Acidosis-sensing glutamine pump SNAT2 determines amino acid levels and mammalian target of rapamycin signalling to protein synthesis in L6 muscle cells

Evans, K., Nasim, Z., Brown, J. , Butler, H., Kauser, S., Varoqui, H., Erickson, J.D., Herbert, T.P. and Bevington, A. (2007) Acidosis-sensing glutamine pump SNAT2 determines amino acid levels and mammalian target of rapamycin signalling to protein synthesis in L6 muscle cells. Journal of the American Society of Nephrology, 18 (5). pp. 1426-1436. ISSN 1046-6673

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Wasting of lean tissue as a consequence of metabolic acidosis is a serious problem in patients with chronic renal failure. A possible contributor is inhibition by low pH of the System A (SNAT2) transporter, which carries the amino acid L-glutamine (L-Gln) into muscle cells. The aim of this study was to determine the effect of selective SNAT2 inhibition on intracellular amino acid profiles and amino acid–dependent signaling through mammalian target of rapamycin in L6 skeletal muscle cells. Inhibition of SNAT2 with the selective competitive substrate methylaminoisobutyrate, metabolic acidosis (pH 7.1), or silencing SNAT2 expression with small interfering RNA all depleted intracellular L-Gln. SNAT2 inhibition also indirectly depleted other amino acids whose intracellular concentrations are maintained by the L-Gln gradient across the plasma membrane, notably the anabolic amino acid L-leucine. Consequently, SNAT2 inhibition strongly impaired signaling through mammalian target of rapamycin to ribosomal protein S6 kinase, ribosomal protein S6, and 4E-BP1, leading to impairment of protein synthesis comparable with that induced by rapamycin. It is concluded that even though SNAT2 is only one of several L-Gln transporters in muscle, it may determine intracellular anabolic amino acid levels, regulating the amino acid signaling that affects protein mass, nucleotide/nucleic acid metabolism, and cell growth.

Cachexia, the wasting of soft tissue, particularly skeletal muscle, is a frequent occurrence in patients with ESRD and is particularly severe in patients with diabetic nephropathy (1). It is a serious clinical problem because of its strong association with morbidity and mortality. An important cause is uremic metabolic acidosis (2), and there is good evidence that correction of acidosis decreases both weight loss and morbidity (3,4). Depletion of intramuscular free amino acids is thought to be an important early step in muscle wasting in uremia (5), and depletion is reversed if acidosis is corrected (6). Intracellular amino acid depletion is sensed through mammalian target of rapamycin (mTOR), a well-documented mechanism through which amino acids stimulate protein synthesis (7). Impaired protein synthesis is an early consequence of metabolic acidosis in humans (8) and is partly compensated for by harvesting of amino acids at the expense of protein degradation (9,10), but precisely how acidosis leads to the initial amino acid depletion is poorly understood.

Availability of the free amino acid L-glutamine (L-Gln) limits protein synthesis (11), protein degradation (12), and nucleotide and nucleic acid biosynthesis (13) in some cell types. Consequently, L-Gln has been proposed as a key factor in growth and maintenance of mammalian tissues (14), and L-Gln availability and losses from muscle may be important contributors to wasting illness and clinical outcome in seriously ill patients (15). A crucial factor is active transport of L-Gln across the plasma membrane (15), maintaining an intracellular concentration at least 20 times higher than the 0.5 to 1 mM found in extracellular fluid (16). In muscle the molecular identity of the transporter(s) involved has been obscure, but studies in cultured L6 skeletal muscle cells (17,18) have implicated System A neutral amino acid transporters of the SNAT/slc38 transporter family, in particular SNAT2 (19). SNAT2 is also a possible mediator of the effects of acidosis because it is strongly inhibited at low pH (20).

This study investigated the SNAT2 transporter in L6 muscle cells as a potential target through which catabolic stimuli trigger protein wasting. We demonstrate, for the first time, that inhibition of SNAT2 alone is sufficient to deplete intracellular L-Gln and other anabolic amino acids, that metabolic acidosis gives similar depletion, and that SNAT2 is consequently a potential regulator of amino acid–dependent signaling to mTOR and hence to protein synthesis.

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Keywords:glutamine, SNAT2, L6
Subjects:A Medicine and Dentistry > A300 Clinical Medicine
ID Code:28230
Deposited On:25 Jul 2018 13:20

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