H OH OH metabolites Article Effects of a Calorie-Restricted Cafeteria Diet and Oleuropein Supplementation on Adiposity and mRNA Expression of Energy Balance Related Genes in Obese Male Rats Alex Subias-Gusils 1,2 , Adam Álvarez-Monell 1,3 , Noemi Boqué 4 , Antoni Caimari 4 , Roger Mariné-Casadó 4 , Rosa M. Escorihuela 1,2, * and Montserrat Solanas 1,3, * 1 Institut de Neurociències, Universitat Autònoma de Barcelona, 08193 Bellaterra, Spain 2 Departament de Psiquiatria i Medicina Legal, Facultat de Medicina, Universitat Autònoma de Barcelona, 08193 Bellaterra, Spain 3 Department of Cell Biology, Physiology and Immunology, Faculty of Medicine, Universitat Autònoma de Barcelona, 08193 Bellaterra, Spain 4 Eurecat, Centre Tecnològic de Catalunya, Biotechnology Area and Technological Unit of Nutrition and Health, 43204 Reus, Spain * Correspondence: rosamaria.escorihuela@uab.cat (R.M.E.); montserrat.solanas@uab.cat (M.S.); Tel.: +34-93-5813296 (R.M.E.); +34-93-5811373 (M.S.) Abstract: Supplementation with natural bioactive compounds has been proposed to be a comple- mentary tool to the calorie-restricted diets and physical exercise programs used to tackle human overweight, obesity and Metabolic syndrome. Herein, we evaluated the effects of 14 weeks of calorie-restricted cafeteria diet either alone or combined with oral administration of the polyphenol oleuropein in obese adult male rats, compared with a control group fed standard chow and a group fed cafeteria diet. Animals were sacrificed at the age of 26 weeks and several tissues of interest were removed. The results showed that both dietary interventions reduced the adiposity index (p < 0.05 Citation: Subias-Gusils, A.; and p < 0.01, respectively), and specifically the abdominal fat depots (mesenteric: p < 0.01 and p < 0.01, Álvarez-Monell, A.; Boqué, N.; respectively; and epididymal: both diets p < 0.001) and restored the decreased soleus skeletal muscle Caimari, A.; Mariné-Casadó, R.; Escorihuela, R.M.; Solanas, M. Effects mass. Both interventions decreased leptin mRNA expression in mesenteric white adipose tissue of a Calorie-Restricted Cafeteria Diet (p < 0.05) and normalized hypothalamic Agrp mRNA expression compared to cafeteria-fed obese rats and Oleuropein Supplementation on (p < 0.05). However, only the calorie-restricted cafeteria diet supplemented with oleuropein induced Adiposity and mRNA Expression of additional lower retroperitoneal adipose accretion (p < 0.05) and increased hypothalamic leptin Energy Balance Related Genes in receptor mRNA levels (p < 0.05). Experiments with female animals, at different doses and longer Obese Male Rats. Metabolites 2023, 13, intervention periods, are needed to better determine the potential benefits of this dietary treatment. 147. https://doi.org/10.3390/ metabo13020147 Keywords: adiponectin; calorie restriction; dietary supplements; hypothalamus; leptin; metabolic Academic Editors: M. Dulce Estêvão syndrome; white adipose tissue and Manuela Meireles Received: 9 December 2022 Revised: 9 January 2023 1. Introduction Accepted: 16 January 2023 Published: 18 January 2023 Obesity is a chronic metabolic disease characterized by an increase of body fat stores due to a chronic positive energy balance [1]. It is associated with numerous comorbidities such as cardiovascular disease, hypertension, type 2 diabetes and certain cancers [2,3]. The acute increase in the prevalence of obesity reported during the recent decades may be Copyright: © 2023 by the authors. associated with the increasing presence of energy dense foods, reduced physical activity Licensee MDPI, Basel, Switzerland. and the sedentary lifestyle mainly in developed countries [4]. This article is an open access article The hypothalamus (HPT) plays a key role in the regulation of food intake and en- distributed under the terms and ergy balance. In the arcuate nucleus region, anorexigenic (POMC/CART) and orexigenic conditions of the Creative Commons (AGRP/NPY) neuron populations activate neuronal pathways that suppress or increase Attribution (CC BY) license (https:// food intake, respectively [5]. Leptin from adipocytes and insulin from the pancreas are creativecommons.org/licenses/by/ implicated in this regulation, acting as peripheral signals of energy status to the central 4.0/). Metabolites 2023, 13, 147. https://doi.org/10.3390/metabo13020147 https://www.mdpi.com/journal/metabolites Metabolites 2023, 13, 147 2 of 13 nervous system