Arginine Regulates TOR Signaling Pathway through SLC38A9 in Abalone Haliotis discus hannai

Arginine plays an important role in the regulation of the target of the rapamycin (TOR) signaling pathway, and Solute Carrier Family 38 Member 9 (SLC38A9) was identified to participate in the amino acid-dependent activation of TOR in humans. However, the regulations of arginine on the TOR signaling pathway in abalone are still unclear. In this study, slc38a9 of abalone was cloned, and the slc38a9 was knocked down and overexpressed to explore its function in the regulation of the TOR signaling pathway. The results showed that knockdown of slc38a9 decreased the expression of tor, ribosomal s6 protein kinase (s6k) and eukaryotic translation initiation factor 4e (eif4e) and inhibited the activation of the TOR signaling pathway by arginine. Overexpression of slc38a9 up-regulated the expression of TOR-related genes. In addition, hemocytes of abalone were treated with 0, 0.2, 0.5, 1, 2 and 4 mmol/L of arginine, and abalones were fed diets with 1.17%, 1.68% and 3.43% of arginine, respectively, for 120 days. Supplementation of arginine (0.5–4 mmol/L) increased the expressions of slc38a9, tor, s6k and eif4e in hemocytes, and abalone fed with 1.68% of dietary arginine showed higher mRNA levels of slc38a9, tor, s6k and eif4e and phosphorylation levels of TOR, S6 and 4E-BP. In conclusion, the TOR signaling pathway of abalone can be regulated by arginine, and SLC38A9 plays an essential role in this regulation.


Introduction
Arginine (Arg) is an essential amino acid for fish and neonatal mammals [1,2]. It is not only used as raw materials for protein synthesis but also as signal molecules to effectively regulate the synthesis and degradation of proteins [3,4]. Arginine is also involved in the secretion of several hormones, such as stimulating the somatotropic axis manifested by the secretion of growth hormone and insulin-like growth factor I [5,6]. In addition, it promotes the secretion of insulin, which could promote the absorption of glucose and amino acids [7,8]. In addition, arginine has several physiological functions, including the synthesis of urea, glutamic acid, creatine, proline, polyamines and nitric oxide (NO), inflammation and innate immune responses [1,9].
Protein synthesis is the foundation and key process of animal growth and development, which is limited by translation initiation [10,11]. The target of the rapamycin (TOR) signaling pathway is one of the most important pathways regulating protein metabolism by amino acids [12]. TOR regulates protein synthesis and initiates translation through stimulating ribosomal s6 protein kinase (S6K) activity and simultaneously inhibiting the binding of the eukaryotic translation initiation factor 4E-binding protein (4E-BP) to eukaryotic translation initiation factor 4E (eIF4E) [13][14][15]. In mammals, arginine regulates Then RNA was treated with DNase and reverse transcribed into cDNA using the Prime ScriptTM RT reagent Kit (RR047A; Takara Biotech, Beijing, China).

Molecular Cloning of slc38a9
The method of slc38a9 cloning was referred to Wang et al. [34]. The mRNA sequence of slc38a9 was obtained from the transcriptome database (unpublished data), and the primers were designed by Oligo 7 software (Molecular Biology Insights, Inc., Colorado Springs, CO) and synthesized by Sangon Biotech (Shanghai, China) ( Table 1). cDNA of abalone muscle was used as a template for PCR, and the products were detected by 1% agarose gel electrophoresis. The target band was recovered using the SanPrep Column DNA Gel Extraction Kit (Sangon Biotech, Shanghai, China). The recovered product was linked to a blunt cloning vector and transferred to Trans1-T1 phage resistant chemically competent cells (TransGen Biotech, Beijing, China). The bacteria solution was culture expanded and applied evenly on LB solid medium containing ampicillin. Positive colonies were selected after overnight culture and verified using colony PCR, the correct colonies were selected and sequenced by Sangon Biotech (Shanghai, China).

