Skip to main content

Advertisement

SpringerLink
Log in

Combination Metabolomics Approach for Identifying Endogenous Substrates of Carnitine/Organic Cation Transporter OCTN1

  • Research Paper
  • Published:
Pharmaceutical Research Aims and scope Submit manuscript

Abstract

Purpose

Solute carrier SLC22A4 encodes the carnitine/organic cation transporter OCTN1 and is associated with inflammatory bowel disease, although little is known about how this gene is linked to pathogenesis. The aim of the present study was to identify endogenous substrates that are associated with gastrointestinal inflammation.

Methods

HEK293/OCTN1 and mock cells were incubated with colon extracts isolated from dextran sodium sulfate-induced colitis mice; the subsequent cell lysates were mixed with the amino group selective reagent 3-aminopyridyl-N-hydroxysuccinimidyl carbamate (APDS), to selectively label OCTN1 substrates. Precursor ion scanning against the fragment ion of APDS was then used to identify candidate OCTN1 substrates.

Results

Over 10,000 peaks were detected by precursor ion scanning; m/z 342 had a higher signal in HEK293/OCTN1 compared to mock cells. This peak was detected as a divalent ion that contained four APDS-derived fragments and was identified as spermine. Spermine concentration in peripheral blood mononuclear cells from octn1 gene knockout mice (octn1−/−) was significantly lower than in wild-type mice. Lipopolysaccharide-induced gene expression of inflammatory cytokines in peritoneal macrophages from octn1−/− mice was lower than in wild-type mice.

Conclusions

The combination metabolomics approach can provide a novel tool to identify endogenous substrates of OCTN1.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6

Similar content being viewed by others

Abbreviations

APDS:

3-aminopyridyl-N-hydroxysuccinimidyl carbamate

CD:

Crohn’s disease

DSS:

dextran sodium sulfate

ERGO:

ergothioneine

IBD:

inflammatory bowel diseases

LPS:

lipopolysaccharide

PBMC:

peripheral blood mononuclear cells

SNP:

single nucleotide polymorphisms

TEA:

tetraethylammonium

References

  1. Lin L, Yee SW, Kim RB, Giacomini KM. SLC transporters as therapeutic targets: emerging opportunities. Nat Rev Drug Discov. 2015;14(8):543–60.

    Article  CAS  Google Scholar 

  2. Rioux JD, Silverberg MS, Daly MJ, Steinhart AH, McLeod RS, Griffiths AM, et al. Genomewide search in Canadian families with inflammatory bowel disease reveals two novel susceptibility loci. Am J Hum Genet. 2000;66(6):1863–70.

    Article  CAS  Google Scholar 

  3. 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(4):341–8.

    Article  CAS  Google Scholar 

  4. Hou X, Mao J, Li Y, Li J, Wang W, Fan C, et al. Association of single nucleotide polymorphism rs3792876 in SLC22A4 gene with autoimmune thyroid disease in a Chinese Han population. BMC Med Genet. 2015;16:76.

    Article  Google Scholar 

  5. Santiago JL, Martinez A, de la Calle H, Fernandez-Arquero M, Figueredo MA, de la Concha EG, et al. Evidence for the association of the SLC22A4 and SLC22A5 genes with type 1 diabetes: a case control study. BMC Med Genet. 2006;7:54.

    Article  Google Scholar 

  6. 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(5):471–5.

    Article  CAS  Google Scholar 

  7. Xuan C, Zhang BB, Yang T, Deng KF, Li M, Tian RJ. Association between OCTN1/2 gene polymorphisms (1672C-T, 207G-C) and susceptibility of Crohn's disease: a meta-analysis. Int J Color Dis. 2012;27(1):11–9.

    Article  Google Scholar 

  8. 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(1):107–11.

    Article  CAS  Google Scholar 

  9. Grundemann D, Harlfinger S, Golz S, Geerts A, Lazar A, Berkels R, et al. Discovery of the ergothioneine transporter. Proc Natl Acad Sci U S A. 2005;102(14):5256–61.

    Article  Google Scholar 

  10. Grigat S, Harlfinger S, Pal S, Striebinger R, Golz S, Geerts A, et al. Probing the substrate specificity of the ergothioneine transporter with methimazole, hercynine, and organic cations. Biochem Pharmacol. 2007;74(2):309–16.

    Article  CAS  Google Scholar 

  11. Pochini L, Scalise M, Galluccio M, Pani G, Siminovitch KA, Indiveri C. The human OCTN1 (SLC22A4) reconstituted in liposomes catalyzes acetylcholine transport which is defective in the mutant L503F associated to the Crohn's disease. Biochim Biophys Acta. 2012;1818(3):559–65.

    Article  CAS  Google Scholar 

  12. Drenberg CD, Gibson AA, Pounds SB, Shi L, Rhinehart DP, Li L, et al. OCTN1 is a high-affinity carrier of nucleoside analogues. Cancer Res. 2017;77(8):2102–11.

