Effects of periodic intake of a high-caloric diet on body mass and leptin resistance

Physiology & Behavior 88 (2006) 191 – 200

Effects of periodic intake of a high-caloric diet on body mass and leptin resistance Mauricio Berriel Díaz ⁎, Sandra Eiden, Carolin Daniel, Alexandra Steinbrück, Ingrid Schmidt Max-Planck-Institut fuer Herz- und Lungenforschung, W.G. Kerckhoff-Institut, Parkstr. 1, D-61231 Bad Nauheim, Germany Received 22 July 2005; received in revised form 13 March 2006; accepted 29 March 2006

Abstract The effects of continuous or intermittent access to a high-caloric (HC) diet, always offered in addition to standard chow, on body mass and leptin resistance were analyzed in female C57BL/6J mice. Susceptibility for diet-induced obesity (DIO) was apparent from the marked preference for the HC diet. Continuous HC diet feeding of mice at 4 weeks of age induced leptin resistance within 2 weeks and massive gains in body mass, although with increasing inter-individual variability in the inbred strain considered to be isogenic. In adult mice receiving HC diet for the first time, leptin treatment failed to reduce energy intake first after 11 days of HC diet feeding, but became effective again within 3 days after HC diet withdrawal. In mice with a history of several preceding periods of access to the HC diet totalling N 30 days, supplementary HC diet abolished the anorectic effect of leptin treatment within only 3 days and it reappeared not earlier than 11 days after HC diet withdrawal. Thus, in the investigated DIO-prone mouse strain both, the loss of responsiveness to leptin under HC diet and its recovery after HC diet withdrawal strongly depended on the dietary history. Recovery from leptin resistance during periods of intermittent chow feeding was associated with losses of body mass that did not completely compensate for the obesity-inducing effect of the preceding HC diet. © 2006 Elsevier Inc. All rights reserved. Keywords: Energy intake; Dietary history; C57BL/6J mice; Diet-induced obesity

1. Introduction The incidence of obesity has increased dramatically, representing a worldwide public health problem [1,2]. Investigations of mutant and transgenic mouse models led to the identification of a variety of genetic factors involved in the regulation of energy homeostasis and, thus, being crucial for the development of obesity [3]. However, the rapidly increased prevalence of obesity emphasizes the importance of environmental factors, e.g. energy content and composition of food, for the development of obesity [4,5]. In this regard, the analysis of diet-induced obesity (DIO) in laboratory rodents receives increasing attention, as it more closely resembles the pathophysiological situation in most cases of human obesity and metabolic syndrome [6]. Mouse strains differ in their susceptibility for diet-induced obesity [7], and in their preference for the different macronutrient components [8]. The C57BL/6J mouse strain has been described to ⁎ Corresponding author. Tel.: +49 6221 423593; fax: +49 6221 423595. E-mail address: [email protected] (M. Berriel Díaz). 0031-9384/$ - see front matter © 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.physbeh.2006.03.028

be highly susceptible for diet-induced obesity and diabetes [7,9]. Like other mouse strains sensitive for dietary obesity, these mice are further characterized by a marked preference for dietary fat [8]. In the present study, C57BL/6J mice were used as a model for the analysis of the influence of diet composition and feeding patterns on the efficiency of leptin to regulate food intake and body mass. Leptin is primarily produced and secreted by adipocytes and represents an afferent endocrine signal providing information to the brain about the energy status of the body. Among other functions, circulating leptin is mainly involved in the regulation of food intake and energy expenditure. As part of a negative-feedback loop system, it acts via the long form of the leptin receptor (LEPRb) on hypothalamic neurons, where it influences expression and release of several neuropeptides involved in the regulation of energy homeostasis [10,11]. In ad libitum fed humans and mice, plasma leptin levels are positively correlated with adipose tissue mass and adipocyte size [12–14]. Despite the constitutive secretion of leptin depending on body fat mass, serum levels can be regulated by various physiological states. Leptin levels fall during fasting, disproportional to the

