Inhibition of SNAT2 by Metabolic Acidosis Enhances Proteolysis in Skeletal Muscle
Kate Evans*,
Zeerak Nasim*,
Jeremy Brown*,
Emma Clapp,
Amin Amin*,
Bin Yang*,
Terence P. Herbert and
Alan Bevington*
* Department of Infection, Immunity and Inflammation, University of Leicester, John Walls Renal Unit, Leicester General Hospital, and Department of Cell Physiology and Pharmacology, University of Leicester, Maurice Shock Medical Sciences Building, Leicester, and School of Sport and Exercise Sciences, Loughborough University, Loughborough, United Kingdom
Correspondence: Dr. Alan Bevington, Department of Infection, Immunity and Inflammation, University of Leicester, John Walls Renal Unit, Leicester General Hospital, Leicester LE5 4PW, UK. Phone: +44-116-258-8041; Fax: +44-116-258-4764; E-mail: ab74{at}leicester.ac.uk
Received for publication October 16, 2007.
Accepted for publication May 26, 2008.
Insulin resistance is a major cause of muscle wasting in patientswith ESRD. Uremic metabolic acidosis impairs insulin signaling,which normally suppresses proteolysis. The low pH may inhibitthe SNAT2 l-Glutamine (L-Gln) transporter, which controls proteinsynthesis via amino acid–dependent insulin signaling throughmammalian target of rapamycin (mTOR). Whether SNAT2 also regulatessignaling to pathways that control proteolysis is unknown. Inthis study, inhibition of SNAT2 with the selective competitivesubstrate methylaminoisobutyrate or metabolic acidosis (pH 7.1)depleted intracellular L-Gln and stimulated proteolysis in culturedL6 myotubes. At pH 7.1, inhibition of the proteasome led togreater depletion of L-Gln, indicating that amino acids liberatedby proteolysis sustain L-Gln levels when SNAT2 is inhibitedby acidosis. Acidosis shifted the dose-response curve for suppressionof proteolysis by insulin to the right, confirming that acidincreases proteolysis by inducing insulin resistance. BlockingmTOR or phosphatidylinositol-3-kinase (PI3K) increased proteolysis,indicating that both signaling pathways are involved in itsregulation. When both mTOR and PI3K were inhibited, methylaminoisobutyrateor acidosis did not stimulate proteolysis further. Moreover,partial silencing of SNAT2 expression in myotubes and myoblastswith small interfering RNA stimulated proteolysis and impairedinsulin signaling through PI3K. In conclusion, SNAT2 not onlyregulates mTOR but also regulates proteolysis through PI3K andprovides a link among acidosis, insulin resistance, and proteinwasting in skeletal muscle cells.
There is now strong evidence that, even in patients withoutdiabetes, insulin resistance in ESRD is a major cause of musclewasting1,2 with its attendant morbidity and increased risk formortality. An important contributor to this clinically seriousproblem is uremic metabolic acidosis,3 suggesting that low pHhas a significant impact on insulin signaling in uremic muscle.
The SNAT2 amino acid transporter in the plasma membrane of mammaliancells is strongly inhibited by low extracellular pH.4 We previouslyshowed, using cultured skeletal muscle cells (L6 myotubes),that inhibition of SNAT2 rapidly depletes intracellular aminoacids and thereby strongly impairs insulin signaling to proteinsynthesis through mammalian target of rapamycin (mTOR), whichis a key sensor of amino acid availability.5 Although this providesa plausible explanation for the inhibition of muscle proteinsynthesis that occurs during acute metabolic acidosis in humans,6the response of muscle to chronic uremic metabolic acidosisin renal patients usually involves increased proteolysis.7,8A possible rationale for this chronic proteolysis is that itis an adaptation to the initial amino acid depletion, wherebyamino acids are harvested from muscle protein to restore intracellularamino acid levels,9 thereby minimizing impairment of proteinsynthesis but at the expense of chronically elevated proteolysis.
The stimulation of proteolysis by low pH in L6 myotubes hasbeen attributed to a defect in insulin signaling through insulinreceptor substrate 1 (IRS-1)-associated phosphatidylinositol-3-kinase(PI3K), leading to impaired activation of protein kinase B (PKB),10and a similar defect has been demonstrated in acidotic and uremicrat skeletal muscle in vivo.11 Unlike mTOR signaling, type IPI3K signaling to PKB is not traditionally regarded as an aminoacid–sensitive pathway, suggesting that inhibition ofSNAT2 by acidosis is not responsible for this effect; however,a type III PI3K has been implicated in amino acid sensing bymTOR.12 There is also evidence from amino acid–starvedL6 myotubes that extracellular amino acid concentration is sensedthrough SNAT2, which acts as a signaling protein in its ownright—a so-called "transceptor"13 that signals to SNAT2gene expression.13 This signal is blunted by inhibitors of PI3K,13suggesting that coupling exists between SNAT2 and this enzyme.It is not known, however, whether such coupling influences PI3Ksignaling to PKB and proteolysis.
