Both Dietary Supplementation with Monosodium L-Glutamate and Fat Modify Circulating and Tissue Amino Acid Pools in Growing Pigs, but with Little Interactive Effect

Background The Chinese population has undergone rapid transition to a high-fat diet. Furthermore, monosodium L-glutamate (MSG) is widely used as a daily food additive in China. Little information is available on the effects of oral MSG and dietary fat supplementation on the amino acid balance in tissues. The present study aimed to determine the effects of both dietary fat and MSG on amino acid metabolism in growing pigs, and to assess any possible interactions between these two nutrients. Methods and Results Four iso-nitrogenous and iso-caloric diets (basal diet, high fat diet, basal diet with 3% MSG and high fat diet with 3% MSG) were provided to growing pigs. The dietary supplementation with fat and MSG used alone and in combination were found to modify circulating and tissue amino acid pools in growing pigs. Both dietary fat and MSG modified the expression of gene related to amino acid transport in jejunum. Conclusions Both dietary fat and MSG clearly influenced amino acid content in tissues but in different ways. Both dietary fat and MSG enhance the absorption of amino acids in jejunum. However, there was little interaction between the effects of dietary fat and MSG.


Introduction
As an umami flavor, monosodium L-glutamate (MSG) has been widely used as a food additive. The worldwide production and consumption of MSG has recently increased, and it is even expected to further increase in the next years (http://www.ihs.com/ products/chemical/planning/ceh/monosodium-glutamate.aspx). MSG is considered as safe among food additives by the Joint Expert Committee on Food Additives of the United Nations Food and Agriculture Organization and World Health Organization [1]. When MSG is consumed, L-glutamate (Glu) is released in the small intestine lumen and metabolized notably by the enterocytes [2]. The regular daily consumption of MSG presumably modify the metabolism of nutrients, notably the metabolism of AAs in the body [3], and protein synthesis regulation and proteolysis [4,5]. Only few studies have reported the effects of daily oral MSG supplementation on AA metabolism.
The Chinese population, including children and adolescents, has been undergoing a rapid transition to a high-fat diet. This change has occurred faster than that in western children over the last century [6,7]. MSG is also currently widely added to food on a daily basis in China. Thus, it would be interesting to evaluate the metabolic effects of the combination of dietary fat and MSG. Pigs are considered to be a suitable animal model for studying human nutrition due to their apparent similarities with humans; i.e., both species are omnivorous, and they share nutritional and digestive characteristics [8]. In the present study, MSG was provided to growing pigs at a dose that was only slightly higher than that found in human food, without or with a plausible amount of dietary fat content. The aim of the present study was to determine the effect of dietary supplementation with MSG on the AA balance in tissues and to clarify whether the interaction between fat and MSG influence AA metabolism.
Since rodents may be more sensitive to MSG than humans [9], the pig model was used in the present study. A total of 32 growing pigs (crossbred population composed of York, maternal Landrace, paternal Landrace, and Duroc breeds; average body weight 2561.3 kg) from 4 litters in a pig farm located in Changsha were used. The pigs were randomly divided into four groups (8 animals per group with half males and half females). Pigs were raised in individual cages and provided with water and four different diets containing different amounts of fat and MSG (basal diet, high fat diet, basal diet with 3% MSG and high fat diet with 3% MSG). The basal diet group was used as control. According to previous experiments [10], the four diets were prepared to be iso-caloric and alanine was used to allow iso-nitrogenous content of all diets [11,12]. The dose of MSG and dietary fat contents were designed according to the NRC 1998 and the consumption in human food. The detailed composition of the four diets are shown in Table 1. The total AAs in each diet were determined and the results are shown in Table 2. The four diets were provided to the pigs ad libitum. In the day time, natural light was provided, and in the dark period, fluorescent lamps were used and turned down at 8:00 pm. Water was provided freely during the entire experiment. Blood samples were firstly collected into 10-mL heparin-coated tubes, and then centrifuged (3,0006g for 10 min at 4uC). The supernatants (plasma) were immediately separated and stored at 270uC until analysis. The sampled pigs were sacrificed by jugular puncture under general anaesthesia via the intravenous injection of 4% sodium pentobarbital solution (40 mg/kg BW) and immediately eviscerated. Samples of the longissimus dorsi, liver, kidney and segments of the intestine including the duodenum, jejunum, ileum and colon (cleaned by ice-cold normal saline) were collected, immediately frozen in liquid nitrogen and stored at 270uC until analysis. All experimental procedures used in the present study were approved by the Animal Care and Use Committee of the Chinese Academy of Sciences [13].

