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Proteomic analysis reveals altered expression of proteins related to glutathione metabolism and apoptosis in the small intestine of zinc oxide-supplemented piglets

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Abstract

Zinc is an important dietary factor that regulates intestinal amino acid and protein metabolism in animals. Recent work with the piglet, an established animal model for studying human infant nutrition, has shown that supplementing high levels of zinc oxide (ZnO) to the diet ameliorates weaning-associated intestinal injury and growth retardation. However, the underlying mechanisms are largely unknown. This study tested the hypothesis that zinc supplementation affects expression of proteins related to glutathione metabolism and oxidative stress in the gut. Using two-dimensional gel electrophoresis and mass spectrometry, we identified 22 up-regulated and 19 down-regulated protein spots in the jejunum of weanling piglets supplemented with ZnO (3,000 mg/kg Zn) compared with the control pigs (100 mg/kg Zn). These proteins are related to energy metabolism (increased level for succinyl-CoA transferase and decreased level for creatine kinase M-type); oxidative stress (decreased levels for 78 kDa glucose-regulated protein and glutathione-S-transferase-ω); and cell proliferation and apoptosis (increased levels for A-Raf-1 and calregulin). Consistent with the changes in protein expression, the ratio of reduced glutathione to oxidized glutathione was increased, whereas glutathione-S-transferase and glutathione peroxidase activities as well as the protein level of active caspase-3 were reduced in ZnO-supplemented piglets. Collectively, these results indicate that ZnO supplementation improves the redox state and prevents apoptosis in the jejunum of weaning piglets, thereby alleviating weaning-associated intestinal dysfunction and malabsorption of nutrients (including amino acids).

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Abbreviations

ER:

Endoplasmic reticulum

ERK:

Extracellular signal-regulated kinase

GRP78:

78 kDa Glucose-regulated protein

GSH:

Reduced glutathione

GSSG:

Oxidized glutathione

GST:

Glutathione-S-transferase

GSH-PX:

Glutathione peroxidase

IEF:

Isoelectric focusing

MEK:

Mitogen-activated protein kinase kinase

SDS-PAGE:

Sodium dodecyl sulfate-polyacrylamide electrophoresis

References

  • Aw TY (2005) Intestinal glutathione: determinant of mucosal peroxide transport, metabolism, and oxidative susceptibility. Toxicol Appl Pharmacol 204:320–328

    Article  PubMed  CAS  Google Scholar 

  • Baek HY, Lim JW, Kim H et al (2004) Oxidative-stress-related proteome changes in Helicobacter pylori-infected human gastric mucosa. Biochem J 379:291–299

    Article  PubMed  CAS  Google Scholar 

  • Beeram M, Patnaik A, Rowinsky EK (2005) Raf: a strategic target for therapeutic development against cancer. J Clin Oncol 23:6771–6790

    Article  PubMed  CAS  Google Scholar 

  • Bhandari N, Mazumder S, Taneja S et al (2008) Effectiveness of zinc supplementation plus oral rehydration salts compared with oral rehydration salts alone as a treatment for acute diarrhea in a primary care setting: a cluster randomized trial. Pediatrics 121:e1279–e1285

    Article  PubMed  Google Scholar 

  • Bhutta ZA, Bird SM, Black RE et al (2000) Therapeutic effects of oral zinc in acute and persistent diarrhea in children in developing countries: pooled analysis of randomized controlled trials. Am J Clin Nutr 72:1516–1522

    PubMed  CAS  Google Scholar 

  • Capone G, De Marinis A, Simone S et al (2008) Mapping the human proteome for non-redundant peptide islands. Amino Acids 35:209–216

    Article  PubMed  CAS  Google Scholar 

  • Carlson MS, Hill GM, Link JE (1999) Early- and traditionally weaned nursery pigs benefit from phase-feeding pharmacological concentrations of zinc oxide: effect on metallothionein and mineral concentrations. J Anim Sci 77:1199–1207

    PubMed  CAS  Google Scholar 

  • Chandra J, Samali A, Orrenius S (2000) Triggering and modulation of apoptosis by oxidative stress. Free Radic Biol Med 29:323–333

    Article  PubMed  CAS  Google Scholar 

  • Duee PH, Pegorier JP, Quant PA et al (1994) Hepatic ketogenesis in newborn pigs is limited by low mitochondrial 3-hydroxy-3-methylglutaryl-CoA synthase activity. Biochem J 298:207–212

