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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online December 20, 2004 as doi:10.1096/fj.04-2284fje. |
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Division of Molecular Physiology, School of Life Sciences, University of Dundee, Dundee, UK
1Correspondence: E-mail: h.s.hundal{at}dundee.ac.uk
SPECIFIC AIMS
Our aim was to determine the effects of ceramide on System A amino acid transport, protein synthesis, and intracellular signaling pathways involved in the regulation of protein synthesis in skeletal muscle.
PRINCIPAL FINDINGS
1. Ceramide inhibits basal and insulin-stimulated System A activity
Rat L6 myotubes were incubated with C2-ceramide for up to 2 h before determining System A activity (measured with 10 µM [14C] 
-methylