Spexin2 Is a Novel Food Regulator in Gallus gallus

Spexin2 (SPX2), a paralog of SPX1, is a newly identified gene in non-mammalian vertebrates. Limited studies in fish have evidenced its important role in food intake and energy balance modulation. However, little is known about its biological functions in birds. Using the chicken (c-) as a model, we cloned the full-length cDNA of SPX2 by using RACE-PCR. It is 1189 base pair (bp) in length and predicted to generate a protein of 75 amino acids that contains a 14 amino acids mature peptide. Tissue distribution analysis showed that cSPX2 transcripts were detected in a wide array of tissues, with abundant expression in the pituitary, testis, and adrenal gland. cSPX2 was also observed to be ubiquitously expressed in chicken brain regions, with the highest expression in the hypothalamus. Its expression was significantly upregulated in the hypothalamus after 24 or 36 h of food deprivation, and the feeding behavior of chicks was obviously suppressed after peripheral injection with cSPX2. Mechanistically, further studies evidenced that cSPX2 acts as a satiety factor via upregulating cocaine and amphetamine regulated transcript (CART) and downregulating agouti-related neuropeptide (AGRP) in hypothalamus. Using a pGL4-SRE-luciferase reporter system, cSPX2 was demonstrated to effectively activate a chicken galanin II type receptor (cGALR2), a cGALR2-like receptor (cGALR2L), and a galanin III type receptor (cGALR3), with the highest binding affinity for cGALR2L. Collectively, we firstly identified that cSPX2 serves as a novel appetite monitor in chicken. Our findings will help clarify the physiological functions of SPX2 in birds as well as its functional evolution in vertebrates.


Introduction
Chicken is a popular domestic poultry species and its appetite regulation is closely associated with food consumption and growth performance [1]. However, feed intake is a complex process influenced by both individual and environmental variables [2]. These signals are mainly received and integrated in the hypothalamus, wherein the proopiomelanocortin (POMC), neuropeptide Y (NPY), and agouti-related peptide (AGRP) neurons respond to release their corresponding neuropeptides, and subsequently regulate feeding behavior and energy expenditure [3]. Over the last three decades, research in this field has largely improved our understanding of the appetite regulation with the ongoing identification of new appetite regulatory peptides, such as cocaine and amphetamine regulated transcript (CART) [4], orexin [5], and ghrelin [6]. Further investigation and the identification of novel food intake regulators will not only shed light on the appetite regulation mechanisms, but also provide potential strategies in dealing with abnormal food consumption in chickens [1,7]. Spexin (SPX), a novel neuropeptide composed of 14 amino acids, is highly conserved from fish to mammals [8], suggesting its essential physiological roles in vertebrates. Consistent with its wide tissue distribution, SPX has been confirmed to participate in multiple physiological functions, such as food intake [9][10][11][12][13][14], reproduction [15][16][17][18], and anxiety [19,20]. It is worth noting that SPX can serve as a satiety factor universally in 2 of 11 mice [11,12], chickens [13,14,21], goldfish [9], zebrafish [10], and Siberian sturgeon [22], implicating that its function of food regulation is well-conserved across vertebrates. Further research shows that SPX suppresses appetite through the modulation of central orexigenic and anorexigenic signals, such as NPY, AgRP, and POMC [9,12,21,23]. SPX, as a tetradecapeptide, exerts its biological functions via activating its cell-surface receptors, such as galanin receptor 2 (GALR2) and GALR3, but not GALR1 [8,16]. Interestingly, apart from the above, two more GALR-like receptor (i.e., GALR1L and GALR2L) subtypes were identified to exist in chicken [24,25]. Our recent studies showed that chicken (c-) SPX (cSPX) had the highest affinity for GALR2L to stimulate the MAPK/ERK cascade in chickens [21]. These studies strongly evidenced that GALR2L is a key functional receptor of SPX as well.
Recently, an SPX paralog, namely SPX2, has been identified in non-mammalian vertebrates, therefore the original SPX is now designated as SPX1 [8,26,27]. Compared with SPX1, little information is available regarding either the expression pattern or the physiological roles of SPX2. To the best of our knowledge, so far there are only a few studies regarding SPX2 which are all limited to fish. Similar to SPX1, zebrafish SPX2 is also able to specifically activate human, Xenopus, and zebrafish GALR2 or GALR3 [8], suggesting that the two different peptides possibly share some similarity in their physiological functions. Using whole-mount in situ RNA hybridization, SPX2 was evidenced to be restrictively expressed in the hypothalamic preoptic area of zebrafish [28]. Subsequently, using qRT-PCR, SPX2 was detected to be widely expressed in tissues including the central nervous system and peripheral tissues [26,27], with high expression in testes and the brain [27]. Intriguingly, the expression of SPX2 in hypothalamus was altered under different feeding states in zebrafish [27], implying its potential role in feeding control. Indeed, intraperitoneal injection of SPX2 inhibited food intake, while the disruption of this gene via TALENs markedly increased food consumption in zebrafish [27]. Similarly, the brain SPX2 was also reported to be regulated by food deprivation in half-smooth tongue sole [26], reinforcing the concept that SPX2 is a novel moderator of appetite. However, whether SPX2 has similar function in birds is unknown.
Unlike SPX1, the functioning of SPX2 has not yet been fully elucidated, and the prior research has only focused on fish. Though SPX2 has been verified to exist in chickens, its gene structure, expression pattern, and functionality are all largely unknown. Thus, in this study, we aimed to clone the full-length cDNA of SPX2 and investigate its tissue distribution and potential functions, especially appetite regulation in chickens.

Cloning and Sequence Analysis of Chicken (c-) SPX2
According to the partial cDNA sequence of cSPX2 (GenBank accession No.: KF601213) deposited in GenBank, we cloned its full-length cDNA from an adult chicken brain using RT-and RACE-PCR. As shown in Figure 1A, the cloned cSPX2 has 1189 nucleotide bases, and its 5 -untranslated region (5 -UTR), CDS, and 3 -UTR are 293 bp, 228 bp, and 668 bp, respectively, in length. It was predicted to encode a precursor protein of 75 amino acids (aa), which could generate a 14-aa mature peptide after proteolytic processing at putative cleavage sites (R33, G48R49R50). Comparison of the cSPX2 cDNA sequence with chicken genomes (https://asia.ensembl.org/Gallus_gallus/Info/Index (accessed on 8 January 2023), cSPX2 consists of four exons separated by three introns ( Figure 1B).
Sequence alignment revealed that the precursors of SPX2 shares high sequence similarity in birds, but comparatively lower identities among different other vertebrate classes, with an obvious sequence variation noted at its NH 2 -terminal and COOH-terminal region ( Figure 2). It is noteworthy that cSPX2 mature peptide displays a high degree of amino acid sequence identity to that of ring-necked pheasant (100%), rock ptarmigan (100%), greater sage-grouse (100%), green anole (92.86%), Xenopus laevis (78.57%), coelacanth (92.86%), and zebrafish (78.57%). Despite the fact that the sequence of cSPX2 mature peptide is four amino acids different from cSPX1, they are highly conserved (64.29%). Sequence alignment revealed that the precursors of SPX2 shares high sequence similarity in birds, but comparatively lower identities among different other vertebrate classes, with an obvious sequence variation noted at its NH2-terminal and COOH-terminal region ( Figure 2). It is noteworthy that cSPX2 mature peptide displays a high degree of amino acid sequence identity to that of ring-necked pheasant (100%), rock ptarmigan (100%), greater sage-grouse (100%), green anole (92.86%), Xenopus laevis (78.57%), coelacanth (92.86%), and zebrafish (78.57%). Despite the fact that the sequence of cSPX2 mature peptide is four amino acids different from cSPX1, they are highly conserved (64.29%).   Sequence alignment revealed that the precursors of SPX2 shares high sequence similarity in birds, but comparatively lower identities among different other vertebrate classes, with an obvious sequence variation noted at its NH2-terminal and COOH-terminal region ( Figure 2). It is noteworthy that cSPX2 mature peptide displays a high degree of amino acid sequence identity to that of ring-necked pheasant (100%), rock ptarmigan (100%), greater sage-grouse (100%), green anole (92.86%), Xenopus laevis (78.57%), coelacanth (92.86%), and zebrafish (78.57%). Despite the fact that the sequence of cSPX2 mature peptide is four amino acids different from cSPX1, they are highly conserved (64.29%).

Synteny Analysis
To confirm whether the cloned cSPX2 is orthologous to the gene identified in other vertebrate species, we performed synteny analysis by searching its neighboring genes in the genomic regions of chicken and other vertebrate species. As shown in Figure 3, SPX2 is located on a syntenic region conserved in chicken, green anole, Xenopus laevis, coelacanth, and zebrafish, suggesting the cloned gene is an ortholog of SPX2. In addition, SPX2 and GAL are on the same chromosome in these species. However, SPX2 is absent in mammals, such as humans and mice.

Synteny Analysis
To confirm whether the cloned cSPX2 is orthologous to the gene identified in other vertebrate species, we performed synteny analysis by searching its neighboring genes in the genomic regions of chicken and other vertebrate species. As shown in Figure 3, SPX2 is located on a syntenic region conserved in chicken, green anole, Xenopus laevis, coelacanth, and zebrafish, suggesting the cloned gene is an ortholog of SPX2. In addition, SPX2 and GAL are on the same chromosome in these species. However, SPX2 is absent in mammals, such as humans and mice.

Tissue Distribution of SPX2 in Adult Chickens
To explore the physiological functions of SPX2 in chickens, we detected its tissue distribution in adult chickens using qRT-PCR. As shown in Figure 4, cSPX2 was widely expressed in all tissues except gizzard. It was abundantly expressed in the anterior pituitary, testis, and adrenal gland, and moderately expressed in the jejunum, ileum, and pancreas. In the central nervous system (CNS), cSPX2 was found to be ubiquitously expressed in the spinal cord and all the brain regions examined, with the highest expression in the hypothalamus.

Tissue Distribution of SPX2 in Adult Chickens
To explore the physiological functions of SPX2 in chickens, we detected its tissue distribution in adult chickens using qRT-PCR. As shown in Figure 4, cSPX2 was widely expressed in all tissues except gizzard. It was abundantly expressed in the anterior pituitary, testis, and adrenal gland, and moderately expressed in the jejunum, ileum, and pancreas. In the central nervous system (CNS), cSPX2 was found to be ubiquitously expressed in the spinal cord and all the brain regions examined, with the highest expression in the hypothalamus.

Effects of cSPX2 on Chicken Feed Intake
The mature peptide of cSPX2 shares high sequence similarity with cSPX1, which can act as a satiety factor in chicken [14,21]. We thus speculated that cSPX2 also has the anorexigenic properties. In order to verify this hypothesis, we detected its transcriptional response to fasting in the hypothalami of two-week-old chicks. As shown in Figure 5A, 24-

Effects of cSPX2 on Chicken Feed Intake
The mature peptide of cSPX2 shares high sequence similarity with cSPX1, which can act as a satiety factor in chicken [14,21]. We thus speculated that cSPX2 also has the anorexigenic properties. In order to verify this hypothesis, we detected its transcriptional response to fasting in the hypothalami of two-week-old chicks. As shown in Figure 5A, 24-or 36-h fasting could obviously elevate (p < 0.01) the levels of cSPX2 mRNA in the chick hypothalamus. Then, the feed consumption was measured after cSPX2 injection. The cumulative feed intake of chicks did not alter (p > 0.05) by three different doses of cSPX2 after 6 h treatment, but it was effectively inhibited (p < 0.05) at 12 h post-injection ( Figure 5B). Moreover, after 24 h post-injection, a moderate (50 ng/g BW) and a high (100 ng/g BW) dosage of cSPX2 could still markedly decrease (p < 0.05) the food intake in chicks. Figure 4. The expression profiles of SPX2 in adult chickens. Quantitative real-time PCR assay of cSPX2 in chicken various tissues, including the telencephalon (Tc), midbrain (Mb), cerebellum (Cb), hindbrain (Hb), hypothalamus (Hp), spinal cord (Sc), anterior pituitary (Pi), crop (Cp), proventriculus (Pr), gizzard (Gi), duodenum (Du), jejunum (Je), ileum (IL), caecum (Ce), colon (Co), testes (Te), ovary (Ov), heart (He), liver (Li), spleen (Sp), lung (Lu), kidney (Ki), adrenal gland (Ad), muscle (Mu), pancreas (Pa), abdominal fat (Fa), skin (Sk), and lymph (Ly). The mRNA levels of cSPX2 were normalized to that of β-actin and expressed as the fold difference compared with that of telencephalon (Tc). Each data point represents the mean ± SEM of 6 adult chickens (n = 6).

