High-Fat Diet-Induced Obesity Primes Impaired Fatty Acid β-Oxidation and Consequent Ovarian Dysfunction During Early Pregnancy

doi:10.21203/rs.3.rs-395617/v1

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High-Fat Diet-Induced Obesity Primes Impaired Fatty Acid β-Oxidation and Consequent Ovarian Dysfunction During Early Pregnancy

https://doi.org/10.21203/rs.3.rs-395617/v1

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Background: Obesity is associated with many adverse effects on female fertility. Obese women are more likely to have ovulatory dysfunction due to dysregulation of the hypothalamic-pituitary-ovarian axis. However, ovarian function in obese women during early pregnancy still needs further assessment.

Methods: Obesity was induced in C57BL6/J mice using high-fat diets (HFD) for 12 weeks; In vitro high-fat model was established with KGN cells treated with Oleate acid and Palmitic acid. Ovarian morphology of obese mice in early pregnancy was assessed by Hematoxylin and Eosin staining and its function was assessed using ELISA, Western blotting and Immunohistochemistry. The Oil Red O staining and Transmission electron microscopy were used to detect fatty acid accumulation and specific markers relating to ovarian functional mechanism were assessed by Real time PCR, Western blotting, Lactate detection, ATP detection, Biochemical analyses and ELISA.

Results: The results of this study showed that during early pregnancy, the number of corpus luteum, serum estradiol and progesterone levels, and the expression of genes CYP19A1, CYP11A1 and StAR, which are related to steroid biosynthesis, were significantly increased in HFD female mice. HFD-fed mice also showed a significant increase in ovarian lipid accumulation on day 7 of pregnancy. Genes involved in fatty acid synthesis (Acsl4 and Elovl5) and fatty acid uptake and transport (Slc27a4), together with the β-oxidation rate-limiting enzyme (Cpt1a) were significantly upregulated in HFD-fed mice. Specifically, there was abnormal elevation of ATP level and aberrant expression of tricarboxylic acid cycle (TCA) and electron transport chain related genes in the ovary of HFD pregnant mice. Treatment of KGN cells with etomoxir targeting β-oxidation of fatty acid, showed decrease tricarboxylic acid cycle and electron transport chain. The elevated ATP level and the increased estradiol and progesterone levels were reversed.

Conclusions: This study indicated that during early pregnancy, high-fat diet and induced-obesity increased fatty acid β-oxidation, which in turn increase the tricarboxylic acid cycle and the electron transport chain, and consequently increases ATP production and ovarian dysfunction.

Endocrinology & Metabolism

Cellular Metabolism

ovary

obesity

early pregnancy

fatty acid β-oxidation

glycolysis

The prevalence of obesity has increased dramatically around the world. It has been reported that more than 650 million adults worldwide are affected by obesity [1]. Obesity is an increase in body weight due to excessive accumulation of fat tissue, which contributes to an increased prevalence of obesity-related metabolic dysfunction, cardiovascular diseases, diabetes, musculoskeletal disorders and some cancers [2] . In addition, obesity exerts a negative influence on female fertility; and it is associated with many adverse maternal and fetal effects prenatally [3]. The rates of many pregnancy complications – such as hypertensive disorders, gestational diabetes, preterm births and the other health challenges that warrant cesarean delivery – are higher in obese women [3]. However, the underlying mechanisms of obesity’s adverse effect on female fertility have not been well elucidated.

The ovary is the female gonad and an important endocrine organ. The main functions of the ovary include production, maturation and release of the oocyte, and synthesis of female sex steroid and peptide hormones that regulate reproductive and non-reproductive functions [4]. Environmental exposures can exhaust the oocyte pool and cause depletion of follicular cells, premature ovarian failure, early menopause, and infertility. Obese women are more likely to suffer ovulatory dysfunction due to dysregulation of the hypothalamic-pituitary-ovarian axis [5, 6]. However, the impact of obesity on ovarian functions, during early pregnancy, is less clear.

In women, about 15% of miscarriages occur during early pregnancy [7]. During early pregnancy, female sex steroid hormones, especially estrogen and/or progesterone, directly regulate the expression of several genes in the uterus to help maintain pregnancy. Appropriate levels of estrogen and progesterone reduce the risk of miscarriages [7-9]. The regulatory effect of estrogen and progesterone on the ovaries and the uterus, during early pregnancy, renders the endometrium more receptive to blastocysts by inducing endometrial secretory changes that are essential for successful implantation and decidualization in mice. Hence, investigations of the impact of obesity on ovarian functions can help to clarify the mechanistic bases of the association between obesity and female infertility.

