Abstract Zeerak Nasim

The acidosis-sensing amino acid pump SNAT2 determines intracellular levels of the anabolic amino acids L-Gln and L-Leu in L6 muscle cells.
¹Z. Nasim
¹Dept. of Infection, Immunity, and Inflammation, Leicester General Hospital, University of Leicester (United Kingdom)

Introduction
Cachexia arising from metabolic acidosis in uraemia has been attributed to inhibition by low pH of the System A (SNAT2) transporter which carries the anabolic amino acid L-Gln into muscle cells. However SNAT2 is only one of several proteins transporting L-Gln into cells. The aim of this study was therefore to determine the effect of selective SNAT2 inhibition on intracellular amino acid profiles determined by high performance liquid chromatography
in L6 skeletal muscle cells.

Material and Methods
After 2h at an extra cellular pH of 7.1 to model acidosis (which halves SNAT2 activity), intracellular L-Gln fell to 20.2±0.2 nmol/35mm well compared with 38.6±0.9 at control pH (7.4) (P<0.05). Complete inhibition of SNAT2 at pH 7.4 with a saturating dose of the selective competitive substrate methylaminoisobutyrate (MeAIB) further depleted intracellular L-Gln (10.2±0.2, P<0.05). As the L-Gln concentration gradient across the plasma membrane may also drive uptake of anabolic branched chain amino acids (BCAA) notably L-Leu, corresponding effects on BCAA were studied. SNAT2 blockade with MeAIB strongly depleted L-Leu (0.91±0.02 nmol/well, compared with 2.1±0.1, P<0.05 in control cultures). Similar results were seen for L-Ile and L-Val. The role of the L-Gln gradient was confirmed by complete removal of extracellular L-Gln. This imposes a steep inside/outside gradient and, as predicted, elevated L-Leu (to 3.2±0.1, P< 0.05).

Conclusion
It is concluded that even though SNAT2 is only one of several L-Gln transporters in muscle, it has the dominant role in active accumulation of L-Gln and other anabolic amino acids. In view of their importance in regulating protein mass, nucleotide and nucleic acid metabolism, SNAT2 is a key target for anabolic and nutritional therapy in uraemic cachexia.

Keyword(s): Glutamine, Metabolic acidosis, System A, proteolysis