Sequence Analysis of slc38a9
Blast retrieval and comparison of the complete sequence were performed on National Center for Biotechnology Information (Available online: https://www.ncbi.nlm.nih.gov/ orffinder/, accessed on 8 May 2020). The amino acid sequence of abalone slc38a9 was deduced and analyzed by ExPASy expert protein analysis system (Available online: http:// web.expasy.org/protparam/, accessed on 8 May 2020), and sequences of other species were downloaded from the NCBI database. Multiple alignment of these amino acid sequences was performed by DNAMAN 6.0 (Lynnon Biosoft, San Ramon, CA, USA). Phylogenetic analysis was carried out by the neighbor-joining method using MEGA X, and bootstrap values (%) of 1000 replicates were calculated for each node of the consensus tree obtained. SignalP 4.1 (Available online: http://www.cbs.dtu.dk/services/SignalP/, accessed on 8 October 2020) was used to detect the signal peptide, and transmembrane domains were predicted using the TMHMM Server v.2.0 (Available online: http://www.cbs.dtu. dk/services/TMHMM/, accessed on 21 October 2020). Furthermore, the secondary and three-dimensional structures of the sequence were predicted by PredictProtein (Available online:http://www.predictprotein.org/, accessed on 21 October 2020) and SWISS-MODEL (Available online: https://swissmodel.expasy.org/, accessed on 21 October 2020). Six abalones were used for the tissue distribution analysis of slc38a9, the expression levels of slc38a9 in seven tissues (hemolymph, muscle, mental, gill, intestine, gonad and digestive gland) of abalone were detected by quantitative real-time PCR.
2.3. Function Analysis of slc38a9 2.3.1. Synthesis and Injection of slc38a9 siRNA The sequence of negative control siRNA (siRNA-NC) and synthesis of siRNA was conducted by Wang et al. [34]. Four siRNAs (siRNA-108, siRNA-236, siRNA-347 and siRNA-469) targeting different encoding regions of slc38a9 were designed by BLOCK-iT™ RNAi Designer (Available online: http://rnaidesigner.thermofisher.com/rnaiexpress/, accessed on 20 May 2020) ( Table 2). Primers of these siRNAs were synthesized by Sangon Biotech and siRNA duplexes were synthesized using the T7 RNAi Transcription Kit (TR102-01, Vazyme, Nanjing, China). The concentration of all the siRNAs was uniformly adjusted to 300 ng/µL. Abalones (Weight: 19.8 ± 0.3 g) were acclimatized to the laboratory environment for two weeks before the experiment. To explore the efficiency of these siRNAs, six groups (6 abalones each group) of abalones were injected intramuscularly with 100 µL PBS, siRNA-NC, siRNA-108, siRNA-236, siRNA-347 or siRNA-469. Abalones injected with PBS served as the control group. The muscles of each the abalone were sampled 12 h after injection. Then 60 abalones were randomly divided into two groups and injected with 100 µL siRNA-NC or siRNA-347, respectively. The muscles were sampled at 0, 6, 12, 24 and 48 h after injection (6 abalones each time) to explore the efficiency of siRNA at different times.

Overexpression Plasmid Construction and Injection
The overexpression plasmid was constructed according to the protocols described by Liu et al. [35]. The sequence of the slc38a9 open reading frame (ORF) was obtained according to the step in Section 2.2.3. Homologous arms of Bam HI region in pcDNA3.1 were added to the slc38a9 by the PCR of the templates of slc38a9 with the forward primer slc38a9-HR-F (5 cttggtaccgagctcggatccATGGGGAGAGGAAGTCGCA 3 ) and the reverse primer slc38a9-HR-R (5 ccacactggactagtggatccTGTTGTATGGCCGATGATGAGG 3 ). The target gene sequence was linked to the pcDNA3.1 digested with the Bam H I enzyme, and then transferred to Trans-T competent cells. The extracted plasmid is sequenced to verify that the sequence is attached to the vector. The bacteria successfully linked to the target gene were expanded by shaking the flask culture overnight, and adequate plasmid was collected using the EasyPure HiPure Plasmid MaxiPrep Kit (TransGen Biotech, Beijing, China). The concentration of the plasmid was eventually diluted to 300 ng/µL. Abalones (Weight: 19.8 ± 0.3 g) were randomly divided into three groups (6 abalones each group) and injected intramuscularly with 100 µL PBS, pcDNA3.1 or pcDNA3.1-slc38a9. Abalones injected with PBS served as the control group. The muscles of abalone were sampled at 12 h after injection.

Oral Administration of Arginine after siRNA Injection
Two groups (12 abalones each group) of abalones (Weight: 19.8 ± 0.3 g) were injected with 100 µL siRNA-NC or siRNA-slc38a9, respectively. After 12 h, abalones of each group were subdivided into two groups and orally administered with 100 µL of arginine (0.6 M) Cells 2021, 10, 2552 5 of 20 (Sigma, St. Louis, MO, USA) or PBS, and abalones injected with siRNA-NC and fed with PBS served as the control group. Muscles were collected 3 h later and immediately frozen in liquid nitrogen, then stored frozen at −80 • C for the following analysis.

siRNA Injection and Feeding
In regard to the feeding model, 200 abalones (weight: 19.8 ± 0.3 g) were randomly divided into two groups. For one group, abalones were fed with basal diet (1.23% arginine), and the other group was fed with the diet with arginine added (1.72% arginine). The diet formula is shown in Table 3, and abalones fed with the basal diet served as the control (Con) group. After a 2-week feeding, abalones of each group were divided into two sub-groups and injected with 100 µL of siRNA-NC or siRNA-slc38a9. After 12 h, abalones were fed with the original diet again. Muscles of abalone were sampled at 0, 2, 3, 6, 9, 12 and 24 h after feeding, and 6 abalones were sampled from each sub-group at each time point. Table 3. Ingredients and proximate analysis of the experimental diets.

