Introduction
Pregnancy and lactation are crucial stages during which obesity gives rise to important health consequences in the short, medium, and long term for both mothers and their offspring. Reference Zambrano, Ibáñez, Martínez-Samayoa, Lomas-Soria, Durand-Carbajal and Rodríguez-González1 In México, an alarming increase in the prevalence of obesity in adult women has been observed; i.e., in women aged > 20–29 years, the prevalence of obesity was 26%, which increased 46% in women of aged 30–59 years (https://www.inegi.org.mx/contenidos/saladeprensa/aproposito/2020/EAP_Obesidad20.pdf). Therefore, a significant percentage of women of reproductive age have obesity. MO damages skeletal muscle morphology and the muscle fibre composition of offspring during postnatal life; Reference Pileggi, Segovia and Markworth2,Reference Mikovic, Brightwell and Lindsay3 this is important since skeletal muscle is crucial for thermogenesis and energy expenditure. Reference Chen, Hu, Wang and Wang4
FNDC5 encodes a membrane protein that is cleaved and secreted as irisin, which is a myokine that is synthesised mainly by skeletal muscle in response to exercise and is regulated in both humans and rodents by the peroxisome proliferator-activated receptor-gamma coactivator 1 α (PGC1 α). Reference Bostrom, Wu and Jedrychowski5 It has been suggested that irisin in skeletal muscle is capable of increasing energy expenditure via an increase in the expression of genes related to metabolism, such as PGC-1 α, and uncoupling protein 3 (UCP3), among others. Reference Bostrom, Wu and Jedrychowski5,Reference Vaughan, Gannon and Barberena6 In this way, it could lead to an improvement in metabolic dysfunction. Reference Bao, She, Hu, Jia and Li7 In addition, in a model of obesity induced by a high-fat diet (HFD), an increase in Fndc5 and Pgc1-α protein levels in the gastrocnemius muscle was reported, suggesting that this increase might have a compensatory role opposing excess body weight. Reference Guilford, Parson, Grote, Vick, Ryals and Wright8
It is well known that regular exercise and good nutrition are relevant to general health; in this context, identifying specific components in food, such as natural phytochemicals, might help prevent several chronic disorders, including obesity and its comorbidities. Reference Cremonini, Iglesias and Kang9 The flavonoid (-)-epicatechin has been shown to have a beneficial effect on the skeletal muscle of the offspring of obese mothers fed a HFD. Reference De los Santos, Coral-Vázquez and Menjivar10 Furthermore, this flavonoid, considered as an exercise mimetic, induced mitochondrial biogenesis in vitro Reference Moreno-Ulloa, Cid, Rubio-Gayosso, Ceballos, Villarreal and Ramirez-Sanchez11 and in vivo. Reference Nogueira, Ramirez-Sanchez and Perkins12
We previously demonstrated that the offspring of obese mothers are also obese and have metabolic disorders and decreased lean mass; Reference De los Santos, Coral-Vázquez and Menjivar10,Reference De los Santos, Reyes-Castro and Coral-Vázquez13,Reference Rodríguez-González, De los Santos and Méndez-Sánchez14 however, to our knowledge, the implications of obesity programming on muscle energy regulation through the irisin pathway have not been reported. In this context, we aimed to analyse the expression of genes related to the regulation of energy metabolism in skeletal muscle tissue by comparing two age groups (at 110 and 245 pnd) of male offspring of a model of MO induced by a HFD and to determine whether these disorders are modified by the administration of the flavonoid (-)-epicatechin. We hypothesised that the presence of MO would decrease the expression of proteins involved in the thermogenic pathway in musculoskeletal tissue and that postnatal intervention with the flavonoid (-)-epicatechin would be able to restore the expression profile of these proteins. Moreover, we hypothesised that these changes would be more pronounced in mature adult rats (245 days of age).
