GPR75 signaling is dispensable for reproduction but contributes to feeding and body growth in rats on normal chow and is involved in high-fat diet-induced hyperphagia, obesity, and hyperglycemia development.

Gpr75 mRNA expression was highly expressed in the hypothalamus (the POA, AVPV, LHA, PVN, ARC, and VMH), cerebrum, cerebellum, brain stem, and pituitary in both male and female rats, and no significant differences were found in the hypothalamic expression levels between males and females ( Fig. 2 ). However, Gpr75 mRNA levels were very low in peripheral tissues, such as the muscle, heart, lung, stomach, spleen, kidney, small intestine, and colon in both sexes. Additionally, Gpr75 expression was low in the reproductive organs, such as the testis in male rats and the ovary and uterus in female rats ( Fig. 2 ). No significant differences were found in the expression levels in these peripheral tissues and organs except for the kidney (#, p < 0.05, Student’s t -test) between males and females ( Fig. 2 ). BW and cumulative food intake were lower in Gpr75 –/– male and female rats on normal chow than in Gpr75 +/+ control rats. Two-way mixed ANOVA revealed that the BW was comparable among Gpr75 –/– , Gpr75 +/– , and Gpr75 +/+ rats at weaning (3 weeks of age) in both sexes ( Fig. 3 A, B). Conversely, for both sexes, Gpr75 –/– rats showed significantly lower BW (*, p < 0.05, simple main effect analysis) than Gpr75 +/+ rats from 5 to 12 weeks of age. In addition, the cumulative food intake of Gpr75 –/– rats was significantly lower (*, p < 0.05, simple main effect analysis) than that of Gpr75 +/+ rats for both sexes (from 4–5 to 11–12 weeks of age). The BW and cumulative food intake of Gpr75 +/– rats were intermediate between those of Gpr75 +/+ and Gpr75 –/– rats in both sexes ( Fig. 3 A, B). Balano-preputial separation (a pubertal sign in male rodents) and vaginal opening (a pubertal sign in female rodents) were observed at 43–52 and 28–38 days of age, respectively, in Gpr75 –/– , Gpr75 +/– , and Gpr75 +/+ rats. For both sexes, one-way ANOVA revealed no significant differences in the mean age of puberty onset between the groups ( Fig. 3 C, D). Gonadectomized Gpr75 –/– and Gpr75 +/+ male and female rats showed apparent pulsatile LH secretion, and no significant differences were found in the mean LH concentrations and the frequency and amplitude of LH pulses between the Gpr75 –/– and Gpr75 +/+ groups ( Fig. 3 E, F). In addition, testicular and ovarian weights were comparable between Gpr75 –/– (testis, 841.4 ± 40.9 mg/100 g BW, n = 4; ovary, 50.6 ± 4.15 mg/100 g BW, n = 5) and Gpr75 +/+ rats (testis, 786.1 ± 35.9 mg/100 g BW, n = 3; ovary, 45.1 ± 1.15 mg/100 g BW, n = 5). Furthermore, Gpr75 –/– male and female rats were fertile, and no significant difference was found in the litter size between Gpr75 –/– (litter size, 10.8 ± 1.0, n = 6) and Gpr75 +/+ rat pairs (litter size, 11.8 ± 1.5, n = 4). In addition, quantitative RT–PCR revealed no significant differences in the hypothalamic Kiss1 and Gnrh1 mRNA expression levels between Gpr75 –/– ( Kiss1 , 0.865 ± 0.153 and Gnrh1 , 1.101 ± 0.148, n = 6) and Gpr75 +/+ ( Kiss1 , 1.00 ± 0.241 and Gnrh1 , 1.00 ± 0.197, n = 6) male rats. Fig. 4 A, B shows the representative 24-h feeding pattern observed in individual Gpr75 +/+ (A) and Gpr75 –/– (B) male rats on normal chow. Both Gpr75 +/+ and Gpr75 –/– rats primarily showed feeding behavior during the dark phase. The daily average number of bouts was comparable between Gpr75 +/+ and Gpr75 –/– male rats ( Fig. 4 C), whereas two-way factorial ANOVA revealed that the number of bouts in Gpr75 +/+ and Gpr75 –/– male rats was significantly higher (§, p < 0.05, main effect analysis) during the dark phase than during the light phase. Moreover, daily feeding duration ( Fig. 4 D) and food intake ( Fig. 4 E) were significantly lower (#, p < 0.05, Student’s t -test) in Gpr75 –/– male rats than those in Gpr75 +/+ male rats. Furthermore, two-way factorial ANOVA revealed that the feeding duration and food intake were significantly lower (*, p < 0.05, simple main effect analysis) in Gpr75 –/– male rats than those in Gpr75 +/+ control during the dark phase ( Fig. 4 D, E), and these parameters were significantly higher during the dark phase than during the light phase in both genotypes (*, p < 0.05, simple main effect analysis). BW, cumulative food intake, and WAT weight were lower in Gpr75 –/– male rats than in Gpr75 +/+ controls under HFD conditions. Three-way mixed ANOVA revealed that the BW of Gpr75 –/– male rats on HFD were significantly lower (*, p < 0.05, simple-simple main effect analysis) than those of Gpr75 +/+ male rats on HFD from 8 to 28 weeks of age ( Fig. 5 A). On the other hand, no significant differences were found in BW between Gpr75 –/– male rats on HFD and Gpr75 –/– male rats on control diet. The BW of Gpr75 +/+ male rats on HFD was significantly higher (†, p < 0.05, simple-simple main effect analysis) than those of Gpr75 +/+ male rats on control diet from 18 to 28 weeks of age. In addition, the BW of Gpr75 –/– male rats on control diet were significantly lower (‡, p < 0.05, simple-simple main effect analysis) than those of Gpr75 +/+ male rats on control diet from 8 to 28 weeks of age. Three-way mixed ANOVA revealed