biology Article Short-Term Effects of Early Menopause on Adiposity, Fatty Acids Profile and Insulin Sensitivity of a Swine Model of Female Obesity Ana Heras-Molina 1 , José Luis Pesantez-Pacheco 1,2 , Marta Vazquez-Gomez 3,4 , Consolacion Garcia-Contreras 1 , Susana Astiz 1 , Beatriz Isabel 3 and Antonio Gonzalez-Bulnes 1,3, * 1 Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria (INIA), Ctra. De La Coruña Km. 7,5., 28040 Madrid, Spain; delasheras.ana@inia.es (A.H.-M.); jose.pesantez@ucuenca.edu.ec (J.L.P.-P.); congarcon@gmail.com (C.G.-C.); astiz.susana@inia.es (S.A.) 2 School of Veterinary Medicine and Zootechnics, Faculty of Agricultural Sciences, University of Cuenca, Avda. Doce de Octubre, Cuenca 010220, Ecuador 3 Faculty of Veterinary Sciences, Universidad Complutense de Madrid, 28040 Madrid, Spain; martavazgomez@gmail.com (M.V.-G.); bisabelr@ucm.es (B.I.) 4 Facultat de Veterinària, Universitat Autònoma de Barcelona. Edifici V, Trav. dels Turons, 08193 Bellaterra, Spain * Correspondence: bulnes@inia.es; Tel.: +34-91-347-4022; Fax: +34-347-4014  Received: 11 August 2020; Accepted: 3 September 2020; Published: 11 September 2020  Abstract: Menopause strongly increases incidence and consequences of obesity and non-communicable diseases in women, with recent research suggesting a very early onset of changes in lipid accumulation, dyslipidemia, and insulin resistance. However, there is a lack of adequate preclinical models for its study. The present trial evaluated the usefulness of an alternative method to surgical ovariectomy, the administration of two doses of a GnRH analogue-protein conjugate (Vacsincel® ), for inducing ovarian inactivity in sows used as preclinical models of obesity and menopause. All the sows treated with the compound developed ovarian stoppage after the second dose and, when exposed to obesogenic diets during the following three months, showed changes in the patterns of fat deposition, in the fatty acids profiles at the different tissues and in the plasma concentrations of fructosamine, urea, β-hydroxibutirate, and haptoglobin when compared to obese fed with the same diet but maintaining ovarian activity. Altogether, these results indicate that menopause early augments the deleterious effects induced by overfeeding and obesity on metabolic traits, paving the way for future research on physiopathology of these conditions and possible therapeutic targets using the swine model. Keywords: models; fatty-acids; insulin-resistance; menopause; obesity; swine 1. Introduction Obesity is currently declared a global pandemic according to WHO (https://www.who.int/health- topics/obesity#tab=tab_1 and http://www.who.int/mediacentre/factsheets/fs311/en/index.html) because its worldwide incidence has nearly tripled since 1975 and currently at least 4 million people are dying each year as a direct result of the condition. Moreover, overweight and lifestyle are major risk factors for non-communicable diseases (NCDs) such as metabolic syndrome, cardiovascular disease (CVD), type-2 diabetes (T2D), musculoskeletal disorders, and some cancers. In turns, NCDs are the leading cause of sickness and death for women and men, being collectively responsible for almost 70% of all deaths worldwide (https://www.who.int/health-topics/noncommunicable-diseases#tab=tab_1). Biology 2020, 9, 284; doi:10.3390/biology9090284 www.mdpi.com/journal/biology Biology 2020, 9, 284 2 of 16 Menopause is a critical point for incidence and consequences of obesity and NCDs and, specifically, CVD and T2D. In this way, occurrence and severity of CVD and T2D are lower in premenopausal females than in age-matched males, but dramatically increase after menopause [1–3]. The protection against CVD and T2D in women during childbearing age is believed to be related to ovarian estrogens [4,5]. Estrogens prevent visceral adiposity, insulin resistance and glucose intolerance, regulate lipid and free-fatty acids metabolism, increase angiogenesis and vasodilatation, and decrease oxidative stress [5–8]. Estrogen-based replacement therapies in postmenopausal women reduce the incidence of T2D [9,10], but their role in preventing CVD, and their own security, remains highly controversial [5]. Current data highlights