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change in adipose tissue mass [15,16], supporting the idea that one important function of leptin is to mitigate the consequences of acute energy deficiency. Conversely, circulating leptin increases several hours after eating [15,17]. Peripheral administration of recombinant leptin, i.e. supplied via its normal route of action, reduces food intake, offsets reductions of energy expenditure and slightly decreases body fat content in lean animals [18–21]. However, the action of leptin is impaired in obesity. Generally, obese animals and humans have proportionally higher leptin levels than lean individuals, indicating that obesity is associated with a state of leptin resistance [14,21,22]. Different studies emphasize the importance of chronic access to high-fat diets for the development of leptin resistance in mice and rats susceptible for diet-induced obesity [23–25]. Moreover, there is also evidence that the composition of diets per se directly or indirectly affects the feeding response to peripheral leptin administration. In Osborne– Mendel rats, high-fat diet feeding induced leptin resistance within only 5 days and conversely leptin sensitivity was rapidly restored only 4 h after rats were switched back to a low-fat diet, suggesting an influence of the diet independent of changes in adipose tissue mass [26]. Also, Wang et al. showed that rats fed a highly palatable diet develop a severe resistance to the metabolic effects of both leptin and insulin after only 3 days of voluntary overfeeding [27]. Another study in C57BL/6J mice demonstrated that the initial (after 2 days) suppression of the mRNA expression of the orexigenic neuropeptides NPY and AgRP in response to high-fat feeding is abolished after 1 week on this diet, possibly due to a rapid establishment of leptin resistance that precedes the increase in body mass [28]. However, comparison of various studies is difficult due to the different feeding regimes, diet compositions and leptin treatments applied. Although leptin resistance has been reported in obese animals, the factors important for onset and progression of leptin resistance are not well understood. The present study on C57BL/6J mice combined continuous standard chow feeding with continuous or temporary, repetitive admission to high-caloric diets (HC diets) with the aim to assess the resulting voluntary choices of chow and HC diet intakes and to investigate the influence of that feeding regime on the onset of leptin resistance as well as on the re-establishment of leptin sensitivity. In this context, the effect of age and the dietary history of the animals on leptin responsiveness were taken into consideration. Continuous access to standard rodent chow ensured supply with vitamins and minerals, which was compromised intentionally, though only temporarily, during the periods of additional supply with high-fat chocolate-like food. With this feeding regime it was intended to simulate (a) the “unhealthy” human feeding conditions characterized by voluntary ingestion of highly palatable “cafeteria diet” while having the chance to ingest “healthy” basic mixed diet as well, and (b) intermittent periods of “dieting” by eating only the basic mixed diet.

FRG) at 3 weeks of age or born in our own colony founded from the same breeding stock. Adult animals were maintained individually in cages at 22 °C under a 12:12 light/dark cycle. All procedures were carried out according to the German guidelines for animal experimentation and approved by the local veterinary control institution for animal care and use. 2.2. Diet compositions and food supply All animals had continuous access to a pelleted standard rodent diet (chow, Altromin 1324, Altromin, Lage, FRG) consisting of fat/ carbohydrate/protein, F/C/P =4:53:20%, and water was available ad libitum. The pellets were fixed in a device that collected any crumbs, thereby enabling accurate determination of food intake. Experimental animals were additionally offered a high-caloric (HC) supplement diet, either continuously (1 group) or periodically for 2–4 repeated periods of 11 to 22 days (12 groups). The HC diet was based on commercially available products and consisted of, (a) white chocolate (HC1; F/C/P =35:58:5%), and (b) of a substitution in which cacao butter was replaced by coco fat and sugar was replaced by corn starch (HC2; F/C/P = 36:50:9%). Both HC diets had similar energy contents (∼23 kJ/g), and were offered to the mice as the HC diet. The HC supplement diet was given in containers that avoided spillage. At an early stage of the analysis, pilot experiments of one author (C. Daniels) had demonstrated that palatability of the HC diets was similar when either offered separately or combined, and that both HC diets had identical effects on leptin responsiveness. In view of the identical energy contents of the two diets, there was no necessity to make a difference between the two diets in the analysis of energy balance as the only relevant parameter for the HC diet effects on body mass and body composition, as well in the analysis of leptin responsiveness. Therefore, the results obtained with the two diets were pooled for data evaluation. 2.3. Leptin treatment Leptin was administered as 1-day treatments. On the day of treatment, half of the animals were injected with leptin (R&D Systems GmbH, Wiesbaden, FRG, Mw = 16 kDa) and the other half serving as controls received phosphate-buffered saline (PBS). Based on previous studies [18,29], a leptin dose of 200 pmol g− 1 day− 1 was used in the present experiments. This was divided into two equal doses and injected subcutaneously at the beginning and at the end of the light phase. Compared with a single daily leptin injection, administering the daily dose in two portions had been shown to result in the marked increase of the hormone effect [30,31]. Dose calculations were based on individual body masses on the day of treatment. Repeated treatments within the same experimental group were carried out at intervals of at least 4 days with experimental and control animals usually reversed.

2. Material and methods 2.4. Energy intake and evaluation of leptin response 2.1. Animals Female C57BL/6J mice were used for the present study. The animals were either obtained from Harlan Winkelmann (Borchen,

Energy intake was calculated based on energy contents of 13 kJ/g for the standard rodent chow and 23 kJ/g for the HC diet. Values represent metabolizable energy and were used to account

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for differences between the diets in fiber contents and energy loss due to excretion. Metabolizable energy was calculated based on the calorific values of diet components, considering differences in the energy content of the faeces determined by bomb calorimetry and in the energy loss due to urea excretion. The value for the metabolizable energy of the standard diet (Altromin 1324) was confirmed by another study on C57BL/6J mice [32]. On the day of treatment, average food intake for the preceding 2 days and the current body masses were matched as closely as possible between the experimental group of mice receiving leptin and control animals receiving PBS. The treatment effects were evaluated for the 24-h period starting after the second injection (effect period) as former studies revealed that the main anorectic effect of the leptin dose used in this study is restricted to that period [31]. The deviation of the mean energy intake of leptin treated animals from control animals, whose energy intake was set to zero, was used as the measure for leptin responsiveness. The 2 days preceding the treatment day and the 2 days following the effect day served as control days to account for the minor chance of differences between the individual and its control mean. The difference between the average deviation during the control days and the deviation in the effect period was calculated to obtain the individual net treatment effect. 2.5. Determination of body composition and plasma hormone concentrations For final body composition analysis, mice were anesthetized by exposure to CO2 for 30 s and then decapitated. After removing the gastrointestinal tract, body composition–water, fat, and fat-free drymass (FFDM)–was evaluated by drying the carcasses to constant weight and extracting the fat using chloroform in a Soxhlet apparatus. Blood was collected either by retro-orbital puncture or by decapitation and plasma was analyzed with commercially available radioimmunoassay (RIA) kits for mouse leptin (Linco Research Inc., USA) and human insulin (Biochem ImmunoSystems, FRG). Binding capacities of the polyclonal antibodies to mouse leptin or insulin, respectively, were determined by using mouse standards. Plasma measurements were independently duplicated and variability was decreased by correcting the data for inter-assay-variability and buffer dilution using internal correction factors.