that the cumulative food intake of Gpr75 –/– male rats on HFD was significantly lower (*, p < 0.05, simple-simple main effect analysis) than that of Gpr75 +/+ male rats on HFD throughout the experimental period ( Fig. 5 B). Conversely, no significant differences were found in the cumulative food intake between Gpr75 –/– male rats on HFD and Gpr75 –/– male rats on control diet. The cumulative food intake of Gpr75 +/+ male rats on HFD was significantly higher (†, p < 0.05, simple-simple main effect analysis) than that of Gpr75 +/+ male rats on control diet from 10–12 to 26–28 weeks of age. In addition, the cumulative food intake of Gpr75 –/– male rats on control diet was significantly lower (‡, p < 0.05, simple-simple main effect analysis) during 8–10 weeks of age and tended to be lower ( p < 0.1, simple-simple main effect analysis) thereafter than that of Gpr75 +/+ rats on control diet. Two-way factorial ANOVA revealed that the visceral WAT weight of Gpr75 –/– male rats on HFD was significantly lower (*, p < 0.05, simple main effect analysis) than that of Gpr75 +/+ male rats on HFD ( Fig. 5 C). However, no significant difference was found in the visceral WAT weight between Gpr75 –/– male rats on HFD and control diet. The visceral WAT weight was significantly higher (*, p < 0.05, simple main effect analysis) in Gpr75 +/+ male rats on HFD than in Gpr75 +/+ male rats on control diet. In addition, the visceral WAT weight of Gpr75 –/– male rats on control diet was significantly lower (*, p < 0.05, simple main effect analysis) than that of Gpr75 +/+ male rats on control diet. IPGTT revealed that the blood glucose levels of Gpr75 –/– male rats on HFD transiently increased (for 15 min) and subsequently decreased (for 60–120 min). The similar changes in blood glucose levels were found in both Gpr75 –/– and Gpr75 +/+ male rats on control diet ( Fig. 5 D). However, plasma glucose levels were maintained at high levels throughout the experimental period in Gpr75 +/+ rats on HFD. Specifically, three-way mixed ANOVA revealed that plasma glucose levels after 60 min of glucose injection in Gpr75 +/+ rats on HFD were significantly higher than those in Gpr75 –/– male rats on HFD (*, p < 0.05, simple-simple main effect analysis) and Gpr75 +/+ male rats on control diet (†, p < 0.05, simple-simple main effect analysis). No significant differences were found in plasma glucose levels between Gpr75 –/– male rats on HFD and Gpr75 –/– male rats on control diet. Plasma insulin levels were maintained at low levels in Gpr75 –/– male rats, regardless of their diet, even after glucose injection ( Fig. 5 E). The Gpr75 –/– (on HFD or control diet) and Gpr75 +/+ male rats on control diet showed a transient increase in plasma insulin levels 30 min after the intraperitoneal glucose injection, and the insulin levels decreased 60–120 min after the injection. Gpr75 +/+ male rats on HFD showed high plasma insulin levels even before the glucose challenge, and the levels remained high till the end of the experiment. Three-way mixed ANOVA revealed that plasma insulin levels in Gpr75 –/– male rats on HFD were significantly lower after 0, 60, and 120 min of glucose injection than those in Gpr75 +/+ male rats on HFD (*, p < 0.05, simple-simple main effect analysis). Plasma insulin levels in Gpr75 –/– male rats on control diet were significantly lower after 15, 30, and 60 min of glucose injection than those in Gpr75 +/+ male rats on control diet (‡, p < 0.05, simple-simple main effect analysis). Plasma insulin levels in Gpr75 +/+ male rats on control diet were significantly lower at 0 min and higher at 15 min than in Gpr75 +/+ male rats on HFD (†, p < 0.05, simple-simple main effect analysis). No significant differences were found in plasma insulin levels between Gpr75 –/– male rats on HFD and Gpr75 –/– male rats on control diet. The levels of cholesterol and triglycerides, but not of free fatty acids, in the plasma were lower in Gpr75 –/– rats than in Gpr75 +/+ male rats. Two-way factorial ANOVA showed that plasma cholesterol ( Fig. 5 F) and triglyceride ( Fig. 5 G) levels were significantly lower (¶, p < 0.05, main effect analysis) in Gpr75 –/– male rats than those in Gpr75 +/+ male rats. Two-way factorial ANOVA also showed that HFD significantly decreased plasma cholesterol ( Fig. 5 F), triglyceride ( Fig. 5 G), and free fatty acid ( Fig. 5 H) levels (§, p < 0.05, main effect analysis) in male rats. To examine the mechanism underlying GPR75-dependent hyperphagia, the mRNA expression of major orexigenic (NPY and AgRP) and anorexigenic neuropeptides (CART and α-MSH) and a GPR75 ligand candidate (CCL5) were examined in the hypothalamus of Gpr75 –/– and Gpr75 +/+ male rats on either HFD or control diet ( Fig. 6 A). Two-way factorial ANOVA revealed that hypothalamic Ccl5 mRNA expression levels were significantly higher (¶, p < 0.05, main effect analysis) in Gpr75 –/– male rats than in Gpr75 +/+ male rats. In addition, Cartpt mRNA expression levels tended to be higher ( p = 0.15, main effect analysis) in Gpr75 –/– male rats than in Gpr75 +/+ male rats. However, Pomc (encoding anorexigenic α-MSH), Npy , and Agrp mRNA expression levels were comparable among groups. Among genes encoding inflammatory adipokines (CCL2 and TNFα), adiponectin, leptin, enzymes