nutrients Article Impact of Calorie-Restricted Cafeteria Diet and Treadmill Exercise on Sweet Taste in Diet-Induced Obese Female and Male Rats Adam Alvarez-Monell 1,2 , Alex Subias-Gusils 1,3 , Roger Mariné-Casadó 4 , Noemi Boqué 4 , Antoni Caimari 4 , Montserrat Solanas 1,2, * and Rosa M. Escorihuela 1,3, * 1 Institut de Neurociències, Universitat Autònoma de Barcelona, 08193 Bellaterra, Spain 2 Department of Cell Biology, Physiology and Immunology, Faculty of Medicine, Universitat Autònoma de Barcelona, 08913 Bellaterra, Spain 3 Department de Psiquiatria i Medicina Legal, Facultat de Medicina, Universitat Autònoma de Barcelona, 08193 Bellaterra, Spain 4 Eurecat, Centre Tecnològic de Catalunya, Technological Unit of Nutrition and Health, 43204 Reus, Spain * Correspondence: montserrat.solanas@uab.cat (M.S.); rosamaria.escorihuela@uab.cat (R.M.E.); Tel.: +34-93-5811373 (M.S.); +34-93-5813296 (R.M.E.) Abstract: The goal of the present study was to evaluate the sweet taste function in obese rats fed with a 30% calorie-restricted cafeteria diet (CAFR) and/or subjected to moderate treadmill exercise (12–17 m/min, 35 min, 5 days per week) for 9 weeks. A two-bottle preference test, a taste reactivity test, and a brief-access licking test were carried out when animals were aged 21 weeks; biometric and metabolic parameters were also measured along the interventions. Two separate experiments for females and males were performed. Behaviorally, CAF diet decreased sucrose intake and preference, as well as perceived palatability, in both sexes and decreased hedonic responses in males. Compared to the CAF diet, CAFR exerted a corrective effect on sweet taste variables in females by increasing sucrose intake in the preference test and licking responses, while exercise decreased sucrose intake in Citation: Alvarez-Monell, A.; both sexes and licking responses in females. As expected, CAF diet increased body weight and Lee Subias-Gusils, A.; Mariné-Casadó, R.; index and worsened the metabolic profile in both sexes, whereas CAFR diet ameliorated these effects Boqué, N.; Caimari, A.; Solanas, M.; mainly in females. Exercise had no noticeable effects on these parameters. We conclude that CAF Escorihuela, R.M. Impact of diet might diminish appetitive behavior toward sucrose in both sexes, and that this effect could be Calorie-Restricted Cafeteria Diet and partially reverted by CAFR diet in females only, while exercise might exert protective effects against Treadmill Exercise on Sweet Taste in overconsumption of sucrose in both sexes. Diet-Induced Obese Female and Male Rats. Nutrients 2023, 15, 144. https://doi.org/10.3390/ Keywords: energy restriction; physical activity; metabolic syndrome; sex differences; sucrose; nu15010144 wanting; liking Academic Editor: Megan A. McCrory Received: 2 December 2022 1. Introduction Revised: 15 December 2022 Obesity and overweight are two of the biggest health concerns worldwide, with Accepted: 21 December 2022 one-third of the population being overweight or obese [1]. The increase in available Published: 28 December 2022 high-fat and high-sugar food choices has contributed to increased obesity rates [2]. The consumption of these foods and drinks has spiked in recent decades and has been associated with an increase in adiposity, biomarkers of metabolic syndrome (MetS), and associated Copyright: © 2022 by the authors. pathologies [2,3]. Licensee MDPI, Basel, Switzerland. Studies have tried to elucidate how an obese phenotype impacts sweet taste. It has This article is an open access article been reported in humans that a high BMI is associated with lower perceived sweetness, and distributed under the terms and with increased liking for sweetness [4]. Preclinical studies have shown that the adipokine conditions of the Creative Commons leptin has been linked to sweet taste function at several levels, from the taste receptors Attribution (CC BY) license (https:// in the taste buds to the brain reward processing system [5]. It is known that leptin alters creativecommons.org/licenses/by/ sweet taste transduction through the Ob-Rb leptin receptor located in the T1R3 cells of the 4.0/). taste bud that are activated by sweet stimuli [6]. In nonobese humans and rodents, the Nutrients 2023, 15, 144. https://doi.org/10.3390/nu15010144 https://www.mdpi.com/journal/nutrients Nutrients 2023, 15, 144 2 of 21 behavioral responses to a sweet stimulus change with the circadian rhythms of circulating leptin, whilst, in obese individuals, these circadian changes in leptin are absent [7]. Clinically, obesity is treated by improving diet quality and reducing caloric intake, by increasing physical exercise, and by bariatric surgery in severe cases [8]. These interventions have been shown to affect the sweet taste function with contrasting outcomes. For example, weight loss induced through dieting [9] or cognitive therapy [10] has been reported to decrease liking for concentrated sucrose solutions in women, although no differences