Materials and methods
Animal model
The animal research protocol used in the present study was approved by the Animal Experimentation Ethics Committee of the Instituto Nacional de Ciencias Médicas y Nutrición Salvador Zubirán (CINVA: UIO-1892-17/19-1) and by the Research and Ethics Commissions of the Facultad de Medicina, Universidad Nacional Autónoma de México. All procedures followed the Guidelines for the Care and Use of Laboratory Animals of the Institute of Laboratory Animal Resources (http://www.nal.usda.gov/awic/animal-welfare-act), and the Mexican Official Standard Norm NOM-062-ZOO-1999.
The chow diet and the HFD, as well as the standardisation of the phenotype of the Wistar female rats (F0) used to produce the F1 offspring, were described previously. Reference De los Santos, Coral-Vázquez and Menjivar10,Reference De los Santos, Reyes-Castro and Coral-Vázquez13 All female albino Wistar F0 rats were maintained on a diet (chow or HFD) during pregnancy and lactation (Fig. 1). The Chow diet contained 23.9% protein, 5.0% fat, 57.9% carbohydrates, 5.1% fibre, 7.0% minerals and 1.0% vitamins (w/w) (4.07 kcal/g of energy) (Purina 5001, Labdiet, St Louis, MO, USA). In contrast, the HFD contained 23.5% protein, 20.0% animal lard, 5.0% fat, 20.2% polysaccharide, 20.2% simple sugars, 5.0% fibre, 5.0% mineral mix and 1.0% vitamin mix (wt/wt) (4.9 kcal/g of energy). The mineral and vitamin contents of the two diets (chow and HFD) were selected following the American Institute of Nutrition recommendation to support growth, pregnancy, and lactation phases (AIN-93G). Reference Reeves, Nielsen and Fahey15
Timeline for the study of male offspring of obese mothers (F1) of 110 or 245 postnatal days. For the obesity model, six female rats (F0) per group were fed a control or high-fat diet at weaning and during pregnancy and lactation. At postnatal day 21, twelve males per group aged 110 or 245 (C and MO) from different litters were randomly allocated to be administered either water or (-)-epicatechin twice per day for 2 weeks (C, C + Epi, MO, MO + Epi, n = 6 rats per group). Pnds= postnatal days, Epi= (-)-epicatechin; Veh= Vehicle, C = male rat offspring of control mothers at 110 or 245 pnds; C + Epi= male rat offspring of control mothers treated with Epi at 110 or 245 pnds; MO = male rat offspring of obese mothers at 110 or 245 pnds; MO + Epi= male rat offspring of obese mothers treated with Epi at 110 or 245 pnds.
Since sexual dimorphism in skeletal muscle has been described, for this study, we analysed only male offspring. Reference O’Reilly, Ono-Moore and Chintapalli16 To standardise the phenotype of the male offspring (F1), food intake, weight, adiposity index, and (-)-epicatechin administration were described by De los Santos et al. Reference De los Santos, Coral-Vázquez and Menjivar10,Reference De los Santos, Reyes-Castro and Coral-Vázquez13 In brief, male offspring were weaned at postnatal day 21, housed with two rats per cage, and fed a chow diet throughout the duration of the study. Because genotypic and phenotypic changes in various rat age-related muscles have been observed, Reference Sengupta17 in the present study, we analysed rats at 110 (young adult) and 245 pnd (mature adult). Six males per group from different litters were randomly selected to form the control groups or the Epi intervention groups, totalling four groups of male rats for each postnatal age group: male rats descended from mothers fed a control diet (C); male rats treated with Epi descended from mothers fed a control diet (C + Epi); male rats descended from mothers fed a HFD (MO); and male rats treated with Epi descended from mothers fed a HFD (MO + Epi) (Fig. 1).