Ingredients
Dietary Arginine Levels (%) Abalones (Weight: 20.0 ± 0.2 g) were temporarily cultured in the re-circulating water system with an ultraviolet sterilization lamp. After that, abalones were anesthetized with 5% of ethanol. The body surface was wiped and sterilized with 75% ethanol. Foot muscle was gashed with a sterile scalpel, and then the hemolymph was collected and placed into the anticoagulant tube immediately. The complete medium consisted of Leibovitz's L-15 Medium (Thermo Fisher Scientific, Waltham, MA, USA), 15% fetal bovine serum (FBS) (Bioind, Kibbuiz, Israel), and four antibiotics (100 uL/mL penicillin-streptomycin, 250 µg/mL gentamicin and 2 µg/mL amphotericin) (Solarbio, Beijing, China). The hemolymph was mixed with the complete medium in a ratio of 1:3 and plated into 6-well plates (Corning, Lowell, MA, USA) and then incubated at 22 • C.
Before the experiment, cells were washed with phosphate buffer solution (PBS) (Hy-Clone, Logan, UT, USA) for three times and then cultured in the DMEM medium without arginine (Thermo Fisher Scientific, Waltham, MA, USA). After 12 h, cells were washed with PBS for another time and cultured in the DMEM medium with antibiotics and different arginine concentrations (0, 0.2, 0.5, 1, 2 and 4 mmol/L), respectively, (three replicates per concentration). Cells were sampled 9 h later.

Feeding Trial
Abalones (Weight: 15.7 ± 0.03 g) were obtained from a fishery company in Pingtan, Fujian, China. Three experimental diets with different levels of arginine (1.17%, 1.68% and 3.43%) were designed (Table 4). Abalones were randomly assigned into nine tanks (60 abalones per tank, and three tanks for one group) and fed with the experimental diets for 120 days. Abalones were fed every two days (15:00-17:00). During the 120-day feeding trial, the water temperature was 24.4 ± 4.3 • C, pH was 7.63 ± 0.74 and the dissolved oxygen was higher than 6.9 mg·L −1 . After the feeding trial, abalones were fasted for 72 h before sampling. The muscles and digestive gland from each abalone were collected and immediately frozen in liquid nitrogen, then stored at −80 • C. The expressions of slc38a9 and TOR-related genes of abalone were detected by qRT-PCR using the 2× ChamQ Universal SYBR qPCR Master Mix (Q711-02, Vazyme Biotech, Nanjing, China). The cDNA of abalone tissue was used as the template for qRT-PCR. All the primers were designed by Oligo 7 software according to the sequence from NCBI and synthesized by Sangon Biotech (Shanghai, China), and the primer sequences are listed in Table 1. The qRT-PCR condition was as follows: one cycle of 95 • C for 30 s, followed by 40 cycles of 95 • C for 10 s, 58 • C for 30 s, a cycle 95 • C for 15 s and 60 • C for 60 s. β-actin gene was used as the reference gene to normalize the mRNA expression levels. The Ct values were calculated by using 2 −∆∆Ct method to quantitate the relative expression levels of target genes.

Western Blot Analysis
Abalone muscles (30-40 mg) were homogenized and lysed in RIPA (Solarbio Science and Technology Co., Ltd., Beijing, China) with protease inhibitor and phosphatase inhibitor (Thermo Fisher scientific, Waltham, MA, USA). The homogenates were set on ice for 10 min and then centrifuged at 4 • C to collect the supernatant. Protein concentrations were determined using the BCA protein assay kit (Beyotime Biotechnology, Shanghai, China) according to the manufacturer's instructions, and then all the samples were adjusted to the same concentration. Equal amounts of protein were subjected to SDS-PAGE gel electrophoresis and then electrophoretically transferred to PDVF membrane. The PVDF membrane was blocked with 5% non-fat powdered milk for 1h and incubated with the antibody overnight at 4 • C. The primary antibodies used are as follows: phospho-mTOR (Ser2448) (2971), mTOR (2972), phospho-S6 (Ser235/236) (4858), S6 (2217), phospho-4E-BP1 (Thr37/46) (2855) and 4E-BP1 (9644) were purchased from Cell Signaling Technology Inc (Danvers, MA, USA). Then the PVDF membranes were washed 3 times and incubated with HRP-labeled Goat Anti-Rabbit/Mouse IgG (Beyotime Biotechnology, Shanghai, China) for 1 h at room temperature and enhanced chemiluminescence by Beyo ECL Plus reagents (Beyotime Biotechnology, Shanghai, China). The bands were quantified by densitometry using ImageJ software (Ver. 1.53, National Institutes of Health, Bethesda, MD, USA). GAPDH (AB-P-R001, Goodhere Biotechnology, Hangzhou, China) was the reference protein to normalize the target protein abundance and the phosphorylation level was calculated by the intensity ratio of phosphorylated protein to total protein.