were observed in other studies [11]. Physical exercise appears to have differential effects on sweet taste depending on whether the training is acute or chronic. Acute bouts of intense aerobic exercise increased the intake of sweet solutions and the reported liking of sucrose in healthy subjects [12,13], suggesting a compensatory effect of the nutrient depletion due to the exercise bout [14], while an increase in self-reported physical activity over 6 months decreased the perceived intensity of sweet solutions [15]. However, it is unclear whether these changes in sweet taste function are a direct result of the exercise itself or of the weight loss associated with it [14]. Nutritional state appears not to be a strong determinant of sweet taste function [16], and studies comparing sweet taste detection thresholds between individuals consuming vegan, vegetarian, or omnivorous diets for decades [17] or low-sugar diets for months [18] did not show altered thresholds. However, it has also been reported that a low-vegetable meal increases sweetness desire 2 h after meal consumption [19], while another study reported no differences in sweet intensity and hedonics after a meal [20]. Several animal models have been developed to study obesity [21]. Among them, the cafeteria diet (CAF)-induced obesity (DIO) model, in which animals are fed several high-fat and high-sugar items of human consumption [22,23], is considered a robust model with high face validity for human obesity [24]. CAF diet induces obesity through hedonic eating and hyperphagia due to the high palatability of its ingredients; additionally, it generates more severe MetS symptoms than other high-fat and high-sugar diets [25,26]. In animal models of DIO, a decrease in motivational behavior toward sweet stimuli, as measured by increased latency to approach the sweet stimuli and/or decreased consump- tion, has been reported in males [27–29]. It has also been reported that diet-induced weight loss in male rats increases preference for low concentrations of sucrose [28]. Furthermore, a decrease in preference for highly concentrated sweet solutions in males has been reported in models of gastric bypass surgery [30,31]. The effects of exercise in animal models and how it affects sweet taste have not been extensively studied. We previously reported a decrease in sugared milk and carbohydrate intake in female Sprague-Dawley rats after 8 weeks of treadmill exercise [32]. Other authors have shown that female mice selectively bred for a high runner phenotype showed decreased consumption of artificial sweeteners when exercised [33]. Sex differences exist in human taste perception, with women being generally more sensitive than men [34–36]. This sexual dimorphism is also present in rats, in which females show higher intakes of sucrose solutions than males [37,38]. The responses to sweet stimuli in animals can be interpreted in terms of wanting and liking, which are considered two separate processes [39]. Wanting is considered to be related to how much of a reward is consumed, while liking is related to hedonic impact and to which reward is consumed [40]. Wanting is thought to be a process mediated by the dopaminergic system that encompasses several brain structures processing reward value, such as the mesocorticolimbic dopaminergic system. On the other hand, liking is thought to be mediated by small and localized areas, termed hedonic hotspots, mainly modulated by the opioidergic system and overlapping with areas controlling wanting [39]. These two processes are relatively independent, since it has been shown that mesolimbic dopamine depletion impairs the ability to exert motivational effort while not affecting hedonic reactivity [41], and that elevated dopamine levels increase motivational effort while not affecting hedonic reactivity [42]. However, while wanting can be affected without altering liking, modifications that alter liking almost always alter wanting [40,43]. Nutrients 2023, 15, 144 3 of 21 In this study, we aimed to (i) characterize the effects of CAF DIO upon the sweet taste in terms of wanting and liking, (ii) determine the putative changes in wanting and liking of sucrose solutions due to dietary and exercise interventions based on a calorie-restricted CAF diet (CAFR) and treadmill training in DIO animals, and (iii) analyze the changes in food intake, as well as biometric and metabolic parameters, induced by these interventions. We aimed to determine these parameters in Long-Evans rats, both in females (Experiment 1) and in males (Experiment 2). 2. Materials and Methods 2.1. Experiment 1 2.1.1. Animals Sixty female Long-Evans rats (Janvier, France), 23–25 days-old at the time of delivery, were used. Animals were housed in pairs in standard Plexiglas cages, maintained under a 12 h/12 h light/dark cycle (lights on at 8 a.m.) in standard conditions (temperature: 21 ± 1 ◦ C; humidity 50 ± 10%). After 1 week of habituation to the animal room, they were randomly assigned to two groups balancing mean body weight (BW): STD (n = 20, BW = 89.11 ± 1.94 g) and CAF (n = 40, BW = 88.58 ± 1.25 g). The experimental protocol was approved by the Generalitat de Catalunya (DAAM 9978), following the ‘Principles of laboratory animal care’ and was performed in accordance with the European Communities Council Directive (2010/63/EU). 