Effects of cSPX2 on Chicken Feed Intake
The mature peptide of cSPX2 shares high sequence similarity with cSPX1, which can act as a satiety factor in chicken [14,21]. We thus speculated that cSPX2 also has the anorexigenic properties. In order to verify this hypothesis, we detected its transcriptional response to fasting in the hypothalami of two-week-old chicks. As shown in Figure 5A, 24or 36-h fasting could obviously elevate (p < 0.01) the levels of cSPX2 mRNA in the chick hypothalamus. Then, the feed consumption was measured after cSPX2 injection. The cumulative feed intake of chicks did not alter (p > 0.05) by three different doses of cSPX2 after 6 h treatment, but it was effectively inhibited (p < 0.05) at 12 h post-injection ( Figure  5B). Moreover, after 24 h post-injection, a moderate (50 ng/g BW) and a high (100 ng/g BW) dosage of cSPX2 could still markedly decrease (p < 0.05) the food intake in chicks.

Transcriptional Regultion of Appetite-Regulating Regulators by cSPX2
To elucidate the mechanism of cSPX2 in feeding regulation, we monitored the expression of appetite-regulating factors in the hypothalamus. As shown in Figure 6A, cSPX2 could visibly inhibit (p < 0.05) AGRP mRNA levels at 6 h post-injection. Oppositely, the expression of a pro-melanin-concentrating hormone (PMCH) was stimulated (p < 0.05) by cSPX2 administration. CART mRNA levels exhibited downward tendencies, and no significant difference was examined after 6 h injection with cSPX2. Conversely, transcripts of CART in hypothalami of three-week-old chicks were obviously elevated (p < 0.05) after cSPX2 administration for 24 h, and the expressions of NPY, AGRP, POMC, and PMCH were not regulated (p > 0.05) by cSPX2 ( Figure 6B).

Potency of cSPX2 in Activating Chicken Galanin Receptors
To investigate whether cSPX2 is a natural ligand for galanin receptors in chickens, each receptor was transfected into HEK293 cells, and subjected to peptide treatment. Receptor activation was examined by pGL4-SRE-luciferase reporter system. As shown in Figure 7, cSPX2 could not efficiently stimulate luciferase activity in HEK293 cell expressing cGALR1 (accession No.: NP_001121534) and cGALR1L (accession No.: NP_001121533). On the contrary, cGALR2 (accession No.: ACB22016), cGALR2L (accession No.: ACD99708) and cGALR3 (accession No.: ACE60644) could be initiated by cSPX2 in a dose-dependent manner, and the EC 50 values are 218 nM, 53 nM, and 110 nM, respectively. Among them, cSPX2 exhibited the highest binding ability with cGALR2L. cSPX2 could visibly inhibit (p < 0.05) AGRP mRNA levels at 6 h post-injection. Oppositely, the expression of a pro-melanin-concentrating hormone (PMCH) was stimulated (p < 0.05) by cSPX2 administration. CART mRNA levels exhibited downward tendencies, and no significant difference was examined after 6 h injection with cSPX2. Conversely, transcripts of CART in hypothalami of three-week-old chicks were obviously elevated (p < 0.05) after cSPX2 administration for 24 h, and the expressions of NPY, AGRP, POMC, and PMCH were not regulated (p > 0.05) by cSPX2 ( Figure 6B).

Potency of cSPX2 in Activating Chicken Galanin Receptors
To investigate whether cSPX2 is a natural ligand for galanin receptors in chickens, each receptor was transfected into HEK293 cells, and subjected to peptide treatment. Receptor activation was examined by pGL4-SRE-luciferase reporter system. As shown in Figure 7, cSPX2 could not efficiently stimulate luciferase activity in HEK293 cell expressing cGALR1 (accession No.: NP_001121534) and cGALR1L (accession No.: NP_001121533). On the contrary, cGALR2 (accession No.: ACB22016), cGALR2L (accession No.: ACD99708) and cGALR3 (accession No.: ACE60644) could be initiated by cSPX2 in a dose-dependent manner, and the EC50 values are 218 nM, 53 nM, and 110 nM, respectively. Among them, cSPX2 exhibited the highest binding ability with cGALR2L.

