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LIVER AND BILIARY TRACT

Hepatic uptake of -butyrobetaine, a precursor of carnitine biosynthesis, in rats

¹Department of Membrane Transport and Biopharmaceutics, Faculty of Pharmacy, Institute of Medical, Pharmaceutical, and Health Sciences, Kanazawa University, Kanazawa; ²Faculty of Pharmaceutical Sciences, Health Sciences University of Hokkaido, Hokkaido, Japan; and ³Department of Internal Medicine, Ann Arbor Veterans Affairs Healthcare System and University of Michigan School of Medicine, Ann Arbor, Michigan

Submitted June 22, 2009 ; accepted in final form August 6, 2009

-Butyrobetaine (GBB) is a precursor in the biosynthesis of carnitine, which plays an important role in the β-oxidation of fatty acids, and is converted to carnitine by -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 (d₃-GBB) was examined in HEK293 cells expressing rOctn2 (HEK293/rOctn2) and freshly isolated rat hepatocytes. d₃-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 d₃-GBB was taken up by rOctn2 in an Na⁺-dependent manner with K_m value of 13 µM. The apparent K_m value for d₃-GBB transport in freshly isolated rat hepatocytes was 9 µM. d₃-GBB uptake by the rat hepatocytes was inhibited by -aminobutyric acid (GABA) to 30% of the control, whereas it was inhibited by carnitine to 62% of the control, even at 500 µM. Furthermore, d₃-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.

Octn2; transporter; hepatocytes; carnitine; -butyrobetaine; liver; GABA transporter