    PubMed  CAS  Google Scholar 

  • Fang YZ, Yang S, Wu G (2002) Free radicals, antioxidants, and nutrition. Nutrition 18:872–879

    Article  PubMed  CAS  Google Scholar 

  • Formigari A, Santon A, Irato P (2007) Efficacy of zinc treatment against iron-induced toxicity in rat hepatoma cell line H4-II-E-C3. Liver Int 27:120–127

    Article  PubMed  CAS  Google Scholar 

  • Galli F (2007) Amino acid and protein modification by oxygen and nitrogen species. Amino Acids 32:497–499

    Article  CAS  Google Scholar 

  • Gerwins P, Blank JL, Johnson GL (1997) Cloning of a novel mitogen-activated protein kinase kinase kinase, MEKK4, that selectively regulates the c-Jun amino terminal kinase pathway. J Biol Chem 272:8288–8295

    Article  PubMed  CAS  Google Scholar 

  • Görg A, Obermaier C, Boguth G et al (2000) The current state of two-dimensional electrophoresis with immobilized pH gradients. Electrophoresis 21:1037–1053

    Article  PubMed  Google Scholar 

  • Gualano B, Novaes RB, Artioli GG et al (2008) Effects of creatine supplementation on glucose tolerance and insulin sensitivity in sedentary healthy males undergoing aerobic training. Amino Acids 34:245–250

    Article  PubMed  CAS  Google Scholar 

  • Hahn JD, Baker DH (1993) Growth and plasma zinc responses of young pigs fed pharmacologic levels of zinc. J Anim Sci 71:3020–3024

    PubMed  CAS  Google Scholar 

  • Higashida M, Xu S, Kojima-Yuasa A et al (2009) 1′-Acetoxychavicol acetate-induced cytotoxicity is accompanied by a rapid and drastic modulation of glutamine metabolism. Amino Acids 36:107–113

    Article  PubMed  CAS  Google Scholar 

  • Hoque KM, Binder HJ (2006) Zinc in the treatment of acute diarrhea: current status and assessment. Gastroenterology 130:2201–2205

    Article  PubMed  CAS  Google Scholar 

  • Hu CA, Khalil S, Zhaorigetu S et al (2008) Human ∆1-pyrroline-5-carboxylate synthase: function and regulation. Amino Acids 35:665–672

    Article  PubMed  CAS  Google Scholar 

  • Jobgen WS, Fried SK, Fu WJ et al (2006) Regulatory role for the arginine-nitric oxide pathway in metabolism of energy substrates. J Nutr Biochem 17:571–588

    Article  PubMed  CAS  Google Scholar 

  • Kaufman RJ (1999) Stress signaling from the lumen of the endoplasmic reticulum: coordination of gene transcriptional and translational controls. Genes Dev 13:1211–1233

    Article  PubMed  CAS  Google Scholar 

  • Kim SW, Wu G (2008) Regulatory role for amino acids in mammary gland growth and milk synthesis. Amino Acids. doi:10.1007/s00726-008-0151-5

  • Kong XF, Wu GY, Liao YP et al (2007) Effects of Chinese herbal ultra-fine powder as a dietary additive on growth performance, serum metabolites and intestinal health in early-weaned piglets. Livest Sci 108:272–275

    Article  Google Scholar 

  • Lee AS (2001) The glucose-regulated proteins: stress induction and clinical applications. Trends Biochem Sci 26:504–510

    Article  PubMed  CAS  Google Scholar 

  • Lee AS (2005) The ER chaperone and signaling regulator GRP78/BiP as a monitor of endoplasmic reticulum stress. Methods 35:373–381

    Article  PubMed  CAS  Google Scholar 

  • Li X, Yin J, Li D et al (2006) Dietary supplementation with zinc oxide increases Igf-I and Igf-I receptor gene expression in the small intestine of weanling piglets. J Nutr 136:1786–1791

    PubMed  CAS  Google Scholar 

  • Liao XH, Majithia A, Huang XL, Kimmel AR (2008) Growth control via TOR kinase signaling, an intracellular sensor of amino acids and energy availability, with crosstalk potential to proline metabolism. Amino Acids 35:761–770

