, Volume 11, Issue 3, pp 545–558 | Cite as

Explorative study of metabolic adaptations to various dietary calcium intakes and cereal sources on serum metabolome and hepatic gene expression in juvenile pigs

  • Barbara U. Metzler-Zebeli
  • Reinhard Ertl
  • Dieter Klein
  • Qendrim Zebeli
Original Article


Daily calcium (Ca) intake is critical for bone health and energy metabolism, but less is known about its metabolic role at young ages. We used targeted electrospray ionization liquid chromatography–mass spectrometry-based metabolomics and candidate gene expression to compare the effect of dietary Ca level (high vs. adequate intake), as influenced by dietary cereal composition (wheat–barley vs. corn grains), on postprandial serum metabolome profiles and hepatic expression of genes related to glucose, lipid and amino acid metabolism in juvenile pigs (n = 32; 7-weeks of age). Explorative analysis of 132 metabolites demonstrated that postprandial serum of pigs fed high Ca contained greater (p < 0.05) concentrations of hexoses, 13 amino acids, 3 polyamines and 33 phosphatidylcholines compared to pigs fed adequate Ca. Partial-least-squares-discriminant analysis and cereal–Ca interactions indicated that the dietary cereal source may modulate the metabolic Ca response to a meal. Hepatic responses showed trends for upregulation of PPARG and SRBEF1 expression with high versus adequate Ca diets. Present data are consistent with the concept that dietary Ca modifies systemic metabolic processes in young animals. Targeted metabolomics provided greater insight into the complex metabolic responses related to dietary Ca than previously reported.


High-calcium diet Dietary cereals Serum metabolome Hepatic gene expression Juvenile pigs 



This research was supported by the Profile Lines of the University of Veterinary Medicine Vienna (Start-up project “PL Effects of Ca in weaned pigs” to B.U.M.Z.). We thank A. Dockner, M. Wild, R. Rick (Institute of Animal Nutrition and Functional Plant Compounds), E. Mann (Institute of Milk Hygiene, University of Veterinary Medicine Vienna), M. Adib Razavi (VetCore Facility for Research) and the staff of the Clinic for Swine for assistance with sampling and analyses. Our thanks are extended to E. Schneeberger (GARANT Tiernahrung, Pöchlarn, Austria) for diet preparation.

Conflict of interest

All authors declare no conflict of interest.

Supplementary material

11306_2014_714_MOESM1_ESM.pdf (72 kb)
Supplementary material 1 (PDF 71 kb)


