Pharmaceutical Research

, Volume 27, Issue 5, pp 832–840 | Cite as

Gene Knockout and Metabolome Analysis of Carnitine/Organic Cation Transporter OCTN1

  • Yukio Kato
  • Yoshiyuki Kubo
  • Daisuke Iwata
  • Sayaka Kato
  • Tomohisa Sudo
  • Tomoko Sugiura
  • Takashi Kagaya
  • Tomohiko Wakayama
  • Akiyoshi Hirayama
  • Masahiro Sugimoto
  • Kazushi Sugihara
  • Shuichi Kaneko
  • Tomoyoshi Soga
  • Masahide Asano
  • Masaru Tomita
  • Toshiyuki Matsui
  • Morimasa Wada
  • Akira TsujiEmail author
Research Paper



Solute carrier OCTN1 (SLC22A4) is an orphan transporter, the physiologically important substrate of which is still unidentified. The aim of the present study was to examine physiological roles of OCTN1.


We first constructed octn1 gene knockout (octn1 −/− ) mice. Metabolome analysis was then performed to identify substrates in vivo. The possible association of the substrate identified with diseased conditions was further examined.


The metabolome analysis of blood and several organs indicated complete deficiency of a naturally occurring potent antioxidant ergothioneine in octn1 −/− mice among 112 metabolites examined. Pharmacokinetic analyses after oral administration revealed the highest distribution to small intestines and extensive renal reabsorption of [3H]ergothioneine, both of which were much reduced in octn1 −/− mice. The octn1 −/− mice exhibited greater susceptibility to intestinal inflammation under the ischemia and reperfusion model. The blood ergothioneine concentration was also much reduced in Japanese patients with Crohn’s disease, compared with healthy volunteers and patients with another inflammatory bowel disease, ulcerative colitis.


These results indicate that OCTN1 plays a pivotal role for maintenance of systemic and intestinal exposure of ergothioneine, which could be important for protective effects against intestinal tissue injuries, providing a possible diagnostic tool to distinguish the inflammatory bowel diseases.


Crohn’s disease gene knockout metabolome analysis OCTN1 transporter 



Crohn’s disease


capillary electrophoresis time-of-flight mass spectrometry


Organic carnitine/organic cation transporter


ulcerative colitis



We thank Lica Ishida, Kazuhiro Suzuki and Ryutaro Matsuhashi for technical assistance in Kanazawa University. We also thank Maki Sugawara and Naoko Toki for technical assistance in Keio University. We thank Prof. Shoichi Iseki in Kanazawa University for fruitful discussion. This study was supported in part by a Grant-in-Aid for Scientific Research provided by the Ministry of Education, Science and Culture of Japan, and a grant from the Mochida Memorial Foundation (Tokyo, Japan) for Medical and Pharmaceutical Research.

Supplementary material

11095_2010_76_MOESM1_ESM.doc (308 kb)
Supplementary Table I (DOC 99 kb)
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Supplementary Table II (DOC 129 kb)
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Supplementary Table III (DOC 210 kb)
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Supplementary Figure 1 (PDF 446 kb)
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Supplementary Figure 2 (PDF 2058 kb)
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Supplementary Figure 3 (PDF 284 kb)