Statistical Analysis
All data were analyzed by SPSS 25.0 software (IBM Corp., Armonk, NY, USA) and presented as mean ± SEM. Normality test and homogeneity of variances were performed before statistical analysis. T-test was used for the analysis of the two sets of data. Data with more than 2 sets were analyzed by one-way analysis of variance (ANOVA) followed by the Tukey's multiple range test. Probabilities of p < 0.05 were considered statistically significant.

Characterization and Phylogenetic Analysis of the slc38a9
The nucleotide sequence of cDNA and deduced amino acid sequence of slc38a9 are shown in Figure 1. The sequence has been uploaded to NCBI (Accession No. MW390888). The full length of slc38a9 sequence contains 1832 nucleotides, including an ORF of 1578bp. It codes a polypeptide of 525 amino acids with a molecular weight of 58.8 kDa and a theoretical isoelectric point (pI) of 7.20. The secondary structure was mainly alpha-helix, and the deduced protein includes 11 transmembrane domains. No signal peptide was found of the sequence. The predicted tertiary structure of abalone SLC38A9 protein is shown in Figure 2C.
Multiple alignment and phylogenetic tree for slc38a9 and other counterparts are shown in Figure 2A,B. The results showed that slc38a9 of abalone had significant homology with mollusk, including Crassostrea gigas, Crassostrea virginica, Mytilus coruscus, Pecten maximus, Mizuhopecten yessoensis, Pomacea canaliculata. Among these, M. yessoensis had the highest sequence consistency with abalone. All mollusk species formed a clade separate from zebrafish, mice and humans.

Expression Analysis of slc38a9 in Different Tissues of Abalone
Transcript levels of slc38a9 in different tissues including muscle, hemocytes, mantle, gill, gonad, digestive gland and intestine were investigated to determine the tissue-specific expression profile of slc38a9 in abalone. The result showed that slc38a9 was widely expressed in all tested tissues, and it was most highly expressed in the gonad (p < 0.05), followed by intestine, gill, digestive gland and mantle. The lowest expressions were found in hemocytes and muscle ( Figure 2D).

Function Analysis of slc38a9 3.2.1. Expressions of TOR Pathway Related Genes after siRNA Injection
The silencing efficiency of four siRNA was measured to screen the most effective one. Compared with the control group, siRNA-NC had no significant effect on the mRNA level of slc38a9 (p > 0.05), and the relative expression level of slc38a9 was significantly decreased in all groups injected with siRNA-slc38a9 (p < 0.05). The silencing efficiencies of siRNA-108, siRNA-347 and siRNA-469 were about 60% without significant difference among the three (p > 0.05), silencing efficiency of siRNA-236 was 40% ( Figure 3A). To determine the time with the highest efficiency of siRNA, abalones were sampled at different time after injection. The result showed that the relative expression of slc38a9 was significantly inhibited at 6 h after injection, the inhibition efficiency was highest at 12 h after injection, and the relative expression was still significantly inhibited at 48 h (p < 0.05) ( Figure 3B).  The relative expression levels of target of rapamycin (tor), ribosomal s6 protein kinase (s6k), eukaryotic translation initiation factor 4e binding protein (4e-bp) and eukaryotic translation initiation factor 4e (eif4e) in muscle at 12 h after slc38a9 knockdown. Results are represented as mean ± SEM. Different lowercase letters (a < b < c) indicate significant differences among groups (p < 0.05), and asterisk (*) indicates a significant difference between the control and the experimental group (p < 0.05).

Expressions of TOR Pathway Related Genes after Injection with pcDNA3.1-slc38a9
Compared with the control group, intramuscular injection of pcDNA3.1 plasmid had no significant influence on the relative expression of slc38a9 (p > 0.05), and the relative expression of slc38a9 was up-regulated by pcDNA3.1-slc38a9 plasmid injection significantly (p < 0.05). The mRNA levels of tor, s6k and eif4e were significantly increased after the intramuscular injection of pcDNA3.1-slc38a9 plasmid (p < 0.05) (Figure 4). The relative expression levels of target of rapamycin (tor), ribosomal s6 protein kinase (s6k), eukaryotic translation initiation factor 4e binding protein (4e-bp) and eukaryotic translation initiation factor 4e (eif4e) in muscle at 12 h after slc38a9 knockdown. Results are represented as mean ± SEM. Different lowercase letters (a < b < c) indicate significant differences among groups (p < 0.05), and asterisk (*) indicates a significant difference between the control and the experimental group (p < 0.05).
Compared with the control group, the relative expression levels of tor, s6k and eif4e were significantly reduced after the injection of siRNA-slc38a9 (p < 0.05), but there was no significant change in mRNA level of 4e-bp (p > 0.05) ( Figure 3C).

