Inhibitory effects of statins on human monocarboxylate transporter 4

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Abstract

Human MCT4 (SLC16A3) is responsible for the efflux of l-lactic acid from skeletal muscle cells and is essential for muscle homeostasis. However, the effects of monocarboxylate drugs, such as statins on the MCT4-mediated transport of l-lactic acid have not been elucidated. Inhibition of l-lactic acid transport mediated by MCT4 might to lead to collapse of muscle homeostasis. The aim of this study was to establish an MCT4 transfected cell line and to clarify the transport mechanism of l-lactic acid and the effects of statins on this transport system. Results of Western blot analyses and immunohistochemistry studies indicated that the expression of CD147 and MCT4-FLAG protein were observed and was displayed clear plasma membrane localization in CD147 and MCT4-FLAG co-transfected cell line (cm cells). Uptake of l-lactic acid in cm cells was significantly greater than that in cells transfected with a vector alone. l-lactic acid uptake was concentration-dependent with a Km value of 28.43 ± 3.87 mM. The results of a previous study showing a Km value of 28.5 mM in hMCT4-expressed oocytes. Lipophilic statins significantly inhibited [14C] l-lactic acid uptake in a concentration-dependent manner. In contrast, the inhibitory effects of hydrophilic statins were very weak.

Introduction

The monocarboxylate transporter (MCT) family now comprises 14 members, of which the first four (MCT1–MCT4) subtypes have been demonstrated experimentally to catalyse the proton-linked transport of metabolically important monocarboxylates, such as l-lactic acid, but the detailed kinetic mechanisms of MCT2–MCT4 subtypes have not been elucidated (Halestrap and Meredith, 2004).

At present, there is some evidence indicating that MCTs play a role in the transport of some drugs, such as valproic acid and HMG-CoA reductase inhibitors (statins), that have monocarboxylate structures within the molecules. (Hosoya et al., 2001, Wu et al., 2000, Nagasawa et al., 2000). Nagasawa et al. (2003) reported that lovastatin acid, a statin, by rat mesangial cells is handled by proton-coupled monocarboxylate transporter, and the involvement of MCT4 in the uptake was suggested. However, it has not been determined whether MCT4 contributes to the transport of statins.

Statins are the most widely used cholesterol-lowering agents for prevention of obstructive cardiovascular events (Havel and Rapaport, 1995, Jukema et al., 1995, Downs et al., 1998). However, statins have been shown to induce various forms of skeletal muscle abnormalities ranging from mild myopathy to myositis and occasionally rhabdmyolysis and even death (Evans and Rees, 2002).

MCT4 is the major monocarboxylate transporter isoform present in white skeletal muscle and is responsible for the efflux of l-lactic acid (Manning Fox et al., 2000, Wilson et al., 1998), and it is essential for muscle homeostasis. Since it has been reported that over-accumulation of lactic acid led to intracellular acidification and apoptosis (Jeong et al., 2001), we hypothesized that inhibition of lactic acid transport mediated by MCT4 leads to over-accumulation of lactic acid and induction of intracellular acidification and apoptosis in skeletal muscle.

In order to verify this hypothesis, we established a cell line transfected with MCT4 and to clarify the transport mechanism of l-lactic acid and the effects of statins on this transport system.

Section snippets

Chemicals

[14C] l-lactic acid sodium salt was purchased from Amersham Life Science (Buckinghamshire, UK). l-lactic acid sodium salt was purchased from ICN Biomedicals Inc. Cerivastatin Na, simvastatin, atorvastatin Ca, lovastatin, rosuvastatin Ca and pravastatin Na were kindly donated by Sankyo (Tokyo, Japan). Fluvastatin Na was kindly supplied by Novartis Pharma (Tokyo, Japan). Simvastatin acid and lovastatin acid were prepared from their lactone form by hydrolysis in a 0.05N NaOH solution, with

Expression of CD147 and MCT4-FLAG in LLC-PK1 cells

To confirm the expression of CD147 and MCT4-FLAG in transfected cells, we performed Western blot analyses and immunohistochemistry studies. As shown in Fig. 1 (a), CD147 protein was detected at about 40 kDa in cm and cv cells. On the other hand, MCT4-FLAG protein was detected at about 50 kDa in cm and vm cells. No expression of CD147 or MCT4-FLAG was observed in vv cells. As shown in Fig. 1 (b), CD147 and MCT4-FLAG were stained near the nuclei of cv or vm cells, respectively. On the other hand,

Discussion

In the present study, we investigated the interactions of l-lactic acid and statins with MCT4. Firstly, since CD147 interacts with MCT4 and assists MCT4 expression at the cell surface, we established a CD147 and MCT4 co-transfected cell line. Western blot analyses and immunohistochemistry studies indicated that the expression of CD147 and MCT4-FLAG protein were observed and was displayed clear plasma membrane localization in CD147 and MCT4-FLAG co-transfected cell line (cm cells) (Fig. 1). The

Acknowledgement

This work was supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology, Japan.

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