MOLECULAR MEDICINE REPORTS 19: 5087-5096, 2019 Inhibitory effect of 17β‑estradiol on triglyceride synthesis in skeletal muscle cells is dependent on ESR1 and not ESR2 QUAN LIU1*, RUI LI1*, GUANJUN CHEN2, JIANMING WANG3, BINGFENG HU1, CHAOFEI LI4, XIAOHUAN ZHU5 and YUNXIA LU4,6 1Department of Clinical Pharmacy, Class 2014, School of Pharmacy; 2Center of Scientific Research, Anhui Medical University, Hefei, Anhui 230032; 3Dalian Maple International School, Dalian, Liaoning 116100; 4The Comprehensive Laboratory, Anhui Medical University; 5Department of Endocrinology, The First Affiliated Hospital of Anhui Medical University; 6Department of Biochemistry and Molecular Biology, Anhui Medical University, Hefei, Anhui 230032, P.R. China Received July 13, 2018; Accepted March 13, 2019 DOI: 10.3892/mmr.2019.10189 Abstract. The present study aimed to investigate the inhibitory note, treatment with E2 restored the expression levels of the effects and the mechanisms underlying 17β-estradiol (E2) aforementioned factors. In C2C12 cells, treatment with E2 or effects on triglyceride synthesis and insulin resistance PPT reversed the alterations induced by treatment with PA. In in skeletal muscle tissues and cells. Ovariectomy (OVX) contrast, pretreatment with DPN did not influence the effect was performed on 6-month-old female rats treated with or of PA. Collectively, E2 was able to interact with ESR1, thus without E2. Subsequently, various serum biochemical markers activating the CD36-PPARα pathway, decreasing the level of were measured. Additionally, pathological alterations of TG in the muscles and improving insulin resistance in skeletal the uterus, liver and skeletal muscle were analyzed, and muscles and C2C12 cells. the content of triglycerides (TG) in muscle was detected. Differentiated myotubes formed by C2C12 cells were treated Introduction with palmitic acid (PA) or pretreated with E2, estrogen receptor (ESR) 1 agonist propylpyrazoletriol (PPT) and The three principal muscle types in mammals are cardiac, ESR2 agonist diarylpropionitrile (DPN). Subsequently, the smooth and skeletal muscles, with skeletal muscle being mRNA or protein expression levels of ESR1/2, peroxisome the most abundant tissues in the human body (1). Although proliferator activated receptor α (PPARα), CD36 molecule previous studies identified sex‑specific differences in the phys- (CD36), fatty acid synthase (FASN), perilipin 2 (PLIN2), iopathological characteristics of the cardiovascular system (2), phosphorylated acetyl‑CoA carboxylase α (p‑ACACA), p‑AKT the sex-specific physiopathological properties of skeletal serine/threonine kinase (p‑AKT) and p-mitogen-activated muscles remain unclear. A previous study demonstrated that protein kinase 8 (p-MAPK8) were analyzed in skeletal muscle >3,000 genes were differentially expressed between male and or in C2C12 cells by reverse transcription-semi-quantitative female skeletal muscle (3). polymerase chain reaction and western blotting. The present In mammals, there are three principal types of estrogens: results suggested that treatment with E2 inhibited OVX-induced Estrone, 17β-estradiol (E2) and estriol. E2, which exhibits body weight gain, TG accumulation and insulin resistance. increased biological activity compared with other estrogens, The protein or mRNA expression levels of ESR1, CD36, is secreted primarily by the growing follicles in the ovaries PPARα, p‑ACACA and p‑AKT were decreased, whereas during the ovarian cycle (4). Notably, skeletal muscles are the protein or mRNA expression levels of ESR2, PLIN2, targeted by E2 (5). During perimenopausal and postmenopausal FASN and p-MAPK8 were increased in the OVX group. Of periods, there is a significant decrease in muscle strength that may be increased by hormonal replacement therapy (HRT), suggesting that estrogens are important modulators of muscle physiology and are able to affect gene expression and mito- chondrial function, thus maintaining the bioenergetic status of Correspondence to: Dr Yunxia Lu, Department of Biochemistry muscle cells (6). and Molecular Biology, Anhui Medical University, 81 Meishan In addition to the regulation of reproductive func- Road, Hefei, Anhui 230032, P.R. China E‑mail: [email protected] tions, estrogens may affect various physiological functions, including cellular metabolism (7,8). Decreased levels of E2 *Contributed equally following menopause or ovariectomy (OVX) are associated with hyperphagia, obesity, hepatic steatosis and triglycerides Key words: estrogen, fatty acid metabolism, ovariectomy, skeletal (TG) accumulation in skeletal muscle cells (9-11); however the muscle, estrogen receptor molecular mechanisms underlying E2 function remain unclear. Due to their lipophilic properties, estrogens are able to diffuse