the importance of factors related to the treatment (protocol, route, type of steroids) and, mainly, to the age of the woman and the time of initiation of replacement therapy after menopause [11]. Epidemiological studies indicate that beneficial effects are more frequently found with short-term therapies starting at the early post-menopause [12–14], which suggest a very early onset of changes in the metabolism of glucose, lipids and fatty acids. Therefore, it is necessary to increase the knowledge about the interplay between obesity and menopause and its early effects on women’s health. Interventional research is obviously limited in humans, so preclinical research in animal models is needed. Most of the studies in obesity and menopause have been carried out on laboratory rodents despite the marked differences in metabolism and adipose tissue biology between rodents and humans [15,16]. In this sense, some large animal species offer more profitable characteristics [17]. Among them, pig constitutes a largely known, robust and reliable model for studies on obesity and metabolism, because of omnivorous habits, inclination to sedentarism and metabolic traits similar to humans and mainly because of the high propension for fat deposition of the species [18–21]. The most commonly used swine breeds for research on obesity and metabolic diseases are Göttingen, Yucatan, and Ossabaw Island pigs. All these breeds have a common ancestor, the Iberian pig, which is characterized by a polymorphism of the leptin receptor gene (LEPR). Leptin and LEPR are involved not only in the regulation of feed intake but also in an ample set of physiological actions and are not only modulated by the energy content of the diet but also by other dietary effects [22]. The LEPR polymorphism of the Iberian pig has been related to effects on food intake, body mass, and fat deposition [23] which resemble leptin resistance in humans [24], but other effects need to be investigated. Iberian pigs, likewise humans, develop obesity, insulin resistance and dyslipidemia in case of food excess and sedentarism during juvenile development [25], adulthood [26], and senescence [27]. Aging and inherent menopause in this model, similarly to humans, are related to sarcopenia and a trend for higher adiposity; in the case of overt obesity, there is increased visceral fattening, dyslipidemia, insulin resistance, lipotoxicity in non-adipose tissues and even obesity-induced renal damage [27,28]. The present study, having in mind these previous considerations about possible metabolic changes during menopause transition and early menopause and about the need of adequate animal models, aimed to characterize a large-animal model based on the use of mature Iberian sows with hormonally-induced menopause. Usually, the estrogen withdrawal associated to menopause is experimentally induced in animal models by surgical ovariectomy [29]. However, abrupt loss of ovarian hormones usually causes more drastic symptoms than spontaneous menopause and, excepting surgical menopause, only about 10% of women cease menstruating abruptly and nearly 90% show a prior period of menstrual abnormality [30]. Moreover, cessation of reproductive competence is primarily driven by the ovarian hormones but there is also a pivotal role of the hypothalamus and hypophysis through changes in the secretion and effects of gonadotrophin-releasing hormone (GnRH) and gonadotrophins FSH and LH [31]. Hence, instead of surgical ovariectomy, we used a treatment based on a GnRH analogue-protein conjugate (Vacsincel® , Zoetis, Madrid, Spain) to induce a progressive suppression of the function of the hypothalamus-hypophysis-ovary axis and, thus, of ovarian estrogens secretion. Biology 2020, 9, 284 3 of 16 2. Experimental Section 2.1. Ethics Statement The experiment was performed according to the Spanish Policy for Animal Protection RD1201/05, which meets the European Union Directive 86/609, after being specifically assessed and approved by the INIA Committee of Ethics in Animal Research on 11 June 2012 (ethic code CEEA 2012/012). The sows were housed at the animal facilities of the INIA, which meet local, national and European requirements. 