Serum analyses
Plasma total concentrations of albumin, alanine transaminase, aspartate aminotransferase, c-glutamyl transpeptidase, creatine kinase, total protein and serum urea were determined with an automatic analyzer (Beckman Instruments, Inc., Fullerton, CA) using reagents purchased from Beijing Leadman Biochemistry Co., LTD.

Determining of AA concentrations using the stable isotope dilution HPLC-electrospray ionization (ESI)-MS/MS method
To determine contents of AAs in experimental diet, 5 g samples of diets were dissolved in hydrochloric acid solution (6 mol/L) at a concentration equal to 20 mg/ml, and hydrolized for 24 h, before centrifugation at 13200 rpm for 5 min. The supernatants were filtered with hydrophilic membrane, diluted 40 folds with methanol, and then were dried under nitrogen at 50uC. The samples were finally dissolved in 100 ml methanol solution (v/ v = 1:1) for the derivatization procedure. Ten ml of the extracted solution were transferred to a new centrifuge tube, and mixed with 90 ml of the stable isotope solution (the stable isotope being

Quantitative real-time reverse transcription-polymerase chain reaction
Total RNA was extracted from samples using TRIzolH Reagent (Invitrogen-Life Technologies, CA, USA) following the manufacturer's suggested protocol and finally dissolved in DEPC-treated water. Quality and concentration of the extracted RNA were checked by spectrophotometry using NanoDropH ND2000 (NanoDrop Technologies Inc., DE, USA), respectively. Then, 1.0 mg of total sample RNA was incubated with DNase I (Fermentas), and reverse-transcribed with oligo dT and random primers using reverse transcription using First-Strand cDNA Synthesis Kit according to the manufacturer's instructions (TAKARA, Dalian, China). Finally, the cDNAs were stored at 270uC before further processing. qPCR was performed with an ABI PRISM 7900 HT (Applied Biosystems, USA) using 384-well plates. Duplicate sample analysis was routinely performed in a total volume of 10 ml using SYBRHPremix Ex TaqTM II (TAKARA, Dalian, China). The primers were designed using Primer 5.0 software and be listed in Table 3. Data were analyzed

Statistical analyses
The data regarding gene expression were expressed as means 6 SEM, and those regarding AA concentrations were expressed as means 6 SD. Differences between groups were assessed by ANOVA. Statistical analyses were performed with SAS 9.2 (SAS Institute Inc., North Carolina, USA). The difference were considered as statistically significant at P,0.05.

MSG and fat modify serum biochemical parameters
Serum albumin was firstly tested, and the results are shown in Fig. 1A. Dietary fat clearly increased the serum albumin concentration (p = 0.0228), while MSG had no effect, and there was no interaction between the effect of dietary fat and MSG on serum albumin. To clarify the changes in AA metabolism, the activities of serum alanine transaminase and aspartate amino-transferase were measured, and the results are shown in Fig. 1B and Fig. 1C. Dietary fat significantly reduced the activity of aspartate aminotransferase (p,0.0001). Dietary fat and MSG synergistically reduced the activities of serum alanine transaminase (p = 0.0510) and aspartate aminotransferase (p = 0.0359). There were no significant differences between treatments for the other serum biochemical parameters, including creatine kinase, total protein and serum urea.