Glucose and free fatty acids (FFAs) are the major energy sources for the body [10]. Mitochondrial β-oxidation of FFAs is also a major source of ATP production [11]. Whenever the metabolism of glucose and FFAs are disturbed – as a result of inappropriate dieting, unhealthy lifestyle or other factors – the functions of tissues and organs are dysregulated. It is well established that lots of tissues and organs, such as the skeletal muscle, exhibits dysregulation of glucose uptake, lipid metabolism, and mitochondrial function in obese individuals [12, 13]. However, whether ovarian glucose and fatty acid homeostasis is dysregulated in diet-induced obesity, during early pregnancy, has not been extensively investigated.

Thus, in this study, we investigated ovarian functions and assessed the integrity of ovarian glucose and fatty acid homeostasis in high-fat diet-induced obesity in maternal mice during early pregnancy. Our findings would provide theoretical bases for exploring the effects and mechanisms of ovarian dysfunction in obese maternal mice during early pregnancy, and offer a scientific basis for a comprehensive evaluation of obesity as a risk factor for female infertility.

Animals

C57BL6/J mice (4-week-old) were purchased from Charles River Laboratories. The animals were housed in cages with a 12:12 h light:dark cycle set at ambient temperature (22°C-25°C). After acclimation of ordinary food for one-week, female mice were fed either chow diet (Research Diets, D12450J) or high-fat diet (HFD) (Research Diets, D12492) for 12 weeks respectively. Caloric composition of chow diet consisted of 20% protein, 10% fat and 70% carbohydrates, while high-fat diet had 20% protein, 60% fat and 20% carbohydrates. The mice were weighed weekly. After 12 weeks, the female mice were mated with fertile males of the same strain, which were fed with normal diet. The appearance of a vaginal plug was marked as day 1 of pregnancy (D1). The serum and tissue samples on D7 were collected and stored for further studies. All animal procedures were approved by the Ethics Committee of Chongqing Medical University, China.

Cell culture

The human ovarian granulosa cell line KGN was purchased from EK-Bioscience (Shanghai, China) and authenticated through STR analysis by Cellcook Biotech (Guangzhou, China). The cells were cultured at 37°C in DMEM/F-12 medium (Gibco, USA) supplemented with 10% fetal bovine serum (Gibco, USA) and 1% penicillin-streptomycin (Beyotime, Shanghai, China). In order to establish the high-fat cell model, we treated the cells with 400μM Oleate acid (OA, Sigma, USA) and 200μM Palmitic acid (PA, Sigma, USA) for 24h. After that, luteinization was induced in the cells with 1IU human Chorionic Gonadotropin (hCG, Merck, Germany) for 12h.

Real time PCR

Total RNA was extracted from the ovaries and KGN cells with the Trizol reagent (Invitrogen, USA). cDNA was synthesized using PrimeScript™ RT reagent Kit with gDNA Eraser (TaKaRa, Dalian, China). Real-time RT-PCR reactions were performed using GoTaq® qPCR Master Mix Technical Manual (Promega, USA) and ABI ViiATM7 real-time fluorescence quantitative PCR instrument. Data was analyzed using the ΔΔCt method; and were normalized to β-actin. Primer sequences are listed in Supplemental Table 1 and 2.

Western blot

Tissues and cells were homogenized in RIPA buffer (Beyotime, Shanghai, China) with protease and phosphatase inhibitor cocktail (Thermo Fisher, USA). The BCA kit (Beyotime, Shanghai, China) was used to measure the protein concentration. Equal amounts of proteins were separated using 10% SDS-PAGE and then transferred onto a PVDF membrane. The nonspecific binding sites on the membranes were blocked with 5% non-fat milk at room temperature for 1 h. Then, the membranes were incubated with primary antibodies (Supplemental Table 3) at 4 °C overnight. After washing with PBST, the membranes were incubated with anti-rabbit or anti-mouse IgG secondary antibody conjugated with horseradish peroxidase. Finally, the bands on the membranes were visualized using an ECL detection kit (Millipore, Germany). The light density and grey level were analyzed and calculated by using the ImageJ software. β-actin was used as a loading control.

Biochemical analyses

Blood was centrifugated at 3000 rpm for 10 min under 4 °C to obtain serum. Serum levels of triglyceride (TG) and total cholesterol (TC) were determined using specific assay kits (Nanjing Jiancheng, Nanjing, China) through glycerol-3-phosphate oxidase p-aminophenol (GPO-PAP) and cholesterol oxidase-peroxidase coupling (COD-PAP) methods. Ovarian tissue and KGN cells were homogenated with absolute ethyl alcohol over ice, then TG was tested according to Triglyceride test kit (Nanjing Jiancheng, Nanjing, China) protocols, by GPO-PAP method. TG level in tissues and cells were expressed as mol/g protein. Protein concentration was detected with the BCA protein assay kit.