At 95 or 230 days of age, male offspring were administered a vehicle (water) or Epi (1 mg/kg body weight) (Sigma-Aldrich, St Louis, MO, USA) by oral gavage twice daily (morning and afternoon) for 15 days. As previously described, Reference De los Santos, Coral-Vázquez and Menjivar10,Reference De los Santos, Reyes-Castro and Coral-Vázquez13 the interventions were carried out at the same time and by the same individual. No adverse effects were recorded due to this dietary intervention, and no rats died before the end of the study. At postnatal day 110 or 245, after 6 hours of fasting, the rats were anaesthetized with isoflurane (based on the instructions for the use of the isoflurane inhaler of the brand: Stoelting Co., Cat. No. 50,207 regarding animal weight) and euthanized; we subsequently obtained the gastrocnemius muscle tissues and stored them at −80°C for subsequent analysis. The gastrocnemius muscle was chosen as a muscle model, since it has been reported that this muscle modifies its thermogenesis capacity under different conditions. Reference Barbosa, Guerra-Sá and De Castro18
Total RNA extraction and gene expression analysis
Total RNA was extracted from eighty milligrams of gastrocnemius tissue using TRIzol reagent (Invitrogen, California, U.S.A.) according to the manufacturer’s protocol. A nanophotometer (Implen GmBH, M FC;nchen, Germany) was used to determine RNA purity and concentration. RNA quality was assessed by determining the integrity of the 28S and 18S ribosomal RNA bands following electrophoresis on a 1.5% agarose gel. After RNA isolation, all the samples were frozen and stored at −80°C until use.
RNA (100 ng) was used for DNA synthesis (cDNA) using the AgPath-ID™ One-Step RT-PCR Kit Reagents (Applied Biosystems™, Thermo Fisher Scientific) following the instructions provided by the manufacturer.
The gene expression of fibronectin type III domain containing 5 (Fndc5) (Rn01519161_m1), the transcription coactivator peroxisome proliferator-activated receptor gamma (Ppar-γ) type 1 alpha (also known as Pgc-1α) (Rn00580241_m1), Ucp3 (Rn00565874_m1), and sarcolipin (Sln) (Rn02769377_s1) was analysed in gastrocnemius muscle tissue by reverse transcription–quantitative polymerase chain reaction (RT–qPCR) using the TaqMan tool from the Demand Gene Expression Probes of Thermo Fisher Scientific. The values were normalised to the relative amounts of β-actin (assay ID: Rn00667869_m1) for each sample.
Reverse transcription-quantitative PCR was performed on a LightCycler® 480 instrument (Roche Diagnostics Ltd., Switzerland), and relative mRNA concentrations were calculated with LightCycler Relative Quantification analysis Software. All the samples were analysed in duplicate.
Western blotting
Between 120 and 150 mg of gastrocnemius muscle tissue was homogenised in RIPA lysis buffer (Santa Cruz Biotechnology, Inc., Dallas, U.S.A.) (sc-2494A) supplemented with protease and phosphatase inhibitors. Fifty milligrams of protein was separated via SDS-PAGE and transferred to a nitrocellulose membrane (Bio-Rad Laboratories, Cat. #1620115, Germany); after being blocked with 5% fat-free milk, the membranes were incubated with different primary antibodies. We used anti-Fndc5/irisin (ab174833 Abcam, dilution 1:1000), anti-Pgc1 α (ab3242 EMD Millipore, dilution 1:750), anti-Ucp3 (ab3046 EMD Millipore, dilution 1:1000), and anti-Sln (18,395-1-AP Proteintech, dilution 1:500) antibodies. For the loading control, anti-Gadph (PA1-988; dilution 1:14000) (Thermo Fisher Scientific, Rockford, U.S.A.) was used.
Afterwards, we incubated the sections with HRP-conjugated goat anti-rabbit IgG (111-095-003) and anti-mouse IgG (715-035-150) (Jackson ImmunoResearch Laboratories, I.N.C.) as secondary antibodies. The proteins were detected with a SuperSignaltm West Femto Maximum Sensitivity Substrate Kit (Thermo Fisher Scientific, Rockford, U.S.A.). The band intensities were digitally quantified using LI-COR Image Studio software (http://www.licor.com/bio/products/software/image_studio_lite/) and ImageJ software (U.S. National Institutes of Health, Bethesda, Maryland, U.S.A.).