and receptors controlling lipid metabolism (VLDLR, FAS, DGAT1, and HSL), and CCL5, the mRNA expression of Ccl2 decreased and that of Ccl5 increased in the gonadal WAT of Gpr75 –/– male rats ( Fig. 6 B). Specifically, two-way factorial ANOVA revealed that Ccl2 mRNA expression levels, but not Tnf , were significantly lower (¶, p < 0.05, main effect analysis) in the gonadal WAT of Gpr75 –/– male rats than in the gonadal WAT of Gpr75 +/+ male rats. Two-way factorial ANOVA revealed that Ccl5 mRNA expression levels were significantly higher (¶, p < 0.05, main effect analysis) in the gonadal WAT of Gpr75 –/– male rats than in the gonadal WAT of Gpr75 +/+ male rats. However, Adipoq , Ob , Vldlr , Fasn , Dgat1 , and Lipe mRNA expression levels were comparable among the groups. Gpr75 KO did not significantly affect the gene expression of enzymes and receptors associated with energy metabolism, such as Fasn , Dgat1 , Hmgcr , Cyp7a1 , and Ldlr , or of transcription factors associated with energy metabolism, such as Srebf1 , Srebf2 , and Nr1h3 , as well as of Ccl5 in the liver of male rats on HFD and control diet ( Fig. 6 C). On the other hand, HFD significantly decreased Fasn mRNA expression levels (§, p < 0.05, main effect analysis) in the liver of male rats. HFD did not significantly affect the mRNA expression levels of Dgat1 , Hmgcr , Cyp7a1 , Ldlr , Srebf1 , Srebf2 , and Nr1h3 , or Ccl5 in both Gpr75 +/+ and Gpr75 –/– male rats.

Iar:Wistar-Imamichi male and female rats (Institute for Animal Reproduction, Kasumigaura, Japan) were kept in an air-conditioned room (22 ± 2°C) under a 14/10 h light/dark cycle (lights on at 0500 h). All rats had free access to normal chow (CE-2; CREA Japan, Tokyo, Japan) and tap water unless otherwise specified. Female rats (9–12 weeks old) with at least two consecutive 4-day estrous cycles were used for zygote collection or as pseudopregnant recipients for embryo transfer. Rats carrying CRISPR/Cas9-induced Gpr75 gene modifications and their progeny ( Gpr75 –/– , Gpr75 +/– , and Gpr75 +/+ rats) were also kept under the same conditions. All animal experiments described here were approved by the Committee on Animal Experiments of the Graduate School of Bioagricultural Sciences, Nagoya University. Unless otherwise specified, surgeries were conducted under aseptic conditions and anesthesia induced by injecting a mixture of ketamine (26.7 mg/kg; Fujita Pharmaceutical, Tokyo, Japan) and xylazine (5.3 mg/kg; Bayer, Leverkusen, Germany) intraperitoneally followed by the inhalation of isoflurane (1–3% in air; Pfizer Japan, Tokyo, Japan). Wistar-Imamichi WT male rats ( n = 5) and female rats ( n = 5) on the second day of diestrus were euthanized by decapitation, and brain and peripheral tissue samples were immediately collected. The brains were washed with saline, and two brain slices were prepared using a brain blocker to isolate the following hypothalamic areas: the preoptic area (POA) and the anteroventral periventricular nucleus (AVPV) from slice 1 (from 1.3 mm anterior to 1.3 mm posterior to bregma) and the lateral hypothalamic area (LHA), paraventricular nucleus (PVN), arcuate nucleus (ARC) and ventromedial nucleus (VMH) from slice 2 (from 1.3 mm to 4.3 mm posterior to bregma) according to the brain atlas [ 17 ]. In addition, the following tissues were excised (to approximately 5 mm × 5 mm × 5 mm in size): the cerebrum, cerebellum, brain stem, pituitary gland, muscle, heart, lung, stomach, spleen, kidney, small intestine, colon, and reproductive organs—ovary and uterus in females and testis in males. The small intestine was subsequently washed with saline. The collected tissues were rapidly frozen in liquid nitrogen, stored at –80°C, and subjected to gene expression analysis as described later. Nucleotide sequences corresponding to the rat Gpr75 gene (Accession nos. NM_001109096 and NC_051349 ) were retrieved from the National Center for Biotechnology Information database ( https://www.ncbi.nlm.nih.gov/ ). Two guide RNA (gRNA) target sites (gRNA1: 5'-gccttcagaaagttcagaac-3' and gRNA2: 5'-gatggtgatggggaagcagc-3') in exon 1 of the rat Gpr75 gene were designed using CRISPR direct ( https://crispr.dbcls.jp/ ) to delete approximately 220 bp of DNA coding the first and second intracellular loops of rat GPR75 by Cas9 nuclease ( Fig. 1 A). The number of potential off-target sequences was estimated using GGGenome ( http://gggenome.dbcls.jp/ ). Each gRNA sequence was introduced into the Cas9 expression plasmid pX330-U6-Chimeric_BB-CBh-hSpCas9 (pX330; http://www.addgene.org/42230/ ; RRID: Addgene_42330) at the BbsI restriction enzyme site [ 18 ]. A total of 178 pronuclear-stage zygotes were collected from female rats superovulated by intraperitoneal injection of equine chorionic gonadotropin (150 U/kg; Aska Pharmaceutical, Tokyo, Japan), followed 48 h later by human chorionic gonadotropin (75 U/kg; Mochida Pharmaceutical, Tokyo, Japan), and mated overnight with stud males, as previously described [ 19 ]. Two linearized pX330 vectors carrying gRNAs targeting Gpr75 (2.5 ng/μL each) were co-injected into the pronucleus of zygotes. The zygotes were cultured overnight in M16 medium at 37°C under 5% CO 2 in air. Eighty-three surviving embryos were transferred into the oviductal