ELISA

Serum and cell medium estradiol and progesterone levels were determined using commercial enzyme-linked immunosorbent assay kits (ELISA, Yanhui Biotech, Shanghai, China) according to the manufacturer’s protocols. Absorbance was read at 450 nm.

Hematoxylin and eosin (H&E) staining

Ovarian tissues were fixed with 4% paraformaldehyde and embedded in paraffin. Embedded tissues were sectioned at 5μm and stained with H&E prior to the assessment of the morphological changes in the condition of high-fat diet.

Immunohistochemistry

Paraffin-embedded sections (5μm thickness) were deparaffinized and treated with citrate repair solution in microwave oven for 5 min at high fire and 15 min at low fire. After repairing, the rabbit SP kit for immunohistochemistry (ZSGB-BIO, Beijing, China) was used for the subsequent procedures. Firstly, endogenous peroxidases were inhibited via incubation with 3% hydrogen peroxide for 10 min at room temperature. Secondly, the nonspecific binding sites were blocked with 10% goat serum for 30 min at 37°C and then incubated with primary antibodies (Supplemental Table 3) overnight at 4℃. Then, the sections were incubated with horseradish peroxidase-conjugated secondary antibody for 30 min at 37°C. Finally, peroxidase activity was detected using the 3,3-diaminobenzidine substrate (DAB, ZSGB-BIO, Beijing, China). The sections were counterstained with hematoxylin. Positive area quantification was calculated by using ImageJ.

Transmission electron microscopy

Specimens were fixed in 2.5% glutaraldehyde, dehydrated in graded alcohol and stained with OsO4 and uranyl acetate. The sections were polymerized and cut to prepare for use on a Hitachi H-7650 transmission electron microscope.

Oil Red O staining

Ovarian tissues were fixed in 4% paraformaldehyde for 24 h and 30% sucrose for 1 h, then embedded in OCT and serially sectioned at 4μm. KGN cells were cultured on 13mm round glass coverslips. After incubating in 60% isopropyl alcohol for 5 min, both sections and cells were stained with freshly prepared 0.15% Oil Red O (Solarbio, Beijing, China) for 10 min. Then, sections and cells were washed in 60% isopropyl alcohol to remove unspecific attachments. Finally, the sections and cells were counterstained with hematoxylin and mounted with glycerol jelly.

Lactate detection

Ovarian tissue was homogenized on ice, centrifugation at 12000g at 4℃ for 5 min and the supernatant taken for lactate detection. For KGN cells, culture medium was directly used for lactate detection. Lactate content was determined by a lactate test kit (Nanjing Jiancheng, Nanjing, China) according to the instructions of the manufacturer. Briefly, 1 ml enzyme reagent, and 0.2 ml Color-substrate solution were added to the samples, mix well and incubated in 37 ° C water bath for 10 min. 2 ml stop buffer was then added and the absorbance measured at 530nm. The lactate content was then normalized to total protein content.

ATP detection

ATP levels were measured using ATP Assay Kit (Beyotime, Shanghai, China). Tissue and cells were homogenized on ice, centrifugation at 12000g at 4℃ for 5 min and the supernatant taken. ATP detection reagent add to the supernatant and measured with luminometer at 10 s RLU and concentration calculated based on an ATP standard curve and expressed as mol/mg. Protein concentration was determined using the BCA protein assay kit.

CCK-8 assay

KGN cells were seeded on a 96-well microtiter plate (5.0×10³ cells/well) and the Cell Counting Kit-8 (CCK-8) (Dojindo, Japan) was used to determine the number of viable cells. The CCK-8 reagent was added to each well and incubated for 2 h. Then, the absorbance of each well was measured at 450 nm.

Statistical analysis

Differences between two groups were determined using unpaired Student’s t-test (2-tailed). Differences among three groups were calculated using one-way analysis of variance (ANOVA), while differences within groups were assessed using the Mann-Whitney test. All data are shown as mean ± standard error of the mean (SEM). GraphPad Prism version 5.4 was used for all statistical analyses, and differences were considered significant when P < 0.05. All experiments were repeated, at least, 3 times.

Ovarian function during early pregnancy is impaired by high-fat diet-induced obesity.