The aims of this study were, first, to determine whether theeffect of metabolic acidosis or SNAT2 inhibition on amino acidlevels in L6 myotubes shows an adaptive response consistentwith compensatory harvesting of amino acids by proteolysis;second, to determine whether metabolic acidosis or SNAT2 inhibitionin the presence of insulin activates proteolysis by signalingthrough mTOR or PI3K; and, third, to determine whether couplingbetween SNAT2 and PI3K/PKB signaling is detectable when theactivity or expression of SNAT2 is impaired.
Time Course of Intracellular l-Glutamine Depletion by SNAT2 Inhibition
As demonstrated previously,5 the 40% inhibition of the activityof the SNAT2 transporter that occurs on lowering the pH to 7.15leads within 2 h to a fall in the concentration of many aminoacids in L6-G8C5 myotubes, most notably a 25% fall in the largel-Glutamine (L-Gln) pool (Figure 1A); however, in longer incubationsof 7 or 24 h, the L-Gln–depleting effect of low pH wasblunted, no longer achieving statistical significance (Figure 1,B and C). When amino acid influx through SNAT2 was completelyinhibited with a saturating competing dose of the SNAT2 substratemethylaminoisobutyrate (MeAIB), strong L-Gln depletion was observedat 2 h5 (Figure 1A), but no subsequent recovery occurred (Figure 1,B and C). The cells therefore apparently possess mechanism(s)to bolster L-Gln levels in the face of partial SNAT2 inhibitionin chronic acidosis but not when SNAT2 is saturated with MeAIB.
Figure 1. (A through C) Time course of the effect of low pH or MeAIB (10 mM) on the intracellular concentration of L-Gln in L6-G8C5 myotubes, with MG132 (10 µM) or methionine sulfoximine (MSO; 1 mM). Media were based on MEM and contained 2 mM L-Gln with 2% dialyzed FBS. Pooled data from three independent experiments are shown (with at least three replicate culture wells for each treatment). In 24-h incubations, MG132 and MSO were present only for the last 7 h. (D) l-Glutamine synthetase catalytic activity in lysates from L6-G8C5 myotubes incubated for 48 h at pH 7.4 without L-Gln or with 2 mM L-Gln at the specified pH or with 2 mM L-Gln at pH 7.4 with 10 mM MeAIB or 1 mM MSO. Fresh medium of the same composition was added after 24 h. Pooled data from five independent experiments are shown (with at least three replicate culture wells for each treatment). *P < 0.05 versus the corresponding control value at pH 7.4.
Role of SNAT2 Upregulation and L-Gln Synthetase in Restoring L-Gln Levels during Acidosis
Complete amino acid starvation of L6 myotubes leads within afew hours to upregulation of SNAT2.13 A possible explanationtherefore for the apparent L-Gln adaptive response to acidosis(and its failure on complete SNAT2 inhibition with MeAIB) isthat intracellular amino acid depletion during acidosis upregulatesSNAT2, thereby increasing L-Gln influx; however, no significantincrease in expression of the SNAT2 protein was detected inresponse to 6 h at low pH, even though a clear increase wasobserved on complete amino acid starvation (Figure 2, A andB). SNAT2 transporter activity across the plasma membrane wasalso strongly activated by amino acid starvation (Figure 2C),but, again, no significant increase was observed in responseto 6 h of exposure to acid when transport was assayed immediatelyafter restoring the pH from 7.1 to 7.4 (Figure 2C).
Figure 2. Effect of 6 h of incubation in MEM with 2% dialyzed FBS at the specified pH or at pH 7.4 with no amino acids (No AA), on SNAT2 expression in L6-G8C5 myotubes. (A) Immunoblots of proteins separated by SDS-PAGE from a 170,000 x g membrane preparation, probing with SNAT2-specific antibody (or 1-Na,K-ATPase antibody or Annexin II antibody as loading controls). (B) Quantification by densitometry of the principal 65-kD SNAT2 band in blots as in A. Pooled data are presented from four independent experiments. (C) Assay of SNAT2 transporter activity. After 6 h of incubation, cultures were rinsed with HEPES-buffered balanced salt solution (at pH 7.4 for all cultures), and uptake of 14C-labeled MeAIB was measured at 25°C (see the Concise Methods section). Representative assay from three independent experiments. *P < 0.05 versus the pH 7.4 control value.
Complete removal of L-Gln from the extracellular medium activatesthe enzyme L-Gln synthetase in these cells (Figure 1D); however,no such effect occurred in response to partial depletion ofL-Gln with acid or MeAIB (Figure 1D). The L-Gln synthetase inhibitormethionine sulfoximine, at a dosage that abolishes activityof the enzyme (Figure 1D), also gave no detectable depletionof intracellular L-Gln during acidosis (Figure 1, B and C),confirming that L-Gln synthesis is not a major factor sustainingL-Gln levels in acidosis.