MSG and fat modify serum AA profiles
Serum is the main circulating AA pool and the serum AA profile is often used as an index for the diagnosis of several diseases [14].
To determine the effects of fat and MSG on serum AAs, the AA profile was measured and the results are shown in Table 4.

Dietary supplementation with both fat and MSG can influence the expression of AA-sensing genes
To document the effects of dietary fat and MSG on AA-related gene expression, the expression of a broad spectrum of AA-sensing genes in the kidney, liver and muscle were determined. The results are shown in Fig. 2. Dietary fat can significantly up-regulated the expression of taste receptor type 1 member 1 (T1R1) in the kidney (p = 0.0036), and down-regulated its expression in muscle (p = 0.0434), and G-protein-coupled receptor family C member 6A (GPRC6A) in the liver (p = 0.0342). MSG significantly upregulated the expression of T1R1 in the kidney (p = 0.0007), and down-regulated the expression of T1R1 in the liver (p = 0.0447). MSG also down-regulated the expression of extracellular Ca 2+sensing receptor (CaR) in the muscle (p = 0.0213), and significantly down-regulated the expression of GPRC6A in the liver (p = 0.002) and muscle (p = 0.0191).

Dietary supplementation with fat and MSG modify AA pools in liver and muscle
The free AA profiles in the liver and muscle were determined, and the results are shown in Table 5 and Table 6. Dietary fat increased the concentrations of Ser (p = 0.0078), Tyr (p = 0.0046), Asn (p = 0.0347), and Cys (p = 0.0493) in the liver, and also increased the concentrations of Gly (p = 0.0155) and Ala (p = 0.0441) in the muscle. MSG had no effect on AA pool in the liver and muscle. Dietary fat and MSG had opposite effects on the concentrations of Cit (p = 0.0004) in the liver and Hyp (p = 0.0092) in the muscle. Interestingly, while dietary fat and MSG each tended to reduce homocysteine (Hcy) in muscle, their combination increased the concentration of Hcy in muscle (p = 0.0258). The free AA pool in the kidney is markedly altered by supplementation with MSG The kidney plays a major role in the homeostasis of AA pools leading us to determine the free AA profile in this tissue. The results are shown in Table 7

Both dietary fat and MSG can modify the expression of the AA transporters in the jejunum
To better understand AA absorption, the expressions of AA transporters in different intestinal segments was measured, and the results are shown in Fig. 3. These AA transporters include: Excitatory amino-acid carrier 1, EAAC1, which is the most abundant Glu and Asp transporter in the intestine; L-type AA transporter 1 (LAT1) and B 0+ AA transporter (B 0+ ), which can transport neutral and alkaline AAs; ASC-like Na + -dependent neutral AA transporter 2 (ASCT2), which can transport neutral AAs; and intestinal H + /peptide co-transporter (PEPT1), which mainly transports peptides in the intestine. While dietary fat had no effect on the expression of AA transporters, MSG significantly reduced the expression of EAAC1 (p = 0.0009) in the duodenum.  synergistic effects on the expression of LAT1 (p = 0.0093). Although the colonic epithelial cells do not transport AAs, except in the neonatal period, amino acid transporter expression in the large intestine was also measured. Dietary fat had no effect on the expression of AA transporters in the colon, while MSG significantly reduced the expression of EAAC1 (p = 0.0199). To determine the consequence of the altered expression of AA transporters, free AA concentrations were measured. The disturbed AA balance in intestinal wall (Tables 8-10) suggests that dietary fat and MSG may modify the absorption and metabolism of AAs in the jejunum mucosa.