Expressions of TOR Pathway Related Genes after Injection with pcDNA3.1-slc38a9
Compared with the control group, intramuscular injection of pcDNA3.1 plasmid had no significant influence on the relative expression of slc38a9 (p > 0.05), and the relative expression of slc38a9 was up-regulated by pcDNA3.1-slc38a9 plasmid injection significantly (p < 0.05). The mRNA levels of tor, s6k and eif4e were significantly increased after the intramuscular injection of pcDNA3.1-slc38a9 plasmid (p < 0.05) (Figure 4).

Expressions of TOR Pathway Related Genes after Injection of siRNA and Oral Administration of Arginine
Compared with the control group, oral administration of arginine significantly increased the expression levels of slc38a9, tor, s6k and eif4e in the muscles of abalone (p < 0.05). siRNA-mediated knockdown of slc38a9 significantly inhibited the activation of the TOR signaling pathway by arginine and decreased the expression of tor, s6k and eif4e (p < 0.05) ( Figure 5).

Expressions of TOR Pathway Related Genes after Feeding and siRNA Injection
Compared with the basal diet, the arginine-added diet increased the transcript levels of slc38a9, tor, s6k and eif4e in abalone (p < 0.05). The expression of slc38a9 had a peak after 6 h of feeding, and the expression levels of other genes reached the peak at 9 h ( Figure 6). Furthermore, feeding after siRNA-slc38a9 injection also up-regulated the expression levels of slc38a9, tor, s6k and eif4e (p < 0.05); however, the expression levels of these genes were significantly lower than that in abalone without siRNA injection (p < 0.05).

Expressions of TOR Pathway Related Genes in Hemocyte Treated with Different Concentrations of Arginine
The relative expression levels of slc38a9, s6k and eif4e were increased significantly when the concentration of arginine was 0.5-4 mmol/L (p < 0.05), and there was no significant difference among them (p > 0.05). Compared with the control group, the addition of arginine up-regulated the relative expression of tor (p < 0.05). Furthermore, 1-4 mmol/L arginine significantly increased the relative expression of 4e-bp (p < 0.05) (Figure 7).

Expressions of TOR Pathway Related Genes after Feeding and siRNA Injection
Compared with the basal diet, the arginine-added diet increased the transcript levels of slc38a9, tor, s6k and eif4e in abalone (p < 0.05). The expression of slc38a9 had a peak after 6 h of feeding, and the expression levels of other genes reached the peak at 9 h ( Figure 6). Furthermore, feeding after siRNA-slc38a9 injection also up-regulated the expression levels of slc38a9, tor, s6k and eif4e (p < 0.05); however, the expression levels of these genes were significantly lower than that in abalone without siRNA injection (p < 0.05).

Expressions of TOR Pathway Related Genes in Hemocyte Treated with Different Concentrations of Arginine
The relative expression levels of slc38a9, s6k and eif4e were increased significantly when the concentration of arginine was 0.5-4 mmol/L (p < 0.05), and there was no significant difference among them (p > 0.05). Compared with the control group, the addition of arginine up-regulated the relative expression of tor (p < 0.05). Furthermore, 1-4 mmol/L arginine significantly increased the relative expression of 4e-bp (p < 0.05) (Figure 7).

Expressions of TOR Pathway Related Genes Affected by Dietary Arginine
As presented in Figure 8A, the relative expression levels of slc38a9 and tor in muscle of abalone fed with 1.68% and 3.43% of dietary arginine were significantly higher than those fed diet with 1.17% of arginine (p < 0.05). Compared with 1.17% and 3.43% of dietary arginine, diet with 1.68% of arginine significantly increased the expression of s6k in abalone (p < 0.05). The relative expression levels of eif4e in muscle of abalone fed with 1.68% of dietary arginine were significantly higher than those fed with 1.17% of dietary arginine (p < 0.05). In addition, there was no significant difference in the mRNA level of 4e-bp in abalone fed with different levels of dietary arginine (p > 0.05).
As for the digestive gland, the relative expression levels of tor and eif4e in abalone fed with 1.68% and 3.43% of dietary arginine were significantly higher than those fed with diet with 1.17% of arginine (p < 0.05). Compared with 1.17% of dietary arginine, 1.68% of dietary arginine significantly improved mRNA levels of slc38a9 and s6k in digestive gland (p < 0.05). Dietary arginine level had no significant effect on the expression of 4e-bp in the digestive gland (p > 0.05) ( Figure 8B). Results are represented as mean ± SEM (n = 3 replicate experiments), and different lowercase letters (a < b < c < d) indicate significant differences among groups (p < 0.05).

