Available online at www.sciencedirect.com Journal of Nutritional Biochemistry 125 (2024) 109549 RESEARCH PAPER Body weight gain and control: beneficial effect of extra virgin olive oil versus corn oil in an experimental model of mammary cancer Raquel Moral a,∗, Garyfallia Kapravelou a, Marta Cubedo b, Montserrat Solanas a, Eduard Escrich a,∗ a Department of Cell Biology, Physiology and Immunology, Physiology Unit, Faculty of Medicine, Universitat Autònoma de Barcelona, Bellaterra, Barcelona, Spain b Department of Statistics, Universitat de Barcelona, Barcelona, Spain Received 31 October 2023; received in revised form 2 December 2023; accepted 13 December 2023 Abstract Obesity is a known risk factor for breast cancer, the most common malignancy among women worldwide. We have previously described different effects of high-fat diets on mammary experimental carcinogenesis. In this work, we analyzed the animal growth data obtained in six experimental assays, in healthy and carcinogen-induced rats undergoing different dietary interventions. The animals were fed with three experimental diets administered at different periods of development: a control low-fat diet, and two isocaloric high-fat diets (rich in corn oil or in extravirgin olive oil -EVOO-). Weekly weight throughout the development of 818 animals have been compiled and reanalyzed using adjusted mathematical models. Molecular mechanisms have been investigated: ethanolamides in small intestine, neuropeptides controlling satiety in hypothalamus, and proteins controlling lipid metabolism in adipose and mammary tissues. The results indicated that the effect of diets depended on type of lipid, timing of intervention and health status. The high corn oil diet, but not the high EVOO diet, increased body weight and mass, especially if administered from weaning, in healthy animals and in those that received a moderate dose of carcinogen. The potential protective effect of EVOO on weight maintenance may be related to anorexigenic neuropeptides such as oxytocin and lipolysis/deposition balance in adipose tissue (increasing phospho-PKA, HSL, MGL and decreasing FAS). In animals with cancer, body weight gain was related to the severity of the disease. Taken together, our results suggest that EVOO has a beneficial effect on body weight maintenance in both health and cancer. © 2023 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/) Keywords: Dietary lipids; Extra virgin olive oil; Body weight; Breast cancer; Molecular mechanisms; Lipolysis. 1. Introduction to the Mediterranean diet and obesity risk, weight gain, and ab- dominal obesity [4–6]. The Mediterranean diet was acknowledged as an Intangible Cul- Several studies have also associated overweight and obesity tural Heritage of Humanity by UNESCO in 2010. This diet includes with an increased risk of breast cancer [7]. This cancer is also an a variety of food patterns from the Mediterranean region and is important public health problem and a leading cause of mortal- characterized, among other factors, by the consumption of olive oil ity in women worldwide [8]. Although the risk for breast cancer is as the principal source of fat [1]. A wealth of evidence supports the multifactorial, there is a substantial contribution of environmental health benefits of this diet, associated with a reduced risk of mor- factors such as diet, attracting great interest since they are modifi- tality, and incidence of major chronic diseases, such as cardiovas- able risk factors [7,9]. Epidemiological and especially experimental cular disease, cancer andobesity [2]. These results are of great im- studies have found a link between dietary lipids and this neoplasia. portance since the prevalence of chronic diseases is a global pub- This influence depends, foremost, on the total amount of fat con- lic health problem. For example, worldwide obesity prevalence has sumed, but also on the specific type of dietary lipid [10]. Diets rich nearly tripled from 1975 to 2016 and continues to rise [3]. Prospec- in n-6 polyunsaturated fatty acids (PUFA), as well as diets rich in tive studies have found an inverse association between adherence saturated or trans fatty acids, enhance experimental tumorigenesis, Abbreviations: AEA, N-arachidonoyl ethanolamide; DMBA, 7,12-dimethylbenz(a)anthracene; EVOO, extravirgin olive oil, Exp, experiment; FA: fatty acids, HCO, high corn oil; HOO, high extravirgin olive oil; LA, linoleic acid; LF, low-fat; MUFA, monounsaturated fatty acids; OA, oleic acid; OEA, oleaoyl ethanolamide; PUFA, polyunsaturated fatty acids. ∗ Corresponding authors at: Edifici M, Avinguda de Can Domènech, Bellaterra 08193 (Cerdanyola del Vallès), Barcelona, Spain. Tel.: +34 93 5811341. E-mail addresses: Raquel.Moral@uab.cat (R. Moral), Eduard.Escrich@uab.cat (E. Escrich). https://doi.org/10.1016/j.jnutbio.2023.109549 0955-2863/© 2023 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/) 2 R. Moral, G. Kapravelou, M. Cubedo et al. / Journal of Nutritional Biochemistry 125 (2024) 109549 whereas n-3 PUFA has shown inhibitory effects, and the effects of monounsaturated fatty acids (MUFA) remains unclear [10]. In hu- mans, different dietary patterns have been associated with cancer risk, and Mediterranean diet has been reported to decrease breast cancer risk and mortality [11,12]. Although it is more difficult to establish associations with dietary components, prospective cohort studies have suggested a protective effect of olive oil consumption in hormone-independent breast tumors [13]. The beneficial effects of healthy dietary patterns on the development of this disease can account, at least in part, by their influence on body composition. Body weight and adiposity have been associated with breast can- cer mortality and recurrence [7,14]. This cancer has been linked as well to other parameters related to nutrition and body growth, such as early menarche, which is an established risk factor for this disease [7]. Thus, nutritional exposures in early life that influence growth, sexual maturation, and adiposity may have an impact on later risk for this neoplasia. Due to the difficulty of obtaining data with controlled vari- ables in humans, animal models are widely used to get an insight into the influence of dietetic factors in health. Using the experi- mental model of mammary cancer chemically induced with 7,12- dimethylbenz(α )anthracene (DMBA) in the female Sprague-Dawley rat [15], we found a differential effect of diets high in seed oils (rich in n-6 PUFA) and in extra virgin olive oil (EVOO) on clini- cal behavior and histopathological features of experimental mam- mary tumors. While a diet rich in n-6 PUFA had a clear stimu- lating effect, the influence of a diet high in EVOO was compati- ble with a potential protective effect [10,16–18]. Such differential influence can be aroused by multiple and complex mechanisms, including body growth and adiposity [19,20]. To get an insight into the effects that diets rich in n-6 PUFA or in EVOO can ex- ert on body mass, in this work we analyzed animal growth data Fig. 1. Experimental