Abstract
The branch chain amino acid leucine is a potent stimulator of protein synthesis in skeletal muscle. Leucine rapidly enters the cell via the L-Type Amino Acid Transporter 1 (LAT1); however, little is known regarding the localisation and distribution of this transporter in human skeletal muscle. Therefore, we applied immunofluorescence staining approaches to visualise LAT1 in wild type (WT) and LAT1 muscle-specific knockout (mKO) mice, in addition to basal human skeletal muscle samples. LAT1 positive staining was visually greater in WT muscles compared to mKO muscle. In human skeletal muscle, positive LAT1 staining was noted close to the sarcolemmal membrane (dystrophin positive staining), with a greater staining intensity for LAT1 observed in the sarcoplasmic regions of type II fibres (those not stained positively for myosin heavy-chain 1, Type II—25.07 ± 5.93, Type I—13.71 ± 1.98, p < 0.01), suggesting a greater abundance of this protein in these fibres. Finally, we observed association with LAT1 and endothelial nitric oxide synthase (eNOS), suggesting LAT1 association close to the microvasculature. This is the first study to visualise the distribution and localisation of LAT1 in human skeletal muscle. As such, this approach provides a validated experimental platform to study the role and regulation of LAT1 in human skeletal muscle in response to various physiological and pathophysiological models.
Highlights
Amino acid transport is an essential component in the survival of all cells [1], providing substrates for processes such as protein synthesis [2,3] and cell division [4]
Characterisation and Validation of L-Type Amino Acid Transporter 1 (LAT1) Immunofluorescence Labelling Approaches In wild type (WT) skeletal muscle samples, positive LAT1 signals were noted at the periphery of many fibres, in addition to displaying a stronger sarcoplasmic staining than seen in muscle-specific knockout (mKO) samples (Figure 1A)
As this amino acid transporter is only active when associated with cellular membranes, this novel method allows a greater depth of investigation into the dynamics of LAT1 in human muscle in response to anabolic stimuli
Summary
Amino acid transport is an essential component in the survival of all cells [1], providing substrates for processes such as protein synthesis [2,3] and cell division [4]. Amino acid transport into cells is of particular importance within skeletal muscle to maintain positive protein balance [2,3]. 1 (LAT1) known as the solute carrier family 7 member 5 (SLC7A5) has received significant attention [9,10,11,12,13,14,15] due to its role in the ingress of amino acids, vital for optimal stimulation of muscle protein synthesis (MPS) [16,17,18]. LAT1 transports leucine (or other branched chain amino acids (BCAAs)) into the cell via a bi-transport system that simultaneously exports glutamine [19].
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