After a 12-week feeding period, female C57BL/6J mice fed with HFD diets showed higher body weights (Fig. 1a and b). Compared with controls, HFD female mice had significantly elevated fasting levels of circulating serum triglycerides and total cholesterol (Fig. 1c and d). Since embryo implantation and decidualization, are related to estrogen and progesterone levels, which are produced by the corpus luteum of the ovary [14]. We determined the effects of HFD-induced obesity on the ovary during early pregnancy. Assessment of the luteal structure on D7 by H&E staining showed that the number of corpus luteum was significantly increased in the HFD group (Fig. 1e and f). Serum E2 and P4 levels on D7 were further measured to explore the effect of HFD-induced obesity on luteal function. As shown in Fig. 1g and h, serum estradiol and progesterone levels were considerably increased by HFD-induced obesity. It is well known that CYP19A1, CYP11A1 and steroidogenic acute regulator (StAR) are linked to the regulation of steroid biosynthesis. The CYP19A1 is a cytochrome P450 enzyme responsible for the aromatization of testosterone into estradiol [15]. StAR and the cholesterol side chain cleavage enzyme (P450scc), which is encoded by CYP11A1, catalyzes the first and rate-limiting steps of steroid biosynthesis [16, 17]. Our results showed a significant increase in CYP19A1, CYP11A1 and StAR on D7 in the HFD-fed pregnant mice when compared with those of the control group (Fig. 1i-j). Taken together, our results indicate that ovarian function was impaired by the HFD-induced obesity.

Ovarian glycolysis is not altered by high-fat diet-induced obesity during early pregnancy.

Hyperglycemia and dyslipidemia are two metabolic alterations of obesity, a condition which is associated with dysregulated glucose and fatty acid metabolism in tissues and organs [18]. To understand the possible mechanism by which high-fat diet-induced obesity impairs ovarian function during early pregnancy, we first explored whether glycolysis, the central pathway of glucose metabolism [19], is affected by obesity. Previous reports showed that highly expressed pyruvate dehydrogenase kinase 1 (PDK1) reduces the activity of pyruvate dehydrogenase (PDH) by inducing the phosphorylation of its E1α subunit (PDHA1) and subsequently, shifts the energy metabolism from oxidative phosphorylation to aerobic glycolysis [20]. In addition, hypoxia-inducible factor-1 (HIF1) can transform cells from aerobic oxidation to anaerobic glycolysis by activating the switch protein: PDK1 [21]. Our results showed that, there was no significant difference in ovarian HIF1α, PDK1, PDP1 and PDHA1 expression between the HFD group and the control group on D7 (Fig. 2b-e). This suggests that the impaired ovary, during early pregnancy, exhibited no obvious energy metabolic shifts between aerobic and anaerobic glycolysis. In addition, the Slc2a1 (code glucose transporter 1 protein) and Slc2a4 (code glucose transporter 4 protein), and the glycolysis key enzymes Hexokinase 2 (Hk2), Pyruvate kinase M2 (Pkm2) and phosphoglycerate kinase 1 (Pgk1) were similarly expressed in the pregnant mice ovary on D7 in both the HFD group and the control group (Fig. 2a). Moreover, the absence of significant changes in ovarian lactate production in the HFD group (Fig. 2f) confirms that ovarian glycolysis is not significantly altered by high-fat diet-induced obesity during early pregnancy.

High-fat diet-induced obesity enhances fatty acid β-oxidation in the ovary during early pregnancy

Based on ATP availability, ATP can trigger different cell responses, including cell proliferation, differentiation and apoptosis [22-24]. In this study, the ovarian tissue concentration of ATP was significantly increased by high-fat diet-induced obesity on D7 (Fig. 3a). Due to the stability of ovarian glycolysis in high-fat diet-induced obesity during early pregnancy, we speculated that the abnormally elevated ATP level in the dysfunctional ovary mainly comes from disordered fatty acid metabolism. Thus, we first examined the effect of high-fat diet-induced obesity on ovarian lipid accumulation. According to our results, high-fat diet caused a significant increase in ovarian lipid accumulation, as evidenced by the quantitative results of lipid content and Oil Red O staining and transmission electron microscopy of the ovary tissue on D7 (Fig. 3b-d). Moreover, genes involved in fatty acid synthesis (Acsl4 and Elovl5) and fatty acid uptake and transport (Slc27a4), together with the β-oxidation rate-limiting enzyme (Cpt1a), were significantly upregulated by high-fat diet-induced obesity on D7 (Fig. 3e-g). This suggests that high-fat diet-induced obesity enhances fatty acid β-oxidation in the ovary during early pregnancy. Mitochondrial fatty acid β-oxidation, the electron transport chain, and the tricarboxylic acid cycle are interrelated pathways involved in the cellular bioenergetics of eukaryotes [25]. Specifically, the expressions of some genes involved in the tricarboxylic acid cycle and electron transport chain were also abnormal on D7 in the HFD pregnant mice ovary (Fig. 3h). Taken together, these results suggest that during early pregnancy, high-fat diet-induced obesity primes fatty acid β-oxidation, which in turn increases the tricarboxylic acid cycle and the electron transport chain, and consequently increases ATP production and ovarian dysfunction.