designs. Female Sprague-Dawley rats were fed the low-fat con- obtained in six experimental assays, in healthy and carcinogen- trol diet (LF), the high-corn oil diet (HCO) or and the high-extra virgin olive oil diet induced rats undergoing different dietary interventions. For this, (HOO). Healthy animals (noninduced) were fed the experimental diets from wean- the weight data throughout the development of 818 animals be- ing (hLF, hHCO, hHOO groups). Induced animals were fed with the experimental longing to those previous experiments have been compiled and re- diets at different timings: with the LF diet from weaning (LF group); with the HCO diet from weaning (HCOi), after induction (HCOp) or from mid-adulthood (HCOd); analyzed, using adjusted mathematical models. Further, molecular and with the HOO diet form weaning (HOOi), after induction (HOOp), or after a pe- mechanisms associated with such effects have also been investi- riod of intervention with the high corn oil diet (HCO-HOO). Exp: experiment. Exp2 gated, including changes in hypothalamic factors controlling satiety and Exp3 had the same design. and lipid metabolism in adipose and mammary tissue. 2. Material and methods the Universitat Autònoma de Barcelona, following the legislation applicable in this country. 2.1. Diets Different independent experimental series were designed in order to study the effects of the diets, administered at differ- Three semisynthetic diets were designed, a low-fat diet (3.71 ent timing, in healthy and DMBA-induced female Sprague-Dawley kcal/g, with 7.3% calories in the form of fat), and two high-fat di- rats (Charles River Lab.; stain Crl: OFA [SD]; Iffa-Credo -Lyon, ets (4.56 kcal/g, with 39.5% calories in the form of fat). The con- France-). Fig. 1 summarizes the design of each experiment (Exp), trol low-fat diet contained 3% (weight/weight, -w/w-) of corn oil, and Supplementary Fig. 1 compiles and depicts the different ex- the high corn oil diet contained 20% of such oil, and a high olive perimental groups depending on the type and time of dietary oil diet contained 3% of corn oil and 17% of extra virgin olive intervention. All animals arrived at 22–23 days of age, were oil. Carbohydrates (dextrose) were 73% calories (67.9% -w/w-) in housed 3–4 per cage in a controlled environment maintained the low-fat diet, and 40.3% calories (45.9% -w/w-) in both high- 22±2ºC, 50% relative humidity and a 12:12-h light to dark cy- fat diets. Protein, in the form of casein, was 18% -w/w- in the cle. Carcinogen-induced rats were gavaged with one single dose low-fat diet (19.4% calories), and 23% -w/w- in both high-fat diet of 5 mg (experiments 1–5) or 10 mg (experiment -Exp- 6) of (20.2% calories). All diets had 5% -w/w- cellulose, 5.9% -w/w- salt- dimethylbenz(a)anthracene (DMBA, Sigma-Aldrich) at 53 days of mixture and 0.24% -w/w- vitamin mixture. The definition, prepara- age (Fig. 1). The experimental diets and water were administered tion and suitability of the experimental diets were previously de- ad libitum. Animals were euthanized by decapitation (Exp 1–5) scribed [21,22,16]. Diets were prepared weekly and stored under or isoflurane anesthetized and exsanguinated by cardiac puncture nitrogen in the dark at 4ºC (Supplementary Table 1). (Exp6). Experiment 1: Animals were randomly distributed into four ex- 2.2. Animals and experimental designs perimental groups (n=20 each): LF, receiving control diet through- out the experiment; and 3 groups receiving the high corn oil diet, All animals received humane care under the protocols approved from weaning (initiation group, HCOi), from induction with DMBA by the Animal and Human Experimentation Ethics Committee at (promotion group, HCOp), or from day 157 once the tumors had R. Moral, G. Kapravelou, M. Cubedo et al. / Journal of Nutritional Biochemistry 125 (2024) 109549 3 appeared (development group, HCOd). All animals were induced 200µL water/methanol (70:30). Sample ethanolamides were puri- with 5 mg of DMBA (Sigma-Aldrich) and they were euthanized at fied in the DISCOVERY DSC-18LT columns (Sigma-Aldrich), eluted an average age of 214 days [23]. with methanol and speed-vac dried. Ethanolamides quantification Experiment 2 and 3: Animals were distributed into five exper- was performed by UPLC MS/MS at the Servei de Metabolòmica imental groups. Two healthy groups were fed with control (hLF, de la UAT (Vall d’Hebron). In brief, acetonitrile-resuspended sam- n=15 -Exp2-, n=17 -Exp3-) or high corn oil diet (hHCO, n=15) ples were analyzed in duplicates (100µL each) in the Waters Ac- from weaning. Three groups were induced with 5 mg of DMBA and quity UPLC system coupled with a Xevo TQ-S triple quadrupole fed with control diet from weaning (LF, n=20), or the high corn oil mass spectrometer with electrospray ionization interface (Waters) diet from weaning (initiation group, HCOi n=20) or from induction using CORTECS UPLC C18 1.6µm columns (Waters). The mobile (promotion group, HCOp n=20). Rats were euthanized at an aver- phase consisted of 0.1% formic acid milliQ-water (v/v, solvent A) age age of 254 days (Exp2) or 274 days (Exp3). and 0.1 formic acid in acetonitrile (v/v, solvent B) with a flow rate Experiment 4: Animals were distributed into four groups (n=20 of 0.3 mL min−1. Optimized parameters for the mass spectrometer each) and induced with 5 mg of DMBA. All rats received the con- detector were spray voltage, 3 kV; capillary temperature, 270°C; trol diet from weaning to induction. From day 54, they were fed collision energy, 17 V; argon as the collision gas. For calibration with control (LF), with high corn oil (HCOp), or with high olive oil and linearity, six-point concentration standards ranging 0.1–100 diet (HOOp) to sacrifice. HCO-HOO group received the high corn ng/mL OEA (Sigma-Aldrich) and AEA (Cayman) were prepared and oil diet from day 54 to day 80, and the high olive oil diet onwards. analyzed in triplicate. Rats were euthanized at an average age of 261 days [16]. Experiment 5: Animals were distributed into five groups: LF 2.5. Analysis of neuropeptides by luminex assay (n=37) receiving control diet throughout the experiment; two groups receiving the high corn oil, from weaning (HCOi, n=37) For the determination of neuropeptides, total protein ex- or from induction (HCOp, n=29); and two groups receiving the tracts from hypothalamus were obtained by tissue lysis in 20mM high olive oil diet, from weaning (HOOi, n=37) or from induction NaHPO4-Na2PO4 pH7.8, 100mM NaCl, 1% Triton X-100, 0.1% (HOOp, n=29). All animals were induced with 5 mg of DMBA. An- sodium dodecyl sulphate, 0.025% sodium azide, 0.5% Na Deoxy- imals were euthanized at several ages of development (36, 51, 100 cholate, 1 mM phenylmthanesulfonyl fluoride, 5mM NaF, 0.1 mM days), and at the end of the experiment (average age 246 days) Na3VO4 and a protease inhibitor cocktail (Sigma-Aldrich), and [24]. centrifugation at 14,0 0 0g. Peptides (orexin A, substance P, alpha- Experiment 6: Animals were distributed into six groups. Three MSH, oxytocin and neurotensin) were analyzed using the Lu- healthy