  1. Bartlett, P. J., Gaspers, L. D., Pierobon, N., & Thomas, A. P. (2014). Calcium-dependent regulation of glucose homeostasis in the liver. Cell Calcium. doi: 10.1016/j.ceca.2014.02.007.PubMedGoogle Scholar
  2. Brundige, D. R., Maga, E. A., Klasing, K. C., & Murray, J. D. (2010). Consumption of pasteurized human lysozyme transgenic goats’ milk alters serum metabolite profile in young pigs. Transgenic Research, 19, 563–574.CrossRefPubMedCentralPubMedGoogle Scholar
  3. Che, T. M., Perez, V. G., Song, M., & Pettigrew, J. E. (2012). Effect of rice and other cereal grains on growth performance, pig removal, and antibiotic treatment of weaned pigs under commercial conditions. Journal of Animal Science, 90, 4916–4924.CrossRefPubMedGoogle Scholar
  4. Chow, J., Panasevich, M. R., Alexander, D., Vester Boler B. M., Rossoni Serao, M. C., Faber, T. A., et al. (2014). Fecal metabolomics of healthy breast-fed versus formula-fed infants before and during in vitro batch culture fermentation. Journal of Proteome Research (in press). doi: 10.1021/pr500011w.
  5. Claessens, M., Saris, W. H., & van Baak, M. A. (2008). Glucagon an dinsulin responses after ingestion of different amounts of intact and hydrolysed proteins. British Journal of Nutrition, 11, 61–69.Google Scholar
  6. Crenshaw, T. D. (1995). Calcium, phosphorus, vitamin D, and vitamin K in swine nutrition. In A. J. Lewis & L. L. Southern (Eds.), Swine nutrition (2nd ed., pp. 187–212). Boca Raton, FL: CRC Press.Google Scholar
  7. Eller, L. K., & Reimer, R. A. (2010). A high calcium, skim milk powder diet results in a lower fat mass in male, energy-restricted, obese rats more than a low calcium, casein, or soy protein diet. Journal of Nutrition, 140, 1234–1241.CrossRefPubMedGoogle Scholar
  8. Ermer, P. M., Miller, P. S., & Lewis, A. J. (1994). Diet preference and meal patterns of weanling pigs offered diets containing either spray-dried porcine plasma or dried skim milk. Journal of Animal Science, 72, 1548–1554.PubMedGoogle Scholar
  9. Fan, Y., Guo, Y., Hamblin, M., Chang, L., Zhang, J., & Chen, Y. E. (2011). Inhibition of gluconeogenic genes by calcium-regulated heat-stable protein 1 via repression of peroxisome proliferator-activated receptor α. Journal of Biological Chemistry, 286, 40584–40595.CrossRefPubMedCentralPubMedGoogle Scholar
  10. Fehlmann, M., & Freychet, P. (1981). Insulin and glucagon stimulation of (Na+–K+)-ATPase transport activity in isolated rat hepatocytes. Journal of Biological Chemistry, 266, 7449–7458.Google Scholar
  11. Fungwe, T. V., Fox, J. E., Cagen, L. M., Wilcox, H. G., & Heimberg, M. (1994). Stimulation of fatty acid biosynthesis by dietary cholesterol and of cholesterol synthesis by dietary fatty acid. Journal of Lipid Research, 35, 311–318.PubMedGoogle Scholar
  12. GfE [Gesellschaft für Ernährungsphysiologie]. (2006). Empfehlungen zur Energie- und Nährstoffversorgung von Schweinen. Frankfurt am Main: DLG-Verlag.Google Scholar
  13. Giacco, R., Costabile, G., Della Pepa, G., Anniballi, G., Griffo, E., Mangione, A., et al. (2014). A whole-grain cereal-based diet lowers postprandial plasma insulin and triglyceride levels in individuals with metabolic syndrome. Nutrition, Metabolism, Cardiovascular Diseases (in press). doi: 10.1016/j.numecd.2014.01.007.
  14. Gloaguen, M., Le Floc’h, N., Primot, E. C., Primot, Y., Val-Laillet, D., Meunier-Salaün, M. C., et al. (2003). Meal patterns in relation to the supply of branched-chain amino acids in pigs. Journal of Animal Science, 91, 292–297.CrossRefGoogle Scholar
