Effects of Alpha-Ketoglutarate on Glutamine Metabolism in Piglet

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Effects of alpha-ketoglutarate on glutamine metabolism in piglet enterocytes in vivo and in vitro Liuqin He, Huan Li, Niu Huang, Junquan Tian, Zhiqiang Liu, xihong zhou, kang yao, tiejun li, and Yulong Yin J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b00433 • Publication Date (Web): 22 Mar 2016 Downloaded from http://pubs.acs.org on March 26, 2016

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Effects of alpha-ketoglutarate on glutamine metabolism in piglet enterocytes in vivo and in

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vitro

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Liuqin He,†,‡ Huan Li,§ Niu Huang,§ Junquan Tian,†,‡ Zhiqiang Liu,† Xihong Zhou,†, * Kang

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Yao,†,§, * Tiejun Li, †,#, * and Yulong Yin†

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Engineering Research Center of Healthy Livestock, Scientific Observing and Experimental

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Station of Animal Nutrition and Feed Science in South-Central, Ministry of Agriculture, Institute

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of Subtropical Agriculture, Chinese Academy of Sciences, Changsha, Hunan , 410125, China

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410128, China

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Key Laboratory of Agro-ecological Processes in Subtropical Region, Hunan Provincial

University of the Chinese Academy of Sciences, Beijing, 10008, China College of Animal Science and Technology, Hunan Agricultural University, Changsha, Hunan,

Hunan Co-Innovation Center of Animal Production Safety, Changsha, Hunan, 410128, China

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ABSTRACT

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Alpha-ketoglutarate (AKG) plays a vital part in the tricarboxylic acid cycle and is a key

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intermediate in the oxidation of L-glutamine (Gln). The study was to evaluate effects of AKG on

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Gln metabolism in vivo and in vitro. A total of twenty-one piglets were weaned at 28 days with a

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mean body weight (BW) of 6.0 ± 0.2 kg, and randomly divided into 3 groups: corn soybean meal

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based diet (CON group); the basal diet with 1% alpha-ketoglutarate (AKG treatment group); and

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the basal diet with 1% L-glutamine (GLN treatment group). Intestinal porcine epithelial cells-1

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(IPEC-1) was incubated to investigate effects of 0.5, 2, and 3 mM AKG addition on Gln

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metabolism. Our results showed that there were no differences (P > 0.05) among the 3 treatments

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in initial BW, final BW and average daily feed intake. However, average daily gain (P = 0.013)

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and the ratio of gain : feed (P = 0.041) of AKG group were greater than the other two groups. In

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comparison with the CON group, the AKG and GLN groups exhibited an improvement in villus

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length, mucosal thickness, and crypt depth in the jejunum of piglets. Serum concentrations of Asp,

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Glu, Val, Ile, Tyr, Phe, Lys, and Arg in the piglets fed the 1% AKG or Gln diet were lower than

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those in the CON group. Compared with CON group, the mRNA expression of jejunal and ileal

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amino acid (AA) transporters in the AKG and GLN groups were significantly increased (P < 0.05).

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Additionally, the in vitro study showed that the addition of 0.5, 2, and 3 mM of AKG

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dose-dependently decreased (P < 0.05) the net utilization of Gln and formulation of ammonia, Glu,

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Ala and Asp by IPEC-1. In conclusion, dietary AKG supplementation, as a replacement for Gln,

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could improve Gln metabolism in piglet enterocytes and enhance the utilization of AA.

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KEY WORDS: alpha-ketoglutarate; glutamine; amino acids; piglet; enterocytes

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INTRODUCTION

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L-glutamine (Gln),as a predominant amino acid (AA) in the body, contributes more than 50%

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of the total intracellular free α-AA pool in skeletal muscle and blood.1 Compelling evidences show

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that Gln plays a key role in the intestinal health by serving as a crucial metabolic fuel for all fast

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dividing cells,2, 3 and as a precursor of glutathione,4 pyrimidines and purines.5 Thus, Gln promotes

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the synthesis of protein and inhibits protein catabolism in enterocytes.5,

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glutamate (Glu), is readily converted to alpha-ketoglutarate (AKG).7 Two other AA that relate

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closely to the tricarboxylic acid (TCA) cycle are alanine (Ala), which is derived from pyruvate,

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and

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metabolite/precursor of Gln is AKG, which plays a pivotal role in intermediate metabolism, and

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enables the redistribution of nitrogen towards anabolic or catabolic pathways.9 Notably, AKG

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serves as an intermediate in the TCA cycle, and the oxidation of Gln to CO2 and water requires the

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formation of AKG.10 Therefore, AKG is considered to be one of the crucial molecules in

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interorgan nitrogen transport, protein metabolism, as well as regulation of gene expression and

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cellular redox state. 11, 12

aspartate (Asp)

is

derived

from oxaloacetate.8

However,

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Briefly, Gln, via

the most

important

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Emerging evidences showed beneficial effects of AKG in animal nutrition, particularly with

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regulating AA transporters gene expression13 and the mammalian target of rapamycin signaling

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pathway in the pig intestine.14 Notably, AKG may have a sparing effect on Glu and Gln in cells by

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serving as a fuel source in growing pigs.15 Furthermore, AKG has been applied to be a gut nutrient

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and a potential inhibitor of Gln catabolism.16 And through the synthesis of Gln, polyamines, and

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arginine (Arg),3 AKG play a major role in the growth and development of small intestine.17 In the

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practical application, although Gln is currently used as a new feed additive to enhance nitrogen

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metabolism and reduce metabolic stress in animal production,18, 19 AKG is more inexpensive,

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soluble and stable than Gln, thus it would potentially reduce feed cost and increase the efficiency

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of utilization. To date, the mechanisms responsible for the action of AKG as Gln replacement on

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intestinal Gln metabolism in piglets remains unknown. Therefore, the present study was to

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determine the effects of AKG on intestinal Gln metabolism in vivo and in vitro.