groups were fed throughout the study with the control minexMAP technology (Milliplex Map Rat/Mouse Neuropeptide (hLF, n=40), the high corn oil (hHCO, n=40) or the high olive oil Magnetic Bead Panel - Neuroscience Multiplex Assay, Millipore). (hHOO, n=40) diet. In parallel, three groups of animals induced Protein extracts were precipitated with acetonitrile, dried and re- with 10 mg of DMBA were also fed from weaning with control (LF, constituted in buffer assay. Samples were run in duplicated. Pep- n=75), the high corn oil (HCO, n=75) or the high olive oil (HOO, tide quantification was performed by immunoassay on the surface n=75) diet. Animals were euthanized at different ages of develop- of fluorescent-coated magnetic beads, following manufacturer’s in- ment, and at the end of the experiment (average age 150 days). structions. Plate was run on MAGPIX with xPONENT software, In all the experiments, 1–2 days before induction, all animals and peptide concentration in samples was calculated by analyzing were fed the control diet so that the type of diet did not in- the median fluorescent intensity data using a 5-parameter logistic fluence the absorption of the carcinogen. Animal monitoring and method. body weight measurement was performed at least once a week. The day of the euthanasia the animals were examined, weighted 2.6. Western blot and body length (nose-to-anus) were measured. The phase of the estrous cycle was also determined by vaginal cytology, and ani- Total protein extracts were obtained from abdominal adipose mals in the diestrus phase were chosen for euthanasia. Depending tissue (visceral fat, perirenal-retroperitoneal white adipose tissue) on the experiment, after sacrifice different organs (hypothalamus, abdominal mammary gland, and hypothalamus. Tissues were ho- small intestine, adipose tissue, mammary gland, in addition to the mogenized in 50mM Tris HCl pH 8.0, 150mM NaCl, 1% Igepal, 0.5% mammary tumors) were collected and flash frozen and stored at deoxycholic acid, 1% sodium dodecyl sulfate and 10 µl/ml pro- -80ºC for the molecular analyses, while blood was centrifuged and tease inhibitor cocktail (Sigma-Aldrich). Determination of relative the plasma was stored at -20ºC. protein levels was performed by Western blot analyses. Briefly, samples were subjected to SDS-PAGE on 7.5–12% acrylamide Mini- Protean TGX Stain Free Gels and electrophoretically transferred to 2.3. Body weight and mass nitrocellulose membranes using Trans-Blot Turbo Transfer system (Bio-Rad). The primary antibodies and the dilutions used were: Growth studies in each experiment were performed from anti-NPY (Cell Signaling, 1:10 0 0), antiphospho PKA (Cell Signal- weekly body weight measurements throughout the assays. On days ing, 1:10 0 0), anti-ATGL (Thermofisher Scientific, 1:150 0 0), anti-HSL of euthanasia body mass was determined using the Lee Index (Thermofisher Scientific, 1:50 0 0), anti-MGL (Abcam, 1:50 0 0). Anti- [10 0 0 0 × (g)⅓ / cm] [25,26] and the Body Mass Index (BMI) [g Cidec (Thermofisher Scientific, 1:10 0 0), anti-Plin (Abcam 1:30 0 0), / cm2]. anti-Fas (Cell Signaling, 1:10 0 0 0). Membranes were incubated with the secondary peroxidase-conjugated antibodies (Sigma, 1:30 0 0 di- 2.4. Ethanolamides quantification by UPLC MS/MS lution) and Luminata Forte Western HRP Substrate (EMD Milli- pore) luminogen. The specific bands were visualized by Chemi- Samples (proximal and distal small intestine) were pre- DocTM XRS+ Imaging system using Image Lab Software 5.1 Beta pared by homogenization of 50 mg of tissue in 1 mL cold (BioRad Laboratories, Hercules, CA, USA). Densitometric values methanol/acetonitrile (1:1) with 5ng deuterated standards (OEA- were first normalized to total protein loaded, and then to an in- d4 and AEA-d8, Cayman) and centrifugation at 10,0 0 0 g. Super- ternal control of pooled samples loaded in duplicate in all the natant was separated, speed-vac desiccated and resuspended in blots. 4 R. Moral, G. Kapravelou, M. Cubedo et al. / Journal of Nutritional Biochemistry 125 (2024) 109549 Fig. 2. Body weight over time in each experiment. Values represent medians of the groups. Data was analyzed with Liniar Mixed-effects Model methodology. The arrows cover the time periods in which differences between groups were found. The groups with significant differences are indicated within the arrows. Exp: experiment. 2.7. Statistical analysis cance considered throughout the study was 0.05 (P-value <.05). The statistical analysis has been carried out with the free soft- Body weight was analyzed using Linear Mixed-effects Model ware R-4.2.1 for Windows and the LME function of the NLME (LMM) methodology. Data was analyzed in several lifespan peri- package [27]. ods with different dietary interventions (before induction, after in- All other analyses (body mass, molecular results) were per- duction, throughout the experiment). LMM considers the fixed ef- formed with the SPSS software (version 15.0). Data distribution fects (common behavior of each group or diet), the random ef- of each variable studied was determined by Kolmogorov-Smirnov fects (effect of each individual) and that each individual has dif- test, and the equality of variances among groups was determined ferent weight measurements over time (repeated measurements). by Levene’s test. Analysis of parametric quantitative data was For each experiment and period studied, LMM has been used to fit performed with ANOVA followed by Tukey’s test. Nonparametric the mathematical model best fitting weight curves values, based quantitative data was analyzed with the Friedman and the Mann- on the evolution of the weight of the individuals over time or Withney’s U test. Qualitative data was analyzed with the Pearson’s on a function of time (transformation used in order to normal- chi-squared test. Correlations were carried out through Kendall’s ize the residuals of the model). With this methodology it is pos- Tau-b and Spearman’s Rho tests. Differences were considered sig- sible to incorporate the autocorrelation structure of the residu- nificant when P-value <.05. als, allowing, in a simple way, to correct heteroscedasticity prob- lems of the model by incorporating an adequate variance. Once 3. Results two or more models have been fitted, to choose the best one we performed the likelihood ratio test to compare nested mod- 3.1. The effect of high-fat diets on body weight depends of type of fat, els two by two. When the models were not nested, the compar- developmental period, time of dietary intervention, and health status ison was made using the Akaike’s Information Criterion (AIC). Af- ter obtaining the best model, we performed analyses to determine Body weight over time of the animals fed the experimental di- if the fixed factor “diet” has a significative effect with an ANOVA ets at different timing in each experiment is shown in Fig. 2. More- test and a Tukey multiple comparisons test. The level of signifi- over, the evolution of each high-fat diet group compared to LF R. Moral, G. Kapravelou, M. Cubedo et al. / Journal of Nutritional Biochemistry 125 (2024) 109549 5 Fig. 3. Body weight change relative to the low-fat group. Body weight change in healthy animals fed the high fat diets throughout the experiment (A). Body weight change in animals induced with 5 mg of carcinogen (B) or 10 mg of carcinogen (C) and fed the high-fat diets throughout the experiment. Body weight change in animals fed the high-fat diets after DMBA-induction (D). Data represent mean+SEM. control is depicted in Supplementary Fig. 2 (healthy animals) and body weight gain was also observed in the animals fed the HCO Supplementary Fig. 3 (carcinogen-induced animals). Analyses were diet than in those fed the high EVOO diet (Exp4). performed comparing groups in different periods (before induction, The results of body weight and body mass indexes (Lee In- after induction, and all lifespan, Supplementary Table 1). Results dex and BMI) at the end of the experiments are shown in Fig. 4. showed differences in body weight depending on the type of diet, We found increased body mass indexes by effect of the HCO diet time of dietary intervention, and health status. Thus, at prepubertal when it was administered from weaning, marginally significant in and pubertal periods (from weaning to induction at 53 days), the healthy animals from Exp2 (hHCO vs. hLF), but more evident in HCO diet, compared to the LF diet, increased body weight in most carcinogen-induced animals (Exp1, Exp2, Exp3, Exp5; HCOi vs. LF). experiments (all assays except Exp1), while the effect of the high EVOO diet was smaller (increased body weight in Exp5 but not in 3.2. Intestinal levels of ethanolamides Exp6). When the long-term diet intake was analyzed (from induc- tion onwards or all lifespan), the two high-fat diets showed more The levels of N-arachidonoyl ethanolamide (AEA) and oleaoyl clearly a differential effect. In healthy animals, body weights from ethanolamide (OEA), potential mediators of the effects of dietary the hHCO group were higher than those from control hLF (Exp2, lipids on body weight control, were determined in the small intes- Exp6) and hHOO group (Exp6). Similar effects were observed in tine (proximal and distal thirds) of healthy rats from Exp6. the animals induced with 5 mg of DMBA, in which dietary inter- The diet high in EVOO increased the OEA levels in proximal and vention with the high corn oil diet from weaning (HCOi group) distal small intestine, especially compared with the high n-6 PUFA resulted in increased body weights when compared with low-fat diet (statistically significant at all ages except at 100 days), and also and with high EVOO diets. In some experiments we also observed in relation to the control group at 52 days of age (Fig. 5A). Com- this effect if the high corn oil diet was administered after induc- parison of curves confirmed the different OEA levels by effect of tion (HCOp group; Exp1, Exp4). In the animals induced with a diets in proximal small intestine (lower in hHCO, intermedium in higher dose of DMBA (10 mg, Exp6) no effect of the high-fat diets control, higher in hHOO), and in distal small intestine (lower val- on body weight was observed. On the other hand, the long-term ues in hHCO). On the other hand, no differences were found in the high EVOO diet intake did not increase body weight (Exp4, Exp5, AEA levels (Fig. 5B). Exp6). Comparison of the two isocaloric high-fat diets also showed a differential effect on body weight evolution, especially in the long- 3.3. Hypothalamic levels of orexigenic and anorexigenic factors term, in the three experiments including both diets (Exp4, Exp5, Exp6, Fig. 2). Fig. 3 shows the body weight change relative to the The levels of the orexigenic factors Orexin A, Susbtance P and low-fat group. In healthy animals, the weight change was clearly NPY (Fig. 6A), and the anorexigenic factors Oxytocin, Neurotensin higher in the animals fed with the HCO diet, but not in those fed and alpha-MSH P (Fig. 6B) were determined in the hypothalamus with the isocaloric high EVOO diet (Exp6). A similar profile was of healthy animals from Exp6. Results showed variable levels de- obtained in animals induced with 5 mg of DMBA (Exp5), but there pending on dietary intervention and age. Some statistical differ- were no differences in body weight with a stronger carcinogenic ences were found in the levels of orexigenic factors, indicating that insult (10 mg of DMBA, Exp6). Although less evident, when the the high EVOO diet decreased the Substance P by 58 days and diet was administered after induction with 5 mg of DMBA, greater NPY by 150 days (Fig. 6A). Regarding the anorexigenic peptides, the 6 R. Moral, G. Kapravelou, M. Cubedo et al. / Journal of Nutritional Biochemistry 125 (2024) 109549 Fig. 4. Body weight and body mass indexes (Lee index and BMI) at the end of the experiments. Mean+SEM, solid lines connecting groups represent statistically significant differences (P<.05), dotted lines represent differences close to significance (P<.1), Tukey test. EVOO diet increased Oxytocin by 100 days, while the HCO diet de- hHOO), and Plin1 was increased in both induced groups fed high- creased neurotensin by 52 and 58 days of age. The effect of the fat diet (HCOi vs. hHCO; HOOi vs. hHOO). EVOO diet on alpha-MSH depended on age (Fig. 6B). Finally, the lipogenesis main protein (FAS, Fig. 7C) was de- creased by the effect of the EVOO diet in healthy animals. More- 3.4. Lipolysis, lipid deposit, and lipogenesis in abdominal adipose and over, cancer disease upregulated FAS in the animals fed the control mammary tissues diet. Thus, the FAS levels were increased in LF group in compari- son to the noninduced control group, and also in comparison to Levels of key proteins controlling lipolysis (phospo-PKA, ATGL, the induced high-fat diet groups. HSL, and MGL), lipid deposit (Cidec, Plin1, and the ratio Fig. 8A depicts the results of lipolysis proteins in mammary pPKA/Plin1) and lipogenesis (FAS) were analyzed in abdominal adi- gland. No effects of diets were found in healthy animals. In in- pose tissue (Fig. 7) and in mammary glands (Fig. 8) from Exp6. duced animals, the high EVOO diet increased the levels of MGL. In adipose tissue of healthy animals, the high EVOO diet in- Comparison of healthy and induced groups showed an effect of the creased most of the lipolysis proteins (marginally for pPKA, signif- disease decreasing in general the levels of these enzymes, except icantly for HSL and MGL). In induced animals, the EVOO diet also for ATGL. In relation to the deposit proteins (Fig. 8B), no differ- increased the levels of MGL. Moreover, the comparison of healthy ences were observed by the effect of diet in the healthy groups, and induced animals showed an effect of the cancer disease in- and we found an increase in Cidec by the EVOO diet in induced creasing the levels of these lipolysis proteins (significantly for pPKA animals. On the other hand, comparison of healthy and induced in control group, for ATGL and HSL in HCOi group, and for MGL in groups indicated a decrease in Cidec (HCOi) and Plin1 (all groups), HOOi group) (Fig. 7A). while an