 7.75% for the leptin kit. The adequacy of the kits to mice had been tested prior to the experimental assays.

Table 1 shows the body composition data for the two groups after 2 and 6 weeks of feeding. Despite no differences in food intake between the two groups, body weight was slightly reduced in CLA mice after 2 weeks of treatment, but carcass (defined by bones and muscles) and liver weights did not differ. As expected, the white adipose Table 1 Body composition of control mice and mice fed a 1% CLA-augmented semi-purified diet for 2 and 6 weeks Control ðnÞ

CLA ðnÞ

Body weight (g)

2 weeks 6 weeks

22.7 ^ 0.8 (8) 22.9 ^ 0.4 (18)

20.9 ^ 0.8 (7) 23.0 ^ 0.3 (16)

Carcass (g)

2 weeks 6 weeks

11.1 ^ 0.4 (8) 11.3 ^ 0.2 (18)

10.7 ^ 0.3 (7) 11.9 ^ 0.2 (16)

Liver (mg)

2 weeks 902.4 ^ 51.8 (8) 6 weeks 915.2 ^ 20.9 (18)

1046.7 ^ 54.8 (7) 1147.2 ^ 50.1* (16)

WAT (mg)

2 weeks 897.3 ^ 143.7 (8) 6 weeks 906.6 ^ 147.0 (18)

151.0 ^ 20.0* (7) 126.2 ^ 15.0* (16)

IBAT (mg)

2 weeks 6 weeks

69.3 ^ 9.2 (8) 51.2 ^ 5.9 (18)

14.6 ^ 10.3* (7) 1.2 ^ 0.9* (16)

Glycemia (mmol/l) 2 weeks 6 weeks

7.9 ^ 0.3 (8) 9.0 ^ 0.5 (18)

7.0 ^ 0.5 (7) 9.2 ^ 0.7 (16)

Body composition

Insulin (ng/ml)

2 weeks 6 weeks

0.21 ^ 0.03 (8) 0.47 ^ 0.06 (11)

0.17 ^ 0.03 (7) 0.51 ^ 0.05 (11)

Body composition was measured on the 15 mice not devoted to energy expenditure measurement by dissecting and weighing the principal organs (heart, liver, kidney, stomach, intestine, lungs, spleen) and regional adipose depots (epididymal, retroperitoneal, inguinal and other subcutaneous and brown interscapular depots), as previously

Leptin (ng/ml)

2 weeks 6 weeks

0.46 ^ 0.21 (2) 1.58 ^ 0.31 (12)

0.57 ^ 0.07 (4) 0.94 ^ 0.15(12)

Liver TG (mg/g)

2 weeks 6 weeks

24.7 ^ 4.9 (6) 21.3 ^ 8.5 (6)

7.1 ^ 3.1* (6) 8.1 ^ 2.6 (5)

Parameters are expressed as mean values ^ SEM. (*) P , 0:05 CLA versus control.

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tissue mass was markedly reduced in the CLA group. The quantities of brown interscapular adipose tissue were also significantly smaller. The lack of adipose stores was mirrored in the reduced liver triacylglycerol content of CLA-treated mice. Because of the low TG content in the liver of the CLAtreated mice, body composition of mice CLA-treated during six weeks but that did not exercised was added for the sake of verification that the low TG content of the liver was not due to a specific utilization of liver TG in the CLA-treated mice (Table 1). This analysis showed that the weight of the liver, carcass and WAT did not vary much between the 2 and 6 weeks treated mice, and confirmed the low TG content of the liver. Energy expenditure Fig. 1 outlines the changes to EE, glucose and lipid oxidation, corrected for the modifications induced by spontaneous activity in control and CLA mice. The comparisons made during key periods of the study are reported in Table 2. After partial overnight food restriction, both postabsorptive resting-EE, Gox and Lox were similar in CLA and control mice (11:00 to 12:00). The exercise-induced increase in EE values over pre-exercise baseline metabolism did not differ significantly between Control and CLA mice ðP ¼ 0:11Þ: Separate computation of the exercise-induced

