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Reduced expression of intestinal N-acetylglutamate synthase in suckling piglets: a novel molecular mechanism for arginine as a nutritionally essential amino acid for neonates

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Abstract

The objective of this study was to determine developmental changes in mRNA and protein levels for N-acetylglutamate synthase (NAGS; a key enzyme in synthesis of citrulline and arginine from glutamine/glutamate and proline) in the small intestine of suckling piglets. The porcine NAGS gene was cloned using the real-time polymerase-chain reaction (RT-PCR) method. The porcine NAGS gene encoded 368 amino acid residues and had a high degree of sequence similarity to the “conserved domain” of human and mouse NAGS genes. The porcine NAGS gene was expressed in E. coli BL21 and a polyclonal antibody against the porcine NAGS protein was developed. Real-time RT-PCR and western-blot analyses were performed to quantify NAGS mRNA and protein, respectively, in the jejunum and ileum of 1- to 28-day-old pigs. Results indicated that intestinal NAGS mRNA levels were lower in 7- to 28-day-old than in 1-day-old pigs. Immunochemical analysis revealed that NAGS protein was localized in enterocytes of the gut. Notably, intestinal NAGS protein abundance declined progressively during the 28-day suckling period. The postnatal decrease in NAGS protein levels was consistent with the previous report of reduced NAGS enzymatic activity as well as reduced synthesis of citrulline and arginine in the small intestine of 7- to 28-day-old pigs. Collectively, these results suggest that intestinal NAGS expression is regulated primarily at the post-transcriptional level. The findings also provide a new molecular basis to explain that endogenous synthesis of arginine is impaired in sow-reared piglets and arginine is a nutritionally essential amino acid for the neonates.

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Abbreviations

Arg:

Arginine

CPSI:

Carbamylphosphate synthetase I

GAPDH:

Glyceraldehyde 3-phosphate dehydrogenase

IPTG:

Isopropyl-β-d-thiogalactopyranoside

MTS:

Mitochondrial targeting signal

NAG:

N-acetylglutamate

NAGS:

N-acetylglutamate synthase

RT-PCR:

Real-time polymerase-chain reaction

SDS-PAGE:

SDS-polyacrylamide gel electrophoresis

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Acknowledgments

This study was jointly supported by grants from National Natural Science Foundation of China (30928018), Program for the National Basic Research Program of China (30928018), the Chinese Academy of Sciences Knowledge Innovation Project (KSCX2-YW-N-051), the Chinese Academy of Sciences overseas outstanding scholar project (2005-1-4), the CAS/SAFEA International Partnership Program for Creative Research Teams, the Thousand-People-Talent program at China Agricultural University, National Research Initiative Competitive Grants from the Animal Growth & Nutrient Utilization Program (2008-35206-18764) of the USDA National Institute of Food and Agriculture, and Texas AgriLife Research (H-8200).

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Authors and Affiliations

  1. Key Laboratory for Agro-ecological Processes in Subtropical Region, Institute of Subtropical Agriculture, The Chinese Academy of Sciences, Changsha, Hunan, 410125, China

    Meimei Geng, Tiejun Li, Xiangfeng Kong, Xiaoyan Song, Wuying Chu, Ruilin Huang, Yulong Yin & Guoyao Wu

  2. The Graduate School of the Chinese Academy of Sciences, Beijing, 100049, China

    Xiaoyan Song

  3. Department of Animal Science, Texas A&M University, College Station, TX, 77843, USA

    Guoyao Wu

  4. State Key Laboratory of Animal Nutrition, China Agricultural University, Beijing, 100191, China

    Guoyao Wu

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  1. Meimei Geng
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  2. Tiejun Li
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  3. Xiangfeng Kong
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  4. Xiaoyan Song
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  5. Wuying Chu
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  6. Ruilin Huang
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  7. Yulong Yin
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  8. Guoyao Wu
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Correspondence to Yulong Yin.

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Geng, M., Li, T., Kong, X. et al. Reduced expression of intestinal N-acetylglutamate synthase in suckling piglets: a novel molecular mechanism for arginine as a nutritionally essential amino acid for neonates. Amino Acids 40, 1513–1522 (2011). https://doi.org/10.1007/s00726-010-0761-6

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