increase in the ratio pPKA/Plin1 (all groups) by effect of The levels of deposit proteins in adipose tissue (Fig. 7B) did not the disease. Regarding the lipogenesis (Fig. 8C), FAS was decreased change by the effect of diets in healthy animals, while Cidec, and by the EVOO diet in healthy animals, and by both high-fat diets in Plin1 were increased in the induced HOOi group. Cidec was also induced animals. FAS was also decreased in HCOi by effect of the increased by the effect of the disease in the EVOO group (HOOi vs disease. R. Moral, G. Kapravelou, M. Cubedo et al. / Journal of Nutritional Biochemistry 125 (2024) 109549 7 Fig. 5. Ethanothanolamides in proximal and distal small intestine at different ages. A: Levels of oleaoyl ethanolamide (OEA). B: Levels of N-arachidonoyl ethanolamide (AEA). Mean+SEM, ∗ : P<.05 hHOO vs. hHCO; ‡: P<.05 hHOO vs. LF; §: P<.05 LF vs hHCO, Tukey test (n=5 / group / age, Exp6). Curves with different letters indicate significantly different evolution. Fig. 6. Neuropeptides in hipothalamus. A: Levels of orexigenic factors determined by luminex (orexin A, substance P -pg/mL-) or Western blot (NPY -relative levels-). B: Levels of anorexigenic factors determined by Luminex (pg/mL). Mean+SEM, ∗ : P<.05 hHOO vs. hHCO; ‡: P<.05 hHOO vs LF; §: P<.05 LF vs. hHCO, Tukey test (n=5 / group / age, Exp6). 8 R. Moral, G. Kapravelou, M. Cubedo et al. / Journal of Nutritional Biochemistry 125 (2024) 109549 Fig. 7. Lipolysis, lipid deposit, and lipogenesis in abdominal adipose tissue. A: Relative levels of the lipolysis proteins phospho-PKA, ATGL, HSL, and MGL. B: Lipid deposit proteins (Cidec, Plin1) and ratio phospho-PKA/Plin1. C: Relative levels of the lipogenesis protein Fas. Boxplots (median, box 25 and 75 percentile, whisker 10 and 90 percentile); cross (mean). Blue background frame the healthy groups, yellow background frame the DMBA-induced groups. Solid black and red lines connecting groups indicate statistically significant differences (P<.05), dashed lines indicate differences close to significance (P<.1), Tukey test (n=5 / group / age, Exp6). Black lines indicate comparisons between diet groups, red lines indicate comparisons between healthy and DMBA-induced groups. Fig. 8. Lipolysis, lipid deposit and lipogenesis in abdominal mammary gland. A: Relative levels of the lipolysis proteins phospho-PKA, ATGL, HSL, and MGL. B: Lipid deposit proteins (Cidec, Plin1) and ratio phospho-PKA/Plin1. C: Relative levels of the lipogenesis protein Fas. Boxplots (median, box 25 and 75 percentile, whisker 10 and 90 percentile); cross (mean). Blue background frame the healthy groups, yellow background frame the DMBA-induced groups. Solid black and red lines connecting groups indicate statistically significant differences (P<.05), dashed lines indicate differences close to significance (P<.1), Tukey test (n=5 / group / age, Exp6). Black lines indicate comparisons between diet groups, red lines indicate comparisons between healthy and DMBA-induced groups. R. Moral, G. Kapravelou, M. Cubedo et al. / Journal of Nutritional Biochemistry 125 (2024) 109549 9 4. Discussion ethanalamide (AEA) are bioactive molecules derived from fatty acid precursors. OEA is formed from oleic acid and has been proposed In this work we have demonstrated, both in healthy and tumor- as a gut-derived satiety factor involved in lipid oxidation and lipol- bearing animals, a different effect of two isocaloric high fat diets ysis through activation of PPAR-alpha [40]. AEA is derived from the on body weight, with a diet high in corn oil (rich in n-6 PUFA) in- n-6 PUFA arachidonic acid. Here we have observed that the diet creasing body weight and mass, while a diet high in EVOO (rich in high in EVOO (rich in oleic acid) increased the levels of OEA in MUFA and minor bioactive compounds) had no effect. These diets small intestine, while no differences where observed in AEA levels. were not designed to cause obesity, but to demonstrate the specific In hamsters, diets rich in olive oil increased OEA and decreased effects of each lipid, not calories, in promoting experimental car- food intake [41]. Thus, we next investigated hypothalamic factors cinogenesis [21,22]. The percentage of oil used in these isocaloric controlling food intake, including orexigenic (orexin A, substance high-fat diets was 20% (w/w, which corresponds to 39.5% calo- P, NPY) and anorexigenic (oxytocin, neurotensin, alpha-MSH) fac- ries from fat), a percentage similar to human Western dietary pat- tors [42] in healthy animals at several ages. Heterogenous levels terns [28], while diet induced-obesity animal models usually pro- of these neuropeptides were found, with few differences that were vide 45–60% of calories in the form of fat [29]. in line with a decrease in the orexigenic peptides by effect of the The effects of the diets have been tested at different peri- high EVOO diet (substance P, NPY) and a decrease in neurotensin ods of development, highlighting their influence after both short (anorexigenic) by effect of the HCO diet. The high EVOO diet also and chronic dietary intervention. The HCO diet administered from increased the anorexigenic factor oxytocin by 100 days, concordant weaning significantly increased body weight and mass in healthy with previous results [20]. In the literature few studies have ad- animals and in those induced with low doses (5 mg) of DMBA. dressed the effects of this oils in the hypothalamic control of food The different effect of both high-fat diets was more evident af- intake. In this sense, in mice, a diet rich in olive oil increased the ter chronic administration, but it was also observed after short- hypothalamic expression of POMC and CART, neuropeptides con- term administration. Thus, body weight evolution from weaning trolling satiety and energy expenditure [43]. to induction, which corresponds to the puberty development, was On the other hand, we analyzed the effects of these diets on the clearly increased in the animals fed with the HCO diet, while a levels of key proteins in lipid mobilization, deposition, and synthe- slighter effect was observed by the high EVOO diet. The stronger sis processes in visceral abdominal adipose tissue and mammary effect of the HCO diet at early ages of development, though may gland. Complete hydrolysis of triacylglycerides to fatty acids re- be quantitatively small, is of great relevance since it is associated quires the consecutive action of three enzymes: adipose triglyc- to an advance in the puberty onset, as we previously observed eride lipase (ATGL), hormone-sensitive lipase (HSL), and monoa- [19]. Obesity and early pubertal maturation are known risk fac- cylglycerol lipase (MGL). Acute lipolytic response is controlled by tors of human breast cancer [7,30], and our results suggested that PKA. In relation to fat deposition, Perilipin 1 (Plin1) restricts ac- this influence on body weight and maturation are mechanisms un- cess of lipases to lipid droplets, while CIDEC (or fat-specific protein derlying the stronger stimulatory effect of the high corn oil diet, 27, FSP27), enhances