Journal of Reproduction and Development, Vol. 66, No 3, 2020 —Original Article— Gpr120 mRNA expression in gonadotropes in the mouse 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 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 b-subunit (Lhb) and 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. To test the specificity of the anti-GPR120 antibody, we tested II master mix (Takara Bio, Shiga, Japan) containing SYBR® Green antigen preadsorption by incubating 50 μl of 6 μg/ml antibody solution I, and run on the 7500 Real-time PCR System (Applied Biosystems, overnight at 4°C with 30 μg synthetic GPR120 peptide antigen, and Foster City, CA, USA). Denaturation was performed at 95°C (30 then incubating this solution with LβT2 cells. We obtained the peptide sec), amplification was conducted for 40 cycles with denaturation sequence information from a previous study [23], and the peptide

Table 1. List of primer sequences for real-time PCR Gene Forward primer Reverse primer Gpr120 5’-TCGCTGTTCAGGAACGAATG-3’ 5’-CACCAGAGGCTAGTTAGCTG-3’ Lhβ 5’-CTAGCATGGTCCGAGTACTG-3’ 5’-CCCATAGTCTCCTTTCCTGT-3’ Fshβ 5’-CTGCTACACTAGGGATCTGG-3’ 5’-TGACATTCAGTGGCTACTGG-3’ Gnrh-r 5’-CAGGATGATCTACCTAGCAG-3’ 5’-GCAGATTAGCATGATGAGGA-3’ L19 5’-CCAAGAAGATTGACCGCCATA-3’ 5’-CAGCTTGTGGATGTGCTCCAT-3’ FFA REGULATES GPR120 mRNA 251 was synthesized by PH JAPAN, Hiroshima, Japan.

Western blotting Overnight serum-starved LβT2 cells were treated with 100 μM palmitate for 0.5, 1, 2, 5, 10, or 15 min and then lysed in an extraction buffer (50 mM Tris-Cl, 150 mM NaCl, 1 mM EDTA, and 1% Triton X-100) containing 1% protease inhibitors (Nakarai Tesque, Kyoto, Japan) and a phosphatase inhibitor cocktail (Sigma-Aldrich). Total cell lysates were then centrifuged at 15,000 g for 5 min. Supernatants were mixed with 4 × sodium dodecyl sulfate sample buffer, boiled, and then separated in polyacrylamide gels. The proteins were then transferred on to polyvinylidene difluoride membranes (Millipore, San Jose, CA, USA). The membranes were probed with anti-ERK1/2 polyclonal antibody (1:1,000; Cell Signaling Technology, Danvers, MA, USA) or anti-phospho-ERK1/2 polyclonal antibody (1:1,000; Cell Signaling Technology). The membranes were further incubated with anti-rabbit IgG antibody conjugated to horseradish peroxidase (HRP) (1:4,000, Cell Signaling Technology), and developed with ImmunoStar Zeta (Fujifilm Wako Chemicals). Chemiluminescence was recorded using the ImageQuant LAS 500 spectrometer (GE Healthcare, Chicago, IL, USA). Fig. 1. Gpr120 (A), follicle-stimulating hormone b-subunit (Fshb) (B), luteinizing hormone b-subunit (Lhb) (C), and gonadotropin Statistical analysis releasing hormone-receptor (Gnrh-r), (D) mRNA expression All values are expressed as mean ± standard error of the mean levels in immortalized LβT2 cells. The cells were incubated with medium containing 100 μM palmitate (gray columns) and (SEM). One-way ANOVA, Tukey’s Honestly Significant Difference medium with no palmitate, as the control (open columns), for (HSD) post-hoc tests, and Student’s t-test were used to analyze the 24 h. The values are expressed as mean ± SEM (n = 4). Letters data. The results with P-values of < 0.05 were considered significant. indicate significant differences within each column (P < 0.05, Student’s t-test). L19, ribosomal protein L19. Results

