Gpr120 mRNA expression in gonadotropes in the mouse pituitary gland is regulated by free fatty acids.

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.

Dysfunction of the long-chain fatty acid (LCFA) receptor GPR120 leads to obesity in humans and rodents [1], indicating possible functionality as a lipid sensor and modulator of energy balance. GPR120 has been reported to regulate adipogenesis and glucagon-like peptide-1(GLP-1) secretion during lipid metabolism [2], and it was also found to be involved in the anti-inflammatory effects of macrophages [3]. GPR120 has been reported to interact with extracellular signal-regulated kinases 1/2 (ERK1/2) [2, 4]. Furthermore, the expression of GPR120 has been observed in various organs and tissues, including the taste buds [5], muscles [6], pancreas [7], liver [8], intestines [2], and pituitary gland [9, 10]. The anterior pituitary gland contains non-endocrine cells and five types of endocrine cells. In our histological studies, GPR120 expression was observed only in gonadotropes in mice [9], but it was observed in more than 80% of gonadotropes and less than 20% of four other types of endocrine cells in cattle [10]. These results suggest that GPR120 is involved in the mechanism of gonadotropic hormone synthesis and/or secretion and in the regulation of gonadal function. However, the LCFA receptor agonist GW9508 did not affect luteinizing hormone (LH) secretion [11] or the transcription of gonadotropin subunit genes [12] in an LβT2 gonadotropic cell line. Nevertheless, not only fasting but also short-term high fat diet feeding for just 2 days induced an increase in Gpr120 mRNA expression in the mouse pituitary gland [9, 13], suggesting that GPR120 is sensitive to peripheral fatty acid levels and could function as a lipid sensor in pituitary gonadotropes.

Circadian variation has been observed to be involved in a number of physiological functions in animals [14,15,16]. Reproductive functions also display circadian variation [17, 18]. Indeed, it has been reported that the levels of the serum gonadotropic hormones LH and follicle stimulating hormone (FSH) exhibit circadian variation in both female and male rodents [19, 20]. Mammalian cells have an autonomous circadian oscillation period of approximately 24 h, and external stimuli such as light, nutrition, and arousal stimuli are essential for the maintenance of this oscillation [21]. To evoke the circadian variation in reproductive functions, important mechanisms must exist to sense internal and external environmental changes in light, nutrition, and arousal stimuli.

The aim of the present study was to investigate whether GPR120 in the pituitary gonadotropes is activated as a lipid sensor. We investigated the effects of palmitate on Gpr120, follicle-stimulating hormone b-subunit (fshb), luteinizing hormone b-subunit (Lhb), and gonadotropin releasing hormone-receptor (Gnrh-r) mRNA expression, GPR120 immunoreactivity, and ERK1/2 phosphorylation in immortalized gonadotropes LβT2 cells. Furthermore, diurnal variation in Gpr120 mRNA expression in the pituitary gland was measured in mice under ad libitum and night-time restricted feeding conditions.

Materials and Methods

Animals

Eight-week-old male ICR mice were obtained from Japan SLC (Hamamatsu, Japan) and individually housed in a controlled environment (12 h light and 12 h dark; lights on at 0700 h; temperature, 24 ± 2°C). The mice had free access to food (Labo-MR stock, Nihon Nosan Kogyo, Yokohama, Japan) and water for one week for habituation 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 1900 h. The mice in the control group had free access to food at all times. The body weight of time-restricted feeding group mice and control mice were monitored daily throughout the 10 days of experimentation (Supplementary Fig. 1: online only). The Committee on Animal Experiments of Kindai University approved the study. The experiments were performed in accordance with the Guide for the Care and Use of Laboratory Animals.

Cell culture

Mouse gonadotrope cell line LβT2 was cultured in Dulbecco’s modified Eagle’s medium (DMEM; Thermo Fisher Scientific, Waltham, MA, USA) containing 10% fetal calf serum (FCS) and 0.5% Penicillin–Streptomycin solution (Sigma-Aldrich, St. Louis, MO, USA). LβT2 cells were then dispersed into 35 mm culture dishes (AGC techno glass, Shizuoka, Japan) for palmitate exposure and RNA extraction. LβT2 cells were exposed to 100 μM palmitate (Sigma-Aldrich) in the culture medium for 24 h.