Fatty acid β-oxidation contributes to the impairment of ovarian function by high-fat diet-induced obesity.

Palmitic acid (16:0, PA) is the most abundant non-esterified fatty acid in the western diet, and in the blood [26-28], while oleic acid (18:1, OA) is the most abundant monounsaturated non-esterified fatty acid in human serum [27, 28]. Cell loading with free fatty acids have been commonly used to develop in vitro models of obesity. In the present study, a mixture with 400μM OA and 200μM PA was used at a non-cytotoxic concentration to probe further into the role of high-fat diet induced obesity in the compromised ovarian function in vitro (Fig. 4a). We found that exocellular estradiol and progesterone levels significantly increased in OA- and PA-treated KGN cells (Fig. 4b and c). Exposure to OA and PA, significantly increased the protein expressions of steroid biosynthesis regulatory genes, including CYP19A1, CYP11A1 and StAR, in the luteinized KGN cells (Fig. 4d and e).

We further detected the concentration of ATP in the KGN cells, and the results showed that ATP levels were significantly increased following OA and PA treatments (Fig. 6d). Consistent with the in vivo results, both the lactate production and the expression of the factors regulating glycolysis in OA and PA-treated KGN cells were not significantly changed (Fig. 5). This suggests that the glycolysis of luteinized KGN cells is not obviously altered by OA and PA treatment in vitro. Given our earlier observation of the elevated ATP levels and the stable glycolysis, we hypothesized that the abnormally elevated ATP levels in the dysfunctional KGN cells also mainly came from disordered fatty acid β-oxidation. According to the results, the accumulation of lipid droplets and the intracellular TG content significantly increased in OA and PA-treated KGN cells (Fig. 6a-c). Moreover, the β-oxidation rate-limiting enzyme CPT1A in KGN cells was also dramatically upregulated by OA and PA treatments when compared with that of the control group (Fig. 6e and f). Etomoxir is a widely used small molecule that is able to inhibit fatty acid oxidation. It achieves this by exerting an irreversible inhibitory effect on CPT1A [29]. Following interference of β-oxidation of fatty acid with 100μM etomoxir, the compromised tricarboxylic acid cycle and electron transport chain, and the elevated ATP level by OA and PA were also reversed by etomoxir (Fig. 6g and h). Treatment with etomoxir further reversed the high-fat-induced dysfunctional state of the KGN cells, as indicated by a significant decrease in the levels of exocellular estradiol and progesterone than those in the cells treated with OA and PA alone (Fig. 6i and j). Steroid biosynthesis regulatory genes, including CYP19A1, CYP11A1 and StAR, were also downregulated (Fig. 6k and l).

It has been reported that obese women have an increased risk of pregnancy complications, such as miscarriage [3]. However, the underlying mechanisms have not been well elucidated. In early pregnancy, the competent blastocyst adheres to the decidualized endometrium to invade the uterine tissue and vessels and to establish pregnancy. During this time, the corpus luteum of the ovary secretes estrogen and progesterone [30, 31], so as to ensure the success of decidualization. Obese women are more likely to have ovulatory dysfunction due to dysregulation of the hypothalamic-pituitary-ovarian axis[3]. However, ovarian functionality of obese women during early pregnancy is not very clear. In this study, we used high-fat diet to induce obesity in maternal mice and then assessed ovarian functions in them. According to our study, female mice fed HFD diets showed increased numbers of corpus lutea and elevated serum estradiol and progesterone levels during early pregnancy. Significantly increased exocellular estradiol and progesterone levels were also found in luteinized KGN cells after OA and PA treatments. All these in vivo and in vitro results indicate that ovarian function was impaired by high-fat diet induced-obesity during early pregnancy.