ampullae of three pseudopregnant female rats that had been mated with vasectomized male rats one day before. Twenty-seven pups were born alive at term. Genotypes of the offspring were analyzed by polymerase chain reaction (PCR) to detect the DNA deletion between the two target sites. After nested PCR (1st, 5'-cctctgggtgtgtgtttgtg-3' and 5'-tccatttcccttcacagagg-3'; 2nd, 5'-cctgcagggaggaaataaca-3' and 5'-cattcttccgaagggtttga-3'), PCR products were cloned and sequenced using a commercial sequencing kit (Applied Biosystems, Foster City, CA, USA) and a DNA sequencer (Applied Biosystems) according to the manufacturer’s instructions. Four of 27 animals carried an approximately 220-bp deletion between the two target sites in the Gpr75 gene. After mating founder female rats with WT stud males, three of the four animals showed germline transmission at rates greater than 50%. The genomes of the resultant Gpr75 +/– rats from three lines were subjected to sequencing analysis. The genomic sequences of the three Gpr75 KO rat lines (#3, #4, and #13) are shown in Fig. 1 B. The deletions resulted in a frameshift introducing five, six, or one aberrant amino acid(s) and a stop codon in the region encoding the first intracellular loop of GPR75, theoretically producing truncated proteins ( Fig. 1 C). The three lines of Gpr75 +/– females and males were mated to generate Gpr75 –/– rats. Within five days after delivery, the pups’ genotypes were analyzed using PCR (5'-cctgcagggaggaaataaca-3' and 5'-cattcttccgaagggtttga-3'). Litter sizes were adjusted to eight to ten pups to minimize growth variation within and between litters. Rats were weaned at postnatal day 20, housed individually, and fed normal chow (CE-2, 3.402 kcal/g) ad libitum . BW and cumulative food intake of littermate Gpr75 –/– , Gpr75 +/– , and Gpr75 +/+ rats were measured daily from postnatal day 20 to 12 weeks of age. Cumulative food intake was calculated weekly. External signs of puberty onset, such as balano-preputial separation for males or vaginal opening for females, were assessed daily in littermates of Gpr75 –/– ( n = 4 males and 4 females), Gpr75 +/– ( n = 10 males and 10 females), and Gpr75 +/+ rats ( n = 6 males and 8 females) until postnatal days 52 or 39, respectively. Adult Gpr75 –/– male and female rats were mated to evaluate fertility. Other cohorts of adult male and female Gpr75 –/– ( n = 4 males and 5 females) and Gpr75 +/+ ( n = 3 males and 5 females) rats were gonadectomized (GDX) and subjected to blood collection to evaluate pulsatile LH secretion two weeks later, as previously described [ 20 ]. Wet weights of the ovaries and testes were measured. The GDX rats had the advantage of avoiding individual differences in endogenous sex steroids, which modulate LH secretion via negative feedback. A silicone cannula (inner diameter 0.5 mm, outer diameter 1.0 mm; Shin-Etsu Polymer, Tokyo, Japan) was inserted into the right atrium through the jugular vein one day before blood sampling. Blood samples (100 μL) were collected from freely moving conscious GDX rats every 6 min for 3 h (from 1300 h to 1600 h). Plasma samples (50 μL) were collected after centrifugation at 4°C and stored at –20°C until assayed for LH. An equivalent volume of rat red blood cells, taken from donor rats and diluted with heparinized saline, was infused through the atrial catheter after each blood collection to maintain constant hematocrit. Plasma LH concentrations were determined as previously described [ 21 ] using a double-antibody radioimmunoassay (RIA) with a rat LH-RIA kit provided by the National Hormone and Peptide Program (NHPP). Concentrations were expressed in terms of rat LH-RP3. The lowest detectable LH level was 0.078 ng/mL, and the intra- and inter-assay coefficients of variation were 5.6% and 10.3% at 0.92 ng/mL, respectively. LH pulses were identified as described previously [ 22 ] using the PULSAR computer program [ 23 ]. The mean LH concentrations and the frequency and amplitude of LH pulses were calculated during the 3-h sampling period in each rat. Averages of these parameters were then calculated for Gpr75 –/– and Gpr75 +/+ rats. Feeding patterns, such as the number of feeding events (feeding bouts), feeding bout durations, and food intake at each feeding bout, per day or during the light and dark phases, were analyzed in 10–12-week-old Gpr75 –/– and Gpr75 +/+ male rats ( n = 6 per group) using the BioDAQ Food Intake Monitoring System (Research Diets, New Brunswick, NJ, USA), as previously described [ 24 ]. Briefly, animals were housed individually in BioDAQ-equipped cases with free access to normal chow (CE-2) and water. Male rats were acclimated to the BioDAQ cage for 3–4 days, and feeding patterns and food intake were recorded for seven consecutive days. The start and end of a feeding bout were detected by changes in food hopper weight caused by feeding behavior. The date, time, and hopper weight were recorded, and feeding duration and food intake in each bout were calculated. The number of feeding bouts, cumulative feeding duration, and cumulative food intake were calculated daily or separately for the light and dark phases for 7 days, and averages were determined. Male rats were selected for feeding pattern analysis and subsequent HFD experiments