  1. 1.
    Tamai I, Yabuuchi H, Nezu J, Sai Y, Oku A, Shimane M, et al. Cloning and characterization of a novel human pH-dependent organic cation transporter, OCTN1. FEBS Lett. 1997;419:107–11.CrossRefPubMedGoogle Scholar
  2. 2.
    Yabuuchi H, Tamai I, Nezu J, Sakamoto K, Oku A, Shimane M, et al. Novel membrane transporter OCTN1 mediates multispecific, bidirectional, and pH-dependent transport of organic cations. J Pharmacol Exp Ther. 1999;289:768–73.PubMedGoogle Scholar
  3. 3.
    Gründemann D, Harlfinger S, Golz S, Geerts A, Lazar A, Berkels R, et al. Discovery of the ergothioneine transporter. Proc Natl Acad Sci USA. 2005;102:5256–61.CrossRefPubMedGoogle Scholar
  4. 4.
    Tamai I, Ohashi R, Nezu J, Yabuuchi H, Oku A, Shimane M, et al. Molecular and functional identification of sodium ion-dependent, high affinity human carnitine transporter OCTN2. J Biol Chem. 1998;273:20378–82.CrossRefPubMedGoogle Scholar
  5. 5.
    Wu X, Prasad PD, Leibach FH, Ganapathy V. cDNA sequence, transport function and genomic organization of human OCTN2, a new member of the organic cation transporter family. Biochem Biophys Res Commun. 1998;246:589–95.CrossRefPubMedGoogle Scholar
  6. 6.
    Nezu J, Tamai I, Oku A, Ohashi R, Yabuuchi H, Hashimoto N, et al. Primary systemic carnitine deficiency is caused by mutations in a gene encoding sodium ion-dependent carnitine transporter. Nat Genet. 1999;21:91–4.CrossRefPubMedGoogle Scholar
  7. 7.
    Tokuhiro S, Yamada R, Chang X, Suzuki A, Kochi Y, Sawada T, et al. An intronic SNP in a RUNX1 binding site of SLC22A4, encoding an organic cation transporter, is associated with rheumatoid arthritis. Nat Genet. 2003;35:341–8.CrossRefPubMedGoogle Scholar
  8. 8.
    Peltekova VD, Wintle RF, Rubin LA, Amos CI, Huang Q, Gu X, et al. Functional variants of OCTN cation transporter genes are associated with Crohn disease. Nat Genet. 2004;36:471–5.CrossRefPubMedGoogle Scholar
  9. 9.
    Yamazaki K, Takazoe M, Tanaka T, Ichimori T, Saito S, Iida A, et al. Association analysis of SLC22A4, SLC22A5 and DLG5 in Japanese patients with Crohn disease. J Hum Genet. 2004;49:664–8.CrossRefPubMedGoogle Scholar
  10. 10.
    Fisher SA, Hampe J, Onnie CM, Daly MJ, Curley C, Purcell S, et al. Direct or indirect association in a complex disease: the role of SLC22A4 and SLC22A5 functional variants in Crohn disease. Hum Mutat. 2006;27:778–85.CrossRefPubMedGoogle Scholar
  11. 11.
    Shekhawat PS, Srinivas SR, Matern D, Bennett MJ, Boriack R, George V, et al. Spontaneous development of intestinal and colonic atrophy and inflammation in the carnitine-deficient jvs (OCTN2(−/−)) mice. Mol Genet Metab. 2007;92:315–24.CrossRefPubMedGoogle Scholar
  12. 12.
    Asano M, Furukawa K, Kido M, Matsumoto S, Umesaki Y, Kochibe N, et al. Growth retardation and early death of beta-1, 4-galactosyltransferase knockout mice with augmented proliferation and abnormal differentiation of epithelial cells. EMBO J. 1997;16:1850–7.CrossRefPubMedGoogle Scholar
  13. 13.
    Soriano P, Montgomery C, Geske R, Bradley A. Targeted disruption of the c-src proto-oncogene leads to osteoporosis in mice. Cell. 1991;64:693–702.CrossRefPubMedGoogle Scholar
  14. 14.
    Yagi T, Nada S, Watanabe N, Tamemoto H, Kohmura N, Ikawa Y, et al. A novel negative selection for homologous recombination using diphtheria toxin A fragment gene. Anal Biochem. 1993;214:77–86.CrossRefPubMedGoogle Scholar
  15. 15.
    Tamai I, Ohashi R, Nezu JI, Sai Y, Kobayashi D, Oku A, et al. Molecular and functional characterization of organic cation/carnitine transporter family in mice. J Biol Chem. 2000;275:40064–72.CrossRefPubMedGoogle Scholar
  16. 16.
    Soga T, Baran R, Suematsu M, Ueno Y, Ikeda S, Sakurakawa T, et al. Differential metabolomics reveals ophthalmic acid as an oxidative stress biomarker indicating hepatic glutathione consumption. J Biol Chem. 2006;281:16768–76.CrossRefPubMedGoogle Scholar
  17. 17.
    Soga T, Ishikawa T, Igarashi S, Sugawara K, Kakazu Y, Tomita M. Analysis of nucleotides by pressure-assisted capillary electrophoresis mass spectrometry using silanol mask technique. J Chromatogr A. 2007;1159:125–33.CrossRefPubMedGoogle Scholar
  18. 18.
    Malathi P, Preiser H, Fairclough P, Mallett P, Crane RK. A rapid method for the isolation of kidney brush border membranes. Biochim Biophys Acta. 1979;554:259–63.CrossRefPubMedGoogle Scholar
  19. 19.
    Nakamura T, Yoshida K, Yabuuchi H, Maeda T, Tamai I. Functional characterization of ergothioneine transport by rat organic cation/carnitine transporter Octn1 (slc22a4). Biol Pharm Bull. 2008;31:1580–4.CrossRefPubMedGoogle Scholar
  20. 20.
    Brummel MC. In search of a physiological function for L-ergothioneine-II. Med Hypotheses. 1989;30:39–48.CrossRefPubMedGoogle Scholar
  21. 21.
    Fahey RC. Novel thiols of prokaryotes. Annu Rev Microbiol. 2001;55:333–56.CrossRefPubMedGoogle Scholar
  22. 22.
    Tamai I, Nakanishi T, Kobayashi D, China K, Kosugi Y, Nezu J, et al. Involvement of OCTN1 (SLC22A4) in pH-dependent transport of organic cations. Mol Pharm. 2004;1:57–66.CrossRefPubMedGoogle Scholar
  23. 23.
    Chaudière J, Ferrari-Iliou R. Intracellular antioxidants: from chemical to biochemical mechanisms. Food Chem Toxicol. 1999;37:949–62.CrossRefPubMedGoogle Scholar
  24. 24.
    Sakrak O, Kerem M, Bedirli A, Pasaoglu H, Akyurek N, Ofluoglu E, et al. Ergothioneine modulates proinflammatory cytokines and heat shock protein 70 in mesenteric ischemia and reperfusion injury. J Surg Res. 2008;144:36–42.CrossRefPubMedGoogle Scholar
  25. 25.
    Nikolaus S, Schreiber S. Diagnostics of inflammatory bowel disease. Gastroenterology. 2007;133:1670–89.CrossRefPubMedGoogle Scholar
  26. 26.
    Kobayashi D, Aizawa S, Maeda T, Tsuboi I, Yabuuchi H, Nezu J, et al. Expression of organic cation transporter OCTN1 in hematopoietic cells during erythroid differentiation. Exp Hematol. 2004;32:1156–62.CrossRefPubMedGoogle Scholar
  27. 27.
    Wijnholds J, Evers R, van Leusden MR, Mol CA, Zaman GJ, Mayer U, et al. Increased sensitivity to anticancer drugs and decreased inflammatory response in mice lacking the multidrug resistance-associated protein. Nat Med. 1997;11:1275–9.CrossRefGoogle Scholar
  28. 28.
    Jonker JW, Wagenaar E, Van Eijl S, Schinkel AH. Deficiency in the organic cation transporters 1 and 2 (Oct1/Oct2 [Slc22a1/Slc22a2]) in mice abolishes renal secretion of organic cations. Mol Cell Biol. 2003;21:7902–8.CrossRefGoogle Scholar
  29. 29.
    Kawano H, Otani M, Takeyama K, Kawai Y, Mayumi T, Hama T. Studies on ergothioneine. VI. Distribution and fluctuations of ergothioneine in rats. Chem Pharm Bull. 1982;30:1760–5.PubMedGoogle Scholar
  30. 30.
    Melville DB, Horner WH, Otken CC, Ludwig ML. Studies of the origin of L-Ergo in animals. J Biol Chem. 1955;213:61–8.PubMedGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2010