2.2. Animals and Experimental Procedure The experiment involved 13 clinically healthy Iberian sows (5–6 years old). All the sows were maintained in two collective pens, one per experimental group, with individual feeders. Animals were fed during the experimental period (120 days) with an obesogenic diet (6.8% of saturated fat) for inducing obesity [26]; the amount was 4 kg/animal/day for the first 30 days, 5 kg/animal/day for the following 60 days and 6 kg/animal/day for the following 30 days Six sows acted as controls (Group CON) whilst seven sows were used as models for menopause (Group MEN). The sows in the group MEN were treated with two doses of Vacsincel® (Zoetis, Madrid, Spain) at Days 0 and 30 of the study, as per manufacturer’s instructions (2 mL/sow injected subcutaneously just behind and below the base of the ear). In all the sows, body mass and adiposity were measured each 30 days from Day 0 to 120 of the study. Adiposity was evaluated, in terms of subcutaneous back-fat depot at the right-side and at 4 cm from the midline and the head of the last rib, by ultrasonography with a 5–8 MHz lineal-array probe (SonoSite Inc., Bothell, WA, USA). Concomitantly, blood samples were obtained, after around 16 h of fasting, from the orbital sinus using EDTA vacuum tubes (Vacutainer Systems Europe, Becton Dickinson, Meylan Cedex, France). After centrifugation at 1500× g for 15 min, aliquots of plasma were separated and stored at −80 ◦ C. The efficiency of the treatment with Vacsincel® , and therefore induction of ovarian failure, was assessed by determining plasma progesterone concentrations in one of the plasma aliquots. A second aliquot was used for assessment of plasma parameters of metabolic status. At the end of the experimental period (Day 120), all the sows were sequentially euthanized by stunning and exsanguination, in compliance with RD53/2013 standard procedures. Firstly, the ovaries were examined to confirm data from progesterone assays about ovarian activity, by determining the presence or absence of corpora lutea and follicle development. Immediately, samples of subcutaneous and visceral (perirenal) fat, muscle (longissimus dorsi) and left lobe of the liver were collected and stored at −20 ◦ C for assessment of fatty acids composition. 2.3. Assessment of Ovarian Activity The criteria used to determine the decline of ovarian activity was a decrease in progesterone levels under 2.0 ng/mL. Plasma progesterone concentrations were measured in a single analysis using an enzyme immunoassay kit (Demeditec Diagnostics GmbH, Kiel-Wellsee, Germany), as previously described [32]. Assay sensitivity was 0.045 ng/mL and the intra-assay variation coefficient was 5%. The test was validated for its use in sows by comparing the parallelism of the standard curves produced by standards provided in the assay kit and serially diluted porcine plasma samples with known progesterone concentration [33]. 2.4. Evaluation of Metabolic Status The glycemic profile was assessed by determining plasma glucose (GLU) and fructosamine (FRU) concentrations. Lipids metabolism was assessed by determining plasma concentrations of total cholesterol (CHO), high- and low-density lipoproteins cholesterol (HDL-c and LDL-c, respectively), and non-esterified fatty acids (NEFA). Protein metabolism was assessed by determining urea and haptoglobin (HAP). The metabolic state was also assessed through measuring plasma β-hydroxybutyrate (BHB) and lactate (LAC). All metabolites were determined using a clinical Biology 2020, 9, 284 4 of 16 chemistry analyzer (Saturno 300 plus, Crony Instruments s.r.l., Rome, Italy) according to manufacturer’s instructions. 