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MATERIALS AND METHODS

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Animals and Experimental Design

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The animal experiments were approved by the Institutional Animal Care and Use Committee

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of the Institute of Subtropical Agriculture, Chinese Academy of Sciences (2013020). Twenty-one

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piglets with a mean body weight (BW) of 6.0 ± 0.2 kg were weaned at 28 days, and randomly

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assigned into three treatments based on weaning weight (7 piglets/ treatment): one group was fed a

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corn soybean meal based diet (CON group); another was fed the basal diet plus 1%

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alpha-ketoglutarate

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China; purity ≥ 99.2%) (AKG treatment group); and the third was fed the basal diet plus 1%

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L-glutamine6 (Wuhan Yuancheng Gongchuang Technology co., LTD, Wuhan, Hubei, China;

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purity ≥ 99.5%) (GLN treatment group). The composition and nutrient levels of all diets met the

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nutrient specifications for 5 to 10 kg BW pig according to the NRC-recommended requirements

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(NRC, 2012) and showed in Table 1. After 7 days of adaption, piglets were fed their respective

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diets 3 times per day at 8:00, 13:00 and 18:00. During the experiment, piglets were housed

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individually and given free access to water. Average daily weight gain (ADG) and feed intake

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(ADFI) were calculated. The duration of whole experiment was four weeks.

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(Wuhan Yuancheng Gongchuang Technology co., LTD, Wuhan, Hubei,

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Sample Collection and Analytical Methods

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After the whole feeding period, blood samples (10mL) were taken from jugular vein, then

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pigs were anaesthetised with sodium pentobarbital intravenously (50mg/kg BW) and bled by

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exsanguination. Tissue samples from jejunum and ileum, were collected (after being cleaned with

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ice-cold phosphate-buffered saline), immediately frozen in liquid nitrogen, and stored at -80°C.

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And one jejunum and ileum segment were fixed in 10% neutral buffered formalin for examination

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of intestinal morphology. Blood samples were centrifuged at 3,000 ×g for 10 min at 4°C, and then

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stored at -20°C for analysis of free AA by an automatic amino acid analyzer (L-8900; Hitachi

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Global Inc., Hitachi, Japan).

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Intestinal histomorphology

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Paraffin sections (approximately 5 mm) of jejunum and ileum samples were stained with

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hematoxylin and eosin, and villus length and crypt depth were measured using a light microscope

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with a computer-assisted morphometric system (BioScan Optimetric, BioScan Inc., Edmonds, WA,

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USA). Villus length, mucosal thickness, and crypt depth were defined as previous study did. 20

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Relative quantification of mRNA expression of AA transporters by Real-time Quantitative RT-PCR

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The mRNA expression of solute carrier family 1, member 1 (EAAC1), solute carrier family 7,

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member 9 (b0+), solute carrier family 7, member 7 (y+ LAT1), and solute carrier family 1, member

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5 (ASCT2) in the jejunum and ileum were analyzed by real-time quantitative RT-PCR as

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previous study described.21 The primer were shown in Table 2. The relative gene expression was

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expressed as a ratio of the target gene to the control gene using the formula 2 - ( ∆∆Ct), where ∆∆ Ct

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= (Ct Target - Ct GAPDH )treatment - (Ct Target - Ct GAPDH)control.

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Culture of intestinal epithelial cells

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Intestinal porcine epithelial cells-1 (IPEC-1) were given by the lab of the Department of

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Animal Science, Texas A&M University as a gift and cultured as described previously.22 Briefly,

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the cells were grown in uncoated plastic culture flasks (100mm2) in Dulbecco’s modified

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Eagle’s-F12 Ham medium(DMEM-F12). Confluent cells were trypsinized and seeded with

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approximately 1×104 cells per well (6-well cell culture plates) and maintained at 37°C with 5%

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CO2. After incubation for 16 h, the cells were cultured in a medium containing 0.5, 2, and 3 mM

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of AKG respectively for 3 days. This medium contained physiological concentrations of AA

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found in pig plasma.22 There were seven independent replicates AKG dose. The medium was

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changed every day. After 3 days of culture, cells (5×106 /mL) were cultured 3 h with 2mM of

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L-[U-14C] Gln. Briefly, after 3 h culture, medium and cells was collected for the analysis of

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ammonia, Glu, Ala, and Asp using High Performance Liquid Chromatography (Waters 2695;

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Waters Inc., MA, USA). Additionally, 14C-labled Gln was measured as described by Yao. 10

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Statistical analysis

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Data were analyzed by the one-way analysis of variance and a mixed procedure (PROC

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MIXED) using SAS software version 9.2 (SAS Institute Inc., Cary, NC). Additionally, dietary

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treatment is considered as fixed effect and animal as randomized factor. Data were presented as

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Least Squares Means ± SEM. Mean values were considered to be significantly different when P
0.05) among the three treatment groups in initial BW, final BW and

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ADFI. However, ADG and gain: feed (G/F) of AKG group were greater than the other groups (P >

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0.05), and there was difference (P < 0.05) between AKG and CON groups.

