Jeremy Brown

Acidosis-Sensing Glutamine Pump SNAT2 Determines Amino Acid Levels and Mammalian Target of Rapamycin Signalling to Protein Synthesis in L6 Muscle Cells

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.

Inhibition of SNAT2 by Metabolic Acidosis Enhances Proteolysis in Skeletal Muscle

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.

Impaired system A amino acid transport mimics the catabolic effects of acid in L6 cells.

Background Metabolic acidaemia stimulates protein catabolism in skeletal muscle cells, leading to muscle wasting. As this occurs without decreasing cytosolic pH, the initial signal is unclear. A possible explanation is that extracellular pH acts on solute transporters at the cell surface, inhibiting nutrient influx.

Acidosis Downregulates Leptin Production from Cultured Adipocytes through a Glucose Transport-Dependent Post-transcriptional Mechanism

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, P < 0.05). 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 (P < 0.02). 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.

Impaired glycolysis and protein catabolism induced by acid in L6 rat muscle cells.

In skeletal muscle, metabolic acidosis stimulates protein degradation and oxidation of branched‐chain amino acids. This could occur to compensate for impairment of glucose utilization induced by acid.

Inhibition of Protein Synthesis by Acid in L6 Skeletal Muscle Cells: Analogies with the Acute Starvation Response

Impaired protein synthesis (PS) occurs in skeletal muscle during acute starvation. Even though it is well established that uraemic metabolic acidosis (MA) stimulates protein degradation (PD) and is a major contributor to skeletal muscle wasting in chronic renal failure, the accompanying effects of MA on PS are much less clear. Previous work has shown that, in cultured L6 skeletal muscle cells, PD and leucine oxidation are stimulated by acid. The aim of the present study was to determine whether acid (like acute starvation) can also inhibit PS. PS (14C-phenylalanine incorporation) was measured in L6 cells in MEM + 2% serum at acid pH (7.1) or control pH (7.5). After 24 h, acid inhibited PS (7.7 ± 0.2 vs. 8.9 ± 0.1 nmol Phe/4 h/35-mm culture well in controls, p = 0.01) and this was maintained at 72 h. In vitro this could arise because acid only inhibits the rapid PS occurring in dividing cells. However, when division was abolished with 10–5 mol/l cytosine arabinoside, PS inhibition by acid still occurred (6.9 ± 0.1 vs. 8.3 ± 0.2 at control pH, p < 0.05). Acid also had no effect on the specific radioactivity of cellular phenylalanine, suggesting that the impaired PS was not a consequence of inadequate labelling of this pool. Elevated PD and impaired PS together led to loss of 7% of the total protein in only 28 h (–21 ± 3 µg/well, p = 0.004). This combination of impaired PS with increased PD and increased leucine oxidation in response to acid resembles the response of skeletal muscle to acute starvation. These superficial similarities between the starvation state and MA suggest that fundamental metabolic signals may occur which are common to both states.

Leucine suppresses acid‐induced protein wasting in L6 rat muscle cells

Background Metabolic acidosis induces protein wasting in skeletal muscle cells, accompanied by decreased glycolysis and compensatory increased consumption of other metabolic fuels, implying that protein wasting arises from fuel starvation and might be rectified by fuel supplements.

Sialic acid supplementation ameliorates puromycin aminonucleoside nephrosis in rats

Defects in sialylation are known to have serious consequences on podocyte function leading to collapse of the glomerular filtration barrier and the development of proteinuria. However, the cellular processes underlying aberrant sialylation in renal disease are inadequately defined. We have shown in cultured human podocytes that puromycin aminonucleoside (PAN) downregulates enzymes involved in sialic acid metabolism and redox homeostasis and these can be rescued by co-treatment with free sialic acid. The aim of the current study was to ascertain whether sialic acid supplementation could improve renal function and attenuate desialylation in an in vivo model of proteinuria (PAN nephrosis) and to delineate the possible mechanisms involved. PAN nephrotic rats were supplemented with free sialic acid, its precursor N-acetyl mannosamine or the NADPH oxidase inhibitor apocynin. Glomeruli, urine, and sera were examined for evidence of kidney injury and therapeutic efficacy. Of the three treatment regimens, sialic acid had the broadest efficacy in attenuating PAN-induced injury. Proteinuria and urinary nephrin loss were reduced. Transmission electron microscopy revealed that podocyte ultrastructure, exhibited less severe foot process effacement. PAN-induced oxidative stress was ameliorated as evidenced by a reduction in glomerular NOX4 expression and a downregulation of urine xanthine oxidase levels. Sialylation dysfunction was improved as indicated by reduced urinary concentrations of free sialic acid, restored electrophoretic mobility of podocalyxin, and improved expression of a sialyltransferase. These data indicate that PAN induces alterations in the expression of enzymes involved in redox control and sialoglycoprotein metabolism, which can be ameliorated by sialic acid supplementation possibly via its properties as both an antioxidant and a substrate for sialylation.