    Article  PubMed  CAS  Google Scholar 

  • Liu ZP, Wu LY, Wang Y et al (2008) Bridging protein local structures and protein functions. Amino Acids 35:627–650

    Article  PubMed  CAS  Google Scholar 

  • Manna P, Sinha M, Sil PC (2008) Taurine plays a beneficial role against cadmium-induced oxidative renal dysfunction. Amino Acids. doi: 10.1007/s00726-008-0094-x

  • Mannick JB (2007) Regulation of apoptosis by protein S-nitrosylation. Amino Acids 32:523–526

    Article  PubMed  CAS  Google Scholar 

  • Meier R, Burri E, Steuerwald M (2003) The role of nutrition in diarrhoea syndromes. Curr Opin Clin Nutr Metab Care 6:563–567

    Article  PubMed  Google Scholar 

  • Michalak M, Corbett EF, Mesaeli N et al (1999) Calreticulin: one protein, one gene, many functions. Biochem J 344:281–292

    Article  PubMed  CAS  Google Scholar 

  • Opas M, Dziak E, Fliegel L, Michalak M (1991) Regulation of expression and intracellular distribution of calreticulin, a major calcium binding protein of nonmuscle cells. J Cell Physiol 149:160–171

    Article  PubMed  CAS  Google Scholar 

  • Ou D, Li D, Cao Y et al (2007) Dietary supplementation with zinc oxide decreases expression of the stem cell factor in the small intestine of weanling pigs. J Nutr Biochem 18:820–826

    Article  PubMed  CAS  Google Scholar 

  • Phang JM, Donald SP, Pandhare J, Liu YM (2008) The metabolism of proline, a stress substrate, modulates carcinogenic pathways. Amino Acids 35:681–690

    Article  PubMed  CAS  Google Scholar 

  • Ptolemy AS, Lee R, Britz-McKibbin P (2007) Strategies for comprehensive analysis of amino acid biomarkers of oxidative stress. Amino Acids 33:3–18

    Article  PubMed  CAS  Google Scholar 

  • RCN (1998) Nutrients requirements of swine. National Academy Press, Washington DC

  • Rhoads JM, Wu G (2008) Glutamine, arginine, and leucine signaling in the intestine. Amino Acids. doi: 10.1007/s00726-008-0225-4

  • Rider JE, Hacker A, Mackintosh CA et al (2007) Spermine and spermidine mediate protection against oxidative damage caused by hydrogen peroxide. Amino Acids 33:231–240

    Article  PubMed  CAS  Google Scholar 

  • Rouimi P, Anglade P, Benzekri A et al (2001) Purification and characterization of a glutathione S-transferase Omega in pig: evidence for two distinct organ-specific transcripts. Biochem J 358:257–262

    Article  PubMed  CAS  Google Scholar 

  • Sims NR (1996) Energy metabolism, oxidative stress and neuronal degeneration in Alzheimer’s disease. Neurodegeneration 5:435–440

    Article  PubMed  CAS  Google Scholar 

  • Sun FC, Wei S, Li CW et al (2006) Localization of GRP78 to mitochondria under the unfolded protein response. Biochem J 396:31–39

    Article  PubMed  CAS  Google Scholar 

  • Suryawan A, O’Connor PMJ, Bush JA et al (2008) Differential regulation of protein synthesis by amino acids and insulin in peripheral and visceral tissues of neonatal pigs. Amino Acids. doi: 10.1007/s00726-008-0149-z

  • Tom B, Tirra S (1998) 2-D electrophoresis: using immobilized pH gradients. Principles & Methods. Amersham Pharmacia Biotech, Uppsala

    Google Scholar 

  • Wang J, Ying T, Wang H et al (2005) 2-D reference map of Bacillus anthracis vaccine strain A16R proteins. Proteomics 5:4488–4495

    Article  PubMed  CAS  Google Scholar 

  • Wang J, Li D, Dangott LJ, Wu G (2006) Proteomics and its role in nutrition research. J Nutr 136:1759–1762

    PubMed  CAS  Google Scholar 

  • Wang J, Chen L, Li D et al (2008a) Intrauterine growth restriction affects the proteomes of the small intestine, liver, and skeletal muscle in newborn pigs. J Nutr 138:60–66