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Intestinal histomorphology

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The results of intestinal morphology is summarized in Figure 1. In comparison with the CON

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group, the AKG and GLN groups exhibited an increase in villus length, mucosal thickness and

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crypt depth in the jejunum of piglets. And there was difference (P < 0.05) in villus length among

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the three treatment groups. However, in ileum, there were no differences (P > 0.05) in villus

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length, mucosal thickness and crypt depth among the three treatment groups.

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Free AA concentration in serum

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Table 4 shows the effect of AKG and Gln on serum contents of free AA in weaned piglets.

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There were no differences(P > 0.05) in the contents of Asp, Glu, Val, Ile, Tyr, Phe, Lys, and Arg

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between the AKG and the GLN groups, however, these AA concentrations differed from the CON

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group (P < 0.05), and the value in the CON group was the greatest. Notably, the content of Thr in

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the AKG group was the highest among the three groups, Thr concentration was (P < 0.05) higher

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in both the AKG and GLN groups than the CON group.

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The expression of AA transporters mRNA abundance.

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Data on the mRNA abundance of AA transporters in jejunum and in ileum are shown in

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Figure 2 (A and B). In the jejunum (Figure 2A), there were differences (P < 0.01) in the mRNA

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expression of ASCT2, b0+, y+LAT1, and EAAC1 among the three groups, and the mRNA

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abundance of ASCT2, b0+, and y+LAT1 in the GLN group was the highest, followed by the AKG

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and CON groups, in descending order. However, the mRNA abundance of EAAC1 in the AKG

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group was the highest. Furthermore, in the ileum (Figure 2B), remarkable differences (P < 0.05) in

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the mRNA abundance of ASCT2, b0+, y+LAT1, and EAAC1 were also detected among the three

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groups. And the expression of b0+, y+LAT1, and EAAC1 was much greater in the GLN group than

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that in the AKG group, the opposite was observed for ASCT2. Additionally, the mRNA

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abundance of ASCT2, b0+, and y+LAT1 of the AKG group in jejunum was greater than those in

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ileum.

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Effects of AKG on Gln catabolism and its metabolites production in IPEC-1 cells

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As shown in Figure 3 (A, B, C, D, and E), the different concentration of AKG affected the

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utilization of Gln and the production of ammonia, Glu, Ala, and Asp in IPEC-1 cells. Interestingly,

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the increased contents of AKG from 0.5 to 3 mM decreased (P < 0.05) the net utilization of Gln

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and inhibited (P < 0.05) the production of ammonia, Glu, Ala, and Asp. However, no differences

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(P > 0.05) in the contents of Gln, ammonia, Glu, Ala, and Asp were determined between 2 and 3

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mM of AKG groups in IPEC-1.

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DISCUSSION

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Alpha-ketoglutarate, as a replacement for Gln, could improve the growth performance of

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piglets. This may be explained by the reasons that as an intermediate of the TCA cycle, AKG is

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essential for the oxidation of fatty acids, AA, and glucose,12 thus produce enough energy for

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intestinal cell growth and proliferation.23 Furthermore, as a precursor for the synthesis of Gln and

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Glu in multiple tissues,24 AKG bridges carbohydrate and nitrogen metabolism for both

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conservation of AA and ammonia detoxification.12 As an important fuel for all rapidly dividing

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cells, Gln improves the synthesis of protein and inhibits protein catabolism in enterocytes.24

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However, as a feed additive, Gln is easy to be decomposed25, 26 and did not improve the growth

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performance as remarkably as AKG did. Therefore, compared with the GLN treatment, AKG

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could maintain the unremarkable growth performance of piglets under lower ADFI condition.

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Additionally, since G/F affects the economic return in pork production, our work has important

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implications for the sustainability of the swine industry.

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The present study showed an increase in villus length, mucosal thickness and crypt depth in

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the jejunum of AKG and Gln-supplemented piglets. This result suggests a net improvement of the

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intestinal health in the AKG and GLN groups. That is because Gln serves as a critical oxidative

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substrate for the intestinal mucosa and a precursor of vital molecules.27-29 Furthermore, as a

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precursor of Gln and Glu, AKG metabolism via the TCA cycle generates reduced coenzymes used

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by the mitochondria for ATP synthesis.12, 30 And the enterocytes consume a large amount of ATP,

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which is required for nutrient absorption and intestine health maintenance.31, 32 In our current

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study, our results showed that Gln and AKG supplementation beneficially improved the intestinal

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morphology in the jejunum of weaned piglets. These findings indicate that dietary AKG

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supplementation may be also an important factor for the maintenance of intestinal health as well

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as Gln.

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Amino acids are key regulators of intestinal health and metabolic pathways that regulate

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nutrient utilization.33 In the current study, compared with the CON group, the serum

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concentrations of Asp, Glu, Val, Ile, Tyr, Phe, Lys, and Arg in piglets fed the 1% AKG or Gln diet

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were decreased, while the opposite was observed for Thr. These results suggest that dietary AKG

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or Gln supplementation could improve the utilization rate of those AA related with AKG and Gln

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metabolism in serum and other tissues. Notably, the important function of Thr as an essential AA

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is to stimulate protein synthesis and AA abundance, and may be regulate feed intake to some

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degree,34 which may also explain why ADFI in the AKG group and GLN group was higher than

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the CON group. Moreover, unlike with branched-chain AA,35 whose catabolism is initiated in

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extra-hepatic organs and cells, the degradation of Lys, Phe, and Tyr occurs primarily in the liver.9

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It is possible that AKG and Gln enhance the utilization of these AA for tissue protein synthesis or

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promote their oxidation in the liver.36 Tracer studies will be required to examine these

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possibilities.