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increases in Gox and Lox showed that Lox rose less in CLA than in control mice (Table 2). However, during exercise, Lox still accounted for 39 ^ 4% of total energy expenditure in CLA mice, showing that these animals were still able to mobilize significant quantities of lipids in response to increased energy requirements during exercise. During the post-exercise pre-meal period (16:00 to 18:00), resting-EE levels fell progressively in CLA treated mice, so that average values over the 3 h preceding the test meal were significantly lower than in control mice. This resulted mainly from a tendency for Gox to be lower in CLA treated mice ðP ¼ 0:11Þ: Ingestion of the test meal induced a thermogenic response (18:00 to 21:00) that was very similar in both groups. TEF was paralleled by a marked increase in Gox and a clear inhibition of Lox in both groups. However, meal-induced changes in Gox and Lox were of greater amplitude in CT than in CLA mice. After TEF (21:00 to 00:00), the reduction in Gox in CT mice was fully corrected by a parallel increase in Lox, so that resting metabolism stabilized at 0.43 W, i.e. the same value as that recorded during post-exercise periods. In contrast, post-prandial Lox remained low in CLA mice, what induced a decrease in resting-EE levels. This decrease slowed down for a while because of a slower decline in Gox, but as Lox gradually fell, resting-EE levels fell in parallel. Ten hours after the end of the meal (04:00 to 07:00), resting-EE levels were reduced to 0.10 W, i.e. down to 29% of the resting-EE value in control mice. The amplitude of the reduction in resting-EE,

Table 2 Parameters of energy metabolism during the principal periods of recording sessions Control

CLA

P

Energy metabolism Post-absorptive EE (W) d-EE-exercise (W) Post-exercise EE (W) TEF (kJ) Post-TEF EE (W) Fasting EE (W)

0.50 ^ 0.01 (5) 1.01 ^ 0.05 (5) 0.41 ^ 0.02 (5) 1.89 ^ 0.29 (5) 0.43 ^ 0.05 (5) 0.34 ^ 0.04 (5)

0.53 ^ 0.03 (5) 0.85 ^ 0.07 (5) 0.32 ^ 0.03 (4) 1.84 ^ 0.30 (4) 0.31 ^ 0.02 (4) 0.10 ^ 0.02 (4)

0.31 0.11 ,0.04 0.92 0.06 ,0.01

Glucose oxidation Post-absorptive Gox (W) d-Gox-exercise (W) Post-exercise Gox (W) d-Gox-TEF (W) Post-TEF Gox (W) Fasting Gox (W)

0.06 ^ 0.04 (5) 0.47 ^ 0.04 (5) 0.16 ^ 0.05 (5) 0.51 ^ 0.02 (5) 0.02 ^ 0.04 (5) 0.07 ^ 0.04 (5)

0.06 ^ 0.02 (5) 0.51 ^ 0.03 (5) 0.05 ^ 0.03 (4) 0.39 ^ 0.03 (4) 0.12 ^ 0.03 (4) 0.03 ^ 0.02 (4)

0.95 0.53 0.10 ,0.01 0.10 0.52

Lipid oxidation Post-absorptive Lox (W) d-Lox-exercise (W) Lox ratio-exercise (%) Post-exercise Lox (W) d-Lox-TEF (W) Post-TEF Lox (W) Fasting Lox (W)

0.44 ^ 0.05 (5) 0.54 ^ 0.05 (5) 53.3 ^ 3.9 (5) 0.24 ^ 0.06 (5) 20.33 ^ 0.02 (5) 0.41 ^ 0.05 (5) 0.28 ^ 0.01 (5)

0.47 ^ 0.02 (5) 0.35 ^ 0.07 (5) 39.3 ^ 4.4 (5) 0.28 ^ 0.04 (4) 20.20 ^ 0.06 (4) 0.19 ^ 0.02 (4) 0.06 ^ 0.01 (4)

0.58 ,0.04 ,0.05 0.70 .0.05 ,0.01 ,0.00

Data are expressed as mean values ^ SEM for the three mice in the metabolic cage, with n ¼ number of groups of three mice. See Section 2.2 for a definition of the different periods. EE: energy expenditure, Gox: glucose oxidation, and Lox: lipid oxidation; d-EE-, d-Gox-, d-Lox-: increase in EE, Gox and Lox above baseline during the quoted period.