Potency of cSPX2 in Activating Chicken Galanin Receptors
To investigate whether cSPX2 is a natural ligand for galanin receptors in chickens, each receptor was transfected into HEK293 cells, and subjected to peptide treatment. Receptor activation was examined by pGL4-SRE-luciferase reporter system. As shown in

Discussion
As a paralog of SPX1, SPX2 has been discovered in non-mammalian species [8,26,27], but little is known about its physiological functions. In this study, using chicken as an animal model, we firstly cloned the full-length cDNA encoding SPX2. Synteny analysis revealed that it is orthologous to the SPX2 of anole lizards, Xenopus, coelacanths, and zebrafish. Concurring with the previous report that SPX2 is found in a variety of vertebrate species but not in mammals [8,26,27]. We found that cSPX2 and cGAL are closely localized on the same chromosome and possibly coevolutionary in vertebrates, as reported previously [8]. Similar to SPX1, cSPX2 can generate a 14-aa mature peptide flanked by cleavage sites, and the mature peptide is highly conserved, suggesting SPX2 may play similar important physiological roles as SPX1 in non-mammalian vertebrates.
Tissue distribution analysis revealed that cSPX2 mRNA was widely detected in various tissues of a chicken, which is similar with the tissue expression profiles reported in both tongue sole [26] and zebrafish [27]. Remarkably, we found that in chickens the pituitary had the highest expression of cSPX2. As a master hormone secretion center, the pituitary gland controls metabolism, growth, sexual maturation, and reproduction as well as many other vital physiological functions and processes [29]. Therefore, it is likely that the pituitary will operate as a key mediator for SPX2 to regulate various physiological processes in non-mammalian vertebrates. Indeed, it was reported that an intraperitoneal injection of SPX2 could largely alter the expression of the gonadotropin α subunit, the luteinizing hormone β (LHβ), and the follicle-stimulating hormone β subunit (FSHβ) in the pituitary in half-smooth tongue sole [30], and that the knockout of SPX2 could greatly promote body growth in zebrafish by enhancing the expression of pituitary growth hormone (GH) [27]. High amounts of cSPX2 mRNA were also observed in chicken testes and adrenal glands, suggesting it may also directly regulate the functions of these organs, such as the sexual/adrenal steroid secretion, spermatogenesis, etc. Of note, cSPX2 was also moderate in various brain regions, with the highest expression in the hypothalamus, suggesting that SPX2 may be involved in the regulation of food intake in chickens. Taken together, SPX2 is widely distributed in chickens and potentially exerts pleiotropic functions. To better understand its pleiotropic functions in each individually expressed tissue of chickens, more extensive and in-depth investigations are required.
To explore the putative roles of cSPX2 in feeding control in chickens, we firstly detected its hypothalamic mRNA expression in response to food deprivation. Consequently, fasting remarkably increased cSPX2 expression in the hypothalamus, which is analogous to findings reported in half-smooth tongue sole [26]. Conversely, in zebrafish SPX2 mRNA was downregulated by fasting [27]. Despite discrepancies, changes in SPX2 expression under different feeding status conditions across species suggest its potential role in appetite regulation. To further validate its putative roles in feeding control, we performed in vivo SPX administration in chicks. Consequently, SPX2 administration greatly decreased the food intake of chicks, and similar findings were also reported in zebrafish [27]. Fish with SPX2 knockout also exhibited higher food consumption than WT fish [27]. All these results strongly suggest that, like SPX1, SPX2 acts as a novel appetite regulator in chickens as well as some other non-mammalian vertebrates.
To elucidate the underpinning mechanism of cSPX2 in appetite regulation, we further detected its regulatory influence on the expression of key feeding control factors in the hypothalamus of chicks. Following intravenous cSPX2 injection, AGRP, a well-known orexigenic gene, was visibly downregulated at 6 h post injection, whereas the anorexigenic factor, CART, was notably upregulated at 24 h post injection. In parallel, the cumulative food consumption of chicks were substantially reduced onward from 12 h post injection. Consistently, both in vivo SPX2 injection and SPX2 knockout evidenced that SPX2 controls food intake in zebrafish by regulating hypothalamic AGRP and CART expression as well [27]. Unexpectedly, the expression of PMCH, a potential orexigenic factor [31], was increased after cSPX2 treatment for 6 h, similar to what was seen with SPX1 in goldfish [8]. Thus, it appeared that SPXs (both SPX1 and SPX2) could exert an early and quick action on the expression of PMCH in the hypothalamus. However, this early and quick action of SPX2 on PMCH expression cannot essentially change the pattern of chicks' decreased food consumption. SPX2-induced changes of AGRP and CART were still dominant over PMCH to control food intake. Therefore, in chickens, SPX2 acts as a satiety factor to inhibit food intake mainly by downregulating AGRP and upregulating CART in the hypothalamus.
SPX2 exerts its physiological functions by activating its receptors. Previous studies indicated that SPX2 could elevate SRE-luc activity in cells expressing GALR2 and GALR3, but not in cells expressing GALR1 [8,16]. A little surprisingly, in the present study we found cSPX2 could not only activate cGALR2 and cGALR but also cGALR2L. Among them, cSPX2 exhibited the highest binding ability with cGALR2L. Accordingly, it is likely that cSPX2 exerts its feed regulation mainly through binding and activating cGALR2L. Interestingly, cSPX1 [21], but not cSPX2 could inhibit the food intake of chicks after 6 h post-injection at the same treatment dosage, suggesting that cSPX2 is potentially not as effective as cSPX1 in inhibiting the appetite of chicks. This is possibly attributed to the differential potency of SPX1 and SPX2 in activating galanin receptors. In both chickens [21] and humans [8] it has been evidenced that SPX1 has a higher potential to activate GALR2 and GALR3 than SPX2.
In summary, we cloned the full-length cDNA of chicken SPX2 and investigated its expression patterns and functionality. cSPX2 can generate a conserved mature peptide with 14-aa, which can activate GALR2, GALRL2L, and GALR3. Tissue distribution analyses show that cSPX2 is widely expressed in chickens, with abundant expression in the pituitary, testis, and adrenal glands, and its expression in hypothalamus was upregulated by fasting. In vivo administration of cSPX2 could effectively inhibit the feed intake of chickens, by modifying the expression of hypothalamic AGRP and CART. Collectively, our results firstly evidenced that cSPX2 serves as a novel appetite monitor in chickens, which will facilitate the understanding of the physiological functions of SPX2 in birds as well as its functional evolution in vertebrates.