Proteolysis Maintains Intracellular L-Gln Levels during Acidosis
Increased global proteolysis is readily demonstrable in L6-G8C5myotubes after as little as 7 h of exposure to medium at pH7.114 (Figure 3A), and this pH dependence is still observedin the presence of Actinomycin D (Figure 3A), suggesting thatits initiation requires only posttranscriptional events. Thisincreased degradation coincides with the time at which the L-Glndepletion effect is blunted (Figure 1, B and C). Blockade ofproteolysis with the proteasome inhibitor MG132 enhanced theL-Gln–depleting effect of low pH, prolonging it to 7 and24 h (Figure 1, B and C), demonstrating that the stimulationof proteolysis by acidosis has a significant role in maintainingintracellular L-Gln levels at these time points.
Figure 3. (A) Effect of 1 µM Actinomycin D (Act D) on proteolysis rate in L6-G8C5 myotubes during 7-h incubations at the specified pH in MEM + 2 mM L-Phe + 2% dialyzed FBS. Pooled data from four independent experiments are shown (with three replicate culture wells for each treatment). Proteolysis was assessed from the rate of release of radioactivity into the medium from cultures prelabeled with 3H-L-Phe and is expressed as the logarithm of the percentage of the total initial cellular radioactivity per hour (log10 %/h x 1000; see the Concise Methods section). (B) Typical delabeling time course of 3H-L-Phe–prelabeled cells incubated in serum-free MEM + 2 mM L-Phe at the specified pH with 100 nM insulin. Linear regression slopes of plots of this type are presented in A and C and Figure 4. (C) Effect of pH, insulin, and high-dosage IGF-1 on the rate of proteolysis (delabeling) expressed as log10 %/h x 1000 in L6-G8C5 myotubes. Pooled data from three independent experiments are shown (with three replicate culture wells for each treatment). *P < 0.05 versus the corresponding pH 7.4 control value; **P < 0.05 versus the corresponding pH 7.7 value.
Increased Proteolysis in Response to Acid Involves Insulin Resistance and SNAT2
If, as proposed elsewhere,10 proteolysis induced by acidosisarises from insulin resistance, then the dose-response curvefor suppression of proteolysis by insulin or IGF-I should beright-shifted by acid, with the effect of acid disappearingboth in insulin-free medium and when excess insulin or IGF-Iis added to overcome the resistance. In serum-free medium withinsulin or IGF-I as the sole anabolic factor, this hypotheticalright shift was clearly observed (Figure 3, B and C). No pHeffect was observed in serum-free medium alone, and the effectof pH was abolished with high-dosage IGF-I.
The effect of selective inhibition of SNAT2 on proteolysis wastested by selective silencing of SNAT2 expression using a smallinterfering RNA (siRNA) that we characterized previously.5 Treatmentof myotubes with the silencing siRNA reduced SNAT2 transporteractivity by 35% (Figures 4A and 5A), a reduction comparablein magnitude to that observed on lowering extracellular pH to7.15 (Figure 5A). This gave a clear stimulation of global proteolysis(Figure 4B), and, as predicted, the increase in proteolysiswas similar in magnitude to that induced by acidosis (Figure 3C)and yielded net protein wasting (Figure 4C) like that previouslyobserved in acid-treated myotubes.5,14 Furthermore, the previouslyreported stimulation of proteolysis when the amino acid influxthrough SNAT2 was blocked with MeAIB5,14 was also observed inserum-free medium with insulin (Figure 4D).
Figure 4. (A through C) Effect of siRNA silencing of SNAT2 in L6-G8C5 myotubes. Pooled data from three independent experiments are shown (with three replicate culture wells for each treatment). T, cultures incubated with calcium phosphate transfection blank; Scr, scrambled control siRNA; Sil, SNAT2 silencing siRNA. Transfection was followed by an 8-h incubation in DMEM + 10% FBS followed by 16 h in MEM + 2% dialyzed FBS. All measurements were then made in parallel as follows: SNAT2 transporter activity (A) and proteolysis rate during 24 h in MEM + 2 mM L-Phe + 2% dialyzed FBS at pH 7.4 (B). Cultures were prelabeled by incubating with 3H-L-Phe for 72 h (including the transfection incubation) before the delabeling measurements. (C) Protein content of the cultures in B. *P < 0.05 versus Scr control. (D) Effect of 100 nM rapamycin (Rap) and 12.5 µM LY294002 (LY) on proteolysis rate in L6-G8C5 myotubes as in Figure 3B during incubation at pH 7.1, pH 7.4, or pH 7.4 with 10 mM MeAIB in serum-free MEM with 100 nM insulin. *P < 0.05 versus the corresponding pH 7.4 control value; +P < 0.05 versus cultures at pH 7.4 without Rap or LY; #P < 0.05 versus cultures with 10 mM MeAIB without LY. Pooled data from four independent experiments are shown (with three replicate culture wells for each treatment).
