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Effects of glutamate and aspartate on growth performance, serum amino acids, and amino acid transporters in piglets

ABSTRACT This study mainly investigated the effects of different dietary levels of glutamate (Glu) and aspartate (Asp) on growth performance, blood amino acids, and amino acid transporters in piglets. Forty-two healthy piglets were randomly divided into six groups (n = 7): a control group in which piglets were fed 2.9% Glu and 1.5% Asp and other groups in which piglets received 1.3% or 1.7% Asp and 2.6%, 3.2%, or 3.5% Glu for 21 days. Growth performance, serum amino acid profiles from the mesenteric vein, portal vein, and anterior vena cava, and amino acid transporters in the liver were determined. The results showed that lower doses of Asp promoted growth and enhanced the amino acids, while high doses of Asp and Glu reduced growth and the amino acid pool in piglets (P < 0.05). Meanwhile, 3.2% Glu increased branched chain amino acids in the portal vein and anterior vena cava (P < 0.05). 3.5% Glu downregulated SLC7A1, SLC7A7, and SLC6A19 expression in the liver (P < 0.05). Collectively, these results indicated that different dietary doses of Glu and Asp influenced growth performance and serum amino acid.


Introduction
Glutamate (Glu) and aspartate (Asp), two acidic amino acids, are considered as non-essential amino acids in classical nutrition (Wu, 2010). Glu and Asp have been indicated to play important roles in physiological and biochemical processes (Duan et al., 2014;Wang et al., 2016). For example, Glu and Asp serve as major sources of energy and improve intestinal and liver function by alleviating oxidative stress Leng et al., 2014;Lin et al., 2014). Meanwhile, dietary supplementations with Glu and Asp have been reported to enhance growth performance (Yin, Liu, et al., 2015), act as excitatory neurotransmitters in the brain (Wang, Wang, Xia, & Wood, 2014), and mediate feeding behaver (Khropycheva, Andreeva, Uneyama, Torii, & Zolotarev, 2011). Bin et al reported that 1% Asp lowered the ratio of Firmicutes: Bacteroidetes in mice and affected the innate immunity (Bin et al., 2017). Duan et al reported that 2% glutamate acted as a nutritional regulating factor to ameliorate the adverse effects of mycotoxins and improved growth performance in mycotoxins-challenged pigs (Duan et al., 2014) . Although various studies indicate that Glu and Asp protect piglets against different pathological conditions, such as oxidative stress and mycotoxin infection, little is known about the effects of different dietary contents of Glu and Asp on growth performance and amino acid metabolism in healthy piglets. Thus, the present study examined the amino acid metabolic responses to dietary Glu and Asp in piglets and growth performance was also focused.

Animal and diets
All animal procedures were approved by the Committee on Animal Care of the Institute of Subtropical Agriculture, Chinese Academy of Sciences (Changsha, Hunan Province, China). Forty-two piglets (Duroc × Landrace × Yorkshire, male, 13.24 ± 0.25 kg) (Hunan New Wellful Co., Ltd., Hunan Province, China) were randomly assigned into one of six treatment groups (n = 7/group). Piglets in the control group (CG) were fed a basic diet containing 2.9% Glu and 1.5% Asp according to our previous study . Other piglets received 1.3% (LA) or 1.7% (HA) Asp and 2.6% (LG), 3.2% (HG), or 3.5% (HHG) Glu for 21 days, respectively (Table 1). Each animal was housed in a single cage. Piglets had free access to drinking water and diets during the experimental period.

Growth performance
During the experimental period, feed intake was recorded daily and all piglets were weighed individually at day 0 and 21. Average daily feed intake (ADFI), average daily weight gain (ADG), and the ratio of feed intake to weight gain (F/G) were calculated according to the feed consumption and weight of each piglet.

Amino acid determination
Blood samples were obtained from the mesenteric vein, hepatic portal vein, and anterior vena cava after anesthetic treatment using Zoletil 50 (Virbac S.A., France). Serum was prepared via centrifugation at 3000 g and 4°C for 10 min and stored at −80°C until analysis . Eighteen amino acids (Lys, Met, Thr, Trp, Glu, Asp, Val, Ile, Leu, Phe, Arg, Ser, His, Gly, Ala, Pro, Cys, and Tyr) in the serum were detected via 1260 liquid chromatograph (Agilent 1260) according to our previous reports Liao et al., 2017).