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mass and body mass of older animals), one-way ANOVA on ranks was performed. For the evaluation of the anorectic leptin effect on the single treatment days, one-way ANOVA with “treatment” as factor was used. For the groups with periodic access to the HC diet in addition to chow, one-way ANOVA with “day” as factor was used to compare plasma leptin levels during the successive periods of HC diet feeding. Two-way ANOVA with “treatment” and “day on the respective diet” as factors served to test treatment effects within the groups of mice either receiving the HC diet for the first time or having had experienced previous periods of HC diet feeding (N 30 days). 3. Results 3.1. Food intake, body mass and leptin responsiveness depending on the age of mice supplied with standard rodent chow The development of individual body mass of female C57BL/6J mice between 4 and 38 weeks of age as well as the effect of repeated 1-day leptin treatments on energy intake is shown in Fig. 1. With increasing age, chow-fed mice displayed a flat increase in body mass (0.35 g/week) with only small variability between individuals. Until the age of about 20 weeks, this increase mainly reflects a rise in fat-free dry mass (FFDM), rather than the accumulation of fat that starts at older ages (unpublished population data, I. Schmidt). While daily energy intake was slightly higher between 4 and 8 weeks of age (58 ± 1 kJ/day), probably due to adolescence,

2.6. Statistical analysis All analyses were performed with SigmaStat (SPSS Corporation, USA) software. Data are presented as means ± standard errors of the mean (S.E.M.). P values b 0.05 were considered as significant. For the 2 experimental groups continuously supplied with either chow or chow +HC diet from the age of 4 weeks onward, one-way ANOVA with “experimental group” as factor was used for statistical comparison of body mass growth, final body composition and plasma hormone concentrations at the end of the experiment. The Hartley test for heterogeneity of variance [33] was applied to analyze changes in the development of inter-individual variability within the two groups. If data distribution differed in variances (fat

Fig. 1. Body mass and leptin responsiveness depending on age. (A) Development of individual body mass and (B) leptin responsiveness of female C57BL/6J mice supplied with chow only from 4 to 38 weeks of age (N = 10). The effects of subcutaneous 1-day leptin injections (two doses of 100 pmol g− 1 day− 1) given at regular intervals during the treatment period are shown by the average differences in 24-h post-treatment energy intake (black columns, means ± S.E.M.) between leptin treated (N = 5) and control animals (zero line, N = 5). ⁎P b 0.05; ⁎⁎P b 0.01; ⁎⁎⁎P b 0.001.

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average energy intake between 8 and 38 weeks of age remained stable being 54± 2 kJ/day. Leptin treatment of these chow-fed mice resulted in significant reductions in energy intake compared to control animals from 5 to 26 weeks of age (Fig. 1B), the only exception was at 14 weeks of age when the leptin-induced decrease in energy intake just missed significance (P = 0.07). The leptin dose used in the present study (200 pmol g− 1 day− 1) provoked a decrease in daily food intake by 17–24%, thus, exhibiting a distinct but submaximal anorectic effect. However, from 29 to 38 weeks of age, treatment with the same mass-specific leptin dose failed to significantly reduce energy intake demonstrating age-related leptin resistance. At the end of the experiment, average plasma hormone concentrations were still within the normal range with a leptin level of 6.8± 0.7 ng/ml and an insulin concentration of 22± 3 μU/ml. 3.2. Food intake, body mass and leptin responsiveness of mice additionally supplied with the HC diet At the beginning of the experiment at 4 weeks of age, the group of female C57BL/6J mice on HC diet possessed a similar average body mass as the control group supplied with chow only (Fig. 2A: 12.2 ± 0.4 g vs. Fig. 1A: 11.8 ± 0.4 g, both N =10). Continuous free access to the HC diet (both HC diets combined) additionally to chow provoked massive body mass gain (1.35 g/week), leading to a

Fig. 2. Body mass and leptin responsiveness depending on age and continuous HC1 + HC2 diet feeding. (A) Development of individual body mass, and (B) leptin responsiveness of female C57BL/6J mice supplied with HC diet in addition to chow from 4 to 25 weeks of age (N = 10). The effects of subcutaneous 1-day leptin injections (two doses of 100 pmol g− 1 day− 1) given at regular intervals during the treatment period are shown by the average differences in 24-h post-treatment energy intake (black columns, means ± S.E.M.) between leptin-treated (N = 5) and control animals (zero line, N = 5). ⁎P b 0.05; ⁎⁎P b 0.01.