Figure 5. Comparison of the effects of siRNA silencing of SNAT2 and of acidosis on PI3K activity in L6-G8C5 myotubes. Transfection (see Figure 4) was followed by 24 h in DMEM + 10% FBS. Cells were subjected to an additional 2-h incubation in serum-free MEM, at the specified pH with 100 nM insulin before preparing lysates for PI3K assay. (A) SNAT2 transporter activity assayed at the specified pH. (B) Representative autoradiograph of thin-layer chromatography plate showing 32P-labeled phosphatidylinositol-3-phosphate generated by the PI3K lipid kinase reaction (see the Concise Methods section). (C) Quantification by densitometry of data pooled from three independent experiments performed as in B, expressed as percentage of the pH 7.4 Scr control value. *P < 0.05 versus the pH 7.4 Scr control.
Involvement of mTOR and PI3K Signaling in Acid-Induced Proteolysis
In the presence of insulin, the stimulatory effects of acidor MeAIB on proteolysis were mimicked when insulin signalingthrough mTOR and PI3K was inhibited with rapamycin and LY294002,respectively (Figure 4D). In the presence of the stimulatoryeffect on proteolysis of rapamycin or LY294002 alone, the stimulatoryeffect of acid or MeAIB no longer reached statistical significance(Figure 4D). In the presence of rapamycin plus LY294002 combined,acidosis or MeAIB exerted no further stimulatory effect (Figure 4D),consistent with the idea that low pH, acting through SNAT2,signals to proteolysis by impairing insulin signaling to mTORand/or PI3K.
Low pH or selective inhibition of SNAT2 is already known toimpair mTOR signaling in these cells.5 Inhibition by low pHof PI3K and PKB also was reported previously10 and was confirmedin myotubes in this study (Figures 5C; 6, A and B; and 7). Theeffect of selective SNAT2 inhibition on insulin signaling toPI3K and PKB was investigated by silencing SNAT2 expressionwith siRNA in myoblasts that efficiently reduced SNAT2 proteinexpression (Figure 8A) and SNAT2 transporter activity (Figures 8Band 9A). Consistent with the effects of acidosis, silencingof SNAT2 alone significantly impaired both PI3K lipid kinaseactivity (Figure 8, C through E) and PKB activation (Figure 9,B and C). The weaker silencing of SNAT2 obtained in myotubes(Figures 4A and 5A; similar in magnitude to the impairment ofSNAT2 activity observed with acid [Figure 5A]) also gave animpairment of PI3K signaling similar to that observed with acid(Figure 5, B and C), consistent with the similar stimulationof proteolysis observed in myotubes with SNAT2 silencing (Figure 4B)and acid (Figure 3C).
Figure 6. (A and B) Effect on PI3K activity in L6-G8C5 myotubes of 2-h incubations at the specified pH with or without 10 mM MeAIB or 12.5 µM LY294002. All media contained serum-free MEM with 100 nM insulin. Acute, cultures in which MeAIB was present during the 2-h incubation; Preinc, cultures in which MeAIB was absent during the 2-h incubation but had been present in the preceding 4 h in MEM with 2% dialyzed FBS. (A) Representative PI3K assay autoradiograph. (B) Pooled quantification data obtained by densitometry of six independent experiments performed as in A, expressed as percentage of the control value at pH 7.4. *P < 0.05 versus the pH 7.4 control. (C) Effect of incubation of PI3K immunoprecipitates with amino acids or LY294002 only during the kinase assay incubation with 32P-ATP. Representative autoradiograph from one of two experiments.
Figure 7. Effect on PKB activation in L6-G8C5 myotubes of 2-h incubations at the specified pH with or without 10 mM MeAIB as in Figure 6. All media contained serum-free MEM with 100 nM insulin. (A) Representative immunoblot showing PKB activation assessed from phosphorylation of PKB at Ser 473. (B) Pooled quantification data from 18 independent experiments performed as in A expressed as percentage of the control value at pH 7.4. *P < 0.05 versus the pH 7.4 control.
Figure 8. Effect of siRNA silencing of SNAT2 on PI3K activity as in Figure 5 but with L6-G8C5 myoblasts. (A) Confirmation by immunoblotting of SNAT2 silencing. Proteins from a 170,000 x g membrane preparation separated by SDS-PAGE were probed with SNAT2-specific antibody (or 1-Na,K-ATPase antibody as a loading control). Experiments in B through E were run in parallel on the same cells. Pooled data from three independent experiments are presented. (B) SNAT2 transporter activity. (C) Representative autoradiograph of PI3K assay thin-layer chromatography plate. (D) Quantification of pooled experiments performed as in C. (E) Quantification by liquid scintillation counting of 32P spots scraped from the plates in D. *P < 0.05 versus the Scr control.
Figure 9. (A through C) Effect of siRNA silencing of SNAT2 on PKB activation in L6-G8C5 myoblasts as in Figure 8. Experiments were run in parallel on the same cells. Pooled data from five independent experiments are presented. (A) SNAT2 transporter activity. (B) Representative immunoblot showing PKB activation assessed from phosphorylation of PKB at Ser 473. (C) Quantification by densitometry of pooled experiments performed as in B, expressed as percentage of the Scr control value. *P < 0.05 versus the Scr control. (D) Correlation between the degree of silencing of SNAT2 and the accompanying degree of inhibition of the P-PKB and PI3K signals. Data are plotted from the five PKB experiments in A and C (•) and the three PI3K experiments in Figure 8, B and D (). Spearman rank correlation coefficients: All data R = 0.74, P < 0.04; PKB data only R = 0.90, P < 0.04. (E) Representative experiment showing the effect of pH on the residual PI3K activity in myoblasts in which SNAT2 had been silenced as in Figure 8, but the ensuing 2-h incubation with 100 nM insulin was performed either at pH 7.1 or at pH 7.4.