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The absorption of AA requires many transporter systems that differ with respect to their

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substrate specificity and driving force.20 It has been reported that some genes (such as ASCT2,

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EAAC1, bo+, y+LAT1) that are involved in the control of growth or AA metabolism are regulated

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by AA availability.21, 37 An interesting finding of this study is that AKG and Gln increased the

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mRNA expression of ASCT2, EAAC1, b0+, and y+LAT1 in the intestine of weaned piglets. Of

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note, except the expression of EAAC1 in jejunum and ASCT2 in ileum, the expression of other

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AA transporters in the GLN treatment group was higher than that in the AKG treatment group,

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thus the results was consistent with the concentrations of AA in serum. To some extent, AA can

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play a vital role in the control of AA transporters expression by a vast, complex regulatory system,

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which may also affect the energy balance and endocrine system.32 This result can also be

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explained by the extensive catabolism of both Gln and Glu in pig enterocytes.38 Additionally,

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AKG as a precursor of Gln and Glu may have a potent ‘sparing’ effect on endogenous Gln pools.17

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Interestingly, in our data we found that the addition of AKG could affect Gln catabolism and

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the production of its metabolites in IPEC-1. The addition of 0.5, 2, and 3 mM of AKG

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dose-dependently decreased the utilization of Gln and the formulation of Ala, Asp, Glu and

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ammonia. Based on the chemical equilibrium of AKG dehydrogenase,39 AKG may inhibit Glu,

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Asp, Ala and ammonia transaminases in cells, thereby inhibiting the catabolism of these AA and

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Gln.31, 40 Alternatively, AKG may also inhibit cellular Gln transport,41 by affecting the expression

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of phosphate-dependent glutaminase or directly inhibiting the catalytic activity of glutaminase.10,

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and AKG is an important intermediate in the oxidation of Gln, which is largely converted to AKG

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and catabolized to produce ATP in the intestine.12 Therefore, all of these changes can contribute to

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and decrease in Gln catabolism and its metabolites production. In summary, these interesting

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findings may have important implications for the application of AKG as Gln replacement in

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livestock.

Furthermore, to our knowledge, degradation of Gln produces Ala, Asp, Glu, ammonia and CO2,

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ABBREVIATIONS USED

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AKG, alpha-ketoglutarate; Gln, glutamine; AA, amino acid; Glu, glutamate; Ala, alanine; Asp,

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aspartate; BW, body weight; ADFI, average daily feed intake; ADG, average daily gain; G/F,

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gain : feed; IPEC-1, intestinal porcine epithelial cells-1; EAAC1, solute carrier family 1, member

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1; b0+, solute carrier family 7, member 9; y+ LAT1, solute carrier family 7, member 7; ASCT2,

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solute carrier family 1, member 5.

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CORRESPONDING AUTHOR

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*(K.Y.) Fax: +86 84615285.E-mail: [email protected] or (T.L.) Fax: +86 84615285.E-mail:

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[email protected] or (X.Z.) Fax: +86 84615285. E-mail: [email protected]

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FUNDING

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This work was supported by National Basic Research Program of China (2013CB127301 and

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2013CB127306), Chinese Academy of Sciences “Hundred Talent" award for Kang Yao, National

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Natural Science Fundation Project (31472106 and 31472107), National Science and Technology

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Support Project (2013BAD21B04).

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ACKNOWLEDGMENTS

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All of the authors declare no conflicts of interest. Thanks for supporting of Changsha Lvye

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Biotechnology Limited Company Academician Expert Workstation, Guangdong Wangda Group

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Academician Workstation for Clean Feed Technology Research and Development in swine.

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REFERENCES 1.

Nordgren, A.; Karlsson, T.; Wiklund, L. Glutamine concentration and tissue exchange

257

with intravenously administered alpha-ketoglutaric acid and ammonium: A dose-response study in

258

the pig. Nutrition. 2002, 18, 496-504.

259

2.

Palmieri, E. M.; Spera, I.; Menga, A.; Infantino, V.; Iacobazzi, V.; Castegna, A.

260

Glutamine synthetase desensitizes differentiated adipocytes to proinflammatory stimuli by raising

261

intracellular glutamine levels. Febs Lett. 2014, 588, 4807-4814.

262

3.

Fillmann, H.; Kretzmann, N. A.; San-Miguel, B.; Llesuy, S.; Marroni, N.;

263

Gonzalez-Gallego, J.; Tunon, M. J. Glutamine inhibits over-expression of pro-inflammatory genes

264

and down-regulates the nuclear factor kappaB pathway in an experimental model of colitis in the

265

rat. Toxicology. 2007, 236, 217-226.

266

4.

Chellamuthu, V. R.; Ermilova, E.; Lapina, T.; Luddecke, J.; Minaeva, E.; Herrmann, C.;

267

Hartmann, M. D.; Forchhammer, K. A Widespread Glutamine-Sensing Mechanism in the Plant

268

Kingdom. Cell. 2014, 159, 1188-1199.

