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Gpr120 Mrna Expression in Gonadotropes in the Mouse Pituitary

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Gpr120 Mrna Expression in Gonadotropes in the Mouse Pituitary Journal of Reproduction and Development, Vol. 66, No 3, 2020 —Original Article— Gpr120 mRNA expression in gonadotropes in the mouse pituitary gland is regulated by free fatty acids Chikaya DEURA1), Yusuke KIMURA1), Takumi NONOYAMA1) and Ryutaro MORIYAMA1) 1)Laboratory of Environmental Physiology, Department of Life Science, School of Science and Engineering, Kindai University, Higashiosaka 577-8502, Japan Abstract. GPR120 is a long-chain fatty acid (LCFA) receptor that is specifically expressed in gonadotropes in the anterior pituitary gland in mice. The aim of this study was to investigate whether GPR120 is activated by free fatty acids in the pituitary of mice and mouse immortalized gonadotrope LβT2 cells. First, the effects of palmitate on GPR120, gonadotropic hormone b-subunits, and GnRH-receptor expression in gonadotropes were investigated in vitro. We observed palmitate-induced an increase in Gpr120 mRNA expression and a decrease in follicle-stimulating hormone b-subunit (Fshb) expression in LβT2 cells. Furthermore, palmitate exposure caused the phosphorylation of ERK1/2 in LβT2 cells, but no significant changes were observed in the expression levels of luteinizing hormone b-subunit (Lhb) and gonadotropin releasing hormone-receptor (Gnrh-r) mRNA and number of GPR120 immunoreactive cells. Next, diurnal variation in Gpr120 mRNA expression in the male mouse pituitary gland was investigated using ad libitum and night-time restricted feeding (active phase from 1900 to 0700 h) treatments. In ad libitum feeding group mice, Gpr120 mRNA expression at 1700 h was transiently higher than that measured at other times, and the peak blood non-esterified fatty acid (NEFA) levels were observed from 1300 to 1500 h. These results were not observed in night-time-restricted feeding group mice. These results suggest that GPR120 is activated by LCFAs to regulate follicle stimulating hormone (FSH) synthesis in the mouse gonadotropes. Key words: Diurnal variation, Fatty acid, Gonadotrope, GPR120, Pituitary (J. Reprod. Dev. 66: 249–254, 2020) ysfunction of the long-chain fatty acid (LCFA) receptor GPR120 short-term high fat diet feeding for just 2 days induced an increase Dleads to obesity in humans and rodents [1], indicating possible in Gpr120 mRNA expression in the mouse pituitary gland [9, 13], functionality as a lipid sensor and modulator of energy balance. suggesting that GPR120 is sensitive to peripheral fatty acid levels GPR120 has been reported to regulate adipogenesis and glucagon- and could function as a lipid sensor in pituitary gonadotropes. like peptide-1(GLP-1) secretion during lipid metabolism [2], and Circadian variation has been observed to be involved in a number it was also found to be involved in the anti-inflammatory effects of of physiological functions in animals [14–16]. Reproductive func- macrophages [3]. GPR120 has been reported to interact with extracel- tions also display circadian variation [17, 18]. Indeed, it has been lular signal-regulated kinases 1/2 (ERK1/2) [2, 4]. Furthermore, the reported that the levels of the serum gonadotropic hormones LH expression of GPR120 has been observed in various organs and and follicle stimulating hormone (FSH) exhibit circadian variation tissues, including the taste buds [5], muscles [6], pancreas [7], liver in both female and male rodents [19, 20]. Mammalian cells have [8], intestines [2], and pituitary gland [9, 10]. The anterior pituitary an autonomous circadian oscillation period of approximately 24 h, gland contains non-endocrine cells and five types of endocrine cells. and external stimuli such as light, nutrition, and arousal stimuli are In our histological studies, GPR120 expression was observed only in essential for the maintenance of this oscillation [21]. To evoke the gonadotropes in mice [9], but it was observed in more than 80% of circadian variation in reproductive functions, important mechanisms gonadotropes and less than 20% of four other types of endocrine cells must exist to sense internal and external environmental changes in in cattle [10]. These results suggest that GPR120 is involved in the light, nutrition, and arousal stimuli. mechanism of gonadotropic hormone synthesis and/or secretion and The aim of the present study was to investigate whether GPR120 in in the regulation of gonadal function. However, the LCFA receptor the pituitary gonadotropes is activated as a lipid sensor. We investigated agonist GW9508 did not affect luteinizing hormone (LH) secretion the effects of palmitate on Gpr120, follicle-stimulating hormone b- [11] or the transcription of gonadotropin subunit genes [12] in an subunit (fshb), luteinizing hormone b-subunit (Lhb), and gonadotropin LβT2 gonadotropic cell line. Nevertheless, not only fasting but also releasing hormone-receptor (Gnrh-r) mRNA expression, GPR120 immunoreactivity, and ERK1/2 phosphorylation in immortalized Received: December 26, 2019 