  15. Gootenberg, D. B., & Turnbaugh, P. J. (2011). Companion animals symposium: Humanized animal models of the microbiome. Journal of Animal Science, 89, 1521–1537.CrossRefGoogle Scholar
  16. Guilloteau, P., Zabielski, R., Hammon, H. M., & Metges, C. C. (2010). Nutritional programming of gastrointestinal tract development. Is the pig a good model for man? Nutrition Research Reviews, 23, 4–22.CrossRefPubMedGoogle Scholar
  17. He, Y.-H., Li, S.-T., Wang, Y.-Y., Wang, G., He, Y., Liao, X.-L., et al. (2012). Postweaning low-calcium diet promotes later-life obesity induced by a high-fat diet. Journal of Nutritional Biochemistry, 23, 1238–1244.CrossRefPubMedGoogle Scholar
  18. Hooda, S., Matte, J. J., Vasanthan, T., & Zijlstra, R. T. (2010). Dietary oat beta-glucan reduces peak net glucose flux and insulin production and modulates plasma incretin in portal-vein catheterized grower pigs. Journal of Nutrition, 140, 1564–1569.CrossRefPubMedGoogle Scholar
  19. Knudsen, E. K. B. (2011). Triennial growth symposium: Effects of polymeric carbohydrates on growth and development in pigs. Journal of Animal Science, 89, 1965–1980.CrossRefGoogle Scholar
  20. Lechin, F., Dijs, B., & Pardey-Maldonado, B. (2013). Insulin versus glucagon crosstalk: Central plus peripheral mechanisms. American Journal of Therapeutics, 20, 349–362.CrossRefPubMedGoogle Scholar
  21. Lillefosse, H. H., Clausen, M. R., Yde, C. C., Ditlev, D. B., Zhang, X., Du, Z. Y., et al. (2014). Urinary loss of tricarboxylic acid cycle intermediates as revealed by metabolomics studies—an underlying mechanism to reduce lipid accretion by whey protein ingestion? Journal of Proteome Research (in press). doi: 10.1021/pr500039t.
  22. Livak, K. J., & Schmittgen, T. D. (2001). Analysis of relative gene expression data using real-time quantitative PCR and the 2−ddCT method. Methods, 25, 402–408.CrossRefPubMedGoogle Scholar
  23. Lopes Nobre, J., Lisboa, P. C., da Silva Lima, N., Franco, J. G., Firmino Noqueira Neto, J., de Moura, E. G., et al. (2012). Calcium supplementation prevents obesity, hyperleptinaemia and hyperglycaemia in adult rats programmed by early weaning. British Journal Nutrition, 107, 979–988.CrossRefGoogle Scholar
  24. Lynch, C. J., McCall, K. M., Ng, Y. C., & Hazen, S. A. (1996). Glucagon stimulation of hepatic Na(+)-pump activity and alpha-subunit phosphorylation in rat hepatocytes. Biochemistry Journal, 313, 983–989.Google Scholar
  25. Marotte, C., Bryk, G., Gonzales Chaves, M. M. S., Lifshitz, F., Martin de Portela, M. L. P., & Zeni, S. N. (2014). Low dietary calcium and obesity: A comparative study in genetically obese and normal rats during early growth. European Journal of Nutrition, 53, 769–778.CrossRefPubMedGoogle Scholar
  26. Marotte, C., Weisstaub, A., Bryk, G., Olguin, M. C., Posadas, M., Lucero, D., et al. (2013). Effect of dietary calcium (Ca) on body composition and Ca metabolism during growth in genetically obese (β) male rats. European Journal of Nutrition, 52, 297–305.CrossRefPubMedGoogle Scholar
  27. Metzler-Zebeli, B. U., Hooda, S., Mosenthin, R., Gänzle, M. G., & Zijlstra, R. T. (2010). Bacterial fermentation affects net mineral flux in the large intestine of pigs fed diets with viscous and fermentable non-starch polysaccharides. Journal of Animal Science, 88, 3351–3362.CrossRefPubMedGoogle Scholar
  28. Metzler-Zebeli, B. U., Schmitz-Esser, S., Klevenhusen, F., Podstatzky-Lichtenstein, L., Wagner, M., & Zebeli, Q. (2013a). Grain-rich diets differently alter ruminal and colonic abundance of microbial populations and lipopolysaccharide in goats. Anaerobe, 20, 65–73.CrossRefPubMedGoogle Scholar