269

5.

Caballero-Solares, A.; Viegas, I.; Salgado, M. C.; Siles, A. M.; Saez, A.; Meton, I.;

270

Baanante, I. V.; Fernandez, F. Diets supplemented with glutamate or glutamine improve protein

271

retention and modulate gene expression of key enzymes of hepatic metabolism in gilthead

272

seabream (Sparus aurata) juveniles. Aquaculture. 2015, 444, 79-87.

273

6.

Zhong, X.; Li, W.; Huang, X. X.; Wang, Y. X.; Zhang, L. L.; Zhou, Y. M.; Hussain, A.;

274

Wang, T. Effects of glutamine supplementation on the immune status in weaning piglets with

275

intrauterine growth retardation. Arch. Anim Nutr. 2012, 66, 347-356.

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

276

7.

Cooper, A. J. L.; Kuhara, T. alpha-Ketoglutaramate: an overlooked metabolite of

277

glutamine and a biomarker for hepatic encephalopathy and inborn errors of the urea cycle. Metab

278

Brain Dis. 2014, 29, 991-1006.

279 280 281

8.

Neu, J.; Shenoy, V.; Chakrabarti, R. Glutamine nutrition and metabolism: Where do we

go from here? Faseb J. 1996, 10, 829-837. 9.

282

1-17.

283

10.

Wu, G. Y. Amino acids: metabolism, functions, and nutrition. Amino Acids. 2009, 37,

Yao, K.; Yin, Y. L.; Li, X. L.; Xi, P. B.; Wang, J. J.; Lei, J.; Hou, Y. Q.; Wu, G. Y.

284

Alpha-ketoglutarate inhibits glutamine degradation and enhances protein synthesis in intestinal

285

porcine epithelial cells. Amino Acids. 2012, 42, 2491-2500.

286

11.

Filip, R.; Wdowiak, L.; Harrison, A. P.; Pierzynowski, S. G. Dietary supplementation

287

with phytohemagglutinin in combination with alpha-ketoglutarate limits the excretion of nitrogen

288

via urinary tract. Ann. Agr. Env Med. 2008, 15, 309-315.

289

12.

He, L. Q.; Xu, Z. Q.; Yao, K.; Wu, G. A.; Yin, Y. L.; Nyachoti, C. M.; Kim, S. W. The

290

Physiological Basis and Nutritional Function of Alpha-ketoglutarate. Curr Protein Pept Sc. 2015,

291

16, 576-581.

292

13.

Hou, Y. Q.; Wang, L.; Ding, B. Y.; Liu, Y. L.; Zhu, H. L.; Liu, J. A.; Li, Y. T.; Wu, X.;

293

Yin, Y. L.; Wu, G. Y. Dietary alpha-ketoglutarate supplementation ameliorates intestinal injury in

294

lipopolysaccharide-challenged piglets. Amino Acids. 2010, 39, 555-564.

295 296 297

14.

Rhoads, J. M.; Wu, G. Y. Glutamine, arginine, and leucine signaling in the intestine.

Amino Acids. 2009, 37, 111-122. 15.

Junghans, P.; Derno, M.; Pierzynowski, S.; Hennig, U.; Rudolph, P. E.; Souffrant, W. B.

298

Intraduodenal infusion of alpha-ketoglutarate decreases whole body energy expenditure in

299

growing pigs. Clin Nutr. 2006, 25, 489-496.

300

16.

Winkler, S.; Holzenbein, T.; Karner, J.; Roth, E. Kinetics of organ specific metabolism

301

of a bolus injection into the jejunum of glutamine, alpha-ketoglutarate, ornithine and ornithine -

302

alpha-ketoglutarate. Clin Nutr. 1993, 12, 56-7.

ACS Paragon Plus Environment

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Page 13 of 27

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303

17.

Coudray-Lucas, C.; Lasnier, E.; Renaud, A.; Ziegler, F.; Settembre, P.; Cynober, L. A.;

304

Ekindjian, O. G. Is alpha-ketoisocaproyl-glutamine a suitable glutamine precursor to sustain

305

fibroblast growth? Clin Nutr. 1999, 18, 29-33.

306

18.

Haynes, T. E.; Li, P.; Li, X. L.; Shimotori, K.; Sato, H.; Flynn, N. E.; Wang, J. J.; Knabe,

307

D. A.; Wu, G. Y. l-Glutamine or l-alanyl-l-glutamine prevents oxidant- or endotoxin-induced

308

death of neonatal enterocytes. Amino Acids. 2009, 37, 131-142.

309 310 311

19.

Pierzynowski, S. G.; Sjodin, A. Perspectives of glutamine and its derivatives as feed

additives for farm animals. J. Anim. Feed Sci. 1998, 7, 79-91. 20.

He, L. Q.; Yang, H. S.; Hou, Y. Q.; Li, T. J.; Fang, J.; Zhou, X. H.; Yin, Y. L.; Wu, L.;

312

Nyachoti, M.; Wu, G. Y. Effects of dietary l-lysine intake on the intestinal mucosa and expression

313

of CAT genes in weaned piglets. Amino Acids. 2013, 45, 383-391.

314

21.

He, L.; Wu, L.; Xu, Z.; Li, T.; Yao, K.; Cui, Z.; Yin, Y.; Wu, G. Low-protein diets

315

affect ileal amino acid digestibility and gene expression of digestive enzymes in growing and

316

finishing pigs. Amino Acids. 2016, 48, 21-30.