When the myoblast signaling data from Figures 8D and 9C wereplotted against the degree of SNAT2 silencing in those eightexperiments, signaling was found to decline approximately inproportion to the extent of SNAT2 silencing (Figure 9D). Consequently,in strongly SNAT2-silenced myoblasts, there is little residualPI3K signal on which low pH can exert an inhibitory effect (Figure 9E);therefore, at least in myoblasts, inhibition by low pH of SNAT2-independentcontributors to PI3K/PKB signaling seems unlikely to be a majorcontributor to the effect of acid on PI3K/PKB.
Mechanism of SNAT2 Effects on PI3K and PKB
The SNAT2 protein is thought to be able to signal in L6 skeletalmuscle cells by two mechanisms (Figure 10): Through SNAT2 actingas a transporter, influencing intracellular amino acid concentration5(which might then act directly on PI3K), or through the recentlyproposed SNAT2 "transceptor" mechanism, in which a signal isgenerated by a SNAT2/amino acid substrate complex13 acting independentof intracellular amino acid levels. In principle, these twomechanisms can be distinguished by saturating SNAT2 with itssynthetic substrate MeAIB, which should competitively inhibitthe first mechanism and stimulate the second (Figure 10). Theobserved stimulation of both PI3K and PKB by MeAIB (Figures 6and 7) suggests that the second mechanism rather than the firstpredominates. This conclusion of acidosis inhibiting PI3K throughSNAT2 by a pathway independent of the amino acid influx throughSNAT2 is further supported by the failure of amino acids toexert any acute stimulatory effect on the catalytic activityof immunoprecipitated PI3K (Figure 6C), the failure of extracellularamino acid starvation to block pH sensitivity of PI3K (Figure 11),and the apparent blunting of pH sensitivity of PI3K when SNAT2is saturated with MeAIB (Figure 11).
Figure 10. Proposed scheme whereby the pH-sensitive SNAT2 amino acid transporter and the putative SNAT2/amino acid substrate "transceptor" complex influence amino acid signaling and global proteolysis in L6-G8C5 rat skeletal muscle cells. Dashed lines denote the inhibitory effect of low extracellular pH on the SNAT2 transporter and the resulting inhibition of PI3K and depletion of intracellular free amino acid pools. White arrows indicate known or suspected effects of amino acids whose intracellular concentrations are directly (e.g., L-Gln) or indirectly (e.g., L-Leu) regulated by SNAT2 transporter activity. , Naturally occurring metabolizable amino acid substrates of SNAT2; , synthetic nonmetabolizable SNAT2 substrate MeAIB.
Figure 11. Influence of amino acid starvation (No AA) or saturation of SNAT2 with 10 mM MeAIB on the pH sensitivity of PI3K lipid kinase activity in L6-G8C5 myotubes. Cells were incubated for 2 h at the specified pH with or without amino acids or MeAIB. All media contained serum-free MEM with 100 nM insulin. For No AA cultures, this 2-h incubation was preceded by 2 h in MEM without amino acids and with 2% dialyzed FBS. (A) Representative PI3K assay autoradiograph. (B) Pooled quantification data obtained by densitometry of three independent experiments performed as in A. *P < 0.05 versus the corresponding pH 7.4 control.
SNAT2 Regulates mTOR, PI3K, PKB, and Proteolysis
This study is the first to demonstrate that the acidosis-sensitiveSNAT2 transporter is coupled to PI3K, PKB, and proteolysis inskeletal muscle cells. Viewed in conjunction with our previousreport of regulation of mTOR and protein synthesis by SNAT2,5this strongly suggests that this transporter is a key playerin the acid-induced insulin resistance that is regarded as aprime cause of cachexia in patients with acidosis and uremia.
In this study (Figure 4), 35% silencing of SNAT2 activity inmyotubes using siRNA led to a reproducible 11% stimulation ofglobal proteolysis. We showed previously that a similar degreeof silencing also leads to a 12% decrease in global proteinsynthesis.5 Together, these effects impose negative nitrogenbalance on the cells, leading to a significant but nontoxicdecline in the protein content of the cultures5,14 (Figure 4C).From 11C-MeAIB positron emission tomography of skeletal musclein vivo, SNAT2 transport activity in patients with chronic renalfailure has been estimated to be 23% lower than in healthy controlsubjects.15 The effects reported here therefore seem comparableto those in vivo and are of sufficient magnitude to contributeto uremic cachexia.