gonadotropes LβT2 cells. Furthermore, diurnal variation in Gpr120 Accepted: February 18, 2020 mRNA expression in the pituitary gland was measured in mice under Advanced Epub: February 28, 2020 ad libitum and night-time restricted feeding conditions. ©2020 by the Society for Reproduction and Development Correspondence: R Moriyama (e-mail: [email protected]) This is an open-access article distributed under the terms of the Creative Commons Attribution Non-Commercial No Derivatives (by-nc-nd) License. (CC-BY-NC-ND 4.0: https://creativecommons.org/licenses/by-nc-nd/4.0/) 250 DEURA et al. Materials and Methods at 95°C (5 sec), and annealing and amplification were conducted at 60°C (34 sec). Data were analyzed using the standard curve method Animals [22]. The forward and reverse primer set (Nippon EGT, Toyama, Eight-week-old male ICR mice were obtained from Japan SLC Japan) sequences used for each mouse gene are shown in Table 1. (Hamamatsu, Japan) and individually housed in a controlled environ- The expression levels of the target genes were normalized using ment (12 h light and 12 h dark; lights on at 0700 h; temperature, 24 ± L19 housekeeping gene expression levels. All real-time PCR cDNA 2°C). The mice had free access to food (Labo-MR stock, Nihon Nosan amplification samples were monitored for the presence of a single Kogyo, Yokohama, Japan) and water for one week for habituation peak in the dissociation curve. until the start of the experiment. The time-restricted feeding group had access to food for 12 h during the light phase, from 0700 to Measurement of plasma non-esterified fatty acid (NEFA) and 1900 h. The mice in the control group had free access to food at glucose concentration all times. The body weight of time-restricted feeding group mice Plasma NEFA and glucose concentrations were measured to and control mice were monitored daily throughout the 10 days of evaluate the daily energy state of the mice. Plasma was obtained experimentation (Supplementary Fig. 1: online only). The Committee by centrifuging the blood samples obtained from mice at 15,000 on Animal Experiments of Kindai University approved the study. rpm for 15 min at 4°C. Plasma NEFA and glucose concentrations The experiments were performed in accordance with the Guide for were determined using the NEFA-C test and Glucose-CII test kits, the Care and Use of Laboratory Animals. respectively. The NEFA C-test and glucose CII-test had detection levels of 0.05–2 mEq/l and 3.8–700 mg/dl, respectively, and the Cell culture coefficients of variation of the NEFA C-test and glucose CII-test Mouse gonadotrope cell line LβT2 was cultured in Dulbecco’s were < 3% and < 2%, respectively. All test kits were obtained from modified Eagle’s medium (DMEM; Thermo Fisher Scientific, the Fujifilm Wako Pure Chemical, Osaka, Japan. Waltham, MA, USA) containing 10% fetal calf serum (FCS) and 0.5% Penicillin–Streptomycin solution (Sigma-Aldrich, St. Louis, Immunocytochemistry of LβT2 cells MO, USA). LβT2 cells were then dispersed into 35 mm culture For GPR120 immunocytochemistry, LβT2 cells were plated on dishes (AGC techno glass, Shizuoka, Japan) for palmitate exposure poly-l-lysine coated glass coverslips for 48 h before the experiment. and RNA extraction. LβT2 cells were exposed to 100 μM palmitate The cells were exposed to palmitate, and then fixed with Mildform® (Sigma-Aldrich) in the culture medium for 24 h. 10N (Fujifilm Wako Pure Chemical) for 5 min at room temperature. The cells were incubated with 10% normal-donkey serum (NDS) Total RNA extraction and cDNA synthesis in 0.05 M PBS for 1 h at room temperature to block non-specific The mice were sacrificed by decapitation, and the isolated pituitary binding, and then incubated with rabbit anti-GPR120 serum (1:10,000; glands were homogenized with TRI Reagent® (Sigma-Aldrich) in provided by Dr Akira Hirasawa, Kyoto University, Japan) in 0.05 1.5 ml tubes for total RNA extraction. LβT2 cells were washed with M PBS containing 5% NRS and 0.1% Tween 20 at 4°C overnight. sterilized phosphate-buffered saline (PBS), and then dissolved in After three washes in PBS, the cells were incubated in Alexa 488 TRI Reagent. The resulting total RNA of the pituitary gland and conjugated donkey anti-rabbit IgG (1:2,000; Thermo Fisher Scientific) LβT2 cells were treated with RNase-free DNase I (Thermo Fisher in 0.05 M PBS for 90 min at room temperature. The cells were then Scientific) to eliminate genomic DNA contamination, and the cDNA rinsed in 0.05 M PBS. Finally, the coverslips were placed on glass was synthesized using the Superscript II™ kit with an oligo(dT)12-18 slides using VECTASHIELD Mounting Medium with DAPI (Vector primer (Thermo Fisher Scientific). Laboratories, Burlingame, CA, USA) and observed under a BX51 fluorescence microscope (Olympus, Tokyo, Japan). We determined Real-time PCR the number of GPR120 immunoreactive cells out of the 100 DAPI Gpr120, Fshb, Lhb, and Gnrh-r mRNA expression levels were immunoreactive cells in each slide, and counted four slides in each determined by real-time PCR using the SYBR® Premix Ex Taq™ group.
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