because they exhibit stable plasma sex steroid levels, whereas female rats show estrous cycle-dependent fluctuations in plasma estrogen levels. The male rats were then euthanized, and the whole hypothalamus was dissected. The tissues were rapidly frozen in liquid nitrogen, stored at –80°C, and used for Kiss1 and Gnrh1 expression analyses, as described below. Eight-week-old male Gpr75 –/– ( n = 7 for HFD and 7 for control diet) and Gpr75 +/+ rats ( n = 7 for HFD and 8 for control diet) were housed individually and allowed free access to HFD (D12451, 45% energy from fat, 4.73 kcal/g; Research Diet) or control diet (D12450H, 10% energy from fat, 3.85 kcal/g; Research Diet). BW and cumulative food intake were recorded daily for 20 weeks. Cumulative food intake was calculated every 2 weeks. Animals were then subjected to IPGTT as described previously [ 25 , 26 ]. Briefly, a silicone cannula (inner diameter 0.5 mm, outer diameter 1.0 mm, Shin-Etsu Polymer, Tokyo, Japan) was inserted into the right atrium through the jugular vein in the morning one day before the IPGTT. After recovering from surgery, vigorous and conscious rats were subjected to overnight fasting (from 1800 h to 1000 h for 16 h). Animals were then intraperitoneally injected with 20% glucose solution (2 g/kg). Blood samples (100 μL) were collected via the silicone cannula from freely moving conscious rats at 0 min (just before glucose injection), and at 15, 30, 60, and 120 min after injection. Plasma samples were collected after centrifugation at 4°C and stored at –20°C until assayed for glucose and insulin. After a one-week recovery period, animals were euthanized. The whole hypothalamus, liver, and gonadal and retroperitoneal white adipose tissues (visceral WAT) were dissected, and the liver and visceral WAT were weighed. The tissues were rapidly frozen in liquid nitrogen, stored at –80°C, and used for gene expression analyses as described later. Plasma glucose concentrations were determined by the glucose oxidase method using a commercial kit (Glucose C-Test; Fujifilm Wako Pure Chemical, Osaka, Japan), as previously described [ 27 ]. The lowest detectable plasma glucose concentration was 25.0 mg/dL, and the intra- and inter-assay coefficients of variation were 5.6% and 5.1% at 127.0 mg/dL, respectively. Plasma insulin concentrations were determined using a sandwich ELISA commercial kit (LBIS Rat Insulin ELISA Kit; Shibayagi, Gunma, Japan). The lowest detectable concentration of insulin in the 10 μL plasma sample was 0.156 ng/mL, and the intra-assay coefficient of variation was 5.0% at 5.170 ng/mL. Plasma cholesterol concentrations were determined using a commercial kit following the cholesterol oxidase DAOS method (Cholesterol E Test; Fujifilm Wako Pure Chemical). The lowest detectable concentration of cholesterol was 0.4 mg/mL, and the intra- and inter-assay coefficients of variation were 1.6% and 2.0%, respectively, at 1.1 mg/mL. Plasma triglyceride concentrations were determined by the triglycerides GPO-DAOS method using a commercial kit (LabAssay Triglyceride; Fujifilm Wako Pure Chemical). The lowest detectable concentration of triglycerides was 0.4 mg/mL, and the intra- and inter-assay coefficients of variation were 1.0% and 4.7% at 0.6 mg/mL. Plasma free fatty acid concentrations were determined by the ACS-ACOD method using a commercial kit (LabAssay NEFA, Fujifilm Wako Pure Chemical). The lowest detectable concentration of free fatty acids was 0.125 mEq/L, and the intra- and inter-assay coefficients of variation were 0.3% and 10.7% at 0.5 mEq/L. Plasma samples for those measurements were collected at the end of intravenous cannulation. Total RNA was extracted from tissues using ISOGEN (Nippon Gene, Tokyo, Japan). cDNA was synthesized from each sample using oligo (deoxythymidine) primers at 37°C with the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems). Gene expression levels were determined using the 7500 Real-Time PCR System (Applied Biosystems) with Thunderbird SYBR qPCR Mix (TOYOBO, Osaka, Japan), as described previously [ 28 , 29 ]. The cycling protocol was as follows: pre-denaturation for 1 min at 95°C, followed by 40 cycles of amplification (15 sec at 95°C and 1 min at 60°C). The specificity of the amplicons was confirmed by dissociation curve analysis (60–95°C) after 40 amplification cycles. A distinct single peak was considered to indicate that only a single DNA sequence was amplified. Forward and reverse primers for the genes of interest— Kiss1 , Gnrh1 , Agrp (encoding AgRP), Npy (encoding NPY), Pomc (encoding proopiomelanocortin including α-melanocyte-stimulating hormone [α-MSH]), Cartpt (encoding cocaine and amphetamine-regulated transcript prepropeptide [CART]), and Ccl5 in the hypothalamus; Ccl2 (encoding CCL2), Tnf (encoding tumor necrosis factor α [TNFα]), Adipoq (encoding adiponectin), Ob (encoding leptin), Vldlr (very low density lipoprotein receptor [VLDLR]), Fasn (fatty acid synthase [FAS]), Dgat1 (diacylglycerol O-acyltransferase 1 [DGAT1]), Lipe (hormone-sensitive lipase [HSL]), and Ccl5 in the gonadal WAT; and Fasn , Dgat1 , Hmgcr (3-hydroxy-3-methylglutaryl-CoA reductase [HMGCR]), Cyp7a1 (cytochrome P450 family 7 subfamily A member 1 [CYP7A1]), Ldlr (low density lipoprotein receptor [LDLR]), Srebf1 (sterol regulatory element-binding protein 2 [SREBP1]), Srebf2 (SREBP2), Nr1h3 (Liver X receptor α [LXRα]), and Ccl5 in the liver—and a housekeeping gene ( Actb , encoding β-actin) are listed in Table 1 . Relative gene expression levels were normalized to Actb , and fold changes between treatment and control groups were calculated using the 2 –ΔΔCT method. Statistical differences in Gpr75 mRNA expression in the brain and peripheral tissues between WT male and female rats were analyzed using Student’s t -test. Statistical differences in BW and cumulative food intake between Gpr75 +/+ , Gpr75 +/– , and Gpr75 –/– rats were analyzed using two-way mixed ANOVA followed by analysis of simple main effects. Statistical differences in ages at puberty onset between Gpr75 +/+ , Gpr75 +/–, and Gpr75 –/– rats were analyzed using one-way ANOVA. Statistical differences in the mean LH concentrations, frequency and amplitude of LH pulses, ovarian and testicular weights, and litter size between the Gpr75 +/+ and Gpr75 –/– rats were analyzed using Student’s t -test. Statistical differences in Kiss1 and Gnrh1 mRNA expression in the whole hypothalamus and the number of feeding bouts and feeding duration per day between Gpr75 +/+ and Gpr75 –/– male rats were analyzed using Student’s t -test. Statistical differences in the number of feeding bouts and feeding duration during the light and dark phases between Gpr75 +/+ and Gpr75 –/– male rats were analyzed using two-way factorial ANOVA followed by analysis of simple main effects. Statistical differences in BW, cumulative food intake, and plasma glucose and insulin levels between Gpr75 +/+ and Gpr75 –/– male rats fed HFD or control diet were analyzed using three-way mixed ANOVA followed by analysis of simple-simple main effects. Visceral WAT weight, plasma lipid levels, and mRNA expression levels between Gpr75 +/+ and Gpr75 –/– male rats fed HFD or control diet were analyzed using two-way factorial ANOVA followed by analysis of simple main effects. All statistical analyses were performed using SAS OnDemand for Academics ( https://welcome.oda.sas.com/ ).

The present study demonstrated that GPR75 signaling is dispensable for reproduction in rats regardless of the sex. This was evidenced by the absence of any observable effects of Gpr75 KO on reproductive functions, such as puberty onset, pulsatile LH secretion, and litter size in male and female rats. In addition, Gpr75 KO did not affect the gene expression of hypothalamic kisspeptin or GnRH, which are critical regulators of gonadotropin secretion and subsequent reproductive function. In contrast, GPR75 signaling contributes to feeding and body growth, as Gpr75 KO rats showed significantly lower food intake and BW in both sexes fed normal chow. To the best of our knowledge, this is the first report showing that GPR75 signaling is dispensable for reproduction but regulates feeding and BW homeostasis in animals fed normal chow. Previous mouse studies showed that food intake and/or BW were comparable between Gpr75 KO mice and WT control mice fed normal chow [ 6 - 8 ]. The current results, therefore, indicate species differences in GPR75 signaling-mediated regulation of feeding and BW in rats and mice fed normal chow. In addition, the present study demonstrated that GPR75 signaling plays a critical role in regulating feeding, BW, and glucose and lipid metabolism in rats because Gpr75 KO showed significantly lower food intake, BW, visceral WAT weight, and plasma cholesterol and triglyceride levels in male rats than WT controls under HFD conditions, and Gpr75 KO prevented HFD-induced hyperglycemia and hyperinsulinemia in male rats. The observation of anti-obesity phenotype in Gpr75 KO male rats on HFD is largely consistent with the results of previous studies in mice [ 4 - 8 ], in which Gpr75 KO mice showed lower food intake than WT mice, resulting in lower BW and lower body fat mass with normoglycemia than WT obese controls under HFD conditions. Overall, the current study suggests that GPR75 antagonism would serve as a promising therapeutic approach for anti-hyperphagia, anti-obesity, and anti-hyperglycemia/hyperinsulinemia without reproductive toxicity in humans. The present study suggests that Gpr75 KO rats are more prone to satiety than WT control rats, even fed normal chow, because Gpr75 KO male rats showed significantly lower feeding duration and food intake without changing the number of feeding bouts during the dark phase but not during the light phase. The anti-obesity phenotype in Gpr75 KO male rats fed HFD also appears to result primarily from lower food intake, suggesting that Gpr75 KO male rats remain prone to early satiety even under HFD, unlike WT controls, which exhibited hyperphagia. Gpr75 mRNA was highly expressed in the rat brain, including several hypothalamic nuclei (POA, AVPV, LHA, PVN, ARC, and VMH), cerebrum, cerebellum, and brain stem, whereas Gpr75 mRNA expression was limited in peripheral organs in both sexes. These results are consistent with previous findings in humans [ 4 ] and mice [ 7 , 8 ], in which Gpr75 is highly expressed throughout the brain but poorly expressed in most peripheral tissues. Thus, the most probable explanation is that GPR75 signaling in the central nervous system