significant difference in body mass as compared to chow-fed mice after only 2 weeks on HC diet at the age of 6 weeks (Fig. 2A: 18.0 ± 0.4 g vs. Fig. 1A: 14.7 ± 0.3 g, P b 0.001). At the age of 25 weeks, HC diet-fed mice had developed pronounced diet-induced obesity with nearly double the body mass of chow-fed mice at the same age (Fig. 2A: 40.4 ± 1.7 g vs. Fig. 1A: 21.8 ± 0.3 g, P b 0.001). Determination of final body composition confirmed that body mass gain is mainly due to an increase in body fat mass. Compared to the 38-week-old chow-fed mice (Fig. 1), body fat mass had nearly quadrupled (18.1± 0.9 g vs. 4.7± 0.4 g, P b 0.001) while FFDM tended to be slightly, but not significantly higher in mice with access to HC diet (4.9± 0.1 g vs. 4.5 ± 0.1 g). Note the more than 5-fold larger S.E.M. for the mean body mass of the HC diet-fed group at the age of 25 weeks, indicating an unproportional increase in body mass variability within the HC diet-fed group. At a comparable body mass level (Fig. 1A; chow: 24.3 ± 0.6 g at 38 weeks of age vs. Fig. 2A; HC + chow: 25.0 ± 1.2 g at 12 weeks of age), the variability in body mass of HC diet-fed mice was twice that for the chow-fed mice. Statistical analysis revealed that the variances within these two groups matched for body mass differ significantly (P b 0.05, N = 10, Hartley-test for heterogeneity of variance [33]). In contrast to other studies in which high-caloric diets often were offered exclusively, in the present study the intake of the diets with a higher caloric density and fat content was always the result of free choice. Comparison of the two HC diets showed that there was a slight age-dependent difference in preference between the HC diets containing starch or sucrose, being 60:40 in the age period from 4 to 12–16 weeks but switching to 40:60 after that age. However, daily energy intakes of the mice did not systematically change with increasing age being on the average 58 ± 2 kJ/day. Throughout the whole experiment, 90 ± 1% of the ingested calories originated from the intake of the two HC diets, demonstrating a marked preference for the HC diet as compared to chow. Compared to mice receiving only chow, energy intake of the mice with access to the HC diet was only slightly higher by 4 kJ/day (6%), on the average. However, this small daily difference would explain their excessive body weight gain, as it adds up to an energy accumulation of 588 kJ during the period from 4 to 25 weeks of age. Assuming an energy content of 39 kJ/g fat, this excess energy would correspond to a deposition of about 15 g pure fat, as FFDM is only insignificantly changed by the HC diet feeding (see above). Considering the ∼30% water content of adipose tissue, the resulting mass difference of about 20 g is close to the average mass difference of 18.6 g found at 25 weeks of age between mice on chow only and those receiving chow + HC diet. The course of leptin treatment effects in the mice having continuously access to both chow and HC diet (Fig. 2B) differed distinctly from that of the mice receiving only chow (Fig. 1B). Initially the 1-day leptin treatment reduced both, chow and HC diet intake causing a significant decrease in total energy intake. The differences of −10 and −8.7 kJ between leptin-treated and control mice were comparable to those found in chow-fed mice of the same age. However, the third leptin treatment after only 2 weeks of HC diet feeding and all subsequent leptin applications were no longer effective (Fig. 2B) indicating early onset of leptin resistance, due to access to HC diet, in contrast to the persisting leptin responsiveness

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of chow-fed mice during this period of time. It should be noted that leptin doses were adjusted to body mass. Thus, leptin resistance was maintained in the mice on supplementary HC diet, although the absolute amounts of leptin administered to them had approximately doubled, relative to the amounts applied to the chow-fed mice, at 25 weeks of age. Not shown in Fig. 2 are the final levels of plasma leptin (30.3 ± 2.1 ng/ml) and insulin (211 ± 80 μU/ml) which clearly exceeded the levels reported for normal chow-fed mice (P b 0.05).

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significantly reduce energy intake, but the anorectic leptin effect was clearly diminished from day 11 onward (lack of leptin responsiveness on day 15 not shown). However, leptin responsiveness was quickly re-established once the HC diet had been removed, as indicated by the significant reduction of energy intake by leptin treatment after only 3 days on chow again