Total RNA extraction and cDNA synthesis

The mice were sacrificed by decapitation, and the isolated pituitary glands were homogenized with TRI Reagent® (Sigma-Aldrich) in 1.5 ml tubes for total RNA extraction. LβT2 cells were washed with sterilized phosphate-buffered saline (PBS), and then dissolved in TRI Reagent. The resulting total RNA of the pituitary gland and LβT2 cells were treated with RNase-free DNase I (Thermo Fisher Scientific) to eliminate genomic DNA contamination, and the cDNA was synthesized using the Superscript II™ kit with an oligo(dT)12-18 primer (Thermo Fisher Scientific).

Real-time PCR

Gpr120, Fshb, Lhb, and Gnrh-r mRNA expression levels were determined by real-time PCR using the SYBR® Premix Ex Taq™ II master mix (Takara Bio, Shiga, Japan) containing SYBR® Green I, and run on the 7500 Real-time PCR System (Applied Biosystems, Foster City, CA, USA). Denaturation was performed at 95°C (30 sec), amplification was conducted for 40 cycles with denaturation at 95°C (5 sec), and annealing and amplification were conducted at 60°C (34 sec). Data were analyzed using the standard curve method [22]. The forward and reverse primer set (Nippon EGT, Toyama, Japan) sequences used for each mouse gene are shown in Table 1. The expression levels of the target genes were normalized using L19 housekeeping gene expression levels. All real-time PCR cDNA amplification samples were monitored for the presence of a single peak in the dissociation curve.

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’

Measurement of plasma non-esterified fatty acid (NEFA) and glucose concentration

Plasma NEFA and glucose concentrations were measured to evaluate the daily energy state of the mice. Plasma was obtained by centrifuging the blood samples obtained from mice at 15,000 rpm for 15 min at 4°C. Plasma NEFA and glucose concentrations were determined using the NEFA-C test and Glucose-CII test kits, 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 coefficients of variation of the NEFA C-test and glucose CII-test were < 3% and < 2%, respectively. All test kits were obtained from the Fujifilm Wako Pure Chemical, Osaka, Japan.

Immunocytochemistry of LβT2 cells

For GPR120 immunocytochemistry, LβT2 cells were plated on poly-l-lysine coated glass coverslips for 48 h before the experiment. The cells were exposed to palmitate, and then fixed with Mildform® 10N (Fujifilm Wako Pure Chemical) for 5 min at room temperature. The cells were incubated with 10% normal-donkey serum (NDS) in 0.05 M PBS for 1 h at room temperature to block non-specific binding, and then incubated with rabbit anti-GPR120 serum (1:10,000; provided by Dr Akira Hirasawa, Kyoto University, Japan) in 0.05 M PBS containing 5% NRS and 0.1% Tween 20 at 4°C overnight. After three washes in PBS, the cells were incubated in Alexa 488 conjugated donkey anti-rabbit IgG (1:2,000; Thermo Fisher Scientific) in 0.05 M PBS for 90 min at room temperature. The cells were then rinsed in 0.05 M PBS. Finally, the coverslips were placed on glass slides using VECTASHIELD Mounting Medium with DAPI (Vector Laboratories, Burlingame, CA, USA) and observed under a BX51 fluorescence microscope (Olympus, Tokyo, Japan). We determined the number of GPR120 immunoreactive cells out of the 100 DAPI immunoreactive cells in each slide, and counted four slides in each group. To test the specificity of the anti-GPR120 antibody, we tested antigen preadsorption by incubating 50 μl of 6 μg/ml antibody solution overnight at 4°C with 30 μg synthetic GPR120 peptide antigen, and then incubating this solution with LβT2 cells. We obtained the peptide sequence information from a previous study [23], and the peptide 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).