including the hypothalamus primarily controls feeding, BW, and glucose and lipid metabolism in mammals. Indeed, Leeson-Payne et al. [ 8 ] and Jiang et al. [ 7 ] demonstrated that Gpr75 -expressing neurons, widely distributed in the mouse brain, fall into multiple neuronal subtypes (130 and 29 clusters, respectively). Which specific population of Gpr75 -expressing neurons is involved in triggering hyperphagia in WT rats on HFD remains unclear. Notably, the Gpr75 KO male rats in the current study showed higher hypothalamic Ccl5 mRNA expression than WT controls, suggesting that central GPR75 signaling may exert its orexigenic effect by downregulating hypothalamic Ccl5 expression. Indeed, previous studies showed that an intracerebroventricular infusion of CCL5 decreased short-term food intake in male rats [ 12 ], and Ccl5 or Ccr5 KO mice exhibited higher BW than WT mice [ 13 ]. It is speculated that the increase in hypothalamic Ccl5 mRNA expression may result from the absence of GPR75-dependent negative feedback to Ccl5 expression, as CCL5 is a candidate ligand for GPR75 [ 10 ]. In this context, the anorexigenic action of CCL5 may be mediated by CCR5 but not GPR75. Previous studies showed that CCL5 and CCR5 are widely expressed in the rat brain, including the hypothalamus [ 30 , 31 ]. The current results raise questions regarding which populations of Ccl5 - and Ccr5 -expressing cells are involved, and how CCL5-CCR5 signaling decreases food intake in Gpr75 KO rats. Besides, anorexigenic Cartpt mRNA expression tended to be higher in Gpr75 KO male rats than in WT controls, whereas anorexigenic Pomc (encoding α-MSH) and orexigenic Npy and Agrp mRNA expression were comparable between Gpr75 KO male rats and WT controls. Thus, increased hypothalamic anorexigenic CCL5 and CART may partly contribute to the reduced food intake in Gpr75 KO rats. Hossain et al. [ 6 ] showed that both male and female Gpr75 KO mice on HFD maintained comparable energy expenditure, whereas WT mice exhibited reduced energy expenditure. These findings suggest that increased energy expenditure, alongside reduced food intake, may contribute to leanness in Gpr75 KO rats. It is tempting to speculate that Gpr75 KO rats may be less prone to adipose tissue inflammation and insulin resistance. This is because Gpr75 KO male rats exhibited lower circulating cholesterol and triglyceride concentrations and lower mRNA expression of CCL2, a key inflammatory adipokine, in the gonadal WAT than in WT controls under HFD conditions. Previous studies have demonstrated that elevated plasma cholesterol, triglycerides, and free fatty acids lead to macrophage-mediated inflammation, which exacerbates adipocyte inflammation [ 32 - 34 ]. Inflammatory adipokines, such as CCL2, CCL5, and TNFα, also induce insulin resistance in humans and animal models [ 35 , 36 ]. Intriguingly, HFD-fed WT obese rats in the present study did not exhibit increased plasma cholesterol, triglycerides, or free fatty acids, nor elevated Ccl2 and Tnfa mRNA expression in gonadal WAT. Petitt et al. [ 37 ] reported that mice on HFD exhibited adaptive responses, including enhanced lipid absorption capacity in the small intestine and more efficient lipid clearance from the blood than control mice on normal chow. These adaptations may contribute to comparable or even lower plasma lipid levels in WT rats on HFD than in normal chow conditions. Interestingly, gonadal WAT Ccl5 mRNA expression was higher in Gpr75 KO male rats than in WT controls. It is also speculated that the increase in Ccl5 mRNA expression in the gonadal WAT may result from the absence of GPR75-dependent negative feedback to Ccl5 expression in WAT, similar to the brain. Supporting this interpretation, Leeson-Payne et al. [ 8 ] showed that Gpr75 mRNA was expressed in the WAT but not in the liver in mice. In this context, hepatic Ccl5 mRNA expression might be unaffected by Gpr75 deletion because of limited hepatic Gpr75 expression. Regarding plasma free fatty acid levels and liver Fasn mRNA expression, Gpr75 KO failed to affect those parameters, whereas HDF decreased them in both Gpr75 KO and WT male rats. Furthermore, GPR75 deletion in the present study did not affect Ob mRNA expression in the gonadal WAT. This is consistent with the findings of a previous study showing that the absence of Gpr75 did not affect obesity development in leptin-deficient mice under normal chow conditions [ 7 ]. Taken together, these findings suggest that GPR75 is unlikely to directly influence the leptin-leptin receptor signaling pathway that regulates appetite and BW. In conclusion, the present study suggests that central GPR75 signaling regulates feeding and BW homeostasis in rats fed normal chow in both sexes and facilitates HFD-induced hyperphagia, obesity, hyperglycemia, and hyperinsulinemia in male rats. Notably, Gpr75 KO may prevent HFD-induced hyperphagia via central CCL5 signaling in rats. Importantly, the present study revealed that GPR75 signaling is entirely dispensable for reproduction in both sexes. Therefore, antagonists targeting GPR75 may offer a novel therapeutic approach to control appetite and prevent hyperphagia, hyperglycemia, hyperinsulinemia, and obesity without affecting reproductive function in humans ( Graphical Abstract ).