3.3. Body mass and leptin responsiveness during periodic HC diet feeding Periodic access to HC diets led to alternating periods of body mass gain while HC diet was offered and subsequent compensatory down-regulation of body mass during chow feeding (Fig. 3A). As for the group with continuous access to HC diet, the alternating feeding regime revealed marked individual differences in the predisposition for body mass gain during HC diet feeding, as well as for the ability for compensatory body mass loss during periods of exclusive chow feeding. At the end of the last period of supplementary HC diet feeding at an average body mass of 27.8± 4.2 g (Fig. 3A; week 31, N =10) the variance corresponds to that found for the same average body mass (27.7± 4.5 g) at week 14 in the group continuously fed the HC diet (Fig. 2A). Not only body mass but also endogenous plasma leptin concentrations, determined on day 7 of each HC diet period, increased during the experiment, being 4.2 ± 0.4 ng/ml during the first, 5.7 ± 0.8 ng/ml during the second, 12.5± 1.7 ng/ml during the third, and 20.5 ± 2.8 ng/ml during the fourth period of additional HC diet feeding (Pb 0.05). However, only 3 days of exclusive chow feeding after the last HC feeding period distinctly decreased plasma leptin by 50% (10.0± 1.3 ng/ml). Determination of food intake revealed that daily energy intake during periods of additional HC diet feeding slightly increased during the experiment due to increases in both, HC diet and chow intake, being 54 ± 2 kJ/day during the first and 62 ± 3 kJ/day during the fourth HC diet period. This resulted in an increasing weight gain from 0.5 ±0.2 g/week during the first to 1.4 ± 0.4 g/week during the last period of additional HC diet supply (P b 0.05). Intake of the HC diet always accounted for 91 ± 1% of the total energy intake. Removal of the HC diet caused a pronounced increase in chow intake. However, average energy intake of 46 ± 2 kJ/day during the periods of exclusive chow feeding was obviously not sufficient to maintain the increased body mass. The effect of 1-day leptin treatments on energy intake was determined in a total of 9 groups of adult mice (age N 10 weeks) either receiving HC diet for the first time (Fig. 3B; naïve to HC diet) or after having experienced 2–3 episodes of previous HC diet feeding with a total number of 30–43 days on HC diet (Fig. 3C). Leptin responsiveness during the preceding chow period was used as reference. In mice naïve to HC diet feeding (Fig. 3B), leptin administration significantly reduced daily energy intake in comparison to control mice during the initial period when only chow was available. After giving the mice access to the HC diet, leptin treatments on days 3 and 7 of the HC period were still able to

Fig. 3. Body mass and leptin responsiveness during periodic access to a highcaloric (HC) diet. (A) Development of individual body mass of one group of female C57BL/6J mice during periods of either HC1 or HC2 diet feeding (gray bars; first and second period: 11 days; third period: 17 days; fourth period: 21 days) intermitted by periods of chow only feeding (first and second: 9 days; third: 16 days). (B, C) Changes in leptin responsiveness of adult female C57BL/ 6J mice during alternating diet regimes. HC1 or HC2 diet was either offered for the first time (B, 8 experimental groups) or after the mice had previously experienced the HC diet during 2–3 periods with a total duration of N30 days (C, 4 experimental groups). The effects of subcutaneous 1-day leptin injections (two doses of 100 pmol g− 1 day− 1) applied on individual days before, during and after the period of HC diet supply are shown by the average differences in 24-h post-treatment energy intake (black columns, means ± S.E.M.) between leptintreated and control animals (zero line). Each column represents a number of 8– 30 mice from 2 to 6 experimental groups. ⁎P b 0.05; ⁎⁎P b 0.01; ⁎⁎⁎P b 0.001.

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and to a degree not different from that during the period prior to the HC diet feeding (Fig. 3B). Mice having had temporary access to HC diet in addition to chow, separated by periods of chow only (comparable to Fig. 3A), were re-tested for leptin responsiveness after 2–3 periods summing up to N30 days of cumulative HC diet feeding (Fig. 3C). Under these pre-conditions, only 3 days of HC diet feeding were sufficient now to abolish the anorectic effect of leptin, although the mice had been clearly responsive during the preceding chow period (leptin injection after 11–15 days on chow only). Furthermore, and in contrast to mice with a single exposure to the supplementary HC diet, removing the HC diet in these mice with repeated exposures to the HC diet was followed by much slower recovery of their leptin responsiveness. On the average, reduction of energy intake in response to leptin treatment became significant first on day 11 after removal of the HC diet (Fig. 3C). Note that on the first leptin treatment day after HC diet removal (day 3 on chow only) previously naïve mice and mice with previous access to the HC diet differed in body mass (21.3 ± 0.2 g vs. 24.6 ± 0.7 g, P b 0.001), however, the mice with periodic access to the additional HC diet were more obese by just 15%, and the leptin dose had been adjusted accordingly. Moreover, random sampling of the individual responses to leptin within the group of mice with previous access to the HC diet did not reveal a systematic correlation between the latency of recovery of leptin responsiveness and either body mass or endogenous plasma leptin level, which had both increased during later periods of HC diet

Fig 5. Relationship between the increase in body mass during 10-day periods of additional HC1 and/or HC2 diet feeding and the decrease in body during the subsequent 10 days on chow only for individual animals of 3 experimental groups (circles, squares, triangles): y = − 0.77x + 0.46, r = − 0.82, P b 0.0001, N = 70. Dashed line = line of identity.

feeding (see Fig. 3A), although to degrees exhibiting pronounced variability. 3.4. Relationship between body mass and energy intake To further characterize the fluctuations in energy intake and body mass due to the alternating feeding regime, the relationship between these parameters was analyzed with regard to the acute feeding situation. During access to the HC diet, we found a close positive correlation between body mass and daily energy intake (Fig. 4A: r =0.79, P b 0.0001, N = 70). On HC diet, mice with a higher body mass showed higher energy intakes. In contrast, during the intermittent periods of access to chow only, body mass and energy intake were negatively correlated (Fig. 4B: r =−0.44, P b 0.0001, N = 71). Thus, mice with higher body mass and energy intake on HC diet ingested less energy in periods with exclusive access to chow. 3.5. Changes in body mass during the alternating feeding regime

Fig 4. Relationship between body mass and daily energy intake depending on the diet during alternating feeding periods for individual animals of 3 experimental groups (circles, squares, triangles). (A) Correlation between body mass and daily energy intake during additional HC1 and/or HC2 diet feeding: y = 2.75x − 13.3; r = 0.79, P b 0.0001, N = 70. (B) Correlation between body mass and daily energy intake during exclusive chow feeding: y = − 1.49x + 80.4; r = − 0.44, P b 0.0001, N = 71. For body mass and energy intake mean values of days 5–6 of the respective feeding period were used.