Obesity has been found to be associated with dysregulated glucose and fatty acid metabolism in tissues and organs, such as adipose tissues, liver, muscle, pancreas and brain [32, 33]. To understand the mechanisms by which high-fat diet induced-obesity impairs ovarian function during early pregnancy, we first tested whether impaired glycolysis is involved it. According to the in vivo and in vitro results, ovarian glycolysis is not altered by high-fat diet-induced obesity. Also, the impaired ovary exhibited no obvious energy metabolic shifts between aerobic and anaerobic glycolysis. About 90-95% ATP production comes from oxidative phosphorylation of mitochondria and the rest from glycolysis. Mitochondrial fatty acid oxidation is the main source of ATP in many tissues and organs [34]. Fatty acid oxidation is a complex biological process involving mitochondrial β-oxidation, tricarboxylic acid cycle activity, and the electron transport chain [35]. Alterations in fatty acid metabolism can contribute to a variety of pathologies and cell dysfunction [36]. Mitochondrial fatty acid oxidation is the main source of ATP in many tissues and organs [34, 37]. Maintaining lipid homeostasis is a highly complex process which involves lipid storage, synthesis and utilization. During obesity, when the levels of free fatty acids exceed the lipid storage capacity of the adipose tissue, they begin to accumulate in the organs. This condition – known as “ectopic lipid deposition” – has several pathologic consequences [38]. Given our earlier observation of the stability of glycolysis, we hypothesized that dysfunctional ovary mainly results from disordered fatty acid metabolism. When high-fat diet was used to induce obesity in the mice, we found that lipid droplets were accumulated in the ovary during early pregnancy. The levels of triglycerides in the ovary were also elevated. These data indicate that ectopic lipid deposition in the ovary is involved in high-fat diet-induced obesity. To identify the lipid metabolism-associated genes whose expressions are altered in the ovary following the obesity induction, we tested several genes involved in fatty acid synthesis, transportation and β-oxidation. The results showed that ovarian Acsl4 and Elovl5 (which are involved fatty acid synthesis) and Slc27a4 (which is involved in fatty acid transportation) were dramatically up-regulated in the obese mice during early pregnancy. CPT1A is the rate-limiting enzyme that transfers the longchain fatty acyl CoA to the mitochondria for β-oxidation [39]. This enzyme was also observed to be upregulated in the obese mice. The upregulated expression of CPT1A in vivo and in vitro suggests that high-fat diet-induced obesity primes the aberrantly increased fatty acid β-oxidation in the ovary during early pregnancy. Etomoxir is widely used as a potent irreversible inhibitor of CPT1A [40]. In this study, when etomoxir was used to interfere with β-oxidation of fatty acid, the accumulation of lipid droplets and the levels of exocellular estradiol and progesterone levels in the OA- and PA-treated KGN cells were improved. All these suggest that high-fat diet-induced obesity enhances fatty acid β-oxidation in the ovary of mice during early pregnancy.

ATP is the direct energy for all kinds of life activities. Depending on its decrease or increase, ATP can trigger many different cell responses, including cell proliferation, differentiation and apoptosis [18, 22-24]. Here, the ovarian tissue concentration of ATP, during early pregnancy, was significantly increased by high-fat diet-induced obesity. Consistent with the in vivo results, ATP levels were significantly increased in the OA- and PA-treated KGN cells. The mitochondria meet the energy demands of cells through the uptake and oxidation of various substrates. This leads to the generation of acetyl CoA in order to produce ATP via the tricarboxylic acid cycle oxidation [41]. Transfer of electrons via mitochondrial co-factors NAD(H) and FAD(H2) to the electron transport chain creates a proton gradient across the inner mitochondrial membrane generating the mitochondrial membrane potential [41]. This drives oxidative phosphorylation, which generates ATP by the reduction of oxygen to water and the phosphorylation of ADP [41]. In this study, we found that some genes involved in the tricarboxylic acid cycle and electron transport chain were also abnormally expressed in the ovaries of the obese pregnant mice. The compromised tricarboxylic acid cycle and electron transport chain, and the elevated ATP level in the OA- and PA-treated KGN cells were reversed following etomoxir treatment. All these suggest that during early pregnancy, high-fat diet induced obesity can prime impaired fatty acid β-oxidation, which will in turn compromise the tricarboxylic acid cycle and the electron transport chain, and consequently increase ATP production and ovarian dysfunction.

In conclusion, our findings indicate that ovarian functions are impaired by high-fat diet-induced obesity during early pregnancy in mice. Abnormal fatty acid β-oxidation, but not abnormal glycolysis, contributes to impairing ovarian functions during obesity induced by high-fat diet. Our findings help in understanding the mechanistic effect of obesity on the ovary and female fertility; and reinforces the need to regulate the intake of high-fat diets so as to avoid obesity and its associated effects. Nevertheless, the confirmation of our findings in humans is highly recommended.

Ethics approval and consent to participate

The present study was approved by the Chongqing Medical University ethics committee.

Consent for publication

Not applicable.

Availability of data and materials

The datasets used in the present study are available from the corresponding author upon reasonable request.

Competing interests

The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported.

Funding

This work was supported by the National Key Research and Development Program of China (grant number 2018YFC1004401); the Science and Technology Research Program of Chongqing Municipal Education Commission (grant number KJQN201900413); the Natural Science Foundation of Chongqing (grant number cstc2020jcyj-msxmX0041).

Authors' contributions

Conceptualization, R.G. and Q.L.; data curation, Q.L. and S.G.; writing—original draft preparation, Q.L.; writing—review and editing, R.G.; supervision, C.Y., X.L., X.C. and J.H.; project administration, Y.D., Y.G., X.M., T.L. and F.L.; funding acquisition, Y.W. and R.G. All authors have read and agreed to the published version of the manuscript.