triacylglyceride deposition and lipid droplet in comparison to the high EVOO diet, in mammary carcinogene- size. Finally, fatty acid synthase (FAS) catalyzes the de novo syn- sis [24,31,32]. Obesity has been linked with chronic systemic low- thesis of fatty acids [44,45]. Our results in abdominal adipose tis- grade inflammation, driving immune dysfunctions and increased sue and in mammary tissue suggest different scenarios due to the proinflammatory cytokines such as IL-1 [30]. In this sense, al- effect of the diets in health, the effect of the cancer disease, and though the animals were not obese, we have previously reported the effect of dietary lipids in tumor-bearing animals. The results that the HCO diet increased leptin and IL1 plasma levels, while the in adipose tissue of healthy groups suggested greater lipolysis due high EVOO diet increased infiltration of cytotoxic T lymphocytes to the influence of the EVOO diet (higher levels of pPKA, HSL, and in tumors [20,24,31]. Other authors have also reported that olive MGL), while no effect on deposition markers (CIDEC, Plin1). The oil has a beneficial effect in maintaining body weight in models EVOO diet also decreased the lipogenic enzyme FAS. On the other of obesity in rodents [33–35], but there is little evidence of such hand, when comparing the healthy and carcinogen-induced control effects on tumor-bearing animals. groups, cancer disease enhanced both lipolysis (increased ATGL) The effect of the high corn oil diet on body weight also de- and lipogenesis (clear increase of FAS). This effect could be related pended on the health status. It is well known that several hu- to the supply of fatty acids to the tumor, which could send sig- man and experimental cancers elicit a systemic state of the disease nals to adipose tissue to release fatty acids [36]. The effect of high- with loss of adipose tissue and skeletal muscle, inducing inflamma- fat diets in carcinogen-induced animals did not show clear trends, tion and energy imbalance that cause the body to mobilize local since we observed upregulation of both lipolysis (MGL) and fat de- and systemic deposits to maintain tumor growth [36,37]. Concor- position (Plin1 and CICEC) proteins by the high EVOO diet. dant with this, in animals induced with higher doses of carcinogen In mammary gland, the study of these proteins showed hetero- (Exp6), in which we observed a more aggressive manifestation of geneous results. This gland is a complex tissue, formed mainly by the disease (especially in the group fed the HCO diet), no differ- adipose tissue, but with many other cell types such as epithelial ences in body weight were found [38]. On the other hand, with (ductal and alveolar) and myoepithelial cells. The greatest differ- the low dose of carcinogen, the long-term effect of the HCO diet ences found have been due to cancer disease, that decreased the was mainly observed if dietary intervention started at early ages expression of most of the proteins studied. In this sense, Plin1 was (HCOi groups), but some experiments also indicated an effect if the down-regulated in all induced groups, which is in agreement with administration started after induction (HCOp groups). Interestingly, published results in humans [46]. Moreover, the higher ratio acti- in Exp5, in which the HCOp showed no differences in body weight vated PKA (pPKA) / Plin1 suggested an increased lipid mobilization with the control group, HCOp tumors displayed the most aggres- in all groups, concordant with a generalized upregulation of lipol- sive degree of malignancy [39]. ysis induced by the disease [36]. Several mechanisms could be at the basis of the different effects Taken together, our results suggest that EVOO has a benefi- of high-fat diets on body weight, such as control of food intake cial effect on body weight maintenance in both health and can- and fat deposition. Oleoyl ethanolamide (OEA) has been suggested cer. In healthy animals, and despite being administered in high as a mediator of the beneficial influence of olive oil on body com- quantities, this oil could have effects on satiety control (e.g., more position [40]. OEA and other ethanolamides such as arachidonoyl anorexigenic hypothalamic neuropeptides such as Oxytocin), and 10 R. Moral, G. Kapravelou, M. Cubedo et al. / Journal of Nutritional Biochemistry 125 (2024) 109549 on lipolysis/deposition balance facilitating the former (increased CF611.084); “Agencia para el Aceite de Oliva del Ministerio de Agri- pPKA, HSL, MGL, decreased FAS) in abdominal adipose tissue. In cultura, Alimentación y Medio Ambiente” (AAO2008-165.471); “Or- animals with cancer, body weight would be influenced by the ganización Interprofesional del Aceite de Oliva Español (OIAOE)” severity of the disease. High-fat diets could decrease the need to (OIP2009-CD165.646), “Departaments de Salut i d’Agricultura, Al- mobilize depots by providing dietary lipids. The beneficial effect of imentació i Acció Rural de la Generalitat de Catalunya” (GC2010- olive oil on body weight can be due to both its high oleic acid con- 165.0 0 0), FPCO and OIAOE (FPCO-OIP2016-CF614.087). tent and its minor bioactive compounds. EVOO minor compounds such as polyphenols has shown to induce reduced body weight Acknowledgments gain in several experimental models [47–49], and in vitro elicit ac- tivation of the AMPK-ATGL-HSL pathway and suppression of FAS The sponsors had no role in the study design, data collection, [50–52]. In agreement with this, our results showed a clear effect and analysis and interpretation of the results. The authors want to of the EVOO-rich diet in the downregulation of FAS in healthy and thank all the investigators and staff involved in animal care and tumor bearing animals, both in adipose and mammary tissue. research. Finally, the present study has several strengths, mainly related to the large number of data used (from six experiments involving a total of 818 animals) and the long term of dietary intervention Supplementary materials (up to 8 months). Furthermore, there is very little data in the lit- erature on the effect of these diets in general, and the olive oil Supplementary material associated with this article can be diet in particular, on a disease such as breast cancer, tightly linked found, in the online version, at doi:10.1016/j.jnutbio.2023.109549. with body mass and obesity. However, these results have several limitations. This cohort is a compilation of independent cohorts References with their own hypothesis, although all experiments shared a main variable (the experimental diets) and outcome (body weight). Fur- [1] Bach-Faig A, Berry EM, Lairon D, Reguant J, Trichopoulou A, Dernini S, et al. thermore, between different experiments there may be some vari- Mediterranean diet pyramid today. Science and cultural updates. Public Health Nutr 2011;14:2274–84. doi:10.1017/S1368980011002515. ations in significances due to methodological issues, specific com- [2] Dominguez LJ, Di Bella G, Veronese N, Barbagallo M. Impact of mediter- position of the oils, or even the genetic drift