through the cellular membranes, interacting with intracellular 5088 LIU et al: EFFECT OF 17β-ESTRADIOL ON FATTY ACID METABOLISM IN SKELETAL MUSCLE CELLS receptors (12). In total, there are two types of estrogen recep- temperature and humidity (22±1˚C, 60‑70%) under a 12‑h tors (ESR): ESR1 and ESR2. light/dark cycle for one week. All animal experiments were Previous studies demonstrated that the effects of E2 on reviewed and approved by The Ethics Committee of Anhui energy homeostasis are primarily mediated by ESR1 (10,13). Medical University. Activating ESR1 using propylpyrazoletriol (PPT) may affect metabolism, insulin resistance and mitochondrial function Experimental protocol. Rats were randomly divided into three in OVX mice with metabolic syndrome (14); however, the groups: i) Sham surgery (SHAM; n=10); ii) rats that under- molecular mechanism underlying ESR1 function remains went ovariectomy without treatment (OVX; n=10); and iii) rats unclear. that underwent ovariectomy and treated with E2 (OVX + E2; Skeletal muscles are able to oxidize non‑esterified fatty acids, n=10). All groups underwent laparotomy; however, the OVX or to store fatty acids (FAs) as TG, accumulating intramuscular and OVX + E2 groups underwent bilateral ovariectomy. triacylglycerol (IMTAG) (15). Although physiological levels The OVX + E2 rats were injected with E2 (Sigma‑Aldrich; of IMTAG do not impair the metabolic functions of skeletal Merck KGaA, Darmstadt, Germany) at a concentration of muscles, the accumulation of IMTAG may be associated with 0.06 mg/kg/day for 3 months following surgery. Body weight promoted insulin resistance, due to increased levels of lipid and food intake were measured every week. intermediates and the subsequent activation of inflammatory or stress-associated pathway (16). Estrogen increases the levels Glucose tolerance test (GTT) and insulin tolerance test of the FA transporter CD36 molecule (CD36) in the plasma (ITT). Insulin sensitivity was assessed using GTT and ITT as membrane of cardiomyocytes, thus serving cardioprotective previously described (21). Briefly, for the GTT assay, all rats roles (17). Therefore, in the present study, it was hypothesized were fasted overnight and administered glucose (2 g/kg) by that E2 may increase CD36-mediated FA uptake, inhibiting the intraperitoneal injection. Blood samples were obtained from synthesis of FA and TG in skeletal muscle. the tail veins at 0, 30, 60, 90 and 120 min following injection. The transcription factor peroxisome proliferator activated The blood glucose was measured with a glucometer (OneTouch receptor α (PPARα) is expressed primarily in tissues exhibiting Ultra; Johnson & Johnson, New Brunswick, NJ, USA). ITT high rates of FA oxidation, including cardiac and skeletal assays were performed by the intraperitoneal injection of muscles (18). PPARα may regulate the protein expression neutral insulin (1 U/kg) following fasting for 4-6 h. The blood levels of factors involved in FA catabolism. PPARα activation glucose was measured at 0, 30, 45, 60, 90 and 120 min. All was identified to improve the musculoskeletal effects of data were plotted as blood glucose concentration over time. exercise during estrogen deficiency (19); however, its roles in Subsequently, the area under curve (AUC) was calculated. CD36-mediated FA uptake and the synthesis of acetyl‑CoA carboxylase α (ACACA), fatty acid synthetase (FASN) and Serum lipid levels and E2 assays. After 6 months, rats were perilipin 2 (PLIN2) remain unclear. fasted for 12 h and anesthetized by intraperitoneal injection Although the potential health risks and benefits of long‑term of sodium pentobarbital (60 mg/kg). Blood (~10 ml) was HRT remain unclear, future studies using short-term HRT or collected in tubes and serum was separated by centrifuging specific ER agonists including PPT and diarylpropionitrile the samples at 1,000 x g for 10 min at room temperature and (DPN), may provide novel insights into the effects of E2 on stored at ‑80˚C prior to biochemical analysis. Fasting TG, skeletal muscles, thus benefiting postmenopausal patients (20). total cholesterol (TC), low-density lipoprotein cholesterol Therefore, it was hypothesized that short-term HRT may (LDL‑C) and high-density lipoprotein cholesterol (HDL‑C) inhibit TG synthesis in the skeletal muscles of female rats levels were measured using an automatic biochemical analyzer following OVX, and this effect may be associated with the (AU640; Olympus Corporation, Tokyo, Japan). The serum role of FA in promoting the activity of PPARα following the levels of E2 were determined by radioimmunoassay (Beijing interaction between E2, and ESR1 or 2. To investigate this North Institute of Biological Technology, Beijing, China) and hypothesis, in vivo experiments