Acknowledgements

Not applicable.

Obesity. and overweight https://www.who.int/news-room/fact-sheets/detail/obesity-and-overweight.
Kannan B, Chelliah R, Rubab M, Banan-Mwine E, Oh DH. Gut Microbiome Modulation Based on Probiotic Application for Anti-Obesity: A Review on Efficacy and Validation. Microorganisms. 2019;7(10):456.
Broughton DE, Moley KH. Obesity and female infertility: potential mediators of obesity's impact. Fertility Sterility. 2017;107(4):840.
Ning D, D HS, Randolph JJF, Rita LC, Kyun PS. Perfluoroalkyl and polyfluoroalkyl substances (PFAS) and their effects on the ovary. Human Reproduction Update. 2020;26:724–52.
Pasquali R, Pelusi C, Genghini S, Cacciari M, Gambineri A. Obesity and reproductive disorders in women. Hum Reprod Update. 2003;9(4):359–72.
Definitions of infertility and recurrent pregnancy loss. Fertility Sterility. 2008;90(5 Suppl):60.
Ye Q, Cai S, Wang S, Zeng X, Ye C, Chen M, et al. Maternal short and medium chain fatty acids supply during early pregnancy improves embryo survival through enhancing progesterone synthesis in rats. J Nutr Biochem. 2019;69:98–107.
El-Zibdeh MY. Dydrogesterone in the reduction of recurrent spontaneous abortion. J Steroid Biochem Mol Biol. 2005;97(5):431–4.
Duan L, Yan D, Zeng W, Yang X, Wei Q. Effect of progesterone treatment due to threatened abortion in early pregnancy for obstetric and perinatal outcomes. Early Human Dev. 2010;86(1):41–3.
Melkonian EA, Schury MP. Biochemistry. Anaerobic Glycolysis. In StatPearls. Treasure Island (FL)2020.
Stremmel W, Pohl L, Ring A, Herrmann T. A new concept of cellular uptake and intracellular trafficking of long-chain fatty acids. Lipids. 2001;36(9):981–9.
Brendan M, Gabriel, Juleen R, Zierath. Circadian rhythms and exercise - re-setting the clock in metabolic disease. Nature reviews Endocrinology. 2019;15:197–206.
Cervera IP, Gabriel BM, Aldiss P, Morton NM. The phospholipase A2 family's role in metabolic diseases: Focus on skeletal muscle. Physiological Reports. 2021;9(1):e14662.
Liu M, Deng T, He J, Ding Y, Liu X, Xu H, et al. Exposure to Benzo[a]pyrene impairs the corpus luteum vascular network in rats during early pregnancy. Environ Pollut. 2020;259:113915.
Shimodaira M, Nakayama T, Sato I, Sato N, Izawa N, Mizutani Y, et al. Estrogen synthesis genes CYP19A1, HSD3B1, and HSD3B2 in hypertensive disorders of pregnancy. Endocrine. 2012;42(3):700–7.
Hu MC, Hsu HJ, Guo IC, Chung BC. Function of Cyp11a1 in animal models. Mol Cell Endocrinol. 2004;215(1–2):95–100.
Terry K, McGrath M, Lee IM, Buring J, De Vivo I. Genetic variation in CYP11A1 and StAR in relation to endometrial cancer risk. Gynecol Oncol. 2010;117(2):255–9.
Wang JD, Chen WY, Li JR, Lin SY, Wang YY, Wu CC, et al. Aspirin Mitigated Tumor Growth in Obese Mice Involving Metabolic Inhibition. Cells. 2020;9(3):569.
Mulukutla BC, Yongky A, Le T, Mashek DG, Hu WS. Regulation of Glucose Metabolism – A Perspective From Cell Bioprocessing. Trends Biotechnol. 2016;34(8):638–51.
Kwak CH, Jin L, Han JH, Han CW, Ha KT. Ilimaquinone Induces the Apoptotic Cell Death of Cancer Cells by Reducing Pyruvate Dehydrogenase Kinase 1 Activity. Int J Mol Sci. 2020;21(17):6021.
Li X, Li Y, Bai S, Zhang J, Liu Z, Yang J. NR2F1-AS1/miR-140/HK2 Axis Regulates Hypoxia-Induced Glycolysis and Migration in Hepatocellular Carcinoma. Cancer Manag Res. 2021;13:427–37.
Nicotera P, Leist M, Ferrando-May E. Intracellular ATP. a switch in the decision between apoptosis and necrosis. Toxicology Letters. 1998;102–103:139 – 42.
Atlante A, Giannattasio S, Bobba A, Gagliardi S, Petragallo V, Calissano P, et al. An increase in the ATP levels occurs in cerebellar granule cells en route to apoptosis in which ATP derives from both oxidative phosphorylation and anaerobic glycolysis. Biochim Biophys Acta. 2005;1708(1):50–62.