Overweight and obesity are often associated with impaired fertility in humans. Campbell et al. [ 1 ] reported that couples with male partner with obesity were significantly more likely to experience infertility than couples with normal-weight men. Recently, Santi et al. [ 2 ] reported that men who are overweight had lower sperm counts than normal-weight men, and men with obesity had lower sperm counts and motility than men without obesity. Similarly, overweight and obesity in women increase the prevalence of reproductive disorders, such as polycystic ovary syndrome, endometriosis, and infertility [ 3 ]. GPR75 has emerged as a potential therapeutic target for obesity, since a large-scale exome-wide discovery analysis of body mass index demonstrated that loss-of-function variants in the GPR75 gene were associated with leanness, e.g. , lower body mass index, lower body weight (BW), and decreased odds of obesity in humans in the heterozygous state [ 4 ]. In the study, tissue enrichment analysis using the Genotype-Tissue Expression portal revealed that GPR75 mRNA is highly expressed in the human brain [ 4 ]. In addition, Gpr75 knockout (KO) mice reproduced leanness under high-fat diet (HFD) conditions, where wild-type (WT) control mice showed hyperphagia and obesity [ 4 - 8 ]. On the other hand, food intake and/or BW were comparable between Gpr75 KO and WT control mice fed normal chow [ 6 - 8 ]. Recently, Leeson-Payne et al. [ 8 ] showed that Gpr75 was widely distributed throughout the mouse brain, including—but not limited to—hypothalamic neurons such as orexigenic neuropeptide Y (NPY)/agouti-related peptide (AgRP) neurons. These findings suggested that central GPR75 signaling may promote overweight and obesity in animals on HFD. However, it remains unclear whether GPR75 signaling is dispensable for reproduction and feeding/metabolism in animals fed normal chow. If Gpr75 gene deletion does not impair reproductive function, GPR75 antagonists may be advantageous in the development of anti-obesity therapies for humans. Endogenous ligands for GPR75 in the context of feeding and BW homeostasis remain controversial [ 9 ]. Ignatov et al. [ 10 ] showed that C-C motif chemokine ligand 5 (CCL5, encoded by the Ccl5 gene) activated the phospholipase C pathway in cells transfected with Gq-coupled GPR75. Thus, CCL5 is an endogenous ligand candidate for GPR75. CCL5 is also known as a promiscuous chemokine that binds several C-C motif chemokine receptors, including CCR1 and CCR5 (encoded by the Ccr5 gene) [ 11 ]. Notably, central CCL5 administration decreased food intake in male rats [ 12 ], and Ccl5 or Ccr5 KO mice exhibited higher BW than WT mice [ 13 ]. In the cited study, Chou et al. [ 13 ] also demonstrated CCL5 and CCR5 immunoreactivity in the mouse hypothalamus. These findings suggested that CCL5, which activates CCR5, is a hypothalamic anorexigenic peptide. On the other hand, the role of 20-hydroxyeicosatetraenoic acid (20-HETE) [ 14 ], another endogenous ligand candidate for GPR75, in regulating feeding and BW remains controversial. Gilani et al. [ 15 ] showed that overexpression of 20-HETE synthase (encoded by the Cyp4a12 gene) exacerbated HFD-induced obesity in doxycycline-activated Cyp4a12 transgenic mice, whereas overexpression of Cyp4a12 alone did not increase BW in control diet-fed conditional Cyp4a12 transgenic mice. The present study aimed to examine whether GPR75 is dispensable for normal reproduction, feeding behavior, and body growth using newly generated Gpr75 KO rats fed normal chow. We first generated Gpr75 KO rats utilizing the CRISPR/Cas9 system. Body growth, food intake, and reproductive function, including puberty onset, fertility, litter size, and pulsatile luteinizing hormone (LH) secretion, were examined in Gpr75 KO male and female rats and their littermate WT control rats fed normal chow. We also analyzed the expression of the kisspeptin gene ( Kiss1 ) and gonadotropin-releasing hormone (GnRH) gene ( Gnrh1 ) in the hypothalamus of Gpr75 KO and WT male rats. Feeding patterns were also analyzed in Gpr75 KO and WT male rats fed normal chow, as Gpr75 KO rats showed lower food intake than WT controls. In addition, hyperphagia, obesity, and plasma levels of glucose, insulin, and lipids were examined in HFD-fed Gpr75 KO male rats as an additional animal model beyond Gpr75 KO mice. This is because male rats were more prone to obesity than female rats and developed diet-induced obesity less severely than mice [ 16 ]. We also examined, in Gpr75 KO and WT male rats on HFD or control diet, the mRNA expression levels of orexigenic and anorexigenic peptides in the hypothalamus, the expression levels of adipokines in the gonadal white adipose tissue (WAT), and enzymes and receptors controlling lipid metabolism in the gonadal WAT and liver, to deepen our understanding on the roles of GPR75 in HFD-induced obesity and hyperglycemia. Furthermore, Ccl5 mRNA expression was examined in the hypothalamus, gonadal WAT, and liver in Gpr75 KO and WT male rats.