2.1.2. Experimental Design We used a 3 × 2 factorial design with diet (STD, CAF, and CAFR) and exercise (control C and exercise E) as factors and six experimental groups. After inducing obesity by the administration of the CAF diet for 8 weeks, animals were randomly allocated in pairs to the following groups, balancing mean body weight: STD-C (n = 10, BW = 217.7 ± 4.11 g), STD-E (n = 10, BW = 216.9 ± 4.40 g), CAF-C (n = 10, BW = 264.7 ± 9.67 g), CAF-E (n = 10, BW = 261.2 ± 7.89 g), CAFR-C (n = 10, BW = 259.5 ± 7.51 g), and CAFR-E (n = 10, BW = 263.3 ± 5.98 g). Interventions were maintained throughout the experiment unless otherwise noted. Every week, the animals were weighed, and food intake was deter- mined as the difference between foods provided on Monday and unconsumed the day  √ 3  after. Nasoanal length (NAL) and Lee index LNBW A × 1000 were recorded every 2 weeks. Behavioral testing started when the animals were 22 weeks old and consisted of the sucrose preference test (week 18), the taste reactivity test (week 19), and the brief-access licking test (weeks 20 and 21). During the 10–12 days after finishing the behavioral testing, the dietary treatment and treadmill sessions occurred as usual. Animals were sacrificed by decapitation at week 23 after 8 h of fasting. Blood was collected, and serum was obtained by centrifugation at 2000× g for 15 min at 4 ◦ C and stored at −80 ◦ C until further analysis. The white adipose tissue (WAT) depots (retroperitoneal, RWAT; mesenteric, MWAT; epididymal, Nutrients 2023, 15, x FOR PEER REVIEW 4 of 23 EWAT; and inguinal, IWAT) were also removed and weighed. Figure 1 shows a schematic design of experiments 1 and 2. Figure 1. Figure Schematicrepresentation 1. Schematic representationofofthe the experimental experimentallayout layoutfor for experiments experiments11 and and 22 showing showing groups and groups and the the time time of of behavioral behavioral testing. testing. 2.1.3. Diets During the obesity-induction period, the STD and CAF groups were fed standard chow and CAF diet ad libitum (Supplementary Table S1). From week 9 onward, the CAF Nutrients 2023, 15, 144 4 of 21 2.1.3. Diets During the obesity-induction period, the STD and CAF groups were fed standard chow and CAF diet ad libitum (Supplementary Table S1). From week 9 onward, the CAF diet consisted of (averaged quantities per rat per day) bacon (7 g), biscuits with pâté (5 g), biscuits with cheese (5 g), muffins (7 g), carrots (7 g), jellied sugared milk (40 g), and standard chow (25 g). Food items were administered in a clay pot inside the cage and prepared fresh each day. The average amounts of food and energy administered per rat per day were 96 g and 233 kcal, respectively, and the caloric distribution of the CAF diet was 10.6% protein, 38.3% fat, and 51.1% carbohydrates. We previously used a similar CAF diet in females and showed that it has obesogenic effects [43]. The caloric content of the standard chow (2014 Teklad Global Rodent Diet, Envigo) was 290 kcal per 100 g, and its caloric distribution was 20% protein, 13% fat, and 67% carbohydrates. As for the CAFR diet, the amount of each item provided was (averaged quantities per rat per day) bacon (2.4 g), biscuits with pâté (2.5 g), muffins (1.8 g), carrots (6.3 g), jellied sugared milk (4.6 g), and standard chow (7.5 g). The average amounts of food and energy administered per rat per day were 25.1 g and 61.5 kcal, respectively, and the caloric distribution of the CAFR diet was as follows: 11.4% protein, 42% fat, and 46.6% carbohydrates. Food items were also administered in a clay pot inside the cage and renewed daily. 2.1.4. Treadmill Training Training was performed in a treadmill (Columbus instruments, Columbus, OH, USA) as reported in [44] with some modifications. This began on week 9 along with dietary intervention, with daily training sessions from Monday to Friday (5 days/week), and with the intensity raising progressively from 7 m/min in session 1 to 17 m/min in session 9 (Supplementary Table S2). From session 9 onward, all sessions started at an intensity of 7 m/min, increasing progressively until 17 m/min at min 6, which was maintained until minute 30 and then lowered to 8 m/min for the last 5 min. Sessions ended at minute 35, at an approximate total distance run of 591 m. 2.1.5. Behavioral Testing • Two-bottle sucrose preference test To determine the preference for sucrose solutions, a two-bottle preference test was performed. Animals were housed individually the day before starting the test to allow for individualized testing. The exercise intervention was maintained throughout the behavioral