Discussion
Dietary fat and protein are two of the main macronutrients required for life. Many published work had tested the effects of each nutrients independently, however, a few studies are available on the effect of combination of dietary fat and protein. The metabolism is a systemic network, and it would be misleading to evaluate the effects of one nutrient while ignoring the effects of others in plausible nutritional situation. As early as 1956, Mellinkoff et al. found that a diet that contained very few shortchain saturated fats and relatively abundant long-chain unsaturated fats could significantly reduce Leu and Val and increase Pro, Cys, Arg and Asp in plasma [15]. Dietary fatty acids, particularly n-3 fatty acids, can spare AAs for protein and peptide synthesis [16]. On the contrary, AAs, especially those containing sulfur, can also modulate lipid metabolism [17]. As the Chinese economy is developing, the Chinese population, including children and adolescents, has undergone a rapid transition to a high-fat diet [6]. In parallel with these trends, there has recently been a large increase in the consumption of MSG as an umami food additive in China. MSG can facilitate the gastric emptying of a protein-rich meal, and plays an important role in protein digestion [18]. Conversely, MSG has been shown to increase the stomach antral area in human volunteers fed with a normo-proteic diet [19] and to slow gastric emptying in preterm piglets [20]; suggesting that the effects of MSG on the stomach physiology depends on nutritional conditions. In the present study, the effects of dietary fat and MSG on AA metabolism in growing pigs were determined, along with the interaction between these two factors. MSG begins to release Glu and sodium in the mouth, while dietary fat begins to be digested in the small intestine. As the alimentary bolus rapidly passes through the duodenum, with dietary fat having little effect there, an increase in the luminal concentration of MSG may be responsible for the reduced expression of EAAC1 in the small intestine mucosa. This may correspond to a regulation of AA transport through the enterocytes. Glu released from MSG, is an important precursor for bioactive molecules, including glutathione (oxidative stress modulator) [21], and this may be related to the down-regulation of PEPT1. However, the mechanisms responsible for the antagonistic effects of dietary fat and MSG on B o+ remains unclear.
Colonization of the gastrointestinal tract by gut microbiota can influence the absorption and metabolism of nutrients, including AAs. Gut microbiota can incorporate and degrade some available AAs, and can also synthesize AAs [22]; even if the net result of these catabolic and anabolic pathmays remains unknown [22]. Factors that alter the composition of the gut microbiota can also disturb AA metabolism. There have been reports showing that both dietary fat [23,24] and MSG can influence the composition of the gut microbiota. In the present study, changes in microbiota composition following dietary fat and MSG ingestion were observed [unpublished data]. Changes in the gut microbiota may participate in the up-regulated expression of AA transporters in the jejunum, but further experiments are required to test this hypothesis. Glu is the main oxidative fuel for the gut microbiota in the upper gastrointestinal tract [25]. Even when Glu is provided in higher (4-fold more) amount than normal dietary quantities, most glutamate molecules are either oxidized or metabolized by the mucosa into other nonessential AAs [26]. Thus, both dietary fat and MSG may enhance AA synthesis by the gut microbiota, and this may be related to the effects of dietary fat and MSG on the expression of AA transporters. In the lower gastrointestinal tract, the effects of dietary fat and MSG were weak, and this is presumably related to the higher density of bacteria in these two intestinal segments than in the jejunum, and to the fact that AAs degraded from the residual undigested luminal proteins and peptides cannot be absorbed in the distal intestine [22]. In the present study, the expression of AA-sensing genes and AA transporters in tissues were also measured. While the determination of AA-sensing genes are likely indirectly related to AA concentrations, the results obtained are compatible with the view that AA concentration and gene expression in tissues may influence each other in both directions.

Conclusion
In conclusion, the effects of dietary fat and MSG, both alone and in combination, on the AA in the circulating and tissue pool were determined. Little interaction was found between the effects of dietary fat and MSG. Both dietary fat and MSG enhanced the absorption of AAs in jejunum. Dietary fat can enhance the AA pool in plasma and muscle, while MSG enhances AA pool in the kidney. The results of the present study will help to further uncover the effects of dietary fat and MSG addition on human amino acid metabolism, with predictable consequences for the optimization of animal feeding and human nutrition.