Inhibition of SNAT2 by Metabolic Acidosis Enhances Proteolysis in Skeletal Muscle

Abstract

Insulin resistance is a major cause of muscle wasting in patients with ESRD. Uremic metabolic acidosis impairs insulin signaling, which normally suppresses proteolysis. The low pH may inhibit the SNAT2 L-Glutamine (L-Gln) transporter, which controls protein synthesis via amino acid-dependent insulin signaling through mammalian target of rapamycin (mTOR). Whether SNAT2 also regulates signaling to pathways that control proteolysis is unknown. In this study, inhibition of SNAT2 with the selective competitive substrate methylaminoisobutyrate or metabolic acidosis (pH 7.1) depleted intracellular L-Gln and stimulated proteolysis in cultured L6 myotubes. At pH 7.1, inhibition of the proteasome led to greater depletion of L-Gln, indicating that amino acids liberated by proteolysis sustain L-Gln levels when SNAT2 is inhibited by acidosis. Acidosis shifted the dose-response curve for suppression of proteolysis by insulin to the right, confirming that acid increases proteolysis by inducing insulin resistance. Blocking mTOR or phosphatidylinositol-3-kinase (PI3K) increased proteolysis, indicating that both signaling pathways are involved in its regulation. When both mTOR and PI3K were inhibited, methylaminoisobutyrate or acidosis did not stimulate proteolysis further. Moreover, partial silencing of SNAT2 expression in myotubes and myoblasts with small interfering RNA stimulated proteolysis and impaired insulin signaling through PI3K. In conclusion, SNAT2 not only regulates mTOR but also regulates proteolysis through PI3K and provides a link among acidosis, insulin resistance, and protein wasting in skeletal muscle cells.

Amino acid transceptors: gate keepers of nutrient exchange and regulators of nutrient signaling

“…Intriguingly, inducing either a "sepsis-like" state in rats by intraperitoneal injection of lipopolysaccharide (66) or muscle atrophy by spinal chord transection (26) markedly reduces the signaling capacity of the TOR pathway in skeletal muscle. Although these latter studies did not examine whether the observed defect in TOR signaling could be attributed to a loss in intramuscular glutamine, reducing the intramyocellular concentration of this amino acid by suppressing the activity or expression of SNAT2 not only enhances cellular proteolysis but impairs mTOR/S6K1 signaling in L6 muscle cells (28,29). It follows from these observations that the fidelity with which amino acids, such as leucine, can exert an influence on cell signaling is critically dependent on not only maintaining cellular hydration but preserving the transmembrane concentration gradient of glutamine and potentially that of other SNAT2 amino acid substrates, which, although regarded as nonessential, are physiologically indispensable from the perspective of preserving the primacy of leucine in terms of TOR signaling (9).…”

“…An additional line of evidence that indicates a potential role for SNAT2 in amino acid signaling comes from the work of Bevington and coworkers, who have recently reported that incubation of muscle cells with a saturating dose of Me-AIB exerts a stimulatory effect on insulin-stimulated PI 3-kinase activity and PKB/Akt phosphorylation (29). In contrast, silencing SNAT2 expression using RNA interference had a suppressive effect on the lipid kinase activity of PI 3-kinase (29).…”

Exercise-induced Biochemical Modifications in Muscle in Chronic Kidney Disease: Occult Acidosis as a Potential Factor Limiting the Anabolic Effect of Exercise

“…CKD It is well documented that myocyte growth is regulated by mTOR, at least partly through the PI3-K/PKB signalling pathway and this is probably the pathway that is stimulated in response to resistance exercise in vivo resulting in muscle hypertrophy 7 . Secondly, possibly through direct inhibitory effects of low pH on active amino acid transporters in skeletal muscle cells 6 , acidosis in CKD significantly depletes intramuscular pools of free amino acids, thus reducing capacity for protein synthesis, and impairing amino acid-dependent anabolic signalling through mTOR.…”

Combined walking exercise and alkali therapy in patients with CKD4–5 regulates intramuscular free amino acid pools and ubiquitin E3 ligase expression

“…L-Leu) by exchanging intracellular non-essential amino acids (e.g. L-Ala) for extracellular L-Leu (Baird et al 2009;Evans et al 2008;Nicklin et al 2009). Indeed, failure of these gradients could explain depletion of a number of the amino acids observed in Figure 1 and Table 5.…”

“…2004), is strongly inhibited by acidosis (Baird et al 2006;Evans et al 2007;Evans et al 2008) and inhibited in CKD (Asola et al 2001). No change was seen in SNAT2 expression (Table 6), but studies in vitro have shown that acidosis directly inhibits this transporter protein (Baird et al 2006;Zhang et al 2011) independent of gene expression (Evans et al 2007;Evans et al 2008). However this inhibition by acid has previously only been studied acutely, and the corresponding chronic effects of acid on this transporter in the context of exercise and mechanical stress merit further investigation.…”