Statistical analysis
The results are expressed as the median ± range of 6 individual experimental observations and were analysed using GraphPad Prism 9.3.1 software (GraphPad Software, San Diego, CA). The sample size and power were calculated according to Dell et al. Reference Dell, Holleran and Ramakrishnan19 We tested the normality of the distribution of the data using the Kolmogorov-Smirnov test, and a Kruskal-Wallis nonparametric test was performed with Dunn’s post hoc test. Differences were considered significant at p < 0.05.
Results
Effect of (-)-epicatechin treatment on the mRNA expression of genes related to the thermogenic pathway in the gastrocnemius muscle tissue of male offspring of obese Wistar rats of 110 and 245 postnatal days
To evaluate the expression of thermogenic genes, the total RNA in the gastrocnemius muscle of male offspring of obese Wistar rats of 110 and 245 pnd was obtained and analysed via qRT-PCR. We did not observe that Epi treatment modified the expression of Fndc5, Pgc-1α, Ucp3, or Sln of the mRNA level in the male offspring at 110 postnatal days in the experimental groups (C, MO, C + Epi, and MO + Epi) (Fig. 2, Panels A–D).
Effect of Epi on the relative mRNA expression of genes related to thermogenesis in the gastrocnemius muscle of male offspring of obese mothers of 110 postnatal days. ( a ) Fndc5/irisin, ( b ) Pgc-1α, ( c ) Ucp3, and ( d ) Sln. We did not observe differences in the experimental groups (C, C + Epi, MO, MO + Epi). The data are expressed as medians and ranges and were analysed by the Kruskal-Wallis test followed by the post hoc Dunn test for pairwise comparisons (n = 6 rats per group). Epi= (-)-epicatechin; C = male rat offspring of control mothers; C + Epi= male rat offspring of control mothers treated with Epi for 2 weeks; MO = male rat offspring of obese mothers; MO + Epi= male rat offspring of obese mothers treated with Epi for 2 weeks.
Regarding the male offspring of 245 pnd, Epi treatment in the MO group (MO + Epi) induced an increase in the expression of Fndc5 mRNA in comparison to that in the MO group (p < 0.006; Fig. 3, Panel A). We did not observe differences in the expression of Pgc-1α, Ucp3, or Sln in the other groups of male offspring of 245 pnd (Fig. 3, Panels B–D).
Effect of Epi on the relative mRNA expression of genes related to thermogenesis in the gastrocnemius muscle of male offspring of obese mothers at 245 postnatal days. ( a ) Fndc5/irisin, ( b ) Pgc-1α, ( c ) Ucp3, and ( d ) Sln. ( a ) Epi significantly increased the expression of Fndc5/irisin in the MO group. The data are expressed as medians and ranges and were analysed by the Kruskal-Wallis test followed by the post hoc Dunn test for pairwise comparisons (n = 6 rats per group). **p < 0.006, MO vs. MO + Epi. Epi= (-)-epicatechin; C = male rat offspring of control mothers; C + Epi= male rat offspring of control mothers treated with Epi for 2 weeks; MO = male rat offspring of obese mothers; MO + Epi= male rat offspring of obese mothers treated with Epi for 2 weeks.
(-)-Epicatechin treatment affects proteins related to the thermogenic pathway in the gastrocnemius muscle tissue of male offspring of obese Wistar rats of 110 and 245 postnatal days
Epi administration to male offspring of the MO model (MO + Epi) of 110 pnd resulted in an increase in the expression of Pgc-1 α protein in comparison to that in the MO group (p < 0.036; Fig. 4, Panels A and C). Regarding Fndc5, Ucp3, and Sln protein levels, we did not find differences in the experimental groups of male offspring of 110 pnd (Fig. 3, Panels A–E).