test, but all animals were fed only chow to control for interferences between experimental diets and the test results. The test consisted of placing two bottles, one containing a sucrose solution and the other one containing tap water. On the first day, the animals were presented with tap water in the two bottles (250 mL each) placed at opposite sides of the food container cover of the animals’ cage to habituate them to the procedure and to measure water intake for 24 h. The day after and over seven consecutive days, seven concentrations of sucrose solutions were presented in ascending order and tested for 24 h (0.01 M, 0.03 M, 0.06 M, 0.1 M, 0.3 M, 0.6 M, and 1 M). Sucrose solutions were prepared daily using tap water. Water and sucrose intakes were recorded daily, and bottles’ positions were randomly switched between concentrations to avoid lateral preference. Sucrose preference ratio was calculated according to the following formula: sucrose solution intake (g) Sucrose pre f erence = × 100 water intake (g) + sucrose solution intake (g) • Taste reactivity test After the two-bottle preference test, the animals were returned to the home cages in pairs. To evaluate hedonic responses to different sucrose solutions, we performed the Nutrients 2023, 15, 144 5 of 21 taste reactivity test according to Shin et al. 2011 [28]. Over four consecutive days, the orofacial responses elicited by four sucrose solutions (0.03 M, 0.1 M, 0.3 M, and 0.6 M) prepared daily using tap water and administered (one each day) in ascending order were recorded. Animals were placed in a cylindrical (29 cm height; 25 cm diameter) Plexiglass cage, and, after 2.5 min, 1 mL of sucrose solution was deposited with a micropipette on the edge of the circular transparent floor. The orofacial expressions were recorded from below using a video camera (JVC, HD Everio GZ-HD10) for 2.5 min from the time the mouth of the animal had the first contact with the solution. Positive hedonic responses (tongue protrusion TP, rhythmic protrusions of the tongue on the midline, and discrete nonrhythmic lateral protrusions of the tongue [45]) were measured as the number of episodes (n) and total duration (s). Behavior was scored by examination of slow-motion videos by a trained experimenter [45]. • Brief-access licking test To determine the sensitivity and reward value of sucrose, animals were tested on a lickometer (Med Associates Inc., St. Albans, VT, USA). Both the dietary and the exercise interventions were suspended for the duration of the test. The testing procedure encom- passed a 4 day habituation period to the apparatus (days 1–4), a habituation session to the test conditions on day 5, and the final test on day 7. The day before starting the habituation period (day 0) the animals were deprived of water. During the habituation period, the animals were placed in the testing chamber and presented with a spout connected to a plastic bottle containing water positioned behind an open slot in the wall of the cage. On days 1 and 2, animals were placed in the chamber and allowed 30 min of uninterrupted water presentation to train them to lick the spout. On days 3 and 4, they were allowed 20 s to lick the spout, and, once licking started, they had 10 s before a shutter blocked access to the spout and a motorized wheel positioned the next spout. Eight bottles containing water were placed in the motorized wheel, and each bottle was presented eight times for a total of 64 trials each session. On day 5, animals were tested with one bottle containing water and the other seven bottles containing sucrose solutions (0.01 M, 0.03 M, 0.06 M, 0.1 M, 0.3 M, 0.6 M, and 1 M). The presentation order was randomized in eight blocks without repetition. When the session ended, the animals were returned to their home cage and allowed access to water. No session was administered on day 6, and the animals were allowed to rest in their home cage. The final test was carried out on day 7 under no water restriction and using the same procedure as day 5. Animals were weighed daily during the test to ensure no animals lost more than 15% of their baseline body weight due to the water restriction. From this test, the number of trials that initiated licking responses, the total number of licks performed in a session, the number of licks performed at each trial, the mean duration of a licking bout (defined as consecutive licks separated by ILIs between 50 and 250 ms), and the interlick interval (ILI) between consecutive licks with a time resolution of 10 ms, which was used to determine the mean ILI distribution for each experimental group, were recorded. 2.1.6. Serum Analyses Analysis of serum samples was performed as in [44]. Briefly, enzymatic colorimetric kits were used for the determination of serum total cholesterol, triglycerides, and glucose (QCA, Barcelona, Spain). Serum insulin and leptin levels were measured using a mouse/rat insulin ELISA kit (Mercodia, Barcelona, Spain) and a rat leptin ELISA kit (Millipore, Barcelona, Spain). The homeostasis model assessment-estimated insulin resistance (HOMA- IR) was calculated using the following formula [46]: HOMA-IR = glucose × insulin/22.5. 