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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. There is now strong evidence that, even in patients without diabetes, insulin resistance in ESRD is a major cause of muscle wasting1,2 with its attendant morbidity and increased risk for mortality. An important contributor to this clinically serious problem is uremic metabolic acidosis,3 suggesting that low pH has a significant impact on insulin signaling in uremic muscle. The SNAT2 amino acid transporter in the plasma membrane of mammalian cells is strongly inhibited by low extracellular pH.4 We previously showed, using cultured skeletal muscle cells (L6 myotubes), that inhibition of SNAT2 rapidly depletes intracellular amino acids and thereby strongly impairs insulin signaling to protein synthesis through mammalian target of rapamycin (mTOR), which is a key sensor of amino acid availability.5 Although this provides a plausible explanation for the inhibition of muscle protein synthesis that occurs during acute metabolic acidosis in humans,6 the response of muscle to chronic uremic metabolic acidosis in renal patients usually involves increased proteolysis.7,8 A possible rationale for this chronic proteolysis is that it is an adaptation to the initial amino acid depletion, whereby amino acids are harvested from muscle protein to restore intracellular amino acid levels,9 thereby minimizing impairment of protein synthesis but at the expense of chronically elevated proteolysis. The stimulation of proteolysis by low pH in L6 myotubes has been attributed to a defect in insulin signaling through insulin receptor substrate 1 (IRS-1)-associated phosphatidylinositol-3-kinase (PI3K), leading to impaired activation of protein kinase B (PKB),10 and a similar defect has been demonstrated in acidotic and uremic rat skeletal muscle in vivo.11 Unlike mTOR signaling, type I PI3K signaling to PKB is not traditionally regarded as an amino acid–sensitive pathway, suggesting that inhibition of SNAT2 by acidosis is not responsible for this effect; however, a type III PI3K has been implicated in amino acid sensing by mTOR.12 There is also evidence from amino acid–starved L6 myotubes that extracellular amino acid concentration is sensed through SNAT2, which acts as a signaling protein in its own right—a so-called “transceptor”13 that signals to SNAT2 gene expression.13 This signal is blunted by inhibitors of PI3K,13 suggesting that coupling exists between SNAT2 and this enzyme. It is not known, however, whether such coupling influences PI3K signaling to PKB and proteolysis. The aims of this study were, first, to determine whether the effect of metabolic acidosis or SNAT2 inhibition on amino acid levels in L6 myotubes shows an adaptive response consistent with compensatory harvesting of amino acids by proteolysis; second, to determine whether metabolic acidosis or SNAT2 inhibition in the presence of insulin activates proteolysis by signaling through mTOR or PI3K; and, third, to determine whether coupling between SNAT2 and PI3K/PKB signaling is detectable when the activity or expression of SNAT2 is impaired

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.

Metabolic acidosis, a common feature of uremia, has a well documented wasting effect on skeletal muscle. In contrast, the effect of metabolic acidosis on adipose tissue is unknown. Serum levels of the adipocyte hormone leptin have been shown to be lower in acidotic uremic rats when compared with uremic controls. This study investigated the effect of acidosis on leptin protein secretion and leptin gene expression. This was studied in vitro by means of 3T3-L1 cultured adipocytes. Leptin secretion was decreased at an acid pH of 7.1 compared with a control pH of 7.5 (1277 versus 1950 pg/well/48 h, P0.05). In contrast, acidosis did not affect leptin mRNA content. Glucose transport was reduced by 39% at pH 7.1 at 24 h, which was comparable in magnitude with the inhibition of leptin secretion at the same pH. The glucose transport inhibitors cytochalasin B (0.5 to 50 micro M) and phloretin (0.05 to 0.25 mM) mimicked the effect of acidosis and reduced leptin secretion in a dose-dependent fashion (P0.02). Dose-response curves for the inhibition of glucose uptake showed that decreasing glucose transport to the same extent as with acid was sufficient to drive down leptin secretion, independently of changes of leptin mRNA. Acid decreases leptin secretion from 3T3-L1 adipocytes through a post-transcriptional mechanism via changes in glucose transport. This starvation-like response may be physiologically important in conditions such as uremia to prevent excessive energy expenditure.