Expressions of TOR Pathway Related Genes Affected by Dietary Arginine
As presented in Figure 8A, the relative expression levels of slc38a9 and tor in muscle of abalone fed with 1.68% and 3.43% of dietary arginine were significantly higher than those fed diet with 1.17% of arginine (p < 0.05). Compared with 1.17% and 3.43% of dietary arginine, diet with 1.68% of arginine significantly increased the expression of s6k in abalone (p < 0.05). The relative expression levels of eif4e in muscle of abalone fed with 1.68% of dietary arginine were significantly higher than those fed with 1.17% of dietary arginine (p < 0.05). In addition, there was no significant difference in the mRNA level of 4e-bp in abalone fed with different levels of dietary arginine (p > 0.05).
As for the digestive gland, the relative expression levels of tor and eif4e in abalone fed with 1.68% and 3.43% of dietary arginine were significantly higher than those fed with diet with 1.17% of arginine (p < 0.05). Compared with 1.17% of dietary arginine, 1.68% of dietary arginine significantly improved mRNA levels of slc38a9 and s6k in digestive gland (p < 0.05). Dietary arginine level had no significant effect on the expression of 4e-bp in the digestive gland (p > 0.05) ( Figure 8B).

Expressions of TOR Pathway Related Proteins Affected by Dietary Arginine
The phosphorylation levels of TOR (Ser 2448 ) in muscle of abalone fed with 1.68% of dietary arginine were significantly increased compared to those in abalone fed with 1.17% of dietary arginine (p < 0.05). Abalone fed 1.68% of dietary arginine showed the highest phosphorylation level of S6 (Ser 235/236 ). Meanwhile, 1.68% of dietary arginine significantly increased the phosphorylation levels of 4E-BP1 (Thr 37/46 ) compared with the other two groups (p < 0.05) ( Figure 8C,D). The abundance of phosphorylated TOR, Ribosomal S6 (S6) and 4E-BP protein in muscle of abalone fed diets with different arginine levels for 120 d. Results are represented as mean ± SEM (N = 3 replicate experiments), and different lowercase letters (a < b < c) indicate significant differences among groups (p < 0.05).

Expressions of TOR Pathway Related Proteins Affected by Dietary Arginine
The phosphorylation levels of TOR (Ser 2448 ) in muscle of abalone fed with 1.68% of dietary arginine were significantly increased compared to those in abalone fed with 1.17% of dietary arginine (p < 0.05). Abalone fed 1.68% of dietary arginine showed the highest phosphorylation level of S6 (Ser 235/236 ). Meanwhile, 1.68% of dietary arginine significantly increased the phosphorylation levels of 4E-BP1 (Thr 37/46 ) compared with the other two groups (p < 0.05) ( Figure 8C, D).

Discussion
SLC38A9 is characterized as a lysosomal membrane amino acid transporter with 11 transmembrane domains and a member of the solute carrier family 38 in human, which has the dual role of transporting amino acids and interacting with other proteins to activate TOR [28,30,36]. The present results showed that the slc38a9 contains an open reading frame of 1578bp and codes a polypeptide of 525 amino acids with a molecular weight of 58.8 kDa. The sequence analysis in the present study confirmed that SLC38A9 protein has 11 transmembrane domains and no signal peptide, which is in agreement with the results in human [28]. The multiple alignment and phylogenetic tree analysis showed that the slc38a9 sequence of abalone shares a higher degree of similarity with mollusks such as

