title: Hepatic uptake of gamma-butyrobetaine, a precursor of carnitine biosynthesis, in rats.
abstract: Gamma-butyrobetaine (GBB) is a precursor in the biosynthesis of carnitine, which plays an important role in the beta-oxidation of fatty acids, and is converted to carnitine by gamma-butyrobetaine dioxygenase (BBD) predominantly in liver. We investigated the molecular mechanism of hepatic uptake of GBB in rat hepatocytes. Cellular localization of rat Octn2 (rOctn2:Slc22A5) was studied by Western blot analysis. Uptake of deuterated GBB (d(3)-GBB) was examined in HEK293 cells expressing rOctn2 (HEK293/rOctn2) and freshly isolated rat hepatocytes. d(3)-GBB was quantified by use of liquid chromatography-tandem mass spectrometry. Western blot analysis demonstrated an expression of OCTN2 protein in hepatic basolateral membrane but not in bile canalicular membrane fraction. Furthermore, we found that d(3)-GBB was taken up by rOctn2 in an Na(+)-dependent manner with K(m) value of 13 microM. The apparent K(m) value for d(3)-GBB transport in freshly isolated rat hepatocytes was 9 microM. d(3)-GBB uptake by the rat hepatocytes was inhibited by gamma-aminobutyric acid (GABA) to 30% of the control, whereas it was inhibited by carnitine to 62% of the control, even at 500 microM. Furthermore, d(3)-GBB uptake by rat hepatocytes was decreased by 45% with rat Gat2 (Slc6A13, a major liver GABA transporter) silenced by the microRNA method. Accordingly, the present study clearly demonstrates that GBB is taken up by hepatocytes for carnitine biosynthesis not only via Octn2 but also via the GABA transporter, possibly Gat2.
authors.affiliation: Dept. of Membrane Transport and Biopharmaceutics, Faculty of Pharmacy, Institute of Medical, Pharmaceutical, and Health Sciences, Kanazawa Univ., Kakuma-machi, Kanazawa, Ishikawa, 920-1192, Japan. tamai@p.kanazawa-u.ac.jp.
authors.forename: Masaharu
authors.initials: M
authors.lastname: Fujita
authors.forename: Takeo
authors.initials: T
authors.lastname: Nakanishi
authors.forename: Yuta
authors.initials: Y
authors.lastname: Shibue
authors.forename: Daisuke
authors.initials: D
authors.lastname: Kobayashi
authors.forename: Richard H
authors.initials: RH
authors.lastname: Moseley
authors.forename: Yoshiyuki
authors.initials: Y
authors.lastname: Shirasaka
authors.forename: Ikumi
authors.initials: I
authors.lastname: Tamai
mesh_terms: D000818:Animals; D001622:Betaine / Q000031:analogs & derivatives* / Q000378:metabolism; D001692:Biological Transport; D015153:Blotting, Western; D002331:Carnitine / Q000096:biosynthesis* / Q000378:metabolism; D002460:Cell Line; D002853:Chromatography, Liquid; D050485:GABA Plasma Membrane Transport Proteins / Q000235:genetics / Q000378:metabolism; D022781:Hepatocytes / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D008099:Liver / Q000378:metabolism*; D008297:Male; D027701:Organic Cation Transport Proteins / Q000235:genetics / Q000378:metabolism; D034622:RNA Interference; D051381:Rats; D017208:Rats, Wistar; D012964:Sodium / Q000378:metabolism; D000074058:Solute Carrier Family 22 Member 5; D053719:Tandem Mass Spectrometry; D014162:Transfection; D005680:gamma-Aminobutyric Acid / Q000378:metabolism
chemical_list: D050485:GABA Plasma Membrane Transport Proteins; D027701:Organic Cation Transport Proteins; C115441:Slc22a5 protein, rat; C092004:Slc6a13 protein, rat; D000074058:Solute Carrier Family 22 Member 5; D001622:Betaine; C002889:gamma-butyrobetaine; D005680:gamma-Aminobutyric Acid; D012964:Sodium; D002331:Carnitine