of the strain. The as- ranean diet on chronic non-communicable diseases and longevity. Nutrients says were designed with special care to avoid stress, so the animals 2021;13:2028. doi:10.3390/nu13062028. [3] Koliaki C, Dalamaga M, Liatis S. Update on the obesity epidemic: is the sharp have not been housed individually and individual consumption is rise of the evil empire truly levelling off? Curr Obes Rep 2023;42:600–8. not known, nor have fasting samples been obtained. Moreover, the doi:10.1007/s13679- 023- 00527- y. number of samples for molecular studies is small. Even so, the [4] Romaguera D, Norat T, Vergnaud A-C, Mouw T, May AM, Agudo A, et al. data set demonstrates a different effect of the two isocaloric diets Mediterranean dietary patterns and prospective weight change in participants of the EPIC-PANACEA project. Am J Clin Nutr 2010;92:912–21. doi:10.3945/ and suggests different pathways and mechanisms of action of a po- ajcn.2010.29482. tential protective effect of EVOO against obesity. Thus, the results [5] Agnoli C, Sieri S, Ricceri F, Giraudo MT, Masala G, Assedi M, et al. Adher- obtained provide evidence for a qualitative effect linking dietary ence to a Mediterranean diet and long-term changes in weight and waist cir- cumference in the EPIC-Italy cohort. Nutr Diabetes 2018;8:22. doi:10.1038/ lipids and body weight beyond the amount of fat consumed. More- s41387- 018- 0023- 3. over, dietary factors are important at all ages, but there are criti- [6] López-Gil JF, García-Hermoso A, Sotos-Prieto M, Cavero-Redondo I, Martínez- cal periods of development that may be relevant for future health Vizcaíno V, Kales SN. Mediterranean diet-based interventions to improve an- thropometric and obesity indicators in children and adolescents: a system- risks. In this regard, our results also show a short-term effect of di- atic review with meta-analysis of randomized controlled trials. Adv Nutr ets, especially at early ages, with a plausible impact on growth and 2023;14:858–69. doi:10.1016/j.advnut.2023.04.011. pubertal development, and a long-term effect, increasing the risk [7] World Cancer Research Fund/American Institute for Cancer Research. Diet nu- trition, physical activity and cancer: a global perspectiveContinuous Update of chronic diseases. Although caution should be exercised when ex- Project Expert Report 2018. Washington DC: World Cancer Research Fund In- trapolating from experimental data, our investigations support that ternational; 2018. EVOO, in the context of a healthy diet from early ages and with [8] Siegel RL, Miller KD, Wagle NS, Jemal A. Cancer statistics, 2023. CA Cancer J Clin 2023;73:17–48. doi:10.3322/caac.21763. a moderate intake, is a healthy choice in relation to obesity and [9] Sung H, Ferlay J, Siegel RL, Laversanne M, Soerjomataram I, Jemal A, et al. breast cancer risk. Global cancer statistics 2020: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA Cancer J Clin 2021;71:209–49. Declaration of competing interests doi:10.3322/caac.21660. [10] Escrich E, Solanas M, Moral R, Escrich R. Modulatory effects and molecular mechanisms of olive oil and other dietary lipids in breast cancer. Curr Pharm No conflicts of interest. Des 2011;17:813–30. doi:10.2174/138161211795428902. [11] van den Brandt PA, Schulpen M. Mediterranean diet adherence and risk of postmenopausal breast cancer: results of a cohort study and meta-analysis. Int CRediT authorship contribution statement J Cancer 2017;140:2220–31. doi:10.1002/ijc.30654. [12] Schwingshackl L, Schwedhelm C, Galbete C, Hoffmann G. Adherence to Raquel Moral: Conceptualization, Supervision, Formal analysis, Mediterranean diet and risk of cancer: an updated systematic review and meta-analysis. Nutrients 2017;9:1063. doi:10.3390/nu9101063. Writing – original draft, Writing – review & editing. Garyfallia [13] Buckland G, Travier N, Agudo A, Fonseca-Nunes A, Navarro C, Lagiou P, et al. Kapravelou: Investigation, Formal analysis, Writing – review & Olive oil intake and breast cancer risk in the Mediterranean countries of the editing. Marta Cubedo: Formal analysis, Writing – review & edit- European Prospective Investigation into Cancer and Nutrition study. Int J Can- cer 2012;131:2465–9. doi:10.1002/ijc.27516. ing. Montserrat Solanas: Formal analysis, Writing – review & edit- [14] Pang Y, Wei Y, Kartsonaki C. Associations of adiposity and weight change ing. Eduard Escrich: Conceptualization, Funding acquisition, Super- with recurrence and survival in breast cancer patients: a systematic vision, Writing – review & editing. review and meta-analysis. Breast Cancer 2022;29:575–88. doi:10.1007/ s12282- 022- 01355- z. [15] Escrich E. Mammary cancer model induced by dimethylbenz(a)anthracene: a Funding good experimental tool for the study of tumour markers. Int J Biol Markers 1987;2:109–19. This work was supported by grants from “Plan Nacional de [16] Solanas M, Hurtado A, Costa I, Moral R, Menéndez JA, Colomer R, et al. Effects of a high olive oil diet on the clinical behavior and histopathological features I+D+I” (AGL2006-07691; AGL2011-24778); “Fundación Patrimo- of rat DMBA-induced mammary tumors compared with a high corn oil diet. nio Comunal Olivarero (FPCO)” (FPCO2008-165.396; FPCO2013- Int J Oncol 2002;21:745–53. R. Moral, G. Kapravelou, M. Cubedo et al. / Journal of Nutritional Biochemistry 125 (2024) 109549 11 [17] Costa I, Moral R, Solanas M, Escrich E. High-fat corn oil diet promotes in high fat diet-induced obese LDLr−/−.Leiden mice without attenuation of the development of high histologic grade rat DMBA-induced mammary ade- steatohepatitis. Sci Rep 2021;11:8250. doi:10.1038/s41598- 021- 87761- 3. nocarcinomas, while high olive oil diet does not. Breast Cancer Res Treat [36] Rybinska I, Agresti R, Trapani A, Tagliabue E, Triulzi T. Adipocytes in breast 2004;86:225–35. doi:10.1023/B:BREA.0000036896.75548.0c. cancer, the thick and the thin. Cells 2020;9:560. doi:10.3390/cells9030560. [18] Moral R, Escrich E. Influence of olive oil and its components on [37] Feng J, Zheng T, Hou Z, Lv C, Xue A, Han T, et al. Luteolin, an aryl hydrocarbon breast cancer: molecular mechanisms. Molecules 2022;27:477. doi:10.3390/ receptor ligand, suppresses tumor metastasis in vitro and in vivo. Oncol Rep molecules27020477. 2020;44:2231–40. doi:10.3892/or.2020.7781. [19] Moral R, Escrich R, Solanas M, Vela E, Costa I, Ruiz de Villa MC, et al. Diets high [38] Manzanares MÁ, de Miguel C, Ruiz de Villa MC, Santella RM, Escrich E, in corn oil or extra-virgin olive oil provided from weaning advance sexual mat- Solanas M. Dietary lipids differentially modulate the initiation of exper- uration and differentially modify susceptibility to mammary carcinogenesis in imental breast carcinogenesis through their influence on hepatic xenobi- female rats. Nutr Cancer 2011;63:410–20. doi:10.1080/01635581.2011.535956. otic metabolism and DNA damage in the mammary gland. J Nutr Biochem [20] Sospedra I, Moral R, Escrich R, Solanas M, Vela E, Escrich E. Effect of high fat 2017;43:68–77. doi:10.1016/j.jnutbio.2017.01.016. diets on body mass, oleylethanolamide plasma levels and oxytocin expression [39] Garcia-Guasch M, Medrano M, Costa I, Vela E, Grau M, Escrich E, et al. Extra- in growing rats. J Food Sci 2015;80:H1425–31. doi:10.1111/1750-3841.12890. virgin olive oil and its minor compounds influence apoptosis in experimen- [21] Escrich E, Solanas M, Segura R. Experimental diets for the study of lipid in- tal mammary tumors and human breast cancer cell lines. Cancers (Basel) fluence on the induced mammary carcinoma in rats: I–diet definition. In Vivo 2022;14:905. doi:10.3390/cancers14040905. 