317

22.

Tan, B.; Yin, Y. L.; Kong, X. F.; Li, P.; Li, X. L.; Gao, H. J.; Li, X. G.; Huang, R. L.;

318

Wu, G. Y. l-Arginine stimulates proliferation and prevents endotoxin-induced death of intestinal

319

cells. Amino Acids. 2010, 38, 1227-1235.

320

23.

Kristensen, N. B.; Jungvid, H.; Fernandez, J. A.; Pierzynowski, S. G. Absorption and

321

metabolism of alpha-ketoglutarate in growing pigs. J. Anim Physiol. Anim Nutr (Berl). 2002, 86,

322

239-245.

323

24.

Wang, L.; Hou, Y. Q.; Yi, D.; Li, Y. T.; Ding, B. Y.; Zhu, H. L.; Liu, J.; Xiao, H.; Wu,

324

G. Y. Dietary supplementation with glutamate precursor alpha-ketoglutarate attenuates

325

lipopolysaccharide-induced liver injury in young pigs. Amino Acids. 2015, 47, 1309-1318.

326 327 328

25.

Contineanu, I.; Neacsu, A.; Perisanu, S. T. The standard enthalpies of formation of

L-asparagine and L-alpha-glutamine. Thermochim Acta. 2010, 497, 96-100. 26.

Ingraham, L.; Li, M. S.; Renfro, J. L.; Parker, S.; Vapurcuyan, A.; Hanna, I.; Pelis, R. M.

329

A Plasma Concentration of alpha-Ketoglutarate Influences the Kinetic Interaction of Ligands with

330

Organic Anion Transporter 1. Mol Pharmacol. 2014, 86, 86-95.

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

331

27.

Wang, B.; Wu, Z.; Ji, Y.; Sun, K.; Dai, Z.; Wu, G. l-Glutamine Enhances Tight Junction

332

Integrity by Activating CaMK Kinase 2-AMP-Activated Protein Kinase Signaling in Intestinal

333

Porcine Epithelial Cells. J. Nutr. 2016, 146, 501-8.

334

28.

Yu, H.; Gao, Q.; Dong, S.; Lan, Y.; Ye, Z.; Wen, B. Regulation of dietary glutamine on

335

the growth, intestinal function, immunity and antioxidant capacity of sea cucumber Apostichopus

336

japonicus (Selenka). Fish Shellfish Immun. 2016, 50, 56-65.

337

29.

Coutinho, F.; Castro, C.; Rufino-Palomares, E.; Ordonez-Grande, B.; Gallardo, M. A.;

338

Oliva-Teles, A.; Peres, H. Dietary glutamine supplementation effects on amino acid metabolism,

339

intestinal nutrient absorption capacity and antioxidant response of gilthead sea bream (Sparus

340

aurata) juveniles. Comp Biochem. Phys. A. 2016, 191, 9-17.

341

30.

Lin, M.; Zhang, B.; Yu, C.; Li, J.; Zhang, L.; Sun, H.; Gao, F.; Zhou, G. L-Glutamate

342

supplementation improves small intestinal architecture and enhances the expressions of jejunal

343

mucosa amino acid receptors and transporters in weaning piglets. Plos One. 2014, 9, e111950.

344

31.

Lambert, B. D.; Filip, R.; Stoll, B.; Junghans, P.; Derno, M.; Hennig, U.; Souffrant, W.

345

B.; Pierzynowski, S.; Burrin, D. G. First-pass metabolism limits the intestinal absorption of enteral

346

alpha-ketoglutarate in young pigs. J. Nutr. 2006, 136, 2779-2784.

347

32.

Wu, G. Y. Intestinal mucosal amino acid catabolism. J. Nutr. 1998, 128, 1249-1252.

348

33.

Jobgen, W. S.; Fried, S. K.; Fu, W. J.; Meininger, C. J.; Wu, G. Y. Regulatory role for

349

the arginine-nitric oxide pathway in metabolism of energy substrates. J. Nutr Biochem. 2006, 17,

350

571-588.

351

34.

352 353

Dworkin, J. Ser/Thr phosphorylation as a regulatory mechanism in bacteria. Curr Opin.

Microbiol. 2015, 24, 47-52. 35.

Morales, A.; Arce, N.; Cota, M.; Buenabad, L.; Avelar, E.; Htoo, J. K.; Cervantes, M.

354

Effect of dietary excess of branched-chain amino acids on performance and serum concentrations

355

of amino acids in growing pigs. J. Anim Physiol. Anim Nutr (Berl). 2016, 100, 39-45.

356

36.

Chaudhry, K. K.; Shukla, P. K.; Mir, H.; Manda, B.; Gangwar, R.; Yadav, N.;

357

McMullen, M.; Nagy, L. E.; Rao, R. Glutamine supplementation attenuates ethanol-induced

358

disruption of apical junctional complexes in colonic epithelium and ameliorates gut barrier

359

dysfunction and fatty liver in mice. J. Nutr Biochem. 2016, 27, 16-26.

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37.

Wu, L.; He, L. Q.; Cui, Z. J.; Liu, G.; Yao, K.; Wu, F.; Li, J.; Li, T. J. Effects of

361

reducing dietary protein on the expression of nutrition sensing genes (amino acid transporters) in

362

weaned piglets. J. Zhejiang Univ. Sci. B. 2015, 16, 496-502.