Direct effects of palmitate on immortalized gonadotropes Gpr120, gonadotropic hormone b subunits, and gnrh-r mRNA expression levels in LβT2 cells are shown in Fig. 1. Gpr120 mRNA expression in the palmitate treatment group was significantly higher than that of the vehicle treatment group (Fig. 1A) and Fshb mRNA expression in LβT2 cells in the palmitate treatment group was significantly lower than that in the control group (Fig. 1B). On the contrary, no significant differences in Lhb and gnrh-r mRNA expression were observed between the two treatment groups (Fig. 1C and D). GPR120 immunoreactivity was observed on LβT2 cell membrane (Fig. 2). Almost all LβT2 cells were GPR120 immunoreactive in both the palmitate treatment group (99.00 ± 0.6%) and vehicle treatment group (98.25 ± 0.5%). Furthermore, no immunoreactivity was observed with preabsorbed antibodies. Western blotting patterns of ERK1/2 and phosphorylated ERK1/2 are shown in Fig. 3. Palmitate enhanced the phosphorylation of ERK1/2, with the peak phosphorylation occurring at 10 min after the administration of palmitate in LβT2 cells. Fig. 2. GPR120 protein expression in immortalized LβT2 cells. Diurnal variation in Gpr120 mRNA expression levels, and GPR120 immunoreactivity (green) was observed with DAPI plasma NEFA and glucose concentrations in ad libitum feeding (blue) in the palmitate-treated and vehicle-treated control group. Immunocytochemistry of LβT2 was started after 24 h mice of exposure to 100 μM palmitate medium or control medium. Diurnal variation in Gpr120 mRNA expression was observed GPR120 immunoreactivity was eliminated with preincubation of for 24 h in the pituitary gland (Fig. 4a). Gpr120 mRNA expression primary antibody with synthetic antigen peptide in LβT2 cells. was significantly higher at 1700 h than at 0900, 1300, and 2100 h. Scale bar = 20 µm. 252 DEURA et al.

The plasma NEFA concentrations presented higher values between 1300 and 1500 h than during the dark period; in ad libitum feeding group mice, a sharp decrease in serum NEFA concentration was observed at 2100 h with relatively low levels observed until 0700 h (Fig. 4b). No significant variation was observed in the plasma glucose concentration of the ad libitum feeding group mice during the 24 h observation period (Fig. 4c).

Diurnal variation in Gpr120 mRNA expression level, and plasma NEFA and glucose concentrations in night-time restricted feeding group mice Gpr120 mRNA expression levels in night-time restricted feeding Fig. 3. Extracellular signal-regulated kinase (ERK) phosphorylation is group mice were observed throughout the 24 h observation period stimulated by palmitate. LβT2 cells were treated with 100 μM (Fig. 5a). Gpr120 mRNA expression at 1500 h was slightly higher palmitate for 0.5, 1, 2, 5, 10, or 15 min. Total cell lysates were than that at other times during the light period, but no significant extracted and subjected to immunoblotting using anti-phospho- ERK1/2 and anti-ERK1/2 antibodies. Densitometric analysis was differences in Gpr120 mRNA expression were observed in night-time performed in three experiments, and phospho-ERK1/2 (pERK1/2) restricted feeding group mice. was normalized for ERK1/2. Data are expressed as mean ± SEM. No significant differences were observed in the plasma NEFA and * P < 0.05 vs. vehicle.

