Lysine, an essential nutrient for swine, is typically the first limiting amino acid (AA) in those common grain-based swine diets. Studies have shown that dietary deficiency in lysine can lead to reduced growth performance in pigs due to decreased body protein synthesis and increased fat deposition (1, 2). Therefore, in the swine industry, the pig diets are commonly supplied with the non-protein-bound free lysine (called crystalline lysine) to ensure that pigs receive enough lysine for their optimal growth (3).

At the molecular biological level, lysine has regulatory effects on gene expression, which can alter animal metabolic and signaling pathways associated with protein and lipid metabolism (4). For example, Wang et al. (2) reported that dietary lysine deficiency may activate the ubiquitination pathway to increase muscle protein degradation and up-regulate the expression of genes associated with lipid biosynthesis in finishing pigs. Additionally, Jin et al. (4, 5) also found that dietary lysine deficiency inhibited the satellite cell proliferation and reduced protein synthesis via inhibiting the mTOR signaling pathway in the longissimus dorsi muscle of weaning pigs. However, the global gene expression profile or the transcriptional responses to the dietary lysine restriction or deficiency has not been reported in growing pigs.

The transcriptome of a specific tissue under a certain environmental, nutritional, and physiological condition comprises all types of RNAs that include coding and non-coding RNAs (6, 7). In recent years, RNA sequencing (RNA-Seq) – using next-generation sequencing technology – has been employed as a powerful tool to perform a comprehensive analysis and quantification of all RNA species expressed in tissues or cells (7–9). The main objective of this study was to use RNA-Seq to investigate the effects of dietary lysine restriction on the global gene expression profile in the longissimus dorsi muscle of young growing pigs. The functions of those differentially expressed genes (DEGs) were annotated as well.

Lysine is typically the first limiting essential AA in grain-based swine diets. It has been known that dietary deficiency in lysine will negatively affect not only pigs’ muscle – the largest part of the body mass – protein deposition, but also the overall heath and growth performance (1, 2). The growth performance data generated from this study have been reported previously, which showed that the average daily weight gain and the gain to feed ratio were both significantly decreased in the pigs fed LDD than fed LAD, although there was no difference in the average daily feed intake between the two groups of the pigs (3). The reduced weight gain observed in this study must be the consequence of pigs’ poor utilization of dietary nutrients and, especially the proteinogenic AA, for body protein biosynthesis and muscle growth, since the skeletal muscle is the largest organ, as well as the largest protein pool, in the body (1, 3). To further explore the molecular mechanism responsible for the reduced growth (mainly the muscle growth) in response to the dietary lysine restriction, we conducted this global gene expression profiling to identify the DEGs that may have diverse molecular functions associated with the growth and development of the skeletal muscle.

Through this study, we found that dietary lysine restriction downregulated some transporter-protein coding genes (Table 4), such as transthyretin (TTR) and albumin (ALB). Although the TTR and ALB genes are highly expressed in the liver according to the human gene annotation, Soprano et al. (13) and Wagatsuma et al. (14) reported that these genes were also expressed in animal skeletal muscle. The TTR gene product is a 55-kDa homotetraeric transporter protein for T3 and T4 (both are thyroid hormones) in the blood (15). According to Monk et al. (16), TTR deficient mice show a slower transition from suckling to weaning, slower growth, less muscle mass, and impaired bone growth. In addition, some other studies demonstrated that TTR plays a role in promoting the differentiation of muscle cells and the repair of muscle tissue after injury (15, 17, 18). The ALB gene product is another acute phase reactant and a non-specific protein transporter for numerous hormones existing in the blood. It has the highest binding affinity for T3 and T4 followed by TTR, and it determines the transportation time and distribution of thyroid hormones to target tissues. Hypoalbuminemia can be caused by poor nutrition and ongoing inflammation in animals (19). A cross-sectional study reported a significant association between low serum albumin and low muscle strength (20). These results suggest that the reduced skeletal muscle protein turnover and altered local muscle metabolism of thyroid hormones may lead to an intracellular diminished T3 availability (21). Therefore, the downregulation of TTR and ALB genes in this study implies that a reduction in the expression of TTR and ALB genes may be a factor in the negative impact of a lysine restricted diet on muscle growth. This could be due to an impact on those proteins responsible for transporting hormones such as thyroid hormones and other signaling molecules.

The expression of several genes coding for the cytochrome P450 family members, such as cytochrome P450 family 1 subfamily A members 1 and 22 (CYP1A1 and CYP1A22), and cytochrome P450 family 2 subfamily E member 1 (CYP2E1), was decreased in pigs fed the lysine-restriction diet (Table 4). It is known that CYP1A1 and CYP2E1 enzymes metabolize polyunsaturated fatty acids to synthesis biologically active intracellular cell-signaling molecules or lipid mediators, such as ω-3 fatty acid derived resolvins and maresins (22). Some of the lipid mediators are well known for their function in maintaining skeletal muscle mass. During nutritional stress (e.g., deficiency), these anti-inflammatory molecules play important roles in reducing skeletal muscle wasting (23). Consequently, the decreased expression of the genes that are associated with polyunsaturated fatty acid metabolism and synthesis of anti-inflammatory molecules, may negatively contribute to skeletal muscle maintenance and homeostasis.