Statistical analysis

All values are expressed as mean ± standard error of the mean (SEM). One-way ANOVA, Tukey’s Honestly Significant Difference (HSD) post-hoc tests, and Student’s t-test were used to analyze the data. The results with P-values of < 0.05 were considered significant.

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).

Fig. 1.

Gpr120 (A), follicle-stimulating hormone b-subunit (Fshb) (B), luteinizing hormone b-subunit (Lhb) (C), and gonadotropin releasing hormone-receptor (Gnrh-r), (D) mRNA expression levels in immortalized LβT2 cells. The cells were incubated with medium containing 100 μM palmitate (gray columns) and medium with no palmitate, as the control (open columns), for 24 h. The values are expressed as mean ± SEM (n = 4). Letters indicate significant differences within each column (P < 0.05, Student’s t-test). L19, ribosomal protein L19.

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.

Fig. 2.

GPR120 protein expression in immortalized LβT2 cells. GPR120 immunoreactivity (green) was observed with DAPI (blue) in the palmitate-treated and vehicle-treated control group. Immunocytochemistry of LβT2 was started after 24 h of exposure to 100 μM palmitate medium or control medium. GPR120 immunoreactivity was eliminated with preincubation of primary antibody with synthetic antigen peptide in LβT2 cells. Scale bar = 20 µm.

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. 3.

Extracellular signal-regulated kinase (ERK) phosphorylation is stimulated by palmitate. LβT2 cells were treated with 100 μM palmitate for 0.5, 1, 2, 5, 10, or 15 min. Total cell lysates were extracted and subjected to immunoblotting using anti-phospho-ERK1/2 and anti-ERK1/2 antibodies. Densitometric analysis was performed in three experiments, and phospho-ERK1/2 (pERK1/2) was normalized for ERK1/2. Data are expressed as mean ± SEM. * P < 0.05 vs. vehicle.

Diurnal variation in Gpr120 mRNA expression levels, and plasma NEFA and glucose concentrations in ad libitum feeding mice

Diurnal variation in Gpr120 mRNA expression was observed for 24 h in the pituitary gland (Fig. 4a). Gpr120 mRNA expression was significantly higher at 1700 h than at 0900, 1300, and 2100 h.

Fig. 4.

Diurnal variation in Gpr120 mRNA expression levels in the pituitary gland (gray columns in Fig. 4a), plasma non-esterified fatty acid (NEFA) (open circles in Fig. 4b), and plasma glucose (black circles in Fig. 4c) concentrations in ad libitum feeding mice. The values are expressed as mean ± SEM (n = 4). Letters indicate significant differences within each column or circle (P < 0.05, Tukey’s HSD). L19, ribosomal protein L19.

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 group mice were observed throughout the 24 h observation period (Fig. 5a). Gpr120 mRNA expression at 1500 h was slightly higher than that at other times during the light period, but no significant differences in Gpr120 mRNA expression were observed in night-time restricted feeding group mice.

Fig. 5.

Diurnal variation in Gpr120 mRNA expression levels (gray columns in Fig. 5a), plasma non-esterified fatty acid (NEFA) (open circles in Fig. 5b), and plasma glucose (crossed circles in Fig. 5c) concentrations in time-restricted feeding mice. The time-restricted feeding group had access to food for 12 h during the light phase, from 0700 to 1900 h. The values are expressed as mean ± SEM (n = 4). L19, ribosomal protein L19.

No significant differences were observed in the plasma NEFA and glucose concentrations of night-time restricted feeding group mice during the 24 h observation period (Fig. 2c and 5b).