Linkage between Protein Synthesis and Degradation during Acidosis
Evidence from inhibition of proteolysis with MG132 (Figure 1)suggests that the previously reported5 SNAT2-mediated depletionof intracellular amino acids (leading to impaired mTOR signalingand impaired protein synthesis during acidosis) may be bluntedby amino acids generated by subsequent degradation of cell protein(Figure 10). Such a mechanism may explain why impaired proteinsynthesis is observed in response to acute metabolic acidosisin humans6 but not during prolonged acidosis (e.g., in chronicrenal failure) in which degradation of large myofibrillar proteinpools and, hence, liberation of amino acids in the cytosol ischronically stimulated.8
This raises the important mechanistic question of whether proteolysisduring metabolic acidosis is stimulated exclusively by intracellularamino acid depletion triggered by inhibition of SNAT2 activity5(which might therefore be amenable to amino acid supplementationtherapy) or through some other mechanism for sensing of lowextracellular pH (e.g., through effects of pH on SNAT2 otherthan inhibition of the amino acid influx through the transporter).Evidence from this and the previous study5 suggests that bothmechanisms may contribute as follows (Figure 10).
SNAT2 Signaling to Proteolysis through Amino Acid Depletion
The inhibitor studies in Figure 4D suggest that both mTOR signalingand PI3K signaling are involved in the regulation of proteolysisin these cells. Inhibition of SNAT2 leads to significant intracellulardepletion of L-Gln and also indirectly to depletion of the branched-chainamino acids L-Leu, L-Ile, and L-Val,5 which are well establishedas activators of mTOR signaling in skeletal muscle cells, includingL6.16 Although mTOR signaling is probably an important contributor,other amino acid–sensing pathways also possibly contribute,for example, the recently described L-Leu suppressible activationof proteolysis in muscle that occurs through the double-strandedRNA-dependent protein kinase PKR.17 The involvement of pathwaysother than PI3K/PKB in regulation of proteolysis is also shownby the effect of MeAIB, which clearly activates proteolysis5,14(Figure 4D), even though it fails to inhibit PI3K and PKB (Figures 6and 7). This failure of MeAIB to generate one of the potentiallycatabolic signals that are generated by low pH, leading to adisproportionately small generation of amino acids from proteolysis,presumably explains the failure of MeAIB-induced proteolysisto rectify L-Gln depletion during treatment with MeAIB in Figure 1,B and C.
Selective silencing of SNAT2 with siRNA led to significant impairmentof PI3K and PKB activation in the presence of insulin (Figures 5,8, and 9). Near-complete silencing of SNAT2 was achieved onlyin myoblasts (Figures 8 and 9), but a proportionate decreasein PI3K activity was also observed with the partial silencingof SNAT2 achieved in myotubes (Figure 5). Unlike mTOR, PI3Kand PKB are not routinely regarded as responsive to amino acids.Nevertheless, precedents do exist for amino acid effects onPI3K. Activation of the type III PI3K hVps34 by amino acidsis well documented,12 and, under PI3K assay conditions suchas those described in this study, PI3K activity in L-Leu–starvedL6 cells was strongly activated in response to 2 mM L-Leu.18Even though L-Leu is a poor SNAT2 substrate, the L-Leu effectwas short-lived, and no PKB activation resulted from it,16,18the previous report is relevant to this study because L-Leuloading of L-Leu–starved L6 cells is a potent activatorof SNAT2.18
SNAT2 Signaling Independent of Amino Acid Transport
Although the intracellular amino acids supplied through SNAT2may have a role in the coupling between SNAT2 and PI3K, thisis unlikely to be the full explanation. Direct addition of aminoacids did not activate immunoprecipitated PI3K (Figure 6C),blocking amino acid influx through SNAT2 with MeAIB did notinhibit PI3K (Figure 6), and amino acid starvation in intactcells has consistently failed to inhibit insulin signaling toPKB19,20 (the last possibly because of the strong countervailingupregulation of SNAT2 that occurs on complete amino acid starvation13[Figure 2]). This leads to the interesting possibility thatthe SNAT2 protein signals to PI3K and PKB through a pathwayindependent of intracellular amino acid levels (Figure 10),analogous to the mechanisms that have been postulated to explainsignal generation by the Ssy1 protein in yeast,21 signalingfrom the Path amino acid transporter to dTOR in Drosophila,22and sensing of amino acid availability in L6 myotubes.13 Ofparticular relevance is the last study, which proposed thatpart of the signal that suppresses SNAT2 gene expression, whenSNAT2 amino acid substrates are added to L6 cells, arises notfrom amino acids carried into the cell by SNAT2 but from theSNAT2/amino acid substrate complex. Signaling through such acomplex (Figure 10) may explain why PI3K and PKB in this studywere inhibited by siRNA silencing of SNAT2 (Figures 5, 8, and9) but activated when the cells were saturated with SNAT2 substrateMeAIB (Figures 6 and 7). A further relevant finding is thatSystem A–type transporters (which include SNAT2) associatewith integrin 3β123 and therefore possibly co-localizewith adhesion kinases that are potent activators of PI3K.24
In conclusion, the SNAT2 amino acid transporter is a major acutedeterminant of insulin sensitivity in cultured skeletal musclecells and exerts functionally significant effects on both proteinsynthesis and proteolysis. In view of its sensitivity to acidosisand its reported inhibition in uremia, it is an important targetfor further research in uremic cachexia. In particular, it willbe of interest to determine whether the effects of SNAT2 inhibitionobserved in this acute culture model are also of importancein vivo in response to chronic inhibition of SNAT2 by acidosis.It will also be interesting to know whether the dominant roleof SNAT2 (and the other SNAT transporters expressed in vivoat lower level) is as net transporters of amino acids5 or asamino acid sensors13 that might exert functionally importanteffects even at low levels of expression.