In Fig. 5, individual increases in body mass during 10-day periods with free access to HC diet are plotted against the decreases in body mass during the subsequent 10-day periods on chow only. The decrease in body mass is closely correlated to its previous rise (r= −0.82, P b 0.001, N =70). However, the regression line deviates clearly from the line of identity indicating that body mass decreases more slowly during chow feeding than it increases during free access to the HC diet. 4. Discussion Obese humans and mice are characterized by high circulating leptin levels that fail to promote body mass loss due to leptin resistance [12,14,21]. Human obesity in developed countries

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results, as a rule, from high-caloric (HC) and easily digestible food that is highly palatable and ingested as snacks and appetisers in addition to standard diet which by itself would not induce obesity. It was the intention of the present study to imitate this condition in an obesity-prone mouse strain by offering a HC diet for voluntary ingestion in addition to regular chow. For this purpose, C57BL/6J mice were exposed to two feeding schedules with either continuous or intermittent HC diet availability and the effects on body mass and body composition as well as the resulting changes in responsiveness to intermittent leptin administration were compared with those in chow-fed control mice. Access to HC-diet provoked enhanced body mass gain by excess fat deposition and rapidly developing leptin resistance combined with rising leptin levels indicating a disturbed regulation of energy balance. However, withdrawal of the HC diet rapidly decreased endogenous plasma leptin concentrations independent of changes in fat mass and restored leptin sensitivity suggesting a short-term influence of changing the dietary schedule. Moreover, the results showed that the time courses of development and reversal of leptin resistance were not only dependent on the acute availability and removal of the HC diet component but were also distinctly influenced by the animals' preceding dietary history. 4.1. Age dependence of leptin responsiveness Leptin responsiveness was shown to be impaired during lateonset obesity in aged rats, and associated with diminished leptin signal transduction [34,35]. We confirmed age as a factor favoring the development of leptin resistance in C57BL/6J mice. In the group of chow-fed control mice, leptin administration first failed to significantly reduce energy intake at the age of about 30 weeks. Interestingly, at this age their average body mass of 23.7 ± 0.4 g was only slightly higher than their weight of 21.8 ± 0.3 g at the age of 24 weeks when leptin responsiveness was still fully preserved. This strengthens the view that, at least in this mouse strain, age itself affects leptin's effects on energy intake, since onset of leptin resistance was neither associated with pronounced age-related obesity nor with levels of circulating leptin beyond the physiological range. Interestingly, among the nine groups of mice exposed repeatedly to periods of additional HC diet availability, recovery of leptin responsiveness after withdrawal of the HC diet was observed in two groups aged 31 to 34 weeks, i.e., at an age when the control mice maintained on standard chow only had definitively become leptin resistant. This unexpected observation suggests two tentative hypotheses. First, since the test dose of leptin was adjusted to body mass, the amount of leptin per unit FFDM and/or extracellular fluid may have been substantially higher in the obese mice with periodic HC diet feeding than in the lean chow-fed control mice and, thus, still effective. Second, withdrawal of the HC diet, while chow remains available, means a switch to relative starvation, as indicated by body mass loss and pronounced decrease in plasma leptin. This condition is different from that of continuously chow-fed mice which never did experience acutely reduced caloric intake, a resulting weight loss or presumably a drop of plasma leptin. Thus, acute caloric restriction, independent of the diet type, might activate leptin responsiveness, even at an age at which chow diet that is low

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in calories and “healthy”, but available continuously, cannot prevent leptin resistance. 4.2. Preference for the HC diet Continuous access to the HC diet resulted in a marked increase in body mass due to a massive increase in adipose tissue mass. This observation is in agreement with previous work demonstrating that C57BL/6J mice are particularly sensitive to diet-induced obesity [7,9,24]. Free choice of diet revealed a marked preference for the HC diet that accounted for about 90% of total energy intake in C57BL/6J mice. This may be most likely due to the high fat content of the HC diet, according to a study in which free choice of macronutrient diet components revealed that 80% of the energy consumed by C57BL/6J mice derived from fat [8]. Thus, high voluntary fat intake contributes to the susceptibility of certain mouse strains for diet-induced obesity. The two HC diets given in parallel in the present study had similar fat content (∼35%) and equal amounts of carbohydrate though as different components (sucrose vs. starch). Although preference for the 2 diets slightly changed during the long-term experiment, there is no evidence that the two HC diets differentially affect leptin's efficiency to reduce food intake (unpublished data, C. Daniel), confirming the central role of dietary fat, rather than the type of carbohydrates for diet preference and the development of leptin resistance [24,26]. 4.3. Body mass variability increases during HC diet feeding In agreement with previous observations, continuous HC diet feeding significantly increased the inter-individual variability in body mass gain. Individual differences in food intake [36], in the development of hyperleptinemia [37] or in glucose metabolism [38] were considered as possible explanations for the heterogenous body weight gain. The observed changes, however, are not attributable to differing genetic predisposition of individual animals, since C57BL/6J inbred mice are considered to be isogenic [39]. The differences in the susceptibility for diet-induced obesity within an inbred strain indicate the modulation of metabolic and neuroendocrine systems by additional environmental impacts, particularly when experienced during critical periods of ontogeny [5]. For instance, factors like the perinatal nutritional status can influence the phenotypic penetration of a genetic predisposition to a metabolic disorder during adulthood [4]. Whether such kinds of environmental factors are responsible for the observed body mass variability in the present study, in which mice partly were littermates, remains an open question. The findings support the idea that for genetically heterogeneous human populations the penetrance of the obesity phenotype depends on genetic background, potential early malprogramming of regulatory systems [4,5] and environmental factors such as unlimited access to a diet with high energy density and fat content. 4.4. Leptin responsiveness affected by HC diet feeding Development of excessive obesity under continuous feeding of a high-fat, energy-dense diet is known to be associated with rising