Discussion
SLC38A9 is characterized as a lysosomal membrane amino acid transporter with 11 transmembrane domains and a member of the solute carrier family 38 in human, which has the dual role of transporting amino acids and interacting with other proteins to activate TOR [28,30,36]. The present results showed that the slc38a9 contains an open reading frame of 1578bp and codes a polypeptide of 525 amino acids with a molecular weight of 58.8 kDa. The sequence analysis in the present study confirmed that SLC38A9 protein has 11 transmembrane domains and no signal peptide, which is in agreement with the results in human [28]. The multiple alignment and phylogenetic tree analysis showed that the slc38a9 sequence of abalone shares a higher degree of similarity with mollusks such as oyster and scallop, and all mollusk species formed a clade separate from other species, which indicated that slc38a9 of abalone has high homology with mollusk. In addition, the present study showed that slc38a9 was expressed in all the analyzed tissues, which is consistent with the expression pattern of the SLC38 family in human and closely related mammals [37]. Transporters of the SLC38 family are particularly expressed in cells with active growth and amino acid metabolism, and SLC38A9 is especially expressed in testis, adrenal gland, thyroid and parathyroid [37,38]. Similarly, this study found that slc38a9 had the highest expression in the gonad of abalone. Moreover, the transport of arginine is one of the important functions of slc38a9; arginine is involved in the sperm formation process and is a basic component of sperm nuclear protein, and it can also improve sperm motility and protect the integrity of sperm structure and function [39,40]. As a metabolite of arginine, NO is essential for sustaining oocyte quality for optimal fertilization and development [41]. The high expression of slc38a9 may suggest the active transport and utilization of arginine in gonads. Furthermore, the intestine is the main organ of amino acid absorption [42,43], and it may be the reason why slc38a9 is highly expressed in the intestine in abalone.
Current research generally believes that amino acids regulate the TOR signaling pathway through the Rag-GTPases complex [26,44]. SLC38A9 was identified as a novel component of the Rag-Ragulator machinery and plays an important role in the regulation of the TOR signaling pathway in human cells [28,30]. In HEK-293T cells, the depletion of SLC38A9 decreased the phosphorylation of S6K and 4EBP1, and the phosphorylation of S6K was increased when SLC38A9 was overexpressed [28]. In the present study, the slc38a9 was artificially regulated by siRNA knockdown or overexpression to confirm the function and the underlying mechanism of SLC38A9 in the regulation of TOR by arginine in abalone. The results showed that knockdown of slc38a9 inhibited the expression of tor, s6k and eif4e, and the overexpression of slc38a9 activated the TOR signaling pathway, which is manifested as the increased relative expression level of tor, s6k and eif4e. These results are consistent with studies in human [28,30], suggesting that SLC38A9 plays a positive role in the regulation of the TOR signaling pathway of abalone. Furthermore, studies in human and mouse cells indicated that SLC38A9 is required for the amino acid-dependent TOR activation, and arginine is one of the main activators of this process [30,36]. In HEK-293T cells, siRNA-mediated knockdown of SLC38A9 inhibited the amino acid-induced mTORC1 activation, and knocked out of SLC38A9 suppressed the activation of mTOR by arginine at all concentrations [29,30]. In human HEK293E, HeLa, LN229 cells and mouse embryonic fibroblasts, overexpression of SLC38A9 could activate mTORC1 and sustain its activity during amino acid starvation [28,30]. Consistent with these studies, the present results showed that knockdown of slc38a9 did partially inhibit the activation of the TOR signaling pathway by arginine, as detected by the mRNA level of tor and its established substrate s6k and eif4e. Therefore, SLC38A9 is essential in the activation of the TOR signaling pathway by arginine.
The TOR signaling pathway plays a crucial role in maintaining cellular and physiological homeostasis [45,46]. Indispensable amino acids have remarkable effects on regulating the TOR pathway, including the regulation of S6K and 4E-BP to promote protein synthesis and initiate translation [47][48][49], and arginine is essential for the activation of TOR [50]. In the present study, 0.5-4 mmol/L arginine significantly increased the expressions of tor, s6k and eif4e in the hemocytes of abalone. It is consistent with the result in bovine mammary epithelial cells [19]. In addition, the present results demonstrated that 1.68% of dietary arginine could activate the TOR signaling pathway in muscle and digestive gland, which is evidenced by the increased relative expression level of tor, s6k and eif4e. Similarly, previous studies in fish species have shown that optimal dietary arginine increased the expression of tor and s6k [11,[21][22][23][24]. Furthermore, dietary arginine levels had no significant effect on the mRNA level of 4e-bp, which is consistent with studies in tiger puffer, hybrid grouper, blunt snout bream and gibel carp [11,22,23,25], but different results have also been reported in juvenile Jian carp [24], as it suggested that the regulation of arginine on 4E-BP in aquatic animals is complex and requires further relevant study. In the present study, the phosphorylation levels of TOR, 4E-BP and S6 were detected for a comprehensive understanding of the regulation of arginine on the TOR signaling pathway. As 4E-BP is the binding protein of eIF4E, phosphorylation of 4E-BP releases eIF4E and initiates translation [51], and dietary arginine supplementation promoted the formation of the eIF4E·eIF4G complex and reduced the amount of the 4E-BP1·eIF4E complex in skeletal muscle of piglets [20]. Arginine increased the abundance of phosphorylated mTOR, S6K and 4E-BP1 proteins in porcine and ovine trophectoderm cells and brown adipocyte precursor cells [16][17][18]. S6 is the primary substrate of S6K, and it is directly regulated by S6K to regulate protein synthesis [52], the abundance of phosphorylated S6 and S6K was increased by arginine in porcine trophectoderm cells [53]. The present study showed that 1.68% dietary arginine increased phosphorylation levels of TOR, S6 and 4E-BP in muscle. It showed a similar trend in mammals [17,18,20], suggesting that 1.68% dietary arginine activated the TOR signaling pathway at the protein phosphorylation level. Furthermore, excessive metabolism of amino acids may result in excessive energy cost and toxicity, 3.43% dietary arginine may exceed the tolerance level of arginine in abalone, resulting in an imbalance of amino acid, thereby inhibiting the activity of related genes and metabolic processes. Taken together, appropriate levels of arginine activated the TOR signaling pathway, which is evidenced by the increased expression levels of tor, s6k and eif4e in hemocytes, muscle and digestive gland. A total of 1.68% of dietary arginine increased the abundance of phosphorylated TOR, 4E-BP and S6 in muscle. These results suggested a promotion of protein synthesis in abalone.
The gene transcription of several members from the SLC superfamily are transcriptionally affected by amino acid levels [51,54]. Ingestion of essential amino acids increased the mRNA levels of SLC38A9 in human [55]. In piglets, the relative expression of neutral amino acid transporter slc1a5 was increased by L-glutamate, and leucine increased the expression of slc6a14, slc6a19 and slc7a9 [56,57]. An optimal level of dietary arginine up-regulated the expressions of cationic amino acid transporters in spotted grouper (Epinephelus coioides) [58] and tiger puffer (Takifugu rubripes) [25]. Similarly, the present study showed that 1.68% and 3.43% of dietary arginine increased the relative expression levels of slc38a9 in muscles and digestive gland of abalone (p < 0.05). The result indicated that appropriate arginine could increase the activation of amino acid transporter, which can promote the transport and utilization of arginine. In addition, the expression of slc38a9 is consistent with the expression trend of TOR-related genes, which supported the regulation of the TOR signaling pathway by SLC38A9.
The TOR signaling pathway has also emerged as a key regulator of autophagy and inflammatory response [49], and deregulation of TOR underlies the pathogenesis of metabolic disorders, cancer, neurodegeneration and aging [59,60]. SLC38A9 mediates the transport of essential amino acids out of lysosomes, which is required for the growth of pancreatic cancer cells, and loss of SLC38A9 or its transport function strongly inhibited tumor formation [61]. Furthermore, pancreatic cancers have more than 90% KRAS mutation and KRAS is one of the most frequently mutated oncogenes of the RAS family, and it is reported that combinatorial therapies with TOR inhibitors will be necessary against RAS-driven cancers [62]. An antagonist of SLC38A9 (NOVELTY) was developed for the treatment of proliferative disease and metabolic disorder associated with TOR activation [63]. The present study in abalone demonstrated that SLC38A9 is conservative in regulating the TOR signaling pathway and provided further evidence that SLC38A9 may be used as a promising anticancer target against related cancers. Moreover, amino acids and TOR were reported to mediate nutritional checkpoints in the cell cycle, which were usually inactivated and/or overridden in cancer cells [64,65]. Arginine depletion is considered as a viable option for cancer treatment, it results in the induction of cell growth arrest at several checkpoints [66,67], and the present study also showed that arginine deficiency inhibited the TOR signaling pathway. Therefore, exploring the role of arginine and the regulation of TOR might have some potential for therapeutic exploitation.