  29. Metzler-Zebeli, B. U., Mann, E., Schmitz-Esser, S., Wagner, M., Ritzmann, M., & Zebeli, Q. (2013b). Gastrointestinal microbiota and metabolites respond differently to high dietary calcium–phosphorus level in weaned pigs fed various cereal sources. Applied and Environmental Microbiology, 79, 7264–7272.CrossRefPubMedCentralPubMedGoogle Scholar
  30. Michal, G. (1999). Biochemical pathways: An atlas of biochemistry and molecular biology. Heidelberg: Spektrum Akademischer Verlag.Google Scholar
  31. Murgas Torrazza, R., Suryawan, A., Gazzaneo, M. C., Orellana, R. A., Frank, J. W., Nguyen, H. V., et al. (2010). Leucine supplementation of a low-protein meal increases skeletal muscle and visceral tissue protein synthesis in neonatal pigs by stimulating mTOR-dependent translation initiation. Journal of Nutrition, 140, 2145–2152.CrossRefPubMedCentralPubMedGoogle Scholar
  32. Nebendahl, C., Krüger, R., Görs, S., Albrecht, E., Martens, K., Hennig, S., et al. (2013). Effects on transcriptional regulation and lipid droplet characteristics in the liver of female juvenile pigs after early postnatal feed restriction and refeeding are dependent on birth weight. PLoS ONE, 8, e76705.CrossRefPubMedCentralPubMedGoogle Scholar
  33. Nørskov, N. P., Hedemann, M. S., Lærke, H. N., & Bach Knudsen, K. E. (2013). Multicompartmental nontargeted LC-MS metabolomics: Explorative study on the metabolic responses of rye fiber versus refined wheat fiber intake in plasma and urine of hypercholesterolemic pigs. Journal of Proteome Research, 12, 2818–2832.CrossRefPubMedGoogle Scholar
  34. NRC (Nutrient Research Council). (1998). Nutrient requirements of swine (10th ed.). Washington, DC: National Academic Press.Google Scholar
  35. O’Connor, P. M., Kimball, S. R., Suryawan, A., Bush, J. A., Nguyen, H. V., Jefferson, L. S., et al. (2003). Regulation of translation initiation by insulin and amino acids in skeletal muscle of neonatal pigs. American Journal of Physiology, Endocrinology and Metabolism, 285, E40–E53.CrossRefGoogle Scholar
  36. Paik, W. K., & Kim, S. (1993). N(G)-methylarginines: Biosynthesis, biochemical function and metabolism. Amino Acids, 4, 267–286.PubMedGoogle Scholar
  37. Perez-Bonilla, A., Frikha, M., Mirzaie, S., Garcia, J., & Mateos, G. G. (2011). Effects of the main cereal and type of fat of the diet on productive performance and egg quality of brown-egg laying hens from 22 to 54 weeks of age. Poultry Science, 90, 2801–2810.CrossRefPubMedGoogle Scholar
  38. Saleem, F., Ametaj, B. N., Bouatra, S., Mandal, R., Zebeli, Q., Dunn, S. M., et al. (2012). A metabolomics approach to uncover the effects of grain diets on rumen health in dairy cows. Journal of Dairy Science, 95, 6606–6623.CrossRefPubMedGoogle Scholar
  39. Schroeder, A., Mueller, O., Stocker, S., Salowsky, R., Leiber, M., Grassmann, M., et al. (2006). The RIN: An RNA integrity number for assigning integrity values to RNA measurements. BMC Molecular Biology, 7, 3.CrossRefPubMedCentralPubMedGoogle Scholar
  40. Shimano, H. (2002). Sterol regulatory element-binding protein family as global regulators of lipid synthetic genes in energy metabolism. Vitamins and Hormones, 65, 167–194.CrossRefPubMedGoogle Scholar
  41. Soares, M. J., Murhadi, L. L., Kurpad, A. V., Chan She Ping-Delfos, W. L., & Piers, L. S. (2012). Mechanistic roles for calcium and vitamin D in the regulation of body weight. Obesity Reviews, 13, 592–605.CrossRefPubMedGoogle Scholar
  42. Sul, H. S., & Wang, D. (1998). Nutritional and hormonal regulation of enzymes in fat synthesis: Studies of fatty acid synthase and mitochondrial glycerol-3-phosphate acyltransferase gene transcription. Annual Review of Nutrition, 18, 331–351.CrossRefPubMedGoogle Scholar