Authors and Affiliations

  • Yukio Kato
    • 1
  • Yoshiyuki Kubo
    • 1
  • Daisuke Iwata
    • 1
  • Sayaka Kato
    • 1
  • Tomohisa Sudo
    • 1
  • Tomoko Sugiura
    • 1
  • Takashi Kagaya
    • 2
  • Tomohiko Wakayama
    • 3
  • Akiyoshi Hirayama
    • 4
  • Masahiro Sugimoto
    • 4
  • Kazushi Sugihara
    • 5
  • Shuichi Kaneko
    • 2
  • Tomoyoshi Soga
    • 4
  • Masahide Asano
    • 5
  • Masaru Tomita
    • 4
  • Toshiyuki Matsui
    • 6
  • Morimasa Wada
    • 7
  • Akira Tsuji
    • 1
    Email author
  1. 1.Division of Pharmaceutical Sciences, Graduate School of Natural Science and TechnologyKanazawa UniversityKanazawaJapan
  2. 2.Department of GastroenterologyKanazawa University HospitalKanazawaJapan
  3. 3.Department of Histology and Embryology, Graduate School of Medical ScienceKanazawa UniversityKanazawaJapan
  4. 4.Institute for Advanced BiosciencesKeio UniversityYamagataJapan
  5. 5.Division of Transgenic Animal Science, Advanced Science Research CenterKanazawa UniversityKanazawaJapan
  6. 6.Department of GastroenterologyFukuoka University Chikushi HospitalFukuokaJapan
  7. 7.Division of Molecular Biology, Faculty of Pharmaceutical SciencesNagasaki International UniversityNagasakiJapan

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