Statistical analysis
All statistical analyses were performed using IBM SPSS 20 software. The data were performed by using the one-way analysis of variance (ANOVA) to test homogeneity of variances via Levene's test and followed with student's t test Data are expressed as mean ± SEM. Values in the same row with * are significant (P < 0.05)

Effect of Asp and Glu on growth performance in piglets
Effect of Asp on growth performance was summarized in Table 3. There was no significant difference in body weight of piglets compared with control group (P > 0.05). Interestingly, ADG in the LA group was higher in the control group and F/G was markedly lower in the LA group (P < 0.05). However, dietary high Asp (1.7%) markedly decreased ADFI and ADG but increased F/G (P < 0.05), suggesting that low Asp improve growth but high dosage of Asp exert negative effect.  Dietary supplementation with different dosages of Glu failed to affect body weight in piglets (P > 0.05; Table 4). However, dietary high Glu significantly reduced ADG and increased F/G (P < 0.05). Moreover, ADFI in the HHG group was markedly lower than that in the control group (P < 0.05). These results suggested that excess Glu intake inhibit growth performance in piglets.

Effect of dietary Asp on the circulating amino acids in piglets
The levels of amino acids in the mesenteric vein, hepatic portal vein, and anterior vena cava were determined to evaluate amino acid metabolic response to dietary amino acids. In the mesenteric pool, different doses of Asp in the diet failed to affect the levels of Asp and other amino acids (P > 0.05; Table 5). In the hepatic portal, Trp and Tyr levels were significantly higher in the LA group than those in the control group (P < 0.05). In the anterior vena cava, Glu level in the HA group was markedly decreased compared with the control group (P < 0.05).

Effect of dietary Glu on the circulating amino acids in piglets
In the mesenteric pool, Met and Tyr were decreased in the HHG group, while Glu was increased in the HG group compared with the control group (P < 0.05; Table 6). In the hepatic portal, Trp, Val, Ile, Leu, and Ser levels were higher in the HG group than those in the control group. Trp in HHG group and Ser in LG group were markedly increased (P < 0.05). In the anterior vena cava, Ile and Tyr were decreased in the LG group while His was increased compared with the control group (P < 0.05). Moreover, Glu and Tyr in the HG group were decreased (P < 0.05) and Ile, Leu, and Tyr in the HHG group were also decreased compared with control group (P < 0.05).

Liver expression of amino acid transporters
In this study, expressions of SLC1A1, SLC7A1, SLC7A7, and SLC6A19 were determined via RT-PCR (Figure 1). Low-dose Asp down-regulated SLC6A19 expression in the liver (P < 0.05). Moreover, expressions of SLC7A1, SLC7A7 and SLC6A19 were markedly down-regulated in the HHG group (P < 0.05) mRNA in the liver. Also, dietary high Glu significantly down-regulated SLC6A19 expression compared with the control group (P < 0.05). Data are expressed as the mean ± SEM (n = 7). *means the difference was significant compared with the control group.