Effect of Epi on the expression of proteins related to thermogenesis in the gastrocnemius muscle of male offspring of obese mothers at 110 postnatal days. ( a ) Representative immunoblotting of Fndc5/irisin, Pgc-1 α, Ucp3, and Sln proteins; GAPDH was used as the loading control. ( b – e ) Densitometry analysis of Fndc5/irisin, Pgc-1 α, Ucp3, and Sln. (C) Epi significantly increased the protein expression of Pgc-1 α in the MO group. The data are expressed as medians and ranges and were analysed by the Kruskal-Wallis test followed by the post hoc Dunn test for pairwise comparisons (n = 6 rats per group). *p < 0.036, MO vs. MO + Epi. Epi= (-)-epicatechin; C = male rat offspring of control mothers; C + Epi= male rat offspring of control mothers treated with Epi for 2 weeks; MO = male rat offspring of obese mothers; MO + Epi= male rat offspring of obese mothers treated with Epi for 2 weeks.
Moreover, we did not observe that MO or Epi treatment modified the protein levels of Fndc5, Pgc-1 α, Ucp3, or Sln in the male offspring of the four experimental groups (C, MO, C + Epi, and MO + Epi) of 245 pnd (Fig. 5, Panels A–E).
Effect of Epi on protein expression related to thermogenesis in the gastrocnemius muscle of male offspring of obese mothers at 245 postnatal days. ( a ) Representative immunoblotting of Fndc5/irisin, Pgc-1 α, Ucp3, and Sln proteins; GAPDH was used as the loading control. ( b – e ). Densitometric analyses of Fndc5/irisin, Pgc-1 α, Ucp3, and Sln. We did not observe differences in the experimental groups (C, C + Epi, MO, MO + Epi). The data are expressed as medians and ranges and were analysed by the Kruskal-Wallis test followed by the post hoc Dunn test for pairwise comparisons (n = 6 rats per group). Epi= (-)-epicatechin; C = male rat offspring of control mothers; C + Epi= male rat offspring of control mothers treated with Epi 2 weeks; MO = male rat offspring of obese mothers; MO + Epi= male rat offspring of obese mothers treated with Epi 2 weeks.
Discussion
Obesity has a detrimental effect on skeletal muscle, as it can lead to muscle atrophy, inflammation, and changes in skeletal muscle fibre composition, among others. Reference Richter-Stretton, Fenning and Vella20 Importantly, studies in animal models have shown that obesity in mothers has an impact on the skeletal muscle development and metabolism of their offspring Reference De los Santos, Coral-Vázquez and Menjivar10,Reference Bayol, Simbi and Stickland21,Reference Salto, Girón and Manzano22 as well as muscle strength in adulthood, despite following a control diet from weaning. Reference Bayol, Macharia, Farrington, Simbi and Stickland23 In addition, the impact of obesity on morphological changes observed in all types of muscles is more accelerated in aged mice than in young mice, with type II glycolytic muscle fibres being the most affected. Reference Jun, Robinson, Geetha, Broderick and Babu24 This is important since, due to the large volume of skeletal muscle tissue, this is the most critical determinant of thermogenic capacity and total body energy expenditure. Reference Fuller-Jackson and Henry25 Furthermore, it is well known that the basal metabolic rate decreases with age due to skeletal muscle loss, which could lead to obesity. Reference De Luise and Harker26,Reference Vásquez-Alvarez, Bustamante-Villagomez and Vazquez-Marroquin27
In addition, it has been described that the fetal skeletal muscle of a non human primate’s descendant of a model of MO presented a reduced mitochondrial content, oxidative capacity, and oxidative phosphorylation efficiency. Reference McCurdy, Schenk and Hetrick28 Besides, Dias-Rocha et al. Reference Dias-Rocha, Almeida and Woyames29 demonstrated that offspring adults of a model of MO by HFD exhibited increased energy metabolism markers and induced mitochondrial alterations in skeletal muscle.