Fig. 5. Diurnal variation in Gpr120 mRNA expression levels (gray Fig. 4. Diurnal variation in Gpr120 mRNA expression levels in the columns in Fig. 5a), plasma non-esterified fatty acid (NEFA) pituitary gland (gray columns in Fig. 4a), plasma non-esterified (open circles in Fig. 5b), and plasma glucose (crossed circles in fatty acid (NEFA) (open circles in Fig. 4b), and plasma glucose Fig. 5c) concentrations in time-restricted feeding mice. The time- (black circles in Fig. 4c) concentrations in ad libitum feeding restricted feeding group had access to food for 12 h during the mice. The values are expressed as mean ± SEM (n = 4). Letters light phase, from 0700 to 1900 h. The values are expressed as indicate significant differences within each column or circle (P < mean ± SEM (n = 4). L19, ribosomal protein L19. 0.05, Tukey’s HSD). L19, ribosomal protein L19. FFA REGULATES GPR120 mRNA 253 glucose concentrations of night-time restricted feeding group mice This hypothesis was supported by previous studies. Unsaturated during the 24 h observation period (Fig. 2c and 5b). LCFAs downregulated the transcription of the rat Fshb gene [12] and mouse Fshb mRNA expression [31] in LβT2 cells. Short-term Discussion high-fat diets induced an increase in Fshb mRNA expression in adult male mice [13]. Furthermore, it has been established that the The present study demonstrated that palmitate induced an increase ERK1/2 pathway is located downstream of GPR120 to regulate in Gpr120 mRNA expression in mouse immortalized LβT2 cells and several physiological functions such as GLP-1 secretion [2] and that diurnal variation in Gpr120 mRNA expression was eliminated cytosolic phospholipase A2 activation [32]. In addition, GPR120 by night-time feeding restriction in the pituitary glands of adult is a Gq/11, Gi family member, or β-arrestin coupled GPCR [33]. male mice. These results indicate that LCFAs directly induced an Among these three intermediate molecules, the Gi proteins have increase in Gpr120 mRNA in the pituitary glands of adult male been shown to decrease the activity of protein kinase A (PKA) by mice. The rate of lipid metabolism was elevated in the afternoon inhibiting adenylyl cyclase activity [34]. The cAMP-dependent PKA in mice under ad libitum feeding conditions [22], because mice are pathway plays a pivotal role in GnRH-induced FSH synthesis [35–37]. nocturnal, and they typically feed during the active night phase and Therefore, the presence of Gi proteins downstream of GPR120 sleep during the passive day phase [22–24]. In a state of hunger, such means that the activation of GPR120 induces the suppression of as during the afternoon, in nocturnal animals, triglycerides hydrolyze FSH synthesis. Taken together, LCFAs may regulate the synthesis to FFAs in adipocytes and are then released into the bloodstream and secretion of FSH as a peripheral energy signal at the pituitary [25–27]. Therefore, we performed a night-time restricted feeding level via GPR120-mediated activation of ERK1/2 cascades through experiment based on the hypothesis that elevated blood NEFA levels the Gi protein in rodent gonadotropes. for metabolic use in the afternoon (1300–1500 h) would induce an In conclusion, the present study demonstrated that palmitate increase in Gpr120 mRNA expression in the pituitary gland in the induced an increase in Gpr120 mRNA expression and decrease current study. The result suggests that Gpr120 mRNA expression in Fshb expression in LβT2 cells. Furthermore, Gpr120 mRNA in the pituitary gland is directly regulated by nutritional status, expression presented diurnal variation that was regulated by plasma particularly by hunger-induced increase in blood NEFA levels. In NEFA concentration in the mouse pituitary. These results suggest addition, it is likely that Gpr120 mRNA expression was elevated in that GPR120 is activated by LCFAs to regulate FSH synthesis in gonadotropes in the mouse pituitary in this study, because GPR120 the mouse gonadotropes. immunoreactivity was specifically observed on gonadotropes in the mouse pituitary in our previous study [9] and in LβT2 cells in Acknowledgments the present study. Taken together, these results suggest that Gpr120 mRNA expression in gonadotropes of the mouse pituitary gland is We thank Dr PL Mellon for providing us LβT2 cells and Dr directly activated by LCFAs. Akira Hiraswa for providing rabbit anit-GPR120 antibody. We also Hirasawa et al. reported that GPR120 protein internalization was thank Mr Kazuhiko Nose for technical assistance. This work was observed after α-linolenic acid stimulation in gut cells [2]. Indeed, supported in part by the Japan Society for the Promotion of Science the GPCR proteins are often internalized and mRNA expression KAKENHI Grants 20780202 and 19K06446 awarded to RM. is upregulated after the binding of the ligand, which controls a number of receptors in the cell membrane [28]. However, we did not References observe similar protein internalization and the number of GPR120 immunoreactive cells was not altered by palmitate stimulation in 1. Ichimura A, Hirasawa A, Poulain-Godefroy O, Bonnefond A, Hara T, Yengo L, LβT2 cells in the present study. Further studies are needed to confirm Kimura I, Leloire A, Liu N, Iida K, Choquet H, Besnard P, Lecoeur C, Vivequin the effect of LCFAs on GPR120 protein levels. 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