Effect of Statins on Functional Expression of Membrane Transporters in L6 Rat Skeletal Muscle Cells

Abstract

Background: Statins reduce LDL-cholesterol and the risk of atherosclerosis. They are generally safe, although statin-induced myopathy is relatively common. Membrane transporters play a crucial role in determining statin side effects. Little is known regarding the interaction of drug transporters in muscle cells with statins. Study aims: The present study aimed to determine the effect of statins on functional expression of monocarboxylate transporters (MCTs) and multidrug resistance-associated proteins (MRPs) in L6 rat skeletal myotube cells. Methods: Relative gene expression at mRNA level was confirmed by RT Profiler Rat Drug Transporter PCR array. The uptake of H-labelled DL-lactate (1 μCi/ml) was measured to functionally expressed MCT function. The inhibition of [H]-DL-lactate uptake was assessed in the presence or absence of statins and compared to that of the MCT inhibitors, phloretin and CHC. Transporter-mediated dye efflux was used as functional assay for the MRP efflux transporters. Results: In L6 rat skeletal myotubes, relatively high mRNA expression level was observed for Mct1and Mrp1for uptake and efflux transporters, respectively. The [H]-DL-lactate uptake was shown to be a concentration-, pH-dependent and Na-independent manner with Michaelis-Menten constant (Km) value of 16.17 ± 2.4 mM vs 15.63 ± 3.0 mM in the presence and absence of Na, respectively. The maximum velocity of substrate binding (Vmax) of the DL-lactate uptake inhibition by lipophilic statins; simvastatin and atorvastatin, were in the same order as phloretin and CHC, while no significant inhibitory magnitude with hydrophilic statins; pravastatin and rosuvastatin. However, the L6 rat skeletal myotubes did not exhibit lactate efflux function. Among four of statins used, only simvastatin showed an affinity inhibition of MRP function in L6 cells. Conclusions: This study has shown that lipophilic statins significantly inhibit functional expression of MCTs, even though they have not shown relatively high inhibition impact on MRPs. Keywords— Lactate, L6 cells, Monocarboxylate transporter, Multidrug resistance-associated protein, Statins Nur Salwani Bakar 3,* Farhad Kamali, Colin D. A. Brown Institute of Cellular Medicine, 4 Floor William Leech Building, Medical School, Newcastle University NE2 4HH, UK. Institute for Cell and Molecular Biosciences, 1 Floor Cookson Building, Medical School, Newcastle University, NE2 4HH, UK. Present address: School of Health Sciences, Universiti Sains Malaysia, 16150 Kubang Kerian, Kelantan, Malaysia. Email: corresponingauthoraddress @email.com Received 19 Oct 2016. Revised 21 Nov 2016. Accepted 25 Nov 2016. Published O line 13 Dec 2016 *Corresponding Author: Nur Salwani Bakar Email: nursalwani@usm.my J. of Biomed. & Clin. Sci. 2016, Vol 1 (1), 17-26 Original Article http://apps.amdi.usm.my/journal/ 18 has been recognised that elevated plasma statin concentration increases the risk of muscle toxicity [6-8], the aetiology of SRM is not well understood. Since many patients develop SRM such as muscle aches and pain, it is very important to determine the factors which control local skeletal muscle statin concentrations. In fact, little is known regarding the molecular determinants of statin distribution into skeletal muscle and its relevance to toxicity. There are several key studies that highlighted muscle symptoms, such as fatigue and cramp, may be due to lactate build-up in muscles. These lactate accumulations may be inducing dysfunctional monocarboxylate transporters (MCTs) possibly MCT1 and MCT4, two of which highly expressed lactate transporters in striated muscle [9, 10] and transcribed by SLC16A1 and SLC16A3 gene, respectively [10]. Other studies, has shown that exposure to monocarboxylate lactate, was found to be associated with the generation of reactive oxygen species (ROS) and the up-regulation of genes related to mitochondrial lactate oxidation complex in both in vitro and in vivo study using rat skeletal muscle cells [11, 12]. Physiologically, the role of MCTs in skeletal muscle is undeniable since the organ is the major site of lactate production and removal in the body. So far, there is no study has been carried out to investigate the association of the membrane transporter with the risk of SRM i.e., myotoxicity happens as a result of inhibition to the MCT function by a statin and subsequently increases muscular lactate levels for which it might be perceived as muscle pain or cramp. Moreover, substrates for MCTs have not been limited to endogenous metabolites but also xenobiotics such as statins, gamma-hydroxybutyrate and valproic acid [13-16] which thus suggest their potential role in predicting SRM. Our group has previously shown that HK-2 cells (a proximal tubule cell line originated from human kidney) express MCT1 at both mRNA and functional levels [17]. A number of studies also have characterised MCT functions in L6 skeletal muscle cells [12, 18, 19], by which MCT1 in particular mediated lactate transport in both mitochondrial and sarcolemma membrane of a striated muscle fibre. However, limited evidence is available to explain how statins