Of the downregulated genes, phenylalanine hydroxylase (PAH) and tyrosine amino transferase (TAT) are associated with AA metabolism. The PAH enzyme catalyzes the conversion of L-phenylalanine to L-tyrosine, which indicates that dietary lysine restriction may suppress the PAH enzyme activity in pigs. It has long been found that a depressed PAH activity is associated with a decreased ability to dispose the phenylalanine load, leading to an impaired tyrosine formation (24). Tyrosine is used not only in protein synthesis but also in the synthesis of some neurotransmitters (e.g., dopamine, epinephrin, and nor-epinephrin) and hormones (e.g., thyroid hormone). In addition, tyrosine acts as a substrate in reaction with oxoglutarate catalyzed by TAT enzyme to produce 4-hydroxyphenylpyruvate and glutamate. Glutamate can act as a precursor for several neurotransmitters (e.g., GABA) (25). Thus, a decrease in the expression of PAH and TAT in response to dietary lysine restriction may indirectly affect the production of some neurotransmitters and hormones associated with animal growth.

The term “keratin” is known as intermediate filament-forming proteins in cell cytoskeleton, which belong to fibrillar proteins. Keratin fundamentally influences the architecture (e.g., cell polarity and cell shape) and mitotic activity of epithelial cells to sustain their mechanical stress, maintain their structural integrity, and establish cell polarity (26, 27). This study revealed that dietary lysine restriction upregulated the expression of several isoforms of keratin genes including keratin 74 (KRT74), keratin 82 (KRT82), keratin 31 (KRT31), keratin 25 (KRT25), and keratin 33A (KRT33A) (Table 5). As is known, most keratin isoforms are annotated as hair and skin specific in humans, a contamination of our muscle samples by hair or skin tissue is most unlikely due to our rigorous sample preparation protocol. Furthermore, some previous studies had reported the presence of several type I and type II intermediate filament proteins (known as keratins) in developing and striated muscle (28, 29). For example, keratin 19 (K19) and keratin 8 (K8) are prominent keratins expressed in mature striated muscle, where they play a pivotal role in upholding the structural integrity of muscle fibers (30).

There is no previous research, however, that demonstrated a regulatory effect of lysine on keratin gene transcription and translation in vivo. It is of interesting in finding that dietary lysine may play a role in keratin structural activity, which warrants further investigation.

The ECM of skeletal muscle – a complex meshwork consisting of collagens, glycoproteins, proteoglycans, and elastin – maintains the tissue structure and integrity (31). The mRNA abundance of several genes that encode collagens, such as collagen type II alpha 1 chain (COL2A1) and collagen type IX alpha 1 chain (COL9A1), proteoglycans, such as hyaluronan and proteoglycan link protein 1 (HAPLN1) and aggrecan (ACAN), and glycoproteins, such as tenascin N (TNN), was increased in this study. The collagen is the major protein in ECM. It has lower fractional synthetic rate than the myofibrillar proteins, and its concentration in the skeletal muscle is only 15 to 20% of the myofibrillar proteins. The suboptimal nutritional condition, such as AA deficiency, may increase in the collagen fractional synthesis rate while decreasing the syntheses of myofibrillar and sarcoplasmic proteins (32). Therefore, the dietary lysine restriction may be a suboptimal nutritional condition that upregulates the collagen synthesis in pigs.

Cartilage proteoglycan link proteins, on the other hand, are the major non-collagenous components of the ECM cartilage (33). In this study, the expression of aggrecan (ACAN) and hyaluronan and proteoglycan link protein 1 (HAPLN1) genes was increased in response to dietary lysine restriction. ACAN is best known as a major, essential, and defining cartilage component quantitatively. According to Mundlos et al. (34), the gene expression patterns of aggrecan and link protein are identical in different tissues, which agrees with our finding. The regulatory mechanism that promotes such drastic alteration in mRNA expression in response to lysine restriction remain uncertain and will be subject to further studies.

The de novo biosynthesis of serine and glycine can generate carbon units that satisfy many metabolic demands including nucleotide precursors, redox maintenance, and substrates for methylation reaction (35, 36). Of the several enzymatic reaction steps in serine biosynthesis pathway, phosphoglycerate dehydrogenase (PHGDH) catalyzes the conversion of 3-phosphoglycerate into 3-phosphohydroxypyruvate. Phosphoserine aminotransferase 1 (PSAT1) is a pivotal enzyme that regulates the production of two metabolites, serine, and α-ketoglutarate (α-KG), which are involved in one carbon metabolism and the TCA cycle, respectively (1, 37). This study shows that both PHGDH and PSAT1 genes were upregulated in the longissimus dorsi muscle of the pigs fed the lysine restricted diet. This is in agreement with one of our previous studies, which revealed that dietary lysine deficiency decreased the expression of PHGDH and PSAT1 in the longissimus dorsi muscle of finishing pigs (2). Overexpression of these genes indicate that dietary lysine level could affect the balance of one carbon metabolism and its associated metabolic pathways.