Chemicals, Peptides and Primers
All chemicals were purchased from Sigma-Aldrich (Shanghai, China) unless stated otherwise. Chicken SPX2 mature peptide (cSPX2, NWGPQSILYLKGRY-NH 2 ) with amidated C-terminus was synthesized by GL Biochem Ltd. (Shanghai, China), and its structure was verified by mass spectrometry. Dulbecco's Modified Eagle Medium (DMEM), fetal bovine serum (FBS), and penicillin-streptomycin were all obtained from Hyclone (Cytiva, Shanghai, China). JetPRIME ® transfection reagent was purchased from Polyplus (Illkirch, France). All primers used in this study were synthesized by Chengdu Tsingke Biotechnology and listed in Supplementary Table S1.

Animals and Samples
Adult chickens (3 males and 3 females, 31-week-old) or chicks (Lohmann layer) used in the present study were obtained from Muxing company in Sichuan, China. Chickens were sacrificed to collect various tissues including different brain regions. All samples were snap-frozen in liquid nitrogen and then stored at −80 • C before RNA extraction. All procedures involving animals were approved by the Ethics Committee of Sichuan Agricultural University.

RNA Extraction and Reverse Transcription (RT)
Total RNA was isolated from various chicken tissues with RNAzol (Molecular Research Center, Cincinnati, OH, USA) according to the manufacturer's instructions, and then 1 µg of total RNA was used as template to reverse transcription by using the PrimeScript ® RT reagent kit with gDNA eraser (Takara, Dalian, China). The generated cDNA samples were diluted and then used as template for subsequent PCR or quantitative real-time PCR (qRT-PCR) for detecting the expressions of target genes.