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together with the fact that the mice were completely inactive (data not shown), suggested that they were in deep torpor (Geiser, Kortner, & Schmidt, 1998; Heldmaier et al., 1999; Snyder & Nestler, 1990). This state lasted until the end of metabolic measurements. When the mice were returned to their home cage with free access to food, they rapidly recovered from this torpid state. Blood sampling Blood samples were collected within 5 min of the end of exercise (Table 1). Plasma glucose levels were not significantly affected by CLA treatment (7.86 ^ 0.28 in controls and 7.33 ^ 0.51 mmol/l in the CLA group). Plasma insulin levels were also similar (0.21 ^ 0.03 in controls and 0.17 ^ 0.02 ng/ml in the CLA group). Leptin levels were very low in all groups (0.455 ^ 0.21 ng/ml in control and 0.565 ^ 0.07 ng/ml in CLA-treated), and consequently could only be assayed in two of the control mice and four of the CLA-treated mice. Because of these low insulin and leptin values in both the control and CLA-treated mice, leptin and insulin were assayed again in non-exercised Control and CLA-treated mice after 6 weeks of treatment. These assays revealed that exercise indeed induced a decrease in blood insulin and leptin in both groups, but the values after 6 weeks of treatment were not significantly different between the control and CLA-treated mice (Table 1).

Discussion Fast-induced torpor has previously been described in AZIP/F-1 mice, a transgenic model containing very reduced adipose tissue levels (Gavrilova et al., 1999). Whether this phenomenon is due to their genetic defects or to their very low lipid stores is still not clear. However, the present observation shows that an 80% reduction in lipid stores by CLA treatment in C57Bl/6 mice was able to reproduce the fast-induced torpor in the same way as in A-ZIP/F-1 mice. This suggests that fat depletion may be the main factor responsible for entry into torpor in both cases. The CLA treatment applied to C57Bl/6mice over a 15day period induced a dramatic reduction in the quantities of adipose tissue, which fell to 16.8% of control values. In parallel, IBAT levels were reduced to 21% of those seen in controls. These characteristics did not change much after six weeks of treatment. Leptin and insulin levels measured at two weeks and after exercise were low in both CT and CLAtreated mice and not different between the two groups, but the analysis done on the non-exercised mice after six weeks of treatment indicated that exercise per se was not responsible for the normalization of the insulin and leptin values between the CLA-treated and control mice. On the other hand, fat-depleted CLA-treated mice differed from AZIP/F-1 mice in several respects (Moitra et al., 1998): there was no clear increase in body weight or organ size, and the

liver TG content, instead of being increased, was also significantly reduced to 28.7% of control values. This low level of liver TG is also in contradiction with what was observed elsewhere after CLA treatments (Tsuboyama-Kasaoka et al., 2000; Ohnuki et al., 2001). Nevertheless, it cannot be attributed to the previous exercise bout because exercise increased lipid oxidation by 0.35 W during 1 h in the CLA mice, which can account for the mobilization of 33 mg of TG. Therefore, even if all the TG were released from the liver, pre-exercise liver TG was at most 40 mg, i.e. only slightly larger than in the control mice. In addition, liver TG was also very low in the mice after 6 weeks of treatment without any exercise prior the sacrifice. Since liver size was not reduced in CLA-treated mice, in fact it was significantly larger at 6 weeks, it can be suggested that the CLA-treated mice may have adapted to a shortage in lipid reserves by increasing their liver glycogen levels. However, because we did not perform any direct measurement of the liver glycogen, and because others reported no changes in liver glycogen content, but in another mouse strain (Std ddY) (Ohnuki et al., 2001), this point remains to be demonstrated. During this study, the signal(s) inducing torpor in CLA mice was obviously latent throughout the periods of measurement. Indeed, resting-EE levels already demonstrated a significant fall between the end of exercise and presentation of the test meal indicating that the CLA-treated mice responded to the energetic challenge of the exercise by decreasing progressively their metabolic rate during the post-exercise period. After a transient restoration of restingEE values, fuelled by the ingestion of the test-meal and the resulting thermogenic effect of the meal, resting-EE then rapidly decreased again in the CLA-treated mice until these mice entered in torpor. This observation is particularly relevant since it is usually difficult to induce torpor in mice, unless both Gox and Lox are markedly inhibited at the same time (Stamper and Dark, 1997; Stamper et al., 1998). On the other hand, the fall in resting-EE levels after exercise and then after the test meal was not accompanied by the same changes in substrate oxidation. Gox was specifically inhibited during the post-exercise period. In contrast, the decrease in resting-EE levels during the post-meal period, and entry into torpor, were entirely due to a decrease in Lox. This qualitative difference between the two periods was probably due to the long-lasting increase in lipid oxidation induced by exercise (Even et al, 1998). Despite their reduced lipid stores, CLA mice were apparently unable to antagonize this stimulation and saved energy by reducing Gox. In contrast, after exercise-induced lipid oxidation was switched off by the ingestion of the test-meal, CLA-treated mice did not increase Lox during fasting as did control mice, but rather maintained a low Lox level and decreased their resting energy expenditure until torpor. This response, in line with the similitude of response to A-ZIP/F-1 mice, suggests that the main aim of this metabolic adaptation was to spare lipid stores.