    PubMed  CAS  Google Scholar 

  • Wang J, Chen L, Li P et al (2008b) Gene expression is altered in piglet small intestine by weaning and dietary glutamine supplementation. J Nutr 138:1025–1032

    PubMed  CAS  Google Scholar 

  • Wang J, Wu G, Zhou H, Wang F (2008c) Emerging technologies for amino acid nutrition research in the post-genome era. Amino Acids. doi: 10.1007/s00726-008-0193-8

  • Wang W, Qiao S, Li DF (2008d) Amino acids and gut function. Amino Acids. doi: 10.1007/s00726-008-0152-4

  • Wu G (1998) Intestinal mucosal amino acid catabolism. J Nutr 128:1249–1252

    PubMed  CAS  Google Scholar 

  • Wu G, Meier SA, Knabe DA (1996) Dietary glutamine supplementation prevents jejunal atrophy in weaned pigs. J Nutr 126:2578–2584

    PubMed  CAS  Google Scholar 

  • Wu G, Fang YZ, Yang S et al (2004) Glutathione metabolism and its implications for health. J Nutr 134:489–492

    PubMed  CAS  Google Scholar 

  • Wu G, Bazer FW, Davis TA et al (2007) Important roles for the arginine family of amino acids in swine nutrition and production. Livest Sci 112:8–22

    Article  Google Scholar 

  • Wu G, Bazer FW, Datta S et al (2008a) Proline metabolism in the conceptus: implications for fetal growth and development. Amino Acids 35:691–702

    Article  PubMed  CAS  Google Scholar 

  • Wu G, Bazer FW, Davis TA et al (2008b) Arginine metabolism and nutrition in growth, health and disease. Amino Acids. doi: 10.1007/s00726-008-0210-y

  • Yan GR, He QY (2008) Functional proteomics to identify critical proteins in signal transduction pathways. Amino Acids 35:267–274

    Article  PubMed  CAS  Google Scholar 

  • Yang F, Wang J, Li X et al (2007) 2-DE and MS analysis of interactions between Lactobacillus fermentum I5007 and intestinal epithelial cells. Electrophoresis 28:4330–4339

    Article  PubMed  CAS  Google Scholar 

  • Yin J, Li X, Li D et al (2008) Dietary supplementation with zinc oxide stimulates ghrelin secretion from the stomach of young pigs. J Nutr Biochem doi:10.1016/j.jnutbio.2008.07.007

  • Zhang SW, Zhang YL, Yang HF et al (2008) Using the concept of Chou’s pseudo amino acid composition to predict protein subcellular localization: an approach by incorporating evolutionary information and von Neumann entropies. Amino Acids 34:565–572

    Article  PubMed  CAS  Google Scholar 

  • Zhao XM, Chen LN, Aihara K (2008) Protein function prediction with high-throughput data. Amino Acids 35:517–530

    Article  PubMed  CAS  Google Scholar 

  • Zhou X, Li D, Yin J et al (2007) CLA differently regulates adipogenesis in stromal vascular cells from porcine subcutaneous adipose and skeletal muscle. J Lipid Res 48:1701–1709

    Article  PubMed  CAS  Google Scholar 

  • Zhu Y, Carvey PM, Ling Z (2006) Age-related changes in glutathione and glutathione-related enzymes in rat brain. Brain Res 1090:35–44

    Article  PubMed  CAS  Google Scholar 

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Acknowledgments

The research was financially supported by the National Natural Science Foundation of China (no. 30600434, 30571343, and 30671517) and Texas AgriLife Research (H#8200).

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Authors and Affiliations

  1. State Key Laboratory of Animal Nutrition, China Agricultural University, 100193, Beijing, China

    Xiaoqiu Wang, Jingdong Yin, Guoyao Wu & Junjun Wang

  2. College of Animal Science of Guizhou University, 550025, Guiyang, Guizhou, China

    Deyuan Ou

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

    Guoyao Wu

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  1. Xiaoqiu Wang
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  2. Deyuan Ou
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  3. Jingdong Yin
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Correspondence to Junjun Wang.

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Wang, X., Ou, D., Yin, J. et al. Proteomic analysis reveals altered expression of proteins related to glutathione metabolism and apoptosis in the small intestine of zinc oxide-supplemented piglets. Amino Acids 37, 209–218 (2009). https://doi.org/10.1007/s00726-009-0242-y

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