Discussion

The present study demonstrated that palmitate induced an increase in Gpr120 mRNA expression in mouse immortalized LβT2 cells and that diurnal variation in Gpr120 mRNA expression was eliminated by night-time feeding restriction in the pituitary glands of adult male mice. These results indicate that LCFAs directly induced an increase in Gpr120 mRNA in the pituitary glands of adult male mice. The rate of lipid metabolism was elevated in the afternoon in mice under ad libitum feeding conditions [22], because mice are nocturnal, and they typically feed during the active night phase and sleep during the passive day phase [22,23,24]. In a state of hunger, such as during the afternoon, in nocturnal animals, triglycerides hydrolyze to FFAs in adipocytes and are then released into the bloodstream [25,26,27]. Therefore, we performed a night-time restricted feeding experiment based on the hypothesis that elevated blood NEFA levels for metabolic use in the afternoon (1300–1500 h) would induce an increase in Gpr120 mRNA expression in the pituitary gland in the current study. The result suggests that Gpr120 mRNA expression in the pituitary gland is directly regulated by nutritional status, particularly by hunger-induced increase in blood NEFA levels. In addition, it is likely that Gpr120 mRNA expression was elevated in gonadotropes in the mouse pituitary in this study, because GPR120 immunoreactivity was specifically observed on gonadotropes in the mouse pituitary in our previous study [9] and in LβT2 cells in the present study. Taken together, these results suggest that Gpr120 mRNA expression in gonadotropes of the mouse pituitary gland is directly activated by LCFAs.

Hirasawa et al. reported that GPR120 protein internalization was observed after α-linolenic acid stimulation in gut cells [2]. Indeed, the GPCR proteins are often internalized and mRNA expression is upregulated after the binding of the ligand, which controls a number of receptors in the cell membrane [28]. However, we did not observe similar protein internalization and the number of GPR120 immunoreactive cells was not altered by palmitate stimulation in LβT2 cells in the present study. Further studies are needed to confirm the effect of LCFAs on GPR120 protein levels.

In the time-restricted feeding experiment, Gpr120 mRNA expression did not display significant variance during the day, but a slight transient increase was observed at 1500 h. The results suggest that diurnal variation in Gpr120 mRNA in gonadotropes is regulated by not only serum free-fatty acid concentration but also clock genes. The expression of clock genes such as period-1 (mPer1) was observed in the rat gonadotropes and in the immortalized gonadotrope cells [29], and they have been reported to be involved in the regulation of gonadotropin-releasing hormone receptor expression [29, 30]. The coordinated actions of nutritional factors and clock genes may influence the regulation of Gpr120 expression in gonadotropes. Further studies are needed to clarify the regulatory mechanism of diurnal variation in GPR120 signaling in gonadotropes.

The present study indicates that diurnal hunger-induced increase in blood NEFA may regulate the transcription or secretion of FSH via GPR120-activated ERK1/2 pathway at the pituitary level in rodents. This hypothesis was supported by previous studies. Unsaturated LCFAs downregulated the transcription of the rat Fshb gene [12] and mouse Fshb mRNA expression [31] in LβT2 cells. Short-term high-fat diets induced an increase in Fshb mRNA expression in adult male mice [13]. Furthermore, it has been established that the ERK1/2 pathway is located downstream of GPR120 to regulate several physiological functions such as GLP-1 secretion [2] and cytosolic phospholipase A2 activation [32]. In addition, GPR120 is a Gq/11, Gi family member, or β-arrestin coupled GPCR [33]. Among these three intermediate molecules, the Gi proteins have been shown to decrease the activity of protein kinase A (PKA) by inhibiting adenylyl cyclase activity [34]. The cAMP-dependent PKA pathway plays a pivotal role in GnRH-induced FSH synthesis [35,36,37]. Therefore, the presence of Gi proteins downstream of GPR120 means that the activation of GPR120 induces the suppression of FSH synthesis. Taken together, LCFAs may regulate the synthesis and secretion of FSH as a peripheral energy signal at the pituitary level via GPR120-mediated activation of ERK1/2 cascades through the Gi protein in rodent gonadotropes.

In conclusion, the present study demonstrated that palmitate induced an increase in Gpr120 mRNA expression and decrease in Fshb expression in LβT2 cells. Furthermore, Gpr120 mRNA expression presented diurnal variation that was regulated by plasma NEFA concentration in the mouse pituitary. These results suggest that GPR120 is activated by LCFAs to regulate FSH synthesis in the mouse gonadotropes.