2.5. Evaluation of Fatty Acids Composition Fat content and fatty acids composition were determined, as previously described, in samples obtained immediately after euthanasia from adipose (subcutaneous and visceral fat) and non-adipose tissues (muscle and liver). Intramuscular and liver fat were extracted as described by Segura et al. [34] after lyophilization and homogenization; fat content in each tissue was calculated and expressed as a percentage. The neutral lipid fraction (triglycerides) and the polar lipid fraction (phospholipids) were separated using aminopropyl minicolumns previously activated with 7.5 mL of hexane [35]. Subcutaneous and visceral fat were extracted directly; in the case of back-fat, outer and inner layers were analyzed separately, having in mind that the outer layer is more related to thermoregulation whereas the inner layer is more metabolically active [36]. The fatty acids composition of all tissues was analyzed using gas chromatography [37]. The quantities of individual fatty acids expressed as g/100 g of total fatty acid content were used to calculate the proportions of saturated fatty acids (SFA), monounsaturated fatty acids (MUFA), polyunsaturated fatty acids (PUFA), and total omega-3 and P P P P omega-6 fatty acids ( n3 and n6, respectively), as well as the ratios n6/ n3 and MUFA/SFA and the unsaturation index (UI; calculated as follows: 1 (% monoenoics) + 2 (% dienoics) + 3 (% trienoics) + 4 (% tetraenoics) + 5 (% pentaenoics) + 6 (% hexaenoics)) [38,39]. Furthermore, the desaturation index was used to determine the activity of the stearoyl-CoA desaturase enzyme 1 (SCD1; the ratio of the enzyme product, MUFA mainly oleic acid (C18:1n-9), to the enzyme substrate, SFA mainly stearic acid (C18:0); [40]). Finally, the activities of the desaturase enzymes for n6 and n3 were estimated from the ratios between arachidonic and linoleic acids (C20:4n6/C18:2n6) and eicosapentaenoic and alpha-linolenic acids (C20:5n3/C18:3n3), respectively. ∆9 desaturase activity (D9) was estimated from the ratio between the sum of palmitoleic and oleic acids and the sum of palmitic and stearic acids (C16:1n7 + C18:1n9)/(C16:0 + C18:0). 2.6. Statistical Analysis Effects of ovarian failure on changes in body mass, adiposity, and metabolic status were assessed by ANOVA for repeated measures (split-plot ANOVA). Effects on fatty acids composition were assessed by one-way ANOVA or by a Kruskal–Wallis test if a Levene’s test showed non-homogeneous variables; Duncan’s post-hoc test was performed to contrast the differences among groups. All results were expressed as mean ± S.E.M. Statistical significance was accepted from p < 0.05 and a statistical trend was considered when 0.05 < p < 0.1. 3. Results The assessment of plasma progesterone concentrations showed evidence of ovarian cyclic activity throughout the entire period of study in the control sows (Group CON). Conversely, the group treated with Vacsincel® (Group MEN) clearly evidenced ovarian failure and inactivity; from Day 30 in three of the seven females (42.8%) and from Day 60 in the remaining sows. These results were confirmed by the direct observation of the ovaries at Day 120; the ovaries in the Group MEN showed absence of corpora lutea and follicles larger than 2 mm in size and even a strong reduction in the ovarian size (Figure 1). Biology 2020, 9, 284 5 of 16 Biology 2020, 9, x 5 of 15 A 35 30 Progesterone (ng(ml) 25 20 15 10 5 0 0 30 60 90 120 a B 35 30 b 25 20 15 1 cm 10 5 0 0 30 60 90 120 Days 1. Individual FigureFigure plasma 1. Individual progesterone plasma progesteroneconcentrations concentrationsover over time ofstudy time of study(left (lefthand) hand) and and picture picture of of the ovaries the ovaries at 120 days (right hand) in sows used as controls (A and a) or treated with two doses of of at 120 days (right hand) in sows used as controls (A and a) or treated with two doses Vacsincel ® for inducing ovarian inactivity (B(B and Vacsincel ® for inducing ovarian inactivity b);arrows andb); arrows indicate timingofoftreatment). indicate timing treatment). 3.1. Changes in Body 3.1. Changes MassMass in Body and and Adiposity Adiposity All theAllsows the fed with sows fedthe obesogenic with diet, either the obesogenic diet, in the groups either CON orCON in the groups MEN, or showed a significant MEN, showed a increasesignificant in bodyincrease in body mass (127.0 mass ± 1.7 to(127.0 208.3±±1.72.2 to kg 208.3 for± Group 2.2 kg for CONGroupandCON113.9and±113.9 1.3 to± 1.3 193.9to 193.9 ± 1.7 kg ± 1.7 kg for Group MEN; p < 0.0001 for Group MEN; pfor < 0.0001 bothfor both groups) groups) and adiposity and adiposity (19.5 (19.5 ± 0.9toto56.2 ± 0.9 56.2 ±±1.51.5mmmm of total of total back-fat depth for Group CON and 17.0 ± 0.6 to 65.6 ± 1.1 mm for Group back-fat depth for Group CON and 17.0 ± 0.6 to 