    Article  CAS  Google Scholar 

  13. Taubert D, Grimberg G, Jung N, Rubbert A, Schomig E. Functional role of the 503F variant of the organic cation transporter OCTN1 in Crohn's disease. Gut. 2005;54(10):1505–6.

    Article  CAS  Google Scholar 

  14. Taubert D, Jung N, Goeser T, Schomig E. Increased ergothioneine tissue concentrations in carriers of the Crohn's disease risk-associated 503F variant of the organic cation transporter OCTN1. Gut. 2009;58(2):312–4.

    Article  CAS  Google Scholar 

  15. Urban TJ, Yang C, Lagpacan LL, Brown C, Castro RA, Taylor TR, et al. Functional effects of protein sequence polymorphisms in the organic cation/ergothioneine transporter OCTN1 (SLC22A4). Pharmacogenet Genomics. 2007;17(9):773–82.

    Article  CAS  Google Scholar 

  16. Futatsugi A, Masuo Y, Kawabata S, Nakamichi N, Kato Y. L503F variant of carnitine/organic cation transporter 1 efficiently transports metformin and other biguanides. J Pharm Pharmacol. 2016;68(9):1160–9.

    Article  CAS  Google Scholar 

  17. Urban TJ, Brown C, Castro RA, Shah N, Mercer R, Huang Y, et al. Effects of genetic variation in the novel organic cation transporter, OCTN1, on the renal clearance of gabapentin. Clin Pharmacol Ther. 2008;83(3):416–21.

    Article  CAS  Google Scholar 

  18. Cheah IK, Halliwell B. Ergothioneine; antioxidant potential, physiological function and role in disease. Biochim Biophys Acta. 2012;1822(5):784–93.

    Article  CAS  Google Scholar 

  19. Shimizu T, Masuo Y, Takahashi S, Nakamichi N, Kato Y. Organic cation transporter Octn1-mediated uptake of food-derived antioxidant ergothioneine into infiltrating macrophages during intestinal inflammation in mice. Drug Metab Pharmacokinet. 2015;30(3):231–9.

    Article  CAS  Google Scholar 

  20. Maeda T, Hirayama M, Kobayashi D, Miyazawa K, Tamai I. Mechanism of the regulation of organic cation/carnitine transporter 1 (SLC22A4) by rheumatoid arthritis-associated transcriptional factor RUNX1 and inflammatory cytokines. Drug Metab Dispos. 2007;35(3):394–401.

    Article  CAS  Google Scholar 

  21. Tang Y, Masuo Y, Sakai Y, Wakayama T, Sugiura T, Harada R, et al. Localization of xenobiotic transporter OCTN1/SLC22A4 in hepatic stellate cells and its protective role in liver fibrosis. J Pharm Sci. 2016;105(5):1779–89.

    Article  CAS  Google Scholar 

  22. Shimbo K, Oonuki T, Yahashi A, Hirayama K, Miyano H. Precolumn derivatization reagents for high-speed analysis of amines and amino acids in biological fluid using liquid chromatography/electrospray ionization tandem mass spectrometry. Rapid Commun Mass Spectrom. 2009;23(10):1483–92.

    Article  CAS  Google Scholar 

  23. Kato Y, Kubo Y, Iwata D, Kato S, Sudo T, Sugiura T, et al. Gene knockout and metabolome analysis of carnitine/organic cation transporter OCTN1. Pharm Res. 2010;27(5):832–40.

    Article  CAS  Google Scholar 

  24. Ishimoto T, Nakamichi N, Hosotani H, Masuo Y, Sugiura T, Kato Y. Organic cation transporter-mediated ergothioneine uptake in mouse neural progenitor cells suppresses proliferation and promotes differentiation into neurons. PLoS One. 2014;9(2):e89434.

    Article  Google Scholar 

  25. Tanaka S, Fujita Y, Parry HE, Yoshizawa AC, Morimoto K, Murase M, et al. Mass++: a visualization and analysis tool for mass spectrometry. J Proteome Res. 2014;13:3846–53.

    Article  CAS  Google Scholar 

  26. Zhang X, Goncalves R, Mosser DM. The isolation and characterization of murine macrophages. Curr Protoc Immunol. 2008;Chapter 14:Unit 14 1.

  27. 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(2):768–73.

    CAS  PubMed  Google Scholar 

  28. Igarashi K, Kashiwagi K. Modulation of cellular function by polyamines. Int J Biochem Cell Biol. 2010;42(1):39–51.

    Article  CAS  Google Scholar 

  29. Krumpochova P, Sapthu S, Brouwers JF, de Haas M, de Vos R, Borst P, et al. Transportomics: screening for substrates of ABC transporters in body fluids using vesicular transport assays. FASEB J. 2012;26(2):738–47.

    Article  CAS  Google Scholar 

  30. van de Wetering K, Feddema W, Helms JB, Brouwers JF, Borst P. Targeted metabolomics identifies glucuronides of dietary phytoestrogens as a major class of MRP3 substrates in vivo. Gastroenterology. 2009;137(5):1725–35.