2.2. Experiment 2 2.2.1. Animals, Timeline, and Experimental Design Experiment 2 followed a similar design to experiment 1. For detail on the animals used and the experimental design, see [44]. Of note, the exercise and CAFR interventions started at week 10, the preference test was performed at week 19, the taste reactivity test Nutrients 2023, 15, 144 6 of 21 was performed at week 20, and the brief-access licking test was performed at weeks 21–22. Figure 1 shows the schematic design of experiment 2. 2.2.2. Dietary and Exercise Interventions A similar CAF diet, CAFR diet, and treadmill exercise to experiment 1 were used in experiment 2. For details on the amounts of food and energy administered per day and treadmill training, see [44]. Of note, the exercise intensity in experiment 2 was set at 12 m/min instead of 17 m/min. This decrease in intensity is because female rats tolerate higher treadmill intensities and are better runners than males [32,47,48]. 2.2.3. Behavioral Testing • Two-bottle sucrose preference test The test was performed at week 19 using a similar methodology as experiment 1. The corresponding dietary and exercise interventions were maintained during testing. • Taste reactivity test The test was performed at week 20 using the same methodology as experiment 1 • Brief-access licking test The test was performed at week 21 using the same methodology as experiment 1. The exercise intervention was interrupted during the habituation and testing, and the dietary intervention was maintained. 2.3. Statistical Analysis Statistical analysis was performed using the “Statistical package for Social Sciences” (SPSS, version 22, IBM). Biometric and intake data for the first period of obesity induction were analyzed separately with Student’s t-test. Data for the second period were analyzed separately using two two-way ANOVA analyses to determine the effects of the factors diet and exercise: a first two-way ANOVA comparing CAF and STD groups [diet (STD, CAF) × exercise (C, E)] and a second two-way ANOVA comparing CAFR and CAF groups [diet (CAF, CAFR) × exercise (C, E)]. Data comprising several timepoints (BW progression, sucrose intake in the two-bottle preference test, lick score, mean burst size, and mean ILI distribution in the brief-access licking test) were also analyzed with two two-way repeated-measures ANOVAs with time or sucrose concentration as within factors, and diet and exercise as between factors. Sucrose preference and the taste reactivity test data were analyzed with Kruskal–Wallis followed by the Mann–Whitney U test for pairwise comparisons due to unequal variances assessed with Levene’s test. The number of animals that contacted the sucrose solution in the taste reactivity test was analyzed using the chi-squared test. Mean lick scores data for each group were curve fitted using the following logistic function [49,50]: a+b F(x) =  , 1 + 10(x−c)d where a is the asymptotic lick response, b is the lower lick response, x is the log10 stimulus concentration, c is the log10 concentration at the inflection point, and d is the slope of the curve. Mean ILI distribution data were curve-fitted to the Gaussian function according to Lin et al. (2013) [51] using the following function: X−Mean )2 ) F(x) = Amp· exp−0.5·(( SD , where amplitude (Amp) is the height at the center of the distribution, mean is the X value at the center of the distribution, and SD is the standard deviation of the distribution. Mean ILI distribution data were curve-fitted to the Gaussian function according to Lin et al. (2013) [51] using the following function: X−Mean 2 F(x) = Amp · exp−0.5·(( SD ) ) , Nutrients 2023, 15, 144 where amplitude (Amp) is the height at the center of the distribution, mean is the X value 7 of 21 at the center of the distribution, and SD is the standard deviation of the distribution. All the results are expressed as the mean ± SEM. The level of statistical significance was All set at thebilateral results 5%. are expressed as the mean ± SEM. The level of statistical significance was set at bilateral 5%. 3. Results 3. Results 3.1. CAFR Diet Exerted Corrective Effects on CAF-Induced Alterations on Biometric Data in 3.1. CAFR Diet Exerted Corrective Effects on CAF-Induced Alterations on Biometric Data Females in Females We first analyzed body weight gain (BWG) (Figure 2A,C) and the Lee index (Figure Weinfirst 2B,D), theanalyzed bodyWe first period. weight gainhigher found (BWG)BWG(Figure 2A,C) and Lee and indexthein Lee index CAF (Figurethan animals 2B,D), in in STD animals in both sexes (p < 0.01). Over the second period, this increase in BWGSTD the first period. We found higher BWG and Lee index in CAF animals than in and animals Lee indexin both sexes in CAF (p < 0.01).to compared Over STDthe second was period, this maintained (p