65.6 ± 1.1 mm for Group MEN; p < 0.0001 for MEN; p < 0.0001 for bothboth groups) from Day 0 to 120 of the study (Figure 2). There were no significant differences between groups) from Day 0 to 120 of the study (Figure 2). There were no significant differences between groups in these parameters, either at the beginning or throughout the study. However, we observed groups in these parameters, either at the beginning or throughout the study. However, we observed significant differences between groups when assessing separately the inner and the outer layers of significant differences between groups when assessing separately the inner and the outer layers of the the subcutaneous back-fat. The sows in the Group MEN showed a trend for a higher deposition at subcutaneous the inner back-fat. layer and The sows a lower in the Group deposition at the MEN showed outer layer fromaDay trend60 for a higher onwards. deposition These differences,at the inner layer andstatistical reaching a lower deposition significance atat the Dayouter 120 (player from < 0.05), were Day more60evident onwards.whenThese differences, comparing reaching the relative statistical significance increases over previous 120 (p < 0.05), at Dayassessments, were more as depicted evident in the inset when graphscomparing of Figure 2.the relative increases Conversely, there were no significant differences when comparing the intramuscular fat content (0.20 over previous assessments, as depicted in the inset graphs of Figure 2. Conversely, there were no ± 0.03 to 0.16 ± 0.04%). significant differences when comparing the intramuscular fat content (0.20 ± 0.03 to 0.16 ± 0.04%). 3.2. Changes in Metabolic Status The intake of a high-fat diet, in both Groups CON and MEN, causes significant increases from Day 0 to 120 of the study (Figure 3) in the mean plasma concentrations of glucose (p < 0.05 and p < 0.01, respectively), total cholesterol and NEFA (p < 0.05 in both groups) and HDL-c CON (p < 0.001 and p < 0.005, respectively). Plasma levels ofCON fructosamine increased in both groups between Days MEN Group CON (p < 0.05) to remain stable in high values 30 and 60, although only significantly in theMEN until Day 120. Plasma concentrations of urea and lactate (Figure 4) also increased in both groups (p < 0.05 in Group CON and p < 0.0001 in Group MEN for urea and p < 0.05 in both groups for lactate), while haptoglobin increased significantly in the Group MEN (p < 0.05). significant differences between groups when assessing separately the inner and the outer layers of the subcutaneous back-fat. The sows in the Group MEN showed a trend for a higher deposition at the inner layer and a lower deposition at the outer layer from Day 60 onwards. These differences, reaching statistical significance at Day 120 (p < 0.05), were more evident when comparing the relative increases over previous assessments, as depicted in the inset graphs of Figure 2. Conversely,6 of Biology 2020, 9, 284 there 16 were no significant differences when comparing the intramuscular fat content (0.20 ± 0.03 to 0.16 ± 0.04%). CON CON MEN MEN Biology 2020, 9, x 6 of 15 CON CON MEN MEN Figure2.2. Mean values Figure values(±(±S.E.M.), S.E.M.), over time over of study, time of body of study, massmass of body (panel A)and (A) anddepth depth of of total total (panel B) (B) and and outer outer and inner and inner layerslayers (C,D)(panels C and D) of of subcutaneous subcutaneous backfat depots inbackfat sows used depots in sows used as controls as controls (discontinuous (discontinuous line) line) two or treated with or treated doses with of Vacsincel ® for two doses of inducing Vacsincel®ovarian for inducing ovarian inactivity inactivityline; (continuous (continuous arrows line; arrows indicate timing of treatment). The inset graphs represent the relative increases indicate timing of treatment). The inset graphs represent the relative increases when compared to the when compared to the previous previous assessment assessment (white CON, (white group groupblack CON,group black MEN); group MEN); units are units the are samethethan samein than in thegraph. the main main graph. Asterisks Asterisks indicateindicate significant significant differences differences between between