Interestingly, in vitro and in vivo studies have demonstrated that Epi stimulates mitochondrial biogenesis, which is essential for energetically supporting the growth, differentiation, and function of skeletal muscle; Reference Moreno-Ulloa, Cid, Rubio-Gayosso, Ceballos, Villarreal and Ramirez-Sanchez11,Reference Ramírez-Sánchez, De los Santos and Gonzalez-Basurto30 moreover, this flavonoid can reduce cellular oxidative stress in skeletal muscle. Reference Ramírez-Sánchez, De los Santos and Gonzalez-Basurto30,Reference Zbinden-Foncea, Castro-Sepulveda, Fuentes and Speisky31 Furthermore, Epi increased the expression of growth-regulating proteins and muscle differentiation in obese mice induced by a HFD. Reference Munguia, Ramirez-Sanchez, Meaney, Villarreal, Ceballos and Najera32
To our knowledge, there are no reports in the literature related to the expression of irisin or genes related to thermogenesis in skeletal muscle in the context of induced obesity.
Previously, we demonstrated that these offspring were obese and had metabolic disorders. Reference De los Santos, Coral-Vázquez and Menjivar10,Reference De los Santos, Reyes-Castro and Coral-Vázquez13 In addition, the Fndc5/irisin protein can induce mitochondrial biogenesis in skeletal muscle. Reference Bostrom, Wu and Jedrychowski5,Reference Trettel, Pelozin and Barros33 Likewise, it has been described that Fndc5/irisin expression only at the mRNA level Reference de Macêdo, Lelis, Mendes, Fraga, Brandi and Feltenberger34 or in both mRNA and protein expression Reference Li, Yi and Li35,Reference Kou, Li and Shi36 are decreased in skeletal muscle of a model of obese animal induced by HFD.
Although we did not find a significant difference in Fndc5/irisin mRNA expression in pups of 245 days of the MO group, we observed that the descendants of this group presented lower expression of this myokine than did those in the control group (Fig. 3, Panel A). Notably, irisin expression is regulated by exercise, Reference Li, Yi and Li35 and Epi is considered a mimetic of exercise. Reference Nogueira, Ramirez-Sanchez and Perkins12 In this context, we found that Epi treatment in the MO pups of 245 pnd significantly increased Fndc5/irisin mRNA expression. This increase was similar to the expression of this gene in the offspring of the control group treated with this flavonoid. Interestingly, differences in the expression of Fndc5/irisin were not maintained at the protein level in any of the groups analysed.
Moreover, we did not detect differences in mRNA or protein levels of Fndc5/irisin in the offspring of any of the four experimental groups of 110 pnd.
On the other hand, the increase in thermogenesis in skeletal muscle caused by exercise was related to the expression of PGC-1 α, which increases irisin secretion and, by this pathway, could stimulate mitochondrial oxidative metabolism. Reference Perakakis, Triantafyllou and Fernández-Real37 Similarly, as described for irisin expression in an obesity model, PGC-1 α mRNA/protein levels are decreased in the skeletal muscle of animals with obesity induced by a HFD. Reference Li, Yi and Li35,Reference Kou, Li and Shi36
Concerning our model of obesity induced by programming, compared with those in the MO group, the offspring of 110 pnd showed a significant increase in Pgc-1 α in the MO + Epi group but only at the protein level. It has been demonstrated that Epi can mitigate oxidative stress by acting on cell signalling of those genes that regulate mitochondrial function in skeletal muscle, including Pgc-1 α, among other proteins. Reference Ramírez-Sánchez, De los Santos and Gonzalez-Basurto30
The discrepancies observed in the correlation between Fndc5/irisin mRNA expression and protein levels or between the Pgc-1 α protein and mRNA could be due to transcriptional, post-transcriptional, translational, or posttranslational regulation Reference Schwanhäusser, Busse and Li38 as well as protein stability. Reference Geiger, Burkhart, Gambaryan, Walter, Sickmann and Zahedi39 At postnatal day 110, there was an increase in the Pgc-1 α protein but not in the irisin protein. In the case of the offspring at postnatal day 245, an increase in irisin but not in Pgc-1 α was observed, which could indicate that the regulation of thermogenesis in skeletal muscle is mediated by another pathway. Reference Sahu, Pani, Swalsingh and Bal40