White N, Burnstock G. P2 receptors and cancer. Trends Pharmacol Sci. 2006;27(4):211–7.
Wang Y, Palmfeldt J, Gregersen N, Makhov AM, Conway JF, Wang M, et al. Mitochondrial fatty acid oxidation and the electron transport chain comprise a multifunctional mitochondrial protein complex. J Biol Chem. 2019;294(33):12380–91.
Ricchi M, Odoardi MR, Carulli L, Anzivino C, Ballestri S, Pinetti A, et al. Differential effect of oleic and palmitic acid on lipid accumulation and apoptosis in cultured hepatocytes. J Gastroenterol Hepatol. 2009;24(5):830–40.
Lopez-de-Andrés A, Jiménez-Trujillo M, Hernández-Barrera V, de Miguel-Yanes J, Méndez-Bailón M, Perez-Farinos N, et al. Trends in the Prevalence of Depression in Hospitalized Patients with Type 2 Diabetes in Spain: Analysis of Hospital Discharge Data from 2001 to 2011. Plos One. 2015;10(2):e0117346.
Yousif MD, Calder MD, Du JT, Ruetz KN, Crocker K, Urquhart BL, et al. Oleic Acid Counters Impaired Blastocyst Development Induced by Palmitic Acid During Mouse Preimplantation Development: Understanding Obesity-Related Declines in Fertility. Reprod Sci. 2020;27(11):2038–51.
O'Connor RS, Guo L, Ghassemi S, Snyder NW, Worth AJ, Weng L, et al. The CPT1a inhibitor, etomoxir induces severe oxidative stress at commonly used concentrations. Sci Rep. 2018;8(1):6289.
Huet-Hudson YM, Andrews GK, Dey SK. Cell type-specific localization of c-myc protein in the mouse uterus: modulation by steroid hormones and analysis of the periimplantation period. Endocrinology. 1989;125(3):1683–90.
Massimiani M, Lacconi V, Civita FL, Ticconi C, Campagnolo L. Molecular Signaling Regulating Endometrium–Blastocyst Crosstalk. Int J Mol Sci. 2019;21(1):23.
Zhuo M, Weizhen Z. Role of mTOR in Glucose and Lipid Metabolism. Int J Mol Sci. 2018;19(7):2043.
Watt MJ, Miotto PM, De Nardo W, Montgomery MK. The Liver as an Endocrine Organ-Linking NAFLD and Insulin Resistance. Endocr Rev. 2019;40(5):1367–93.
Hou T, Ma H, Wang H, Chen C, Ye J, Ahmed AM, et al. Sevoflurane preconditioning attenuates hypoxia/reoxygenation injury of H9c2 cardiomyocytes by activation of the HIF-1/PDK-1 pathway. PeerJ. 2020;8:e10603.
Jin Y, Shen Y, Su X, Cai J, Liu Y, Weintraub NL, et al. The Small GTPases Rab27b Regulates Mitochondrial Fatty Acid Oxidative Metabolism of Cardiac Mesenchymal Stem Cells. Front Cell Dev Biol. 2020;8:209.
Yufeng C, Lu J, Yunhong L, Guobin X, Chun C, Ying Z. Bamboo-shaving polysaccharide protects against high-diet induced obesity and modulates the gut microbiota of mice. Journal of Functional Foods. 2018;49:20–31.
Alice S, Matthew MK. Mitochondrial Fatty Acid Oxidation Disorders Associated with Short-Chain Enoyl-CoA Hydratase (ECHS1) Deficiency. Cells. 2018;7(6):46.
Nishi H, Higashihara T, Inagi R. Lipotoxicity in Kidney, Heart, and Skeletal Muscle Dysfunction. Nutrients. 2019;11(7):1664.
Ruderman NB, Saha AK, Kraegen EW. Minireview: malonyl CoA, AMP-activated protein kinase, and adiposity. Endocrinology. 2003;144(12):5166–71.
Schlaepfer I, Rider L, Rodrigues L, Gijón M, Pac C, Romero L, et al. Lipid Catabolism via CPT1 as a Therapeutic Target for Prostate Cancer. Mol Cancer Ther. 2014;13(10):2361–71.
Bradley J, Swann K. Mitochondria and lipid metabolism in mammalian oocytes and early embryos. Int J Dev Biol. 2019;63(3-4-5):93–103.

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High-Fat Diet-Induced Obesity Primes Impaired Fatty Acid β-Oxidation and Consequent Ovarian Dysfunction During Early Pregnancy

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