Conclusions
In conclusion, the sequence of slc38a9 in abalone was cloned. It shows high sequence homology to other mollusks and is widely expressed in various tissues. SLC38A9 plays a positive role in the regulation of the TOR signaling pathway, and it is essential in the activation of TOR by arginine. Moreover, appropriate levels of arginine increased the activity of the TOR signaling pathway and slc38a9 in vivo and in vitro (Figure 9). This study provides insights into the potential mechanism of the regulation of the TOR signaling pathway by arginine and the molecular basis for the promotion of dietary amino acids on protein synthesis and growth. positive role in the regulation of the TOR signaling pathway, and it is essential in the activation of TOR by arginine. Moreover, appropriate levels of arginine increased the activity of the TOR signaling pathway and slc38a9 in vivo and in vitro (Figure 9). This study provides insights into the potential mechanism of the regulation of the TOR signaling pathway by arginine and the molecular basis for the promotion of dietary amino acids on protein synthesis and growth.  Institutional Review Board Statement: The study was conducted according to the guidelines of the Declaration of Helsinki and approved by the Ethics Committee Ocean University of China (Approval No. OUC-SMP-2020-0126, January, 2020).

Informed Consent Statement: Not applicable.
Data Availability Statement: The data presented in this study are available on request from the corresponding author.

Institutional Review Board Statement:
The study was conducted according to the guidelines of the Declaration of Helsinki and approved by the Ethics Committee Ocean University of China (Approval No. OUC-SMP-2020-0126, January, 2020).

Informed Consent Statement: Not applicable.
Data Availability Statement: The data presented in this study are available on request from the corresponding author.