363 364 365

38.

Reeds, P. J.; Burrin, D. G.; Stoll, B.; Jahoor, F. Intestinal glutamate metabolism. J. Nutr.

2000, 130, 978s-982s. 39.

Jr, W.; Shpun, S.; Dantzler, W. H.; Wright, S. H. Effect of alpha-ketoglutarate on

366

organic anion transport in single rabbit renal proximal tubules. Am J. Physiol-Renal. 1998, 274,

367

F165-F174.

368

40.

Miles, E. D.; McBride, B. W.; Jia, Y.; Liao, S. F.; Boling, J. A.; Bridges, P. J.;

369

Matthews, J. C. Glutamine synthetase and alanine transaminase expression are decreased in livers

370

of aged vs. young beef cows and GS can be upregulated by 17beta-estradiol implants. J. Anim Sci.

371

2015, 93, 4500-9.

372

41.

Tardito, S.; Oudin, A.; Ahmed, S. U.; Fack, F.; Keunen, O.; Zheng, L.; Miletic, H.;

373

Sakariassen, P. O.; Weinstock, A.; Wagner, A.; Lindsay, S. L.; Hock, A. K.; Barnett, S. C.;

374

Ruppin, E.; Morkve, S. H.; Lund-Johansen, M.; Chalmers, A. J.; Bjerkvig, R.; Niclou, S. P.;

375

Gottlieb, E. Glutamine synthetase activity fuels nucleotide biosynthesis and supports growth of

376

glutamine-restricted glioblastoma. Nat Cell Biol. 2015, 17, 1556-68.

377

42.

Szeliga, M.; Cwikla, J.; Obara-Michlewska, M.; Cichocki, A.; Albrecht, J. Glutaminases

378

in slowly proliferating gastroenteropancreatic neuroendocrine neoplasms/tumors (GEP-NETs):

379

Selective overexpression of mRNA coding for the KGA isoform. Exp. Mol Pathol. 2015, 100,

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74-78.

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FIGURES CAPTIONS

390

Figure 1. Effects of alpha-ketoglutarate and glutamine on the histomorphology of small intestine

391

of weaned piglets. CON group, corn soybean meal based diet; AKG group, the basal diet

392

containing 1.0% AKG supplementation; GLN group, the basal diet containing 1.0% Gln

393

supplementation. a,b Values with different letters are significantly different (P < 0.05).

394

Figure 2. Effects of alpha-ketoglutarate and glutamine on the mRNA abundance of AA

395

transporters in jejunum (A) and in ileum (B) of weaned piglets. CON group, corn soybean meal

396

based diet; AKG group, the basal diet containing 1.0% AKG supplementation; GLN group, the

397

basal diet containing 1.0% Gln supplementation. a,b,c Values with different letters are significantly

398

different (P < 0.05).

399

Figure 3. Effects of alpha-ketoglutarate and glutamine catabolism and its metabolites in intestinal

400

porcine epithelial cells-1. a,b Values with different letters are significantly different (P < 0.05). n=7.

401

A: The net utilization of glutamine (Gln) in intestinal porcine epithelial cells-1 (IPEC-1) cultured

402

for 3 h in the presence of 2 mM of Gln plus 0.5, 2, and 3 mM of alpha-ketoglutarate (AKG).

403

Negative data of glutamine denote the net utilization of Gln. B: The production concentration of

404

glutamate (Glu) in IPEC-1 cultured for 3 h in the presence of 2 mM of Gln plus 0.5, 2, and 3 mM

405

of AKG. C: The production concentration of alanine (Ala) in IPEC-1 cultured for 3 h in the

406

presence of 2 mM of Gln plus 0.5, 2, and 3 mM of AKG. D: The production concentration of

407

aspartate (Asp) in IPEC-1 cultured for 3 h in the presence of 2 mM of Gln plus 0.5, 2, and 3 mM

408

of AKG. E: The production concentration of ammonia in IPEC-1 cultured for 3 h in the presence

409

of 2 mM of Gln plus 0.5, 2, and 3 mM of AKG.

410

411

412

413

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Table 1. Feedstuff ingredients and nutrient composition of basal diet Item

Concentration (%)

Feed Ingredient Corn

57.34

Soybean meal

25.62

Rice bran

2.00

Fish meal

5.00

Dried whey

5.00

Soy oil

0.88

CaHPO4

0.88

Vitamin-mineral premix 1

1.00

Limestone

0.80

ZnO Acidifier

0.30 0.30

NaCl

0.30

L-Lysine·HCl

0.17

Choline chloride

0.20

Mould

0.10

inhibitor

DL-Methionine

0.11

Total

100.00

Nutrition Composition Digestible energy (MJ/kg)

14.22

Crude protein

20.00

Lys Met

1.30 0.30

Met+Cys

0.65

Thr

1.05

Trp

0.25

Ca

0.80

P

0.69

Available phosphorus

0.45

NaCl

0.46

415

1

416

40 IU; vitamin K3, 4 mg; vitamin B1, 6 mg; vitamin B2, 12 mg; vitamin B6, 6 mg; vitamin B12,

Supplied per kilogram of finished feed: vitamin A, 10,800 IU; vitamin D3, 4,000 IU; vitamin E,

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0.05 mg; biotin, 0.2 mg; folic acid, 2 mg; niacin, 50 mg; D-Calcium pantothenate, 25 mg; Fe, 100

418

mg as ferrous sulfate; Cu, 150 mg as copper sulphate; Mn, 40 mg as manganese oxide; Zn, 100

419

mg as zinc oxide; I, 0.5 mg as potassium iodide; and Se, 0.3 mg as sodium selenite.

