The role of acidosis sensing in the regulation of chronic inflammation by skeletal muscle

Many chronic inflammatory diseases including Chronic kidney disease (CKD) are characterised by loss of skeletal muscle or cachexia, which leads to decreased mobility and quality of life. Metabolic acidosis is common in CKD and stimulates muscle protein wasting which may further enhance chronic inflammation. In vivo, this muscle wasting by acidosis also requires the presence of glucocorticoid (GC). Metabolic acidosis is thought to act by inhibiting the pH-sensitive System A amino acid transporter protein SNAT2, resulting in impaired global protein synthesis and enhanced global proteolysis. The L6 rat muscle cell model was used to study the effects of glucocorticoid Dexamethasone (DEX) on SNAT2 transport activity. The activity of this transporter was measured from the rate of α-[1-14C]-MeAIB transport into the cells and related to the total protein content of the cultures. To obtain an experimental system in which changes in the SNAT2 protein could be assessed; GFP-tagged human SNAT2 (SNAT2-eGFP) was cloned and expressed in L6 and in the readily transfected cell line HEK293A. DEX significantly inhibited SNAT2 activity in L6 cells. This effect was blunted by vanadate and abolished by vanadate plus amino acid deprivation (AAD). AAD is reported to stabilise SNAT2 protein, and vanadate inhibits both phosphotyrosine phosphatases (PTPs) and proteasomes. More specific PTP inhibitors failed to blunt DEX’s effect on SNAT2 activity. DEX had no effect on SNAT2 mRNA expression but depleted SNAT2 protein assessed by immunoblotting, suggesting that DEX acts through changes in degradation rather than phosphorylation of SNAT2 protein. This was confirmed by blunting the effect of DEX with proteasome inhibitor MG132. Transfection of L6 myoblasts with SNAT2-eGFP cDNA led to protein expression detected by fluorescence, but only a small increase in α-[1-14C]-MeAIB transport. However, expression of SNAT2-eGFP in HEK-293A cells yielded a functionally active transporter protein which was isolated by GFP trapping and confirmed by mass spectrometry to be SNAT2. This isolated protein showed no evidence of phosphorylation, but SNAT2-eGFP fluorescence was significantly reduced by DEX and partly restored by MG132, again suggesting that DEX promotes SNAT2 degradation. SNAT2-eGFP over-expression also significantly activated the amino acid-dependent signalling pathway through mTORC1 to rpS6, a potential stimulator of global protein synthesis; and increased intracellular concentration of the SNAT2 substrate L-Met, a reported stimulator of mTORC1. To test the hypothesis that SNAT2’s transport of L-Met contributes to mTORC1 activation in L6 cells, SNAT2 was inhibited in L6 myoblasts by competitive inhibition with excess MeAIB or by SNAT2 silencing with siRNA. Addition of L-Met alone to L6 cells detectably stimulated mTORC1/rpS6 signalling and MeAIB abolished this effect. However, it was not possible to show by siRNA silencing that SNAT2 alone mediated the effect of L-Met. SNAT2 silencing in L6 did however significantly blunt MAPK signalling (a key mediator of mechanical stress–induced anabolic signals during exercise), suggesting that SNAT2 contributes to anabolic pathways in addition to mTORC1.