Sarcolipin plays a role in metabolism and muscle thermogenesis through Ca2+-ATPase of the endoplasmic reticulum (SERCA), Reference Maurya, Herrera and Sahoo41 and in an animal model of obesity induced by a HFD, sarcolipin/SERCA expression is decreased. However, irisin treatment increased the expression of both genes. Hence, it has been proposed that irisin modulates Sln, which can improve the function and thermogenesis of skeletal muscle. Reference Gheit, Younis and El-Saka42 In addition, in 2022, Son et al. demonstrated in a mouse model of MO that the expression of sarcolipin and Ucp3 in skeletal muscle is decreased in offspring with diet-induced obesity and that this change is due to pathway regulation other than Find5/irisin. According to our results, we did not find that obesity by programming or Epi treatment modified the expression of Sln or Ucp3 in the offspring of 110 or 245 pnd; as mentioned for Fndc5/irisin or Pgc-1 α, the regulation of thermogenesis in skeletal muscle could occur through pathways other than Fndc5/irisin. Reference Sahu, Pani, Swalsingh and Bal40,Reference Son, Chae and Zhao43
Notably, we performed the analysis only in the gastrocnemius muscle since, in obese mice by HDF, a reduced area of muscle myofibers and satellite cells, with a decrease of the expression and activity of mitochondrial enzyme genes in this muscle was observed. Reference Jun, Robinson, Geetha, Broderick and Babu24
In conclusion, (-)-epicatechin treatment increased Fndc5/irisin mRNA expression and Pgc-α protein levels in the gastrocnemius muscle of offspring of obese mother of 245 and 110 postnatal days, respectively. Furthermore, we believe that the effect of this flavonoid in a programming model of obesity and its impact on thermogenesis in skeletal muscle are regulated by a different pathway than that of Fndc5/irisin.
Data availability statement
The datasets generated during and/or analyzed during the current study are available from the corresponding author upon reasonable request.
Acknowledgments
We thank Sebastián De la Rosa, and Jorge Uribe, from the Unidad de Investigación en Obesidad, Facultad de Medicina, Universidad Nacional Autónoma de México (S.D.R.) and from the Departamento de Biología de Reproducción, Instituto Nacional de Ciencias Médicas y Nutrición ‘Salvador Zubirán’ (J.U.), for their technical assistance in this study.
Author contribution
Tejeda M. E.: Conceptualisation; Data curation; Formal analysis; Investigation; Visualisation; Manuscript elaboration.
De los Santos S: Conceptualisation; Data curation; Formal analysis; Investigation; Visualisation.
Coral-Vazquez RM: Conceptualisation; Formal analysis; Investigation; Visualisation. Manuscript elaboration.
Méndez JP: Conceptualisation; funding acquisition; Manuscript elaboration.
Álvarez-Chávez A: Data curation; Formal analysis; Investigation; Visualisation.
Palma-Flores C: Data curation; Formal analysis.
Zambrano E: Conceptualisation; funding acquisition; Manuscript elaboration.
Canto P: Conceptualisation; Formal analysis; Funding acquisition; Investigation; Project administration; Supervision. Manuscript elaboration.
All authors read and approved the final manuscript.
Funding statement
This work was partially supported by the Fondo Sectorial de Investigación para la Educación SEP-CONACYT, Convocatoria de Investigación Científica Básica 2017-2018, CONACyT México, under Grant: A1-S-9740; by the Newton Fund RCUK- CONACYT-2015 (Research Councils UK – CONACyT) and by the Apoyo al Fortalecimiento y Desarrollo de la Infraestructura Científica y Tecnológica (CONACyT) México, under Grant: 250786.
This work was submitted in partial fulfillment of the requirements for the Ph.D. degree of María Elena Tejeda Hernández at the Programa de Doctorado en Ciencias Bioquímicas, Universidad Nacional Autónoma de México.
M. E. Tejeda Hernández was supported by a Consejo Nacional de Ciencia y Tecnología (CONACyT), México, fellowship award.
Sergio De los Santos received a postdoctoral fellowship award from the Programa de Becas Posdoctorales de la Dirección General de Asuntos del Personal Académico (DGAPA), División de Investigación, Facultad de Medicina, Universidad Nacional Autónoma de México.
Competing interests
None.