420 421 422 423 424 425 426 427 428 429 430 431 432 433 434 435 436 437 438 439 440 441 442 443 444

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Table 2. Primers used for quantitative reverse transcription-PCR

Gene

Accession No.

Primers F:5′-AAGGAGTAAGAGCCCCTGGA-3′

GAPDH

NM_001206359 R:5′-TCTGGGATGGAAACTGGAA-3′ F:5′-GGCACCGCACTCTACGAAGCA-3′

EAAC1

NM_001164649 R:5′-GCCCACGGCACTTAGCACGA-3′ F:5′-GATTGTGGAGATGGAGGATGTGG-3′

ASCT2

XM_003355984 R:5′-TGCGAGTGAAGAGGAAGTAGATGA-3′ F:5′-GAACCCAAGACCACAAATC-3′

b0

+

NM_001110171 R: 5′-CCCAGTGTCGCAAGAAT-3′ F:5′-TTTGTTATGCGGAACTGG-3′

+

y LAT1

NM_001110421 R:5′-AAAGGTGATGGCAATGAC-3′

446

447

448

449

450

451

452

453

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Table 3. Effects of alpha-ketoglutarate and glutamine on growth performance of piglets1

Diet Treatment2 Item

P-value CON group

AKG group

GLN group

Initial body weight , kg

5.92±0.25

5.96±0.20

6.20±0.21

0.642

Final body weight, kg

13.87±1.16

15.65±1.33

14.24±0.72

0.467

Average daily feed intake, g/d

529.34±32.5

556.42±61.4

594.51±43.3

0.372

Average daily gain, g/d

333.02±44.2b

405.21±25.3 a

380.03±17.3a

0.013

Gain: feed, g/g

0.60±0.00b

0.77±0.02a

0.64±0.05a

0.041

455

1

456

differ (P < 0.05).

457

2

458

containing 1.0% AKG supplementation; GLN group, the basal diet containing 1.0% Gln

459

supplementation.

Values are LSMean ± SEM, n=7.

a,b

Values in the same row with different superscript letters

Dietary treatments: CON group, corn soybean meal based diet; AKG group, the basal diet

460

461

462

463

464

465

466

467

468

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Table 4. Effects of alpha-ketoglutarate and glutamine on serum concentrations of amino acids in

470

piglets (µg /mL)1

Diet Treatment2

Item

P-value

CON group

AKG group

GLN group

Ile

25.45±1.89a

20.45±1.98ab

18.89±1.34b

0.042

Leu

39.54±2.93

33.24±3.08

29.81±2.28

0.155

Lys

a

58.33±8.87

b

27.46±7.53

16.95±1.73

b

0.015

Met

15.85±1.55

15.57±0.75

15.04±0.42

0.711

Phe

a

22.48±1.88

b

15.77±1.51

13.88±0.92

b

0.013

Thr

58.81±12.6b

90.44±3.29a

60.31±3.65b

0.022

Val

a

47.59±2.68

b

Ala Arg

47.75±8.78

Asp

20.8±0.53a

35.48±4.45

b

31.8±1.73

0.041

77.29±6.81

79.63±10.25

54.53±2.66

0.057

a

ab

35.52±5.27

27.08±1.10

b

0.024

18.46±0.74ab

15.77±0.80b

0.009

Glu

124.7±10.4

a

b

89.98±7.29

84.35±6.52

b

0.018

Gly

79.88±4.84

94.71±9.02

84.97±3.30

0.650

His

14.72±1.99

13.31±1.23

10.21±0.73

0.337

Pro

38.45±4.41

36.22±3.28

28.49±1.38

0.510

Ser

20.56±2.15

20.03±1.73

18.33±1.25

0.865

Tyr

23.66±2.6a

18.04±1.48b

15.72±0.88b

0.046

471

1

472

differ (P < 0.05).

473

2

474

containing 1.0% AKG supplementation; GLN group, the basal diet containing 1.0% Gln

475

supplementation.

Values are LSMean ± SEM, n=7.

a,b

Values in the same row with different superscript letters

Dietary treatments: CON group, corn soybean meal based diet; AKG group, the basal diet

476

477

478

479

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Figure 1. Effects of alpha-ketoglutarate and glutamine on the histomorphology of small intestine of weaned piglets. 48x34mm (300 x 300 DPI)

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Figure 2. Effects of alpha-ketoglutarate and glutamine on the mRNA abundance of AA transporters in jejunum (A) and in ileum (B) of weaned piglets. 99x50mm (300 x 300 DPI)

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Figure 3. Effects of alpha-ketoglutarate and glutamine catabolism and its metabolites in intestinal porcine epithelial cells-1 . 44x39mm (300 x 300 DPI)

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Figure 3. Effects of alpha-ketoglutarate and glutamine catabolism and its metabolites in intestinal porcine epithelial cells-1 87x43mm (300 x 300 DPI)

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Figure 3. Effects of alpha-ketoglutarate and glutamine catabolism and its metabolites in intestinal porcine epithelial cells-1 87x43mm (300 x 300 DPI)

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TOC 77x63mm (300 x 300 DPI)

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