Cell Culture
L6-G8C5 rat myoblasts were grown to confluence in DMEM with10% vol/vol FBS and fused to form myotubes by culturing in MEMwith 2% vol/vol FBS as described previously.14 For experimentswith unfused myoblasts, cells were seeded in DMEM/10% serumat 3.4 x 104/cm2, fresh medium was added after 24 h, and myoblastswere used after an additional 17 h. Unless otherwise stated,experimental incubations were performed in MEM with 2 mM l-glutamine,penicillin (105 IU/L), streptomycin (100 mg/L), and 2% vol/voldialyzed FBS. At the end of experimental incubations, lysatesfor PI3K and PKB signaling studies and a 170,000 x g total membranefraction containing SNAT2 were prepared as described previously.25,26For determination of l-glutamine synthetase (glutamate-ammonialigase E.C. 6.3.1.2), cultures were washed twice with PBS andstored at –80°C in 50 mM imidazole (pH 6.8) beforecolorimetric determination of enzyme activity with hydroxylamineas described previously.27
Proteolysis was measured by prelabeling of cell protein withl-[2,6 3H]-Phe (Amersham [Little Chalfont, UK] TRK552) and measurementof release of acid-soluble radioactivity into the medium,28expressed as log10 of the percentage of the total initial cellular3H per hour.28 Cell viability was monitored by measurement ofrelease of acid-precipitable radioactivity into the medium (anindicator of intact protein leakage and cell detachment)29 andwas unaffected by the culture conditions described in this study.Total cell protein was determined as described previously.14
SNAT2 transporter activity was assayed from uptake of 14C-methylaminoisobutyrate(MeAIB; NEN-Du Pont, Beaconsfield, UK) into intact myotubesduring 5-min incubations30 with 10 µmol/L MeAIB at 0.5mCi (18.5 MBq)/L. For subconfluent myoblasts, this was increasedto 40 µmol/L at 2 mCi (74 MBq)/L. Other SNAT transporterscapable of carrying 14C-MeAIB are not expressed in these cells.5
PI3K Activity.
PI3K lipid kinase activity was determined as described previously.31Briefly, cells were lysed by scraping in buffer comprising 137mM NaCl, 2.7 mm KCl, 1 mM MgCl2, 1 mM CaCl2, 1% vol/vol IGEPALCA-630, 10% vol/vol glycerol, 1 mg/ml BSA, 20 mM Tris, 0.5 mMNa3VO4, 0.2 mM PMSF, 10 µg/ml leupeptin, 10 µg/mlantipain, 10 µg/ml pepstatin A, and 10 µg/ml aprotininadjusted to pH 8.0 at 4°C. PI3K was immunoprecipitated fromlysates using anti-phosphotyrosine antibody (Santa Cruz PY99;Santa Cruz Biotechnology, Santa Cruz, CA). Washed immunoprecipitateswere then incubated for exactly 5 min at 37°C with assaysubstrates (-32P-ATP and phosphatidylinositol) followed by immediatechilling on ice and extraction of 32P-labeled lipid productinto chloroform/methanol (1:2 vol/vol). 32P-labeled phosphatidylinositol-3-phosphatewas then resolved by thin-layer chromatography and visualizedby autoradiography of the thin-layer chromatography plate. Theproduct was quantified by densitometry of the autoradiograph,and, in some experiments, the result was confirmed by scrapingthe labeled spot off the plate and quantifying the scraped 32Pby liquid scintillation counting. Samples were counted untilat least 1000 counts had accumulated.
Amino Acid Analysis.
Cultures on 35-mm wells were rapidly chilled on ice, rinsedthree times with ice-cold 0.9% wt/vol NaCl, and deproteinizedby scraping in 150 µl of 0.3 M perchloric acid. Precipitatedprotein was sedimented (10 min, 4°C, 3000 x g) and retainedfor total protein assay. Supernatant was neutralized by vortexingwith an equal volume of tri-n-octylamine/1,1,2-trichloro, trifluoro-ethane(22:78 vol/vol).32 The top (neutralized aqueous) phase was storedat –80°C. Amino acids were determined on an Agilent(Wokingham, UK) 1100 high-performance liquid chromatograph withZorbax Eclipse AAA column (4.6 x 75 mm, 3.5 µm) at 40°Cwith o-phthalaldehyde/3-mercaptopropionate/9-fluorenylmethylchloroformateprecolumn derivatization and ultraviolet and fluorimetric postcolumndetection.