    Article  Google Scholar 

  31. van de Wetering K, Sapthu S. ABCG2 functions as a general phytoestrogen sulfate transporter in vivo. FASEB J. 2012;26(10):4014–24.

    Article  Google Scholar 

  32. Chen L, Shu Y, Liang X, Chen EC, Yee SW, Zur AA, et al. OCT1 is a high-capacity thiamine transporter that regulates hepatic steatosis and is a target of metformin. Proc Natl Acad Sci U S A. 2014;111(27):9983–8.

    Article  CAS  Google Scholar 

  33. Shinozaki Y, Furuichi K, Toyama T, Kitajima S, Hara A, Iwata Y, et al. Impairment of the carnitine/organic cation transporter 1-ergothioneine axis is mediated by intestinal transporter dysfunction in chronic kidney disease. Kidney Int. 2017;92(6):1356–69.

    Article  CAS  Google Scholar 

  34. Weiss TS, Herfarth H, Obermeier F, Ouart J, Vogl D, Scholmerich J, et al. Intracellular polyamine levels of intestinal epithelial cells in inflammatory bowel disease. Inflamm Bowel Dis. 2004;10(5):529–35.

    Article  CAS  Google Scholar 

  35. Hong SK, Chaturvedi R, Piazuelo MB, Coburn LA, Williams CS, Delgado AG, et al. Increased expression and cellular localization of spermine oxidase in ulcerative colitis and relationship to disease activity. Inflamm Bowel Dis. 2010;16(9):1557–66.

    Article  Google Scholar 

  36. Hiasa M, Miyaji T, Haruna Y, Takeuchi T, Harada Y, Moriyama S, et al. Identification of a mammalian vesicular polyamine transporter. Sci Rep. 2014;4:6836.

    Article  Google Scholar 

  37. Busch AE, Quester S, Ulzheimer JC, Waldegger S, Gorboulev V, Arndt P, et al. Electrogenic properties and substrate specificity of the polyspecific rat cation transporter rOCT1. J Biol Chem. 1996;271(51):32599–604.

    Article  CAS  Google Scholar 

  38. Watanabe S, Kusama-Eguchi K, Kobayashi H, Igarashi K. Estimation of polyamine binding to macromolecules and ATP in bovine lymphocytes and rat liver. J Biol Chem. 1991;266(31):20803–9.

    CAS  PubMed  Google Scholar 

  39. Takayama T, Tsutsui H, Shimizu I, Toyama T, Yoshimoto N, Endo Y, et al. Diagnostic approach to breast cancer patients based on target metabolomics in saliva by liquid chromatography with tandem mass spectrometry. Clin Chim Acta. 2016;452:18–26.

    Article  CAS  Google Scholar 

  40. Arashida N, Nishimoto R, Harada M, Shimbo K, Yamada N. Highly sensitive quantification for human plasma-targeted metabolomics using an amine derivatization reagent. Anal Chim Acta. 2017;954:77–87.

    Article  CAS  Google Scholar 

Download references

Acknowledgments and Disclosures

We thank Lica Ishida (Kanazawa University) for technical assistance and Prof. Hiroshi Hasegawa at the Laboratory of Analytical and Environmental Chemistry in Kanazawa University for LC-QTOFMS technical consultation. This study was supported in part by Grant-in-Aids for Scientific Research to YK [15H04664] and from the Ministry of Education, Culture, Sports, Science and Technology of Japan to YM [16 K18934], as well as support from a grant provided by the Mochida Memorial Foundation for Medical and Pharmaceutical Research (Tokyo, Japan), the Hoansha Foundation (Osaka, Japan), and Kanazawa University SAKIGAKE project.

Author information

Authors and Affiliations

  1. Faculty of Pharmacy, Institute of Medical, Pharmaceutical and Health Sciences, Kanazawa University, Kakuma-machi, Kanazawa, 920-1192, Japan

    Yusuke Masuo, Yuri Ohba, Kohei Yamada, Aya Hasan Al-Shammari, Natsumi Seba, Noritaka Nakamichi, Munetaka Kunishima & Yukio Kato

  2. Faculty of Pharmacy, Takasaki University of Health and Welfare, Takasaki, Japan

    Takuo Ogihara

Authors
  1. Yusuke Masuo
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Yuri Ohba
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Kohei Yamada
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Aya Hasan Al-Shammari
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Natsumi Seba
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Noritaka Nakamichi
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Takuo Ogihara
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Munetaka Kunishima
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Yukio Kato
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Yukio Kato.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Masuo, Y., Ohba, Y., Yamada, K. et al. Combination Metabolomics Approach for Identifying Endogenous Substrates of Carnitine/Organic Cation Transporter OCTN1. Pharm Res 35, 224 (2018). https://doi.org/10.1007/s11095-018-2507-1

Download citation

  • Received:

  • Accepted:

  • Published:

  • DOI: https://doi.org/10.1007/s11095-018-2507-1

KEY Words

Search

Navigation