affect the function of MCT1 in muscle cells. There are evidences indicating that MCT4, but not MCT1, being the statin target and upregulated during statin-induced cytotoxicity [20, 21]. In terms of blood lactate transport and removal, MCT1 and MCT4 were both considered important mediators for blood lactate removal [22, 23] thus implicating their vital role in maintaining muscle lactate concentration. Therefore in this study, we determined whether SRM could be promoted by the inhibition of MCT1 function by statins primarily looking at their ability to inhibit lactate uptake into the L6 cells. It is possible that the inhibition of MCT1 by statins could advocate myotoxicity due to affected lactate transport. Apart from the above mentioned SLC transcribing membrane transporters, the interplay role by efflux transporters typically ATP-binding cassette (ABC), in muscle such as multi-drug resistance associated protein (MRPs), may also modulate statin local exposure in muscle and eventually intensify muscle toxicity in the event of function inhibition of the transporters. Moreover, our group had also shown that 5-(3-92-(7chloroquinolon-2-yl)ethenyl)phenyl)-8dimethylcarbamyl-4,6-dithiaoctanoic acid (MK571) could be used as a high affinity MRP inhibitor in an MRP-mediated CMFDA efflux assay using HK-2 cells [17]. This dual dye assay was developed to evaluate the impact of two inhibitors, MK571 (MRP inhibitor) and CSA (MDR1 inhibitor); whereby the accumulation of dye provides an indirect measure of efflux inhibition of the ABC transporters. Therefore, the aim of the second part of this study was to evaluate the impact of statins on functional expression of efflux transporters. In order to assess the relative affinity of different statins to the MRPs function, the magnitude of the dye efflux inhibition was compared to MK571. 2 MATERIALS AND METHODS 2.1 Materials and reagents Number RT2 ProfilerTM Rat Drug Transporter PCR array (Catalogue no: PARN-070Z) and reagents were purchased from Qiagen Ltd (Crawley, UK). Simvastatin, atorvastatin, pravastatin, and rosuvastatin were gifts from AstraZeneca (Alderley Park, Cheshire, UK). [2-3H]-DL-lactate (at activity of 20 Ci/mmol) was purchased from Hartmann Analytic (Braunschweig, Germany). SV Total RNA Isolation System was purchased from Promega (Southampton, UK). SYBR Green Dye Master Mix for real-time polymerase chain reaction was purchased from Roche Applied Sciences (Burgess Hill, UK). Reagents for dye assay; 5-chloromethylfluorescein diacetate (CMFDA) was purchased from Invitrogen J. of Biomed. & Clin. Sci. 2016, Vol 1 (1), 17-26 Original Article http://apps.amdi.usm.my/journal/ 19 (Paisley, UK). Unless otherwise stated, all other reagents including phloretin (well-defined MCT1 inhibitor) and α-cyano-4-hydroxycinnamate, CHC (a typical MCT1, 2 and 4 inhibitors) were purchased from Sigma-Aldrich (Dorset, UK). 2.2 L6 rat muscle cell line The L6 rat muscle cell line (ATCC® number: CRL1458TM), supplied at myoblast stage, was kindly provided by Dr Audrey Brown of Newcastle University. L6 exists as myoblasts when cultured in maintenance culture medium, which comprises high glucose DMEM supplemented with 10 % foetal calf serum, 200 units/ml penicillin and 200 μg/ml streptomycin, until 70-80 % confluency. The cells were then introduced to differentiation medium (high glucose DMEM supplemented with 2 % horse serum, 200 units/ml penicillin and 200 μg/ml streptomycin), upon which the cells differentiate into myotube until approximately 80 to 90 % confluent. L6 cells used in this study were within passage numbers 11 through 30. For the uptake and efflux experiments, L6 cells were seeded at 50,000 cells/well and 20,000 cells/well onto 24-well plates and 96-well plates (Coster, Corning Incorporated Corning, UK), respectively, for 2 days or until the indicated confluence. Cells were then differentiated into myotube formation by incubating them with differentiation media. The cell monolayers were fed a fresh differentiation media every 2 days and were used for uptake experiments on day 7. 2.3 Isolation of total RNA and qPCR array for rat membrane transporters On day 7 of differentiation, total RNA was isolated from L6 cells at myotube stage to determine the expression of a series of uptake (both SLC and SLCO sub-groups) and efflux (ABC group) transporters by qPCR array plate. In order to assess whether statins affect gene expression of the uptake transporters in L6 myotubes, the cells were pre-treated on day 5 with simvastatin (2 μM) for 48 hrs prior to RNA extraction and compared with untreated control cells (contained 0.02 % methanol only). The protocols for RNA extraction and qPCR array were carried out as previously described [17]. 2.4 Functional assay of monocarboxylate transporters (MCTs) 2.4.1 3H-DL-lactate uptake assay Lactate uptake experiments by L6 cells seeded on 24-well plates in the presence of statins were carried out according to the previously described

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@inproceedings{Bakar2016EffectOS, title={Effect of Statins on Functional Expression of Membrane Transporters in L6 Rat Skeletal Muscle Cells}, author={Salwani Bakar and Farhad Kamali and Colin D . A . Brown}, year={2016} }