SDS-PAGE and Immunoblotting
Cell lysates or membranes (20 µg protein per lane) weresubjected to SDS-PAGE, and proteins were blotted onto nitrocellulosemembranes (Amersham Hybond ECL). Membranes were blocked for1 h at room temperature with Tris-buffered saline (pH 7.6) and5% wt/vol skim milk and 0.1% vol/vol Tween 20 detergent andthen probed with primary antibodies in blocking buffer at 4°Covernight. Anti-SNAT2 rabbit polyclonal antibody (raised againstthe N-terminal peptide MKKTEMGRFNISPDEDSC of rat SNAT2) wasused at 1:4000 dilution. Anti–1-Na,K-ATPase mouse monoclonal(Abcam, Cambridge, UK) was used at 1:5000, and anti–AnnexinII goat polyclonal (Santa Cruz Biotechnology) was used at 1:4000.Antibody against PKB phosphorylated at Ser 473 (New EnglandBiolabs, Hitchin, UK) was used at 1:1000 dilution. Primary antibodywas detected using horseradish peroxidase–conjugated goatanti-rabbit IgG or rabbit anti-mouse IgG (Dako, Ely, UK) at1:1500 dilution for 2 h in blocking buffer at room temperature.Horseradish peroxidase–labeled bands were detected bychemiluminescence (ECL; Amersham) and quantified with a Bio-RadGS700 densitometer using Molecular Analyst 1.4 software (Bio-Rad,Hemel Hampstead, UK).
siRNA Transfection.
As shown previously,5 efficient silencing of SNAT2 for intracellularsignaling studies can be achieved by transfecting L6-G8C5 myoblastswith siRNA. Myoblasts (40% confluent) were transfected withthe following double-stranded siRNA at a final concentrationof 30 nM for 16 h in DMEM/10% FBS using Profection Calcium Phosphatetransfection (E1200; Promega, Madison, WI): (1) SNAT2 silencingsiRNA (forward sequence 5'-CUGACAUUCUCCUCCUCGUdTdT directedagainst base position 1095 onward in the gene sequence) and(2) scrambled control siRNA (forward sequence 5'-CGCUCUACUCUACUUGUCCdTdT)sharing the same base composition as 1) but in random sequence.After transfection, unless otherwise stated, cultures were incubatedin fresh DMEM/10% FBS for 24 h before commencement of measurements.Myoblasts are unsuitable for measurements of global proteolysisbecause (unlike myotubes) their proteolysis rate is unaffectedby acidosis5; therefore, despite the less efficient silencingof SNAT2 (Figures 4A and 5A), for proteolysis experiments, siRNAsilencing was performed with confluent myotubes using exactlythe same transfection procedure described previously. To observethe effect of siRNA silencing of SNAT2 on proteolysis (whichwas undetectable in our previous study),5 it was also foundnecessary to increase the duration of prelabeling of cellularproteins with 3H-L-Phe to 72 h as explained in the legend toFigure 4B.
Statistical Analysis
Data are presented as means ± SE. Statistical significancewas assessed by ANOVA and post hoc testing with Duncan multiplerange test, using SPSS 11.01 (SPSS, Chicago, IL). Changes wereregarded as significant at P < 0.05. Correlation was expressedas the Spearman rank correlation coefficient.
We gratefully acknowledge grants from Kidney Research UK (RP26/2/2004),Wellcome Trust (059828/Z/99/Z), Jules Thorn Trust (03SC/06A),Peel Medical Research Trust (AGT.R5), and Renal Care and ResearchAssociation. K.F.E. thanks Kidney Research UK and Diabetes UKfor studentship DUK ST3/2004. We thank Dr. L.M. Howells andProf. M.M. Manson for valuable assistance in setting up thePI3K lipid kinase assay.
Footnotes
Published online ahead of print. Publication date availableat www.jasn.org.
Supplemental information for this article is available onlineat http://www.jasn.org/.
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Abstract
Introduction
3β123 and therefore possibly co-localize with adhesion kinases that are potent activators of PI3K.24 
-32P-ATP and phosphatidylinositol) followed by immediate chilling on ice and extraction of 32P-labeled lipid product into chloroform/methanol (1:2 vol/vol). 32P-labeled phosphatidylinositol-3-phosphate was then resolved by thin-layer chromatography and visualized by autoradiography of the thin-layer chromatography plate. The product was quantified by densitometry of the autoradiograph, and, in some experiments, the result was confirmed by scraping the labeled spot off the plate and quantifying the scraped 32P by liquid scintillation counting. Samples were counted until at least 1000 counts had accumulated. 
). Spearman rank correlation coefficients: All data R = 0.74, P < 0.04; PKB data only R = 0.90, P < 0.04. (E) Representative experiment showing the effect of pH on the residual PI3K activity in myoblasts in which SNAT2 had been silenced as in 
, Naturally occurring metabolizable amino acid substrates of SNAT2; 
, synthetic nonmetabolizable SNAT2 substrate MeAIB.