γ-Aminobutyric Acid Transporter 2 Mediates the Hepatic Uptake of Guanidinoacetate, the Creatine Biosynthetic Precursor, in Rats

Guanidinoacetic acid (GAA) is the biosynthetic precursor of creatine which is involved in storage and transmission of phosphate-bound energy. Hepatocytes readily convert GAA to creatine, raising the possibility that the active uptake of GAA by hepatocytes is a regulatory factor. The purpose of this study is to investigate and identify the transporter responsible for GAA uptake by hepatocytes. The characteristics of [14C]GAA uptake by hepatocytes were elucidated using the in vivo liver uptake method, freshly isolated rat hepatocytes, an expression system of Xenopus laevis oocytes, gene knockdown, and an immunohistochemical technique. In vivo injection of [14C]GAA into the rat femoral vein and portal vein results in the rapid uptake of [14C]GAA by the liver. The uptake was markedly inhibited by γ-aminobutyric acid (GABA) and nipecotinic acid, an inhibitor of GABA transporters (GATs). The characteristics of Na+- and Cl−-dependent [14C]GAA uptake by freshly isolated rat hepatocytes were consistent with those of GAT2. The Km value of the GAA uptake (134 µM) was close to that of GAT2-mediated GAA transport (78.9 µM). GABA caused a marked inhibition with an IC50 value of 8.81 µM. The [14C]GAA uptake exhibited a significant reduction corresponding to the reduction in GAT2 protein expression. GAT2 was localized on the sinusoidal membrane of the hepatocytes predominantly in the periportal region. This distribution pattern was consistent with that of the creatine biosynthetic enzyme, S-adenosylmethionine∶guanidinoacetate N-methyltransferase. GAT2 makes a major contribution to the sinusoidal GAA uptake by periportal hepatocytes, thus regulating creatine biosynthesis in the liver

Expression and localization of GAT2 in rat liver.

<p>Red and green fluorescence is defined at the lower left corner of each panel. (A) Immunoblot of GAT2, oatp1a4, and MRP6 in rat liver and heart. Rat heart was used as negative control for oatp1a4 and MRP6 expression. The size of the marker proteins is indicated to the left. (B–C) Preferential distribution of GAT2 (B and C) and GAMT (C) in hepatocytes around the portal vein. (D–E) Distribution of GAT2 and oatp1a4 in the liver. E₃ inset: Localization of GAT2 on the oatp1a4-positive sinusoidal membrane (arrowheads). (F) Plasma membrane localization of GAT2 in GAMT-positive hepatocytes. (G) Double immunofluorescence of GAT2 and MRP6, a marker of the lateral membrane of hepatocytes. Arrowheads indicate the bile canaliculus. Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI, blue). In B–E, single (*) and double (**) asterisks indicate the portal spaces and the central vein, respectively. Scale bars: B–D, 100 µm; E–G, 10 µm.</p

Characteristics of GAA transport in <i>Xenopus laevis</i> oocytes expressing GAT2 (GAT2/oocytes).

<p>(A) Uptake of [¹⁴C]GAA (45 µM), and [¹⁴C]creatine (45 µM) by oocytes injected with water (Water; open column) and GAT2 cRNA (GAT2; closed column) for 1 h at 20°C. Each column represents the mean±SEM (n = 8–14). *p<0.01, significantly different from the control. (B) Time-courses of [¹⁴C]GAA uptake (45 µM) by oocytes injected with water (closed circle) and GAT2 cRNA (open circle) at 20°C. The [¹⁴C]GAA uptake was inhibited by unlabeled GAA (10 mM; open square). Each point represents the mean±SEM (n = 9–15). *p<0.01, significantly different from the control. (C) Concentration-dependence of [¹⁴C]GAA uptake by GAT2/ooccytes at 20°C. The uptake was measured at the indicated concentration for 1 h. Each point represents the mean±SEM (n = 5–15).</p

Characteristics of [¹⁴C]GAA uptake by freshly isolated rat hepatocytes.

<p>(A) Time-course of [¹⁴C]GAA (18 µM) uptake (open circle) and [¹⁴C]creatine (30 µM) uptake (closed circle) by hepatocytes. Inset graph shows no effect of unlabeled creatine (10 mM) on the [¹⁴C]creatine uptake at 2 min. Each point represents the mean±SEM (n = 3–4). (B) Concentration-dependence of GAA uptake by hepatocytes. The uptake was measured at the indicated concentration for 3 min. Each point represents the mean±SEM (n = 4). (C–E) Inhibitory effect of GABA (C), taurine (D), and creatine (E) on [¹⁴C]GAA uptake by freshly isolated rat hepatocytes. [¹⁴C]GAA (18 µM) uptake for 3 min at 37°C was measured in the presence and absence (control) of each compound at the designated concentrations. Each point represents the mean±SEM (n = 3–4). (F) Effect of treatment of shRNA, unrelated or targeted to GAT2, for 24 h on [¹⁴C]GAA (18 µM) uptake by primary cultures of rat hepatocytes at 37°C for 3 min. Immunoblotting (inset) with antibodies to GAT2 and Na⁺, K⁺-ATPase used as an internal standard shows the reduction of GAT2 protein expression in GAT2-silenced hepatocytes. Each column represents the mean±SEM (n = 4). *p<0.01, significantly different from the control.</p

<i>In vivo</i> blood-to-liver transport of GAA.

<p>(A) [¹⁴C]GAA uptake by the liver after its intravenous administration. Each point represents the mean±SEM (n = 5–7). (B) Concentration-dependent uptake of GAA by rat liver after its injection into the portal vein. The extravascular extraction of [¹⁴C]GAA was plotted against the concentration of unlabeled GAA in the injection solution. Each point represents the mean±SEM (n = 3–4). (C) Simultaneous injection of unlabeled creatine had no effect on [¹⁴C]creatine uptake by the liver. Each column represents the mean±SEM (n = 5–6).</p

Effect of several compounds on <i>in vivo</i> [¹⁴C]GAA uptake by rat liver.

<p>Inhibitors dissolved in Ringer's HEPES buffer (pH 7.4) at the indicated concentrations were injected simultaneously with [¹⁴C]GAA (45 µM) into the rat portal vein. Each value represents the mean±SEM (n = 3–19).</p><p>*p<0.01,</p><p>**p<0.05, significantly different from the control.</p

Na⁺- and Cl⁻-dependence and inhibitory effect of several compounds on [¹⁴C]GAA uptake by freshly isolated rat hepatocytes.

<p>[¹⁴C]GAA (18 µM) uptake by hepetocytes was measured at 37°C for 3 min in the absence or presence (control) of Na⁺ and Cl⁻ or in the absence (control) or presence of inhibitors (2 mM). Each value represents the mean±SEM (n = 3–4).</p><p>*p<0.01,</p><p>**p<0.05, significantly different from the control.</p