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plasma leptin concentrations and leptin resistance, hyperinsulinemia and insulin resistance and with multiple neuroendocrine hypothalamic disturbances, especially in the arcuate nucleus [9,11,14,23]. The present study has provided information about the dynamics of onset and of recovery from such disturbances using responsiveness to leptin as an indicator. Under continuous HC diet feeding, with chow being simultaneously available, resistance to peripheral leptin treatment was evident after less than 2 weeks in juvenile mice at the age of 6 weeks, i.e., when their body mass was found first to significantly exceed that of the chow fed control mice. Similarly, Van Heek et al. found that C57BL/6J mice fed a diet containing 45% of fat were still leptin sensitive after 4 days and developed resistance to the anorectic effects of intraperitoneal (i.p.) leptin treatment after 16 days on a high-fat diet [40]. In another study in rats, resistance to the anorectic effects of leptin was evident after only 5 days of highfat diet feeding [26]. Under intermittent HC diet feeding, with chow being continuously available, the time courses of the disappearance of leptin responsiveness during periods of HC diet supply and of the reappearance of leptin responsiveness during periods of access to chow only in adult mice (age N10 weeks) were shown to be strongly influenced by the dietary history. In adult mice receiving HC diet for the first time (naïve to HC diet) leptin treatment failed to significantly reduce energy intake not earlier than on day 11 of HC diet feeding, thus, the time course of the development of leptin resistance in adult mice was similar to that in juvenile mice given access to the HC diet for the first time. After withdrawal of the HC diet and availability of chow only, leptin responsiveness had recovered 3 days after the change of diet. Restoration of leptin responsiveness may even be faster, as it has been demonstrated in Osborne–Mendel rats, in which leptin responsiveness was first evident 4 h after withdrawal of a high-fat diet [26]. The rapid changes in leptin responsiveness suggest that the acute feeding situation modulates leptin effects on energy intake, independent from changes in body fat content. When the time-courses of development and reversal of leptin resistance were re-tested after several periods of intermittent access to the HC diet totalling more than 30 days, however, onset of leptin resistance occurred much faster, within 3 days. Further, recovery from leptin resistance under chow feeding after the end of the last HC diet period was much slower, taking 11 days, on the average. Although it was observed that leptin responsiveness recovered each time during the intermittent periods of chow feeding, the repeated HC diet-induced occurrences of leptin resistance and/or the increasing fat deposition accelerated the onset and delayed the recovery of leptin resistance. Comparison of animals showing the largest and the smallest increase in body mass after several periods of HC diet feeding did not reveal a systematic difference in their reaction to the exogenously applied leptin, neither concerning the time-course of leptin resistance development during the final HC diet period nor concerning the time course of reappearance of leptin responsiveness during the following final period on chow only. Thus, it is conceivable that periods of HC diet feeding and leptin resistance, even if interrupted by periods of chow feeding, may chronically modulate the functions of the neuroendocrine system involved in the control