Cloning the Full-Length cDNA of cSPX2
According to the predicted partial coding sequence (CDS) of cSPX2 (GenBank No.: KF601213), the gene-specific primers were designed to amplify the 5 -cDNA and 3 -cDNA ends by using SMART-RACE cDNA amplification kit (Clontech, Palo Alto, CA, USA). The amplification products were detected by agarose gel electrophoresis, and then ligated into pTA2 vector (Toyobo, Shanghai, China) for sequencing.

Functional Characterization of cSPX2 and Galanin Receptors
The potency of cSPX2 on activating chicken galanin receptors was evaluated by pGL4-SRE-Luciferase system according to our previously established methods [21]. Using JetPRIME ® transfection reagent, HEK293 cells were, respectively, transfected by five galanin receptor subtypes, including GALR1, GALR1L, GALR2, GALR2L, and GALR3, and then were treated with different doses of cSPX2 for an additional 6 h. Whereafter, 1× Passive lysis buffer (Promega, Beingjing, China) was added into cells and the luciferase activity was determined with the luciferase assay kit (Promega).

Quantitative Real-Time PCR (qRT-PCR)
According to our previously established method [21], qRT-PCR was conducted on the CFX96 Real-time PCR Detection System (Bio-Rad, Hercules, CA, USA), and PCR procedures consisted of 40 cycles of 94 • C for 20 s, annealing for 15 s, 30 s extension at 72 • C. To assess the specificity of PCR amplification, melting curve analysis was performed at the end of the reaction. Finally, the mRNA levels of target genes were normalized against β-actin, calculated with the 2 −∆∆CT method, and then expressed as the fold change relative to the chosen tissue or control. Primer sequences of target and reference genes are shown in Supplementary Table S1.

Effects of cSPX2 on Chick Feed Intake
Given that cSPX1 plays a significant role in appetite regulation, its paralog, cSPX2, may perform a similar physiological function. Thus, starvation experiment was conducted in this study. Two-week-old male chicks were divided equally into three groups (n = 24): one control group and two experimental groups. Chicks in control group were fed at regular times, and chicks in the two experimental groups were fasted for 24 and 48 h, respectively. After fasting, chicks were sacrificed and hypothalami were collected for determining cSPX2 expression response to fasting.
To further investigate the effect of cSPX2 on feed intake, similar-weight chicks (~180 g/chick) were randomly assigned to four groups (n ≥ 8), and then were deprived of food for 12 h. Then, chicks in control group were intravenously injected with PBS, and chicks in the three experimental groups were injected with 10 ng/g, 50 ng/g and 100 ng/g body weight of cSPX2, respectively. After that, chicks were re-fed with the pre-weighed diet, and the residual diets were collected and weighed at 6 h, 12 h, and 24 h post-injection, respectively. Food consumption was counted by the total input of food minus the leftover.

Transcriptional Regulation of Appetite-Regulating Factors by cSPX2
Three-week-old male chicks were randomly divided into two groups (n ≥ 4), and then were intravenously injected with PBS (control) or cSPX2 (50 ng/g body weight). After 6 h of administration, the chick hypothalami were collected, and the mRNA expression levels of neuropeptide Y (NPY), agouti gene-related protein (AGRP), cocaine-and amphetamineregulated transcript (CART), proopiomelanocortin (POMC), and pro-melanin-concentrating hormone (PMCH) were detected by qRT-PCR. We also evaluated the expression of the above appetite-related genes in the chick hypothalamus after 24 h injection with cSPX2 by using the same treatment procedure.

Data Analysis
All statistical analysis was performed with GraphPad Prism 9 software (La Jolla, CA, USA). The two-tailed Student's t test was conducted to compare two groups. For comparison between more than two groups, a one-way ANOVA followed by Turkey's test was used. Data were expressed as means ± SEM, and statistical significance was set at p < 0.05.

Informed Consent Statement: Not applicable.
Data Availability Statement: All data generated or analyzed during this study are included in this published article.