groups (* p <(*0.05). groups p < 0.05). There were 3.2. Changes no differences in Metabolic Status in any of the main metabolic parameters between groups CON and MEN at 0 and 30 days of the study (i.e., prior to completion of the treatment and induction of the The intake of a high-fat diet, in both Groups CON and MEN, causes significant increases from ovarian failure in the Group MEN), as depicted in Figures 3 and 4. At 60 days of starting the study, Day 0 to 120 of the study (Figure 3) in the mean plasma concentrations of glucose (p < 0.05 and p < in the first sampling after ovarian hormone stoppage, the sows in the Group MEN showed a trend for 0.01, respectively), total cholesterol and NEFA (p < 0.05 in both groups) and HDL-c (p < 0.001 and p < lower plasma concentrations of β-hydroxybutyrate (p < 0.05) and significantly higher plasma levels of 0.005, respectively). Plasma levels of fructosamine increased in both groups between Days 30 and 60, fructosamine (p < 0.01). The higher fructosamine levels in the Group MEN were maintained at 90 and although only significantly in the Group CON (p < 0.05) to remain stable in high values until Day 120. 120 days of the study (p < 0.05 for both), when plasma urea levels were also significantly higher in the Plasma concentrations of urea and lactate (Figure 4) also increased in both groups (p < 0.05 in Group Group MEN (p < 0.005 for Day 90 and p < 0.01 for Day 120). CON and p < 0.0001 in Group MEN for urea and p < 0.05 in both groups for lactate), while haptoglobin increased 3.3. Changes significantly in Fatty AcidinComposition the Group MEN (p < 0.05). of Adipose Tissues (Subcutaneous and Visceral Fat) There were no differences in any of the main metabolic parameters between groups CON and Most of the significant differences between controls and treated sows (Groups CON and MEN, MEN at 0 and 30 days of the study (i.e., prior to completion of the treatment and induction of the respectively) were found at the visceral fat and at the inner layer of subcutaneous fat rather than at the ovarian failure in the Group MEN), as depicted in Figures 3 and 4. At 60 days of starting the study, outer layer (Table 1 and Tables S1–S3). in the first sampling after ovarian hormone stoppage, the sows in the Group MEN showed a trend At the visceral fat, contents of myristic (C14:0), palmitic (C16:0), palmitoleic (C16:1n-7) and cis-7 for lower plasma concentrations of β-hydroxybutyrate (p < 0.05) and significantly higher plasma hexadecenoic acids (C16:1n-9) were decreased in the Group MEN when compared to Group CON levels of fructosamine (p < 0.01). The higher fructosamine levels in the Group MEN were maintained (p < 0.05 for all). There were also found a higher content of total MUFA and oleic acid (C18:1n-9) at 90 and 120 days of the study (p < 0.05 for both), when plasma urea levels were also significantly and a lower proportion of linoleic acid (C18:2n-6) in the Group MEN (p = 0.08, p < 0.05 and p = 0.08, higher in the Group MEN (p < 0.005 for Day 90 and p < 0.01 for Day 120). respectively). At the subcutaneous fat, the content of palmitoleic acid was also significantly lower at the inner layer of the Group MEN (p < 0.05) and the same trend was found at the outer layer (p = 0.09); both layers also showed a higher content in docohexaenoic (C22:6n-3; p < 0.05 for both) and adrenic acids (C22:4n-6 p < 0.05 for the inner and p = 0.05 for the outer layer). At the inner layer, the Group MEN Plasma concentrations of urea and lactate (Figure 4) also increased in both groups (p < 0.05 in Group CON and p < 0.0001 in Group MEN for urea and p < 0.05 in both groups for lactate), while haptoglobin increased significantly in the Group MEN (p < 0.05). There were no differences in any of the main metabolic parameters between groups CON and MEN at 0 and 30 days of the study (i.e., prior to completion of the treatment and induction of the Biology 2020, 9, 284 7 of 16 ovarian failure in the Group MEN), as depicted in Figures 3 and 4. At 60 days of starting the study, in the first sampling after ovarian hormone stoppage, the sows in the Group MEN showed a trend for alsolower showed plasma concentrations trends for lower content of β-hydroxybutyrate of eicosapentaenoic(p