  43. Suryawan, A., Torrazza, R. M., Gazzaneo, M. C., Orellana, R. A., Fiorotto, M. L., El-Kadi, S. W., et al. (2012). Enteral leucine supplementation increases protein synthesis in skeletal and cardiac muscles and visceral tissues of neonatal pigs through mTORC1-dependent pathways. Pediatric Research, 71, 324–331.CrossRefPubMedCentralPubMedGoogle Scholar
  44. Symonds, M. E., & Gardner, D. S. (2006). Experimental evidence for early nutritional programming of later health in animals. Current Opinions in Clinical Nutrition and Metabolic Care, 9, 278–283.CrossRefGoogle Scholar
  45. Thomas, A. P., Dunn, T. N., Drayton, J. B., Oort, P. J., & Adams, S. H. (2013). A dairy-based high calcium diet improves glucose homeostasis and reduces steatosis in the context of pre-existing obesity. Obesity, 21, E229–E235.CrossRefPubMedGoogle Scholar
  46. Torres-Fuentes, C., Schellekens, H., Dinan, T. G., & Cryan, J. F. (2014). A natural solution for obesity: Bioactives for the prevention and treatment of weight gain. A review. Nutritional Neuroscience. doi: 10.1179/1476830513Y.0000000099.
  47. Vandesompele, J., De Preter, K., Pattyn, F., Poppe, B., van Roy, N., de Paepe, A., et al. (2002). Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes. Genome Biology, 3, RESEARCH0034.Google Scholar
  48. Vaz, F. M., & Wanders, R. J. (2002). Carnitine biosynthesis in mammals. Biochemistry Journal, 361, 417–429.CrossRefGoogle Scholar
  49. VDLUFA. (1976). Methodenbuch. Buch III: Die chemische Untersuchung von Futtermitteln (3rd ed.). Darmstadt: VDLUFA-Verlag.Google Scholar
  50. Wilamil, J., Badiola, I., Devillard, E., Geraert, P. A., & Torrallardona, D. (2012). Wheat–barley–rye or corn-fed growing pigs respond differently to dietary supplementation with a carbohydrase complex. Journal of Animal Science, 90, 824–832.CrossRefGoogle Scholar
  51. Wilson, G. J., Layman, D. K., Moulton, C. J., Norton, L. E., Anthony, T. G., Proud, C. G., et al. (2011). Leucine or carbohydrate supplementation reduces AMPK and eEF2 phosphorylation and extends postprandial muscle protein synthesis in rats. American Journal of Physiology, Endocrinology and Metabolism, 301, E1236–E1242.CrossRefGoogle Scholar
  52. Wu, G. (2009). Amino acids: Metabolism, functions, and nutrition. Amino Acids, 37, 1–17.CrossRefPubMedGoogle Scholar
  53. Xia, J., Mandal, R., Sinelnikov, I., Broadhurst, D., & Wishart, D. S. (2012). MetaboAnalyst 2.0—a comprehensive server for metabolomic data analysis. Nucleic Acids Research, 40, 1–7.CrossRefGoogle Scholar
  54. Zemel, M. B. (2003). Mechanisms of dairy modulation of adiposity. Journal of Nutrition, 133, 252S–256S.PubMedGoogle Scholar
  55. Zemel, M. B. (2004). Role of calcium and dairy products in energy partitioning and weight management. American Journal of Clinical Nutrition, 79, 907S–912S.PubMedGoogle Scholar
  56. Zemel, M. B., & Sun, X. (2008). Calcitriol and energy metabolism. Nutrition Reviews, 66, S139–S146.CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2014

Authors and Affiliations

  • Barbara U. Metzler-Zebeli
    • 1
  • Reinhard Ertl
    • 2
  • Dieter Klein
    • 2
  • Qendrim Zebeli
    • 3
  1. 1.University Clinic for Swine, Department for Farm Animals and Veterinary Public HealthUniversity of Veterinary Medicine ViennaViennaAustria
  2. 2.VetCore Facility for ResearchUniversity of Veterinary Medicine ViennaViennaAustria
  3. 3.Department for Farm Animals and Veterinary Public Health, Institute of Animal Nutrition and Functional Plant CompoundsUniversity of Veterinary Medicine ViennaViennaAustria

Personalised recommendations