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In our opinion, a specific effect of CLAs is unlikely. CLA effects are known to be mediated, at least partially, by nuclear factors involved in the control of lipid homeostasis, such as those which regulate numerous genes involved in adipocyte metabolism (Peters et al., 2001; Roche et al., 2002; Tsuboyama-Kasaoka et al., 2000). In general, however, they have been reported to enhance energy expenditure and triacylglycerol utilization rather than to induce energy sparing (Park et al., 1997; West et al., 1998; Pariza et al., 1996). Moreover, during this study, CLAtreated mice did not enter torpor as long as they were fed ad libitum, and similar measurements of respiratory exchanges performed in mice treated with CLA for only 2 days, and therefore not yet fat-depleted, did not reveal that in this state, mice responded to fasting by entering torpor (data not shown). It has been suggested that low leptin levels initiate and/or facilitate entry into torpor. Indeed, leptin increases the energy expenditure of food-restricted mice (Doring et al., 1998) and of marsupial mammals displaying daily torpor and lacking thermogenic, active, brown adipose tissue (Geiser, Kortner, & Schmidt, 1998). Moreover, leptin supplementation prevents torpor in ob/ob mice with low leptin levels and massive triacylglycerol stores. However, leptin supplementation in A-ZIP/F-1 mice is not able to prevent entry into torpor induced by food restriction (Gavrilova et al., 1999). During the present study, the plasma leptin levels were very low when compared to control values (Tsuboyama-Kasaoka et al., 2000), and similar to those reported in A-ZIP/F-1 mice (Moitra et al., 1998) but exercise per se was partly responsible for these low values. This was true in both control and CLA-treated mice, but only CLA-treated mice entered torpor, thus suggesting that a low leptin level is not a sufficient signal alone to induce torpor. The same probably also applies to insulin levels which were not different between control and CLA-treated mice. However, the metabolic response to the test-meal showed that Gox was increased, and Lox reduced, more markedly in control than in CLA mice. This suggests that either control mice were more sensitive to insulin or that the meal-induced insulin release was greater in control than in CLA mice. In both cases, this observation indicates that elevated insulin levels or an increased antilipolytic response to insulin was not responsible for the reduced lipolysis and subsequent torpor in CLA mice. Other mediators secreted by the adipose tissue can also be involved. A non-exhaustive list can include, for instance (i) perilipins, the most abundant proteins at the surface of lipid droplets in adipocytes that are known to play a major role in regulating triacylglycerol storage and lipolysis (Brasaemle et al., 2000), (ii) adiponectine that is decreased in obesity and has been suggested to increase muscle fatty acid b-oxidation or on the contrary, (iii) resistin that is increased in several rodent models of obesity or (iv) some still undiscovered protein (Guerre-Millo, 2002).

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In conclusion, entry into torpor in the fasted mouse seems to centre on a reduction in lipid oxidation in response to a depletion of their lipid stores. Both the A-ZIP/F-1 mouse and the CLA-induced fat-depleted mouse may thus be interesting models offering a physiological context of in vivo inhibition of lipid oxidation at the whole body level. Analysis in this framework of the status of the numerous molecules released by the adipose tissues may help in discriminating those that can induce significant changes in energy homeostasis at the whole body level, and by extrapolation may be the primary actors regulating body fat.

Acknowledgements This work was partly funded by laboratories Orsinia (Paris, France).

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