The widely expressed activating transcription factor (ATF5) regulates cell cycle, differentiation, homeostasis, and survival (38, 39). The results of this study indicate that the expression of ATF5 was increased in pigs fed the LDD diet compared to the LAD diet. Similar findings in previous studies reported that ATF5 expression was increased in response to cellular stress conditions such as AA limitation, heat stress, or oxidative stress (39, 40). Increased activation of ATF5 may upregulate the protein kinases phosphorylation of EIF2α and lead to a global reduction in protein translation.

Six BP terms were significantly enriched under the lysine restriction status (Figure 3), of which protein polymerization (66-fold), fibrinolysis (79-fold), and plasminogen activation (99-fold) were highly enriched. Based on these terms, it can be seen that some DEGs are mostly associated with the homeostasis, wound healing, inflammation, angiogenesis, and several other biological functions (41, 42). For example, fibrinogen, a proteolytic glycoprotein, composed of three closely linked polypeptides coded from FGA, FGB, and FGG genes. Dietary lysine restriction downregulated these genes that may affect the rate of fibrinogen synthesis and result in abnormalities in blood circulation (42), which in turn may indirectly affect the transportation of oxygenated blood or nutrients to the muscle.

Secondly, there were 4 MF terms that were significantly enriched (Figure 3), of which, the highly enriched one was endopeptidase inhibitor activity (62-fold). Endopeptidases, including trypsin, chymotrypsin, elastase, and pepsin, are proteolytic enzymes that break the peptide bonds of non-terminal AAs (43, 44), and are associated with a wide range of biological roles, such as developmental processes, digestion, fertilization, blood coagulation, apoptosis, fibrinolysis, and immune defense (45). The negative effect of dietary lysine restriction on pig growth performance may be also associated with endopeptidase inhibition.

Thirdly, there were 8 CC terms that were significantly enriched (Figure 3), of which the fibrinogen complex was enriched by 166-fold. The CC terms in GO analysis demonstrate the subcellular location or the structure of macromolecular complex of gene products (46). As shown in Figure 3, a high percentage of gene products among the DEGs are part of fibrinogen complex, intermediate filament, and ECM. Fibrinogen, an important component of ECM, is a large complex protein composed of three polypeptide chains, such as A, B, and G that are encoded by FGA, FGB, and FGG genes, respectively (47, 48). The DEG result of this study indicates that the dietary lysine restriction decreased the fibrinogen gene expression (Table 4) that may lead to a reduction in the amount of fibrinogen available to be incorporated into ECM, leading to a weaker ECM and a reduction in the angiogenesis process, which could affect the integrity of ECM and muscle growth (49, 50).

Finally, the results of the GO terms of KEGG pathway enrichment indicate that the negative impact of feeding lysine restricted diet to pigs on the growth may be mediated via the signaling and metabolic pathways that include AA biosynthesis, cholesterol metabolism, receptor interaction, and complement and coagulation cascades (Figure 3). For example, Carbamoyl l-phosphate synthase 1 (CPS1) gene encode an enzyme involved in nitrogen metabolism that removes nitrogen waste products from the body (51). The decrease in CPS1 expression was observed in this study suggesting that it may cause disruption in the balance of muscle protein synthesis and degradation, leading to muscle wasting. It is important to note that AA metabolism is a very complex metabolic process whose regulatory mechanism warrants further investigation.

At the end, authors would like to point out that the mRNA abundance does not always correlate with protein abundance, even though many scientists use mRNA abundance to indicate protein abundance because in many cases more mRNA would translate out more protein. Also, it should be kept in mind that the global gene expression data in general are not easy to be interpreted simply because of the complexities not only in the data themselves but also in the aspect of animal biological processes.

A transcriptome analysis using RNA-Seq technology revealed that dietary lysine restriction altered the gene expression profile in longissimus dorsi muscle of growing pigs. We found that 80 genes were differentially expressed, of which 46 genes were down-regulated and 34 genes were up-regulated. Some of the down-regulated genes (e.g., TTR, ALB) are associated with transportation of signaling molecules (e.g., hormones and neurotransmitters) and AA metabolism (e.g., PAH, TAT). On the other hand, the down-regulated genes, such as keratin, ACAN, and COL isoforms are associated with the cell structural molecule activities and AA metabolism (e.g., PHGDH and PSAT1). The GO analysis results suggest that the dietary lysine restriction may influences some biological processes, such as blood coagulation and inflammation, some molecular functions, such as endopeptidase inhibitory activity; and some KEGG pathways, such as the complement and coagulation pathway. Collectively, the results generated from this study have provided some critical novel insight regarding the molecular mechanisms of muscle growth that are associated with dietary lysine supply.

The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/Supplementary material.

The animal study was approved by Mississippi State University Institutional Animal Care and Use Committee (IACUC). The study was conducted in accordance with the local legislation and institutional requirements.

SL conceptualized the study, acquired funding for the investigation, and coordinated the whole project. JF and HZ participated in the experiment design and manuscript preparation. MH and SL conducted the animal trial and analyzed data and prepared the manuscript. MH and YW conducted the laboratory sample analyses. All authors approved the final version of the manuscript.