of energy balance, and thereby alter its reactions during subsequent periods of excess energy intake. In several studies, the susceptibility for diet-induced obesity and leptin resistance was shown to be associated with the ability to modulate gene expression of orexigenic and anorexigenic neuropeptides in leptin responsive hypothalamic neurons. In contrast to DIO-prone C57BL/6J mice, 2 weeks of high-fat diet feeding in A/J mice, which are less prone to DIO, resulted in an up-regulation of POMC mRNA expression and a tendency for down-regulation of hypothalamic NPY [41]. The authors concluded that such compensatory responses, in particular the increase in POMC gene expression during high-fat diet feeding, are involved in the protection against diet-induced obesity in DIOresistant mouse strains like A/J mice. Such compensatory changes were not observed in C57BL/6J mice and may be characteristic for the susceptibility for DIO [41]. Similarly, Ziotopoulou et al. demonstrated that C57BL/6J mice display a compensatory downregulation of neuropeptide Y (NPY) and agouti-related protein (AgRP) gene expression during the first 2 days of high-fat diet feeding, but the reductions in the levels of these orexigenic neuropeptides completely disappeared after 1 week on the highfat diet. These data suggest that C57BL/6J mice are characterized by the absence of sustained compensatory responses able to protect against diet-induced obesity [28]. It is possible that the observed precipitated development of diet-induced leptin resistance after repeated periods of HC diet feeding originates from the loss of short-term and transient modulations in neuropeptide gene expression observed by Ziotopoulou et al. in C57BL/6J mice. Whether such mechanisms are responsible for the relative retardation of leptin resistance development in mice naïve to the HC diet in contrast to mice having experienced leptin resistance before, remains to be elucidated. 4.5. Body mass gain versus body mass loss during periodic HC diet feeding The alternating diet regime performed in this study resulted in an overall increase in body mass, despite compensatory body mass loss during periods with exclusive chow feeding. Also, the increase in body mass variability between individual mice was comparable to that observed in continuously HC diet fed mice. During periods of HC diet feeding, body mass was positively correlated with energy intake. Conversely, in periods of exclusive chow diet feeding and re-establishment of leptin responsiveness the correlation between body mass and energy intake was negative, demonstrating that mice with a higher body mass show a stronger reduction in energy intake as compensatory response to higher body mass gain and energy intake during previous HC diet feeding periods. This was also demonstrated in diet-induced obese rats that consumed less food than lean control rats after withdrawal of a high-caloric diet [42]. However, for equal periods of time body mass gain during HC diet feeding exceeds the counter-regulatory decrease during chow feeding, in agreement with the notion that it is easier to gain than to loose fat mass [43]. Based on the comparison of regulatory responses to fasting/ decreased leptin levels and mild hyperleptinemia, it has been suggested that the systems controlling energy homeostasis defend

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more effectively against body mass loss than against body mass gain [16,44]. From the evolutionary perspective, limited access to food is considered to be a stronger selection factor in that it favours defence against energy deficits. A model explaining the differently pronounced compensatory responses to either positive or negative energy balance has proposed that the basal state, defined as neutral energy balance, in which body fat mass remains constant by virtue of “normal” leptin and insulin levels, is characterised by the predominance of catabolic over anabolic signaling and, thus, the former would be stimulated less and the latter inhibited less by a positive energy balance [43]. In summary, the presented results strengthen the view that the development of physiological disturbances associated with the development of obesity is not only modulated by the genetic background but to a great extent by environmental factors such as food composition and the dietary history of the animals. Thus, in attempts to obtain basic information from animal experiments for strategies to treat human obesity, the simulation of humanlike feeding conditions, i.e., free choice of food in combination with repeated phases of dieting, may help to unravel the complex mechanisms underlying the development of diet-induced obesity. Acknowledgements This paper was supported by the BMBF (01GS0118 and 01GS0489) and is part of a German Ph.D. thesis by A. Steinbrück. References [1] Kopelman PG. Obesity as a medical problem. Nature 2000;404:635–43. [2] Friedman JM. Obesity in the new millennium. Nature 2000;404:632–4. [3] Barsh GS, Farooqi IS, O'Rahilly S. Genetics of body-weight regulation. Nature 2000;404:644–51. [4] Levin BE. Metabolic imprinting on genetically predisposed neural circuits perpetuates obesity. Nutrition 2000;16:909–15. [5] Schmidt I. Metabolic diseases: the environment determines the odds, even for genes. News Physiol Sci 2002;17:115–21. [6] Collins S, Martin TL, Surwit RS, Robidoux J. Genetic vulnerability to dietinduced obesity in the C57BL/6J mouse: physiological and molecular characteristics. Physiol Behav 2004;81:243–8. [7] West DB, Boozer CN, Moody DL, Atkinson RL. Dietary obesity in nine inbred mouse strains. Am J Physiol 1992;262:R1025–32. [8] Smith BK, Andrews PK, West DB. Macronutrient diet selection in thirteen mouse strains. Am J Physiol Regul Integr Comp Physiol 2000;278:R797–805. [9] Surwit RS, Kuhn CM, Cochrane C, McCubbin JA, Feinglos MN. Dietinduced type II diabetes in C57BL/6J mice. Diabetes 1988;37:1163–7. [10] Leibowitz SF, Wortley KE. Hypothalamic control of energy balance: different peptides, different functions. Peptides 2004;25:473–504. [11] Ahima RS, Osei SY. Leptin signaling. Physiol Behav 2004;81:223–41. [12] Considine RV, Sinha MK, Heiman ML, Kriauciunas A, Stephens TW, Nyce MR, et al. Serum immunoreactive-leptin concentrations in normalweight and obese humans. N Engl J Med 1996;334:292–5. [13] Maffei M, Halaas J, Ravussin E, Pratley RE, Lee GH, Zhang Y, et al. Leptin levels in human and rodent: measurement of plasma leptin and ob RNA in obese and weight-reduced subjects. Nat Med 1995;1:1155–61. [14] Frederich RC, Hamann A, Anderson S, Lollmann B, Lowell BB, Flier JS. Leptin levels reflect body lipid content in mice: evidence for diet-induced resistance to leptin action. Nat Med 1995;1:1311–4. [15] Saladin R, De Vos P, Guerre-Millo M, Leturque A, Girard J, Staels B, et al. Transient increase in obese gene expression after food intake or insulin administration. Nature 1995;377:527–9.

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