where they inhibit the orexigenic pathways and stimulate the anorexigenic pathways via their corresponding receptors in these hypothalamic neurons [6]. Therefore, by expressing adipocytokines such as leptin or adiponectin, adipose tissue plays a key role in the regulatory cascades involved in obesity and Metabolic Syndrome (MetS) and in the cross-talk between major organs (brain-HPT, liver, muscles) [7]. Leptin acts in a homeostatic loop that regulates body-fat mass and energy balance not only at the central nervous system level, but also directly at the adipocyte level, regulating lipolysis [8]. Interestingly, obesity is associated with selective leptin resistance, which can lead to increased hyperphagia and rapid weight gain [9]. Dietary supplementation with natural bioactive compounds such as polyphenols, combined with calorie-restricted diets and/or physical exercise programs, has emerged as a promising therapeutic tool in the prevention and management of obesity and metabolic disease. Polyphenols have been shown to modulate energy metabolism by stimulating β- oxidation, inhibiting adipocyte differentiation and inflammation, or counteracting oxidative stress [10]. However, the results obtained in meta-analyses of randomized clinical studies are not consistent and more clinical studies with larger numbers of participants, longer times of exposure and different doses must be conducted to validate them as a realistic strategy against obesity [1,11]. One of the polyphenols that might exhibit potential beneficial effects on obesity- related parameters is the secoiridoid oleuropein (OLE), present in olive (Olea europaea) fruits and virgin oils. It is one of the main components of the Mediterranean diet, which is considered the healthiest dietary pattern available to prevent several non-communicable diseases [12]. In experimental animal studies, OLE supplementation was able to reduce dyslipidaemia and improve obesity and diabetes-related alterations such as abdominal fat gain, insulin sensitivity, adipose tissue inflammation and dysbiosis in gut microbiota through its inhibition of the expression of genes related to adipogenesis, and reduction of pro-inflammatory adipokines, among other actions [13–15]. These reported effects of OLE are closely associated with its bioavailability and metabolism once it is absorbed in the gastrointestinal tract. Its bioavailability is influenced by the route of administration, interaction with foods and by the extraction processes [16]. OLE plasma concentrations were observed to peak 20 min after oral ingestion in rodents. In humans, the maximum OLE concentration in plasma was observed after 23–93 min and the peak appeared 64 min after ingestion, on average. In rodents, OLE and its metabolic derivates, such as hydroxytyrosol or elenoic acid, were detected in the liver and kidneys. Regarding its excretion, OLE was detected in urine both in rodents and humans, whereas none was detected in the cecum content or faeces, indicating that it may be mainly excreted via urine [17]. Rodent models, specifically rats and mice, are the most widely used preclinical animal models for the study of metabolic disorders such as obesity or type 2 diabetes [18]. We can distinguish two types of animal models depending on the strategy used to induce obesity in the animals: (i) The diet-induced obesity (DIO) models, which use obesogenic diets such as the high-fat (HFD) or cafeteria (CAF) diets; and (ii) the genetic models, which feature the mutation of one (monogenic) or various genes (polygenic) such as the ob/ob and the C57BL6/J mice, respectively [18–20]. One of the DIO models that is most used to investigate obesity disorders and preclinical treatment strategies is the CAF diet, an experimental obesogenic diet based on the administration of highly energetic and palatable human food items, such as biscuits, cheese, muffins and sugared milk, which induces hyperphagia and increases energy intake [21–24]. In comparison with high-fat/high-sugar diets, it is considered a more robust DIO model in rodents because it better mimics the hedonic hyperphagia observed in obese humans and promotes more severe symptoms [18,25]. In a previous study, we characterized an experimental restricted cafeteria (CAF-R) diet consisting of standard rodent chow supplemented with small portions of CAF items to achieve a 30% calorie-reduction in relation to the total calorie intake of the CAF-fed group [26]. The addition of OLE to the CAF-R diet diminished the hedonic responses to high-sucrose solutions in obese male animals, while preserving the reductions in body Metabolites 2023, 13, 147 3 of 13 weight gain, body mass index (BMI), and daily food and energy intakes; and the improve- ment of several biochemical parameters associated with obesity [27]. The current study aimed to evaluate whether those dietary interventions changed the mass of adipose depots and skeletal muscles, as well as to characterize the mRNA expression levels of genes involved in the above mentioned hypothalamic anorexigenic and orexigenic pathways and the leptin, leptin receptor and adiponectin mRNA expression levels in white adipose tissue. 2. Materials and Methods 2.1. Experimental Design The tissues and samples used in the present study were obtained from the cohort of male Sprague-Dawley rats used in Subias-Gusils et al. [27]. Briefly, two dietary interven- tions based on the CAF-R diet alone or supplemented with OLE (CAF-RO diet) at a dose of 25 mg/kg·day of an Olea europaea leaf extract (Benolea® , Frutarom Health BU, Wädenswil, Switzerland) were administered for 14 weeks to CAF diet-induced obese animals. The CAF and CAF-R diets were composed of the following items: bacon, biscuit with pâté, biscuit with cheese, muffins, carrots, jellied sugared milk and standard chow. The amount of each cafeteria food item administered to the CAF-R and CAF-RO animals was readjusted every week based on a 30% reduction in calories relative to the energy consumed by the CAF group. A control (STD) group was fed standard chow ad libitum during the entire experiment [27]. The CAF diet was provided ad libitum to the animals after weaning, starting at the age of 24 days old and continuing for 8 weeks. When the animals were 80 days-old, the obese CAF-fed animals were subdivided into three groups CAF, CAF-R and CAF-RO according to the diet administered until the end of the experiment. The average daily intake of macronutrients during the dietary treatments, expressed as the percentage of the average total daily energy intake in kilocalories coming from each macronutrient type, as well as the feed efficiency, are indicated in Table S1. The animals were sacrificed at an age of 26 weeks, and the body weights were: STD, 465.9 ± 8.8; CAF, 564.9 ± 15.4; CAF-R, 537.5 ± 15.9; and CAF-RO, 536.6 ± 12.0 (mean ± SEM, grams). Euthanasia was carried out by decapitation after 8 h of fasting, which started in the morning at 6 a.m., leaving 20 min between animals, and sacrifices were performed sequentially between 2 p.m. and 5 p.m. for a total of 3 days. The following tissues were removed: HPT, WAT depots (retroperitoneal -rWAT-, mesenteric -mWAT-, epididymal -eWAT- and inguinal -iWAT-), interscapular brown adipose tissue (iBAT), liver, cecum, gastrocnemius and soleus muscles. Once removed, they were weighed, frozen in liquid nitrogen and stored at −80 ◦ C until further analysis. A macroscopic examination of all tissues was performed before storage. The tissue exeresis was carried out by the same qualified experimenters through the procedure to decrease variability. The adiposity index was determined as the sum of the eWAT, iWAT, mWAT and rWAT weights (in grams) and expressed as a percentage of body weight (g/kg·100). Abdominal WAT referred to the sum of rWAT, mWAT and eWAT weights, while subcutaneous WAT was considered as the iWAT. The experimental protocol was approved by the Generalitat de Catalunya (DAAM 9978), following the ‘Principles of laboratory animal care’ and was carried out in accordance with the EU Directive 2010/63/EU for animal experiments and following ARRIVE guidelines. 2.2. mRNA Expression Analyses The entire HPT and 80 mg of mWAT were each homogenized in 1 mL of QIAzol® Lysis Reagent (Qiagen, Barcelona, Spain) using a tissue homogenizer (Polytron™ PT 2100, KINEMATICA® ) according to the corresponding manufacturers’ protocol (RNeasy Lipid Tissue Mini Kit—Qiagen) to obtain the total RNA from each sample. Quantification and quality (260/280 and 260/230 contamination ratios) of the RNA obtained were analyzed using a NanoDrop™ 1000 Spectrophotometer (Thermo Fisher Scientific, Madrid, Spain). RNA integrity was checked by agarose gel electrophoresis. The cDNA was synthesized Metabolites 2023, 13, 147 4 of 13 from 2 µg/mL of total RNA using the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Madrid, Spain) and the following RT-PCR program: 25 ◦ C for 10 min, 37 ◦ C for 120 min, 85 ◦ C for 5 min and stored at 4 ◦ C. The resulting cDNA was amplified by real-time PCR with TaqMan® methodology in the iCycler MyiQTM Single Color Real- Time PCR Detection System (Bio-Rad Laboratories, Madrid, Spain). Specific Gene TaqMan Assays for Lepr, Insr, Pomc, Cartpt, Npy, Agrp, Lep, Adipoq, and Hprt as the housekeeping control gene, were obtained from Applied Biosystems. The Real-Time PCR program was set as follows: 95 ◦ C for 10 min, 40 cycles of 95 ◦ C for 15 s and 60 ◦ C for 1 min. Each PCR was performed in duplicate. The relative expression of each mRNA was calculated using the 2−∆∆Ct method [28]. The TaqMan® Gene Expression Assays used to amplify the different genes were: Rn01433205_m1 (Lepr); Rn00690703_m1 (Insr); Rn00595020_m1 (Pomc); Rn00567382_m1 (Cartpt); Rn00561681_m1 (Npy); Rn01431702_g1 (Agrp); Rn01433205_m1 (Lepr); Rn00565158_m1 (Lep); Rn00595250_m1 (Adipoq); and Rn01527840_m1 (Hprt1). 2.3. Statistical Analyses Statistical analyses were performed using SPSS Statistics 22 (SPSS, Inc., Chicago, IL, USA). The homoscedasticity among groups was measured using Levene’s test. Grubbs’ test was used to determine significant outlier values, and consequently, discarded them for the analyses. One-way ANOVAs followed by Duncan post-hoc test, or Student’s t test, were performed to evaluate differences among groups. All the results were expressed as mean ± SEM. The level of statistical significance was set at bilateral 5%. 3. Results 3.1. Adipose Depots and Other Tissues The CAF group showed the highest values of mWAT, eWAT, abdominal WAT and adiposity index; the CAF-R and CAF-RO groups displayed intermediate values; and the STD group the lowest ones (p < 0.001; Figure 1B–D,F). The CAF-RO group, but not the CAF-R Metabolites 2023, 13, x FOR PEER group, also showed intermediate values of rWAT, between CAF and STD REVIEW 5 of (p 13 < 0.001; Figure 1A). Moreover, the three cafeteria groups showed increased subcutaneous WAT compared with the STD group (p < 0.001; Figure 1E). A 40 B 20 C 30 *** $ *** $$ *** $$$ $$$ 30 *** 15 $$$ *** *** *** *** mWAT (g/kg) eWAT (g/kg) 20 rWAT (g/kg) ** 20 10 10 10 5 0 0 0 STD CAF CAF-R CAF-RO STD CAF CAF-R CAF-RO STD CAF CAF-R CAF-RO D 80 E F 12 *** $$ 40 *** $ $$ $$ *** Subcutaneus WAT (g/kg) *** *** *** *** 10 *** Abdominal WAT (g/kg) *** Adiposity index (%) 60 30 8 40 20 6 4 20 10 2 0 0 0 STD CAF CAF-R CAF-RO STD CAF CAF-R CAF-RO STD CAF CAF-R CAF-RO Figure 1. Effects of the dietary treatments on the relative weight of the retroperitoneal WAT (A), Figure 1. Effects of the dietary treatments on the relative weight of the retroperitoneal WAT (A), mesenteric WATWAT mesenteric (B), epididymal (B), epididymal WAT WAT (C), (C),abdominal abdominal WAT WAT(D)(D)and and subcutaneous subcutaneous WATWAT (E), and on (E), and the adiposity index (F). on the adiposity Tissue index weights (F). Tissue in grams weights were in grams wererelativized relativizedto to the totalbody the total bodyweight weight in kg. Data in kg. Data are expressed are expressed as the± as the mean mean SEM.± SEM. ** p **< p0.01, < 0.01,*** ***pp < < 0.001 0.001vs. STD vs. STDgroup; $ p < 0.05, group; $ p <$$0.05, p < 0.01, $$ p < 0.01, $$$ p < 0.001 vs. CAF group. $$$ p < 0.001 vs. CAF group. A 2.0 B 40 C 15 r BAT (g/kg) ** *** ** 1.5 30 (g/kg) (g/kg) 10 1.0 20 D 80 E F 12 *** $$ 40 *** $ $$ $$ *** Subcutaneus WAT (g/kg) *** *** *** *** 10 *** Abdominal WAT (g/kg) *** Adiposity index (%) 60 30 8 40 20 6 Metabolites 2023, 13, 147 5 of 13 4 20 10 2 0 0 0 STD CAF CAF-R Besides the changes CAF-RO STD in the CAF WAT CAF-R depots, CAF-RO the three cafeteria STD groups CAF CAF-R displayed CAF-RO a sig- nificant increase in iBAT weight compared with the STD group (p < 0.001; Figure 2A). These groups Figure alsoofshowed 1. Effects the dietarydecreased treatments cecum weight on the relative (p < of0.001; weight Figure 2C),WAT the retroperitoneal whereas (A), no mesenteric differences WATobserved were (B), epididymal WAT in liver (C), abdominal weight (Figure WAT (D) for 2B). As and skeletal subcutaneous WAT (E), muscles, theand weight on the adiposity index (F). Tissue weights in grams were relativized to the total body weight in kg. of gastrocnemius muscles was diminished in the three cafeteria groups compared with the Data are expressed as the mean ± SEM. ** p < 0.01, *** p < 0.001 vs. STD group; $ p < 0.05, $$ p < 0.01, STD$$$group (p