1994;8:1099–105. [40] Bowen KJ, Kris-Etherton PM, Shearer GC, West SG, Reddivari L, Jones PJH. Oleic [22] Escrich E, Solanas M, Ruiz De Villa MC, Ribalta T, Muntane J, Segura R. Exper- acid-derived oleoylethanolamide: a nutritional science perspective. Prog Lipid imental diets for the study of lipid influence on the induced mammary carci- Res 2017;67:1–15. doi:10.1016/j.plipres.2017.04.001. noma in rats: II–suitability of the diets. In Vivo 1994;8:1107–11. [41] Sihag J, Jones PJH. Dietary fatty acid composition impacts plasma fatty acid [23] Escrich E, Solanas M, Soler M, Ruiz de Villa MC, Sanchez JA, Segura R. Dietary ethanolamide levels and body composition in golden Syrian hamsters. Food polyunsaturated n-6 lipids effects on the growth and fatty acid composition of Funct 2018;9:3351–62. doi:10.1039/c8fo00621k. rat mammary tumors. J Nutr Biochem 2001;12:536–49. [42] Vardanyan GS, Harutyunyan HS, Aghajanov MI, Vardanyan RS. Neurochemical [24] Moral R, Escrich R, Solanas M, Vela E, Ruiz de Villa MC, Escrich E. Diets high regulators of food behavior for pharmacological treatment of obesity: current in corn oil or extra-virgin olive oil differentially modify the gene expression status and future prospects. Future Med Chem 2020;12:1865–84. doi:10.4155/ profile of the mammary gland and influence experimental breast cancer sus- fmc- 2019- 0361. ceptibility. Eur J Nutr 2016;55:1397–409. doi:10.10 07/s0 0394- 015- 0958- 2. [43] Cintra DE, Ropelle ER, Moraes JC, Pauli JR, Morari J, de Souza CT, et al. Unsat- [25] Bernardis LL. Prediction of carcass fat, water and lean body mass from Lee’s urated fatty acids revert diet-induced hypothalamic inflammation in obesity. “nutritive ratio” in rats with hypothalamic obesity. Experientia 1970;26:789– PLoS One 2012;7:e30571. doi:10.1371/journal.pone.0030571. 90. doi:10.1007/BF02232553. [44] Wang C-W. Lipid droplets, lipophagy, and beyond. Biochim Biophys Acta [26] Nascimento AF, Sugizaki MM, Leopoldo AS, Lima-Leopoldo AP, Nogueira CR, 2016;1861:793–805. doi:10.1016/j.bbalip.2015.12.010. Novelli ELB, et al. Misclassification probability as obese or lean in hy- [45] Grahn THM, Kaur R, Yin J, Schweiger M, Sharma VM, Lee M-J, et al. Fat- percaloric and normocaloric diet. Biol Res 2008;41:253–9. doi:10.4067/ specific protein 27 (FSP27) interacts with adipose triglyceride lipase (ATGL) S0716-976020 080 0 030 0 0 02. to regulate lipolysis and insulin sensitivity in human adipocytes. J Biol Chem [27] Pinheiro J, Bates DR Core Team. R package version 2022;3:1–158. https://svn. 2014;289:12029–39. doi:10.1074/jbc.M113.539890. r- project.org/R- packages/trunk/nlme/. [46] Fletcher SJ, Sacca PA, Pistone-Creydt M, Coló FA, Serra MF, Santino FE, et al. [28] Cordain L, Eaton SB, Sebastian A, Mann N, Lindeberg S, Watkins BA, et al. Ori- Human breast adipose tissue: characterization of factors that change during gins and evolution of the Western diet: health implications for the 21st cen- tumor progression in human breast cancer. J Exp Clin Cancer Res 2017;36:26. tury. Am J Clin Nutr 2005;81:341–54. doi:10.1093/ajcn.81.2.341. doi:10.1186/s13046- 017- 0494- 4. [29] de Moura e Dias M, dos Reis SA, da Conceição LL, Sediyama CMN de O, [47] Claro-Cala CM, Quintela JC, Pérez-Montero M, Miñano J, Alvarez de So- Pereira SS, de Oliveira LL, et al. Diet-induced obesity in animal models: tomayor M, Herrera MD, et al. Pomace olive oil concentrated in triterpenic points to consider and influence on metabolic markers. Diabetol Metab Syndr acids restores vascular function, glucose tolerance and obesity progression in 2021;13:32. doi:10.1186/s13098- 021- 00647- 2. mice. Nutrients 2020;12:323. doi:10.3390/nu12020323. [30] Devericks EN, Carson MS, McCullough LE, Coleman MF, Hursting SD. The [48] Li X, Wei T, Li J, Yuan Y, Wu M, Chen F, et al. Tyrosol ameliorates the symp- obesity-breast cancer link: a multidisciplinary perspective. Cancer Metastasis toms of obesity, promotes adipose thermogenesis, and modulates the compo- Rev 2022;41:607–25. doi:10.1007/s10555- 022- 10043- 5. sition of gut microbiota in HFD fed mice. Mol Nutr Food Res 2022;66:2101015. [31] Escrich R, Costa I, Moreno M, Cubedo M, Vela E, Escrich E, et al. A high- doi:10.1002/mnfr.202101015. corn-oil diet strongly stimulates mammary carcinogenesis, while a high-extra- [49] Lombardo GE, Lepore SM, Morittu VM, Arcidiacono B, Colica C, Procopio A, virgin-olive-oil diet has a weak effect, through changes in metabolism, im- et al. Effects of oleacein on high-fat diet-dependent steatosis, weight gain, and mune system function and proliferation/apoptosis pathways. J Nutr Biochem insulin resistance in mice. Front Endocrinol (Lausanne) 2018;9:116. doi:10. 2019;64:218–27. doi:10.1016/j.jnutbio.2018.11.001. 3389/fendo.2018.00116. [32] Solanas M, Grau L, Moral R, Vela E, Escrich R, Escrich E. Dietary olive oil and [50] Pacifici F, Farias CLA, Rea S, Capuani B, Feraco A, Coppola A, et al. Tyrosol may corn oil differentially affect experimental breast cancer through distinct mod- prevent obesity by inhibiting adipogenesis in 3T3-L1 preadipocytes. Oxid Med ulation of the p21Ras signaling and the proliferation-apoptosis balance. Car- Cell Longev 2020;2020:1–12. doi:10.1155/2020/4794780. cinogenesis 2010;31:871–9. doi:10.1093/carcin/bgp243. [51] Lepore Maggisano, Bulotta Mignogna, Arcidiacono Procopio, et al. Oleacein pre- [33] Oi-Kano Y, Kawada T, Watanabe T, Koyama F, Watanabe K, Senbongi R, et al. vents high fat diet-induced adiposity and ameliorates some biochemical pa- Extra virgin olive oil increases uncoupling protein 1 content in brown adipose rameters of insulin sensitivity in mice. Nutrients 2019;11:1829. doi:10.3390/ tissue and enhances noradrenaline and adrenaline secretions in rats. J Nutr nu11081829. Biochem 2007;18:685–92. doi:10.1016/j.jnutbio.2006.11.009. [52] Menendez JA, Vazquez-Martin A, Oliveras-Ferraros C, Garcia-Villalba R, [34] Domínguez-Vías G, Segarra AB, Ramírez-Sánchez M, Prieto I. The role of high Carrasco-Pancorbo A, Fernandez-Gutierrez A, et al. Analyzing effects of extra- fat diets and liver peptidase activity in the development of obesity and insulin virgin olive polyphenols on breast cancer-associated fatty acid synthase pro- resistance in Wistar rats. Nutrients 2020;12:636. doi:10.3390/nu12030636. tein expression using reverse-phase protein microarrays. Int J Mol Med [35] Álvarez-Amor L, Sierra AL, Cárdenas A, López-Bermudo L, López-Beas J, Andú- 20 08;22:433–9. doi:10.3892/ijmm_0 0 0 0 0 039. jar E, et al. Extra virgin olive oil improved body weight and insulin sensitivity