Discussion
Glu and Asp play important roles in the nervous system, immunity, and nutrition in animals and humans (Khropycheva et al., 2011;Leng, 2014;Lin et al., 2014;Wang et al., 2016;Yanni et al., 2010). Our previous studies showed that dietary supplementation with Asp and Glu reduced oxidative stress-induced growth suppression Yin, Liu, et al., 2015). Shi et al. also reported that dietary 0.5% and 1% Asp improved ADG in piglets after exposure to lipopolysaccharide (Shi, 2013). Meanwhile, dietary 2% Glu attenuated mycotoxin-induced growth suppression by improving ADFI, ADWG, and the feed/weight ratio in piglets (Wu, 2014). The animals may change in diet and bring about poor growth performance under weaning stress Ren et al., 2018). Thus, we investigated dietary Asp and Glu in healthy piglets with initial weight 10 kg and pre-feeding for a week. In the current study indicated that dietary supplementation with high dosages of Asp and Glu reduced growth performance and diets with low levels of Asp or Glu improved growth performance in piglets. Together, we speculated that low doses of Asp and Glu may satisfy the nutritional requirements in healthy piglets, while supplementations with excess Asp and Glu exhibit a negative effect on growth performance under healthy conditions. Serum amino acid levels are highly associated with animal health and growth performance Liao et al., 2017;Wu et al., 2014;. Previous studies have shown that dietary supplementation with 2.0% Glu, 0.5% −2.0% glutamine, 1.0% Asp or their combinations reduced oxidized stress or a pathological amino acid profile in weaned piglets Ren et al., 2014). Moreover, Glu and Asp can transform alanine, citrulline, glutathione, ornithine and arginine and other precursor substances (Wu et al., 2007;Zhang et al., 2012). A portion of the free amino acids in the intestine are selectively absorbed into the mesenteric vein, and some amino acids, such as Glu and Asp, are used by the intestine to maintain intestinal health (Deutz, Bruins, & Soeters, 1998;Pi et al., 2014;Stoll & Burrin, 2006;Xi et al., 2012). Portal vein blood is Data are presented as mean ± SEM, n = 6. SLC1A1 solute carrier family 1 (neuronal/epithelial high affinity glutamate transporter, system XAG, member 1), SLC7A1 solute carrier family 7 (cationic amino acid transporter, y + system; member 1), SLC7A7 solute carrier family7 (amino acid transporter light chain, y + L system, member 7), and SLC6A19 solute carrier family 6 (neurotransmitter/neutral amino acid transporter, member 19).
collected by the stomach, intestine, spleen, and other organs (extracorporeal tissue, Portal-Drained Viscera, PDV) (Stoll et al., 1998). Studies have shown that the liver contains high levels of Glu and Asp under low crude protein diets (17% crude protein) (Zheng, 2015). Meanwhile, Glu and Asp are absorbed and oxidized in the intestine and liver to maintain the intestinal and liver structure and function (Jinap & Hajeb, 2010;Leng et al., 2014;Wang et al., 2015;Wang et al., 2017). In the current work, only Glu levels in the anterior chamber of the HA and HG group were markedly decreased. The reason may be explained by that low doses of Glu and Asp meet the needs of the body, thus extra Glu and Asp may be oxidized by intestinal epithelial cells and liver cells to provide energy.
Zhenyukh et al. found that pathological conditions increased plasma levels of branched chain amino acids (Zhenyukh et al., 2017). In this study, the hepatic portal vein in the HG group (3.2% Glu group) contained the highest levels of branched chain amino acids, which may explain why 3.2% Glu appeared to have a negative effect on growth performance. Try is a precursor of the neurotransmitter serotonin, which promotes feed intake (Burgoon, Knabe, & Gregg, 1992;Eder, Nonn, Kluge, & Peganova, 2003;Kendall, Gaines, Kerr, & Allee, 2007), reduces energy waste, and enhances feed utilization (Zhang et al., 2013). Meanwhile, tryptophan inhibits xanthine oxidase activity and reactive oxygen, as well as oxidative stress (Lv et al., 2012). The current study showed that the portal vein of the LA, HG, and HHG group showed a high tryptophan level, which might affect growth performance and amino acid metabolism and detailed mechanisms should be further investigated.
Amino acids are mainly sensed and transported by specific amino acid transporters (Burckhardt Birgitta & Burckhardt, 2017;Zuo et al., 2015). For example, SLC1A1 transports Asp and Glu and SLC7A1 contributes to Lys, Arg, and His uptake (Verrey, Meier, Rossier, & Kuhn, 2000). In this study, high dosage of Glu downregulated SLC7A1, SLC7A1, and SLC6A19 expressions, which may be associated with the feedback regulation as excess of amino acids are toxic to cells. SLC6A19 transports all neutral amino acids and is the only transporter of Trp in the small intestine (Bröer et al., 2004). Thus, we speculated that SLC6A19 may mediate Trp metabolism in this study.

Conclusions
Low levels of Glu and Asp in the diet increased growth performance of piglets and the serum tryptophan level. High doses of Glu and Asp, especially 3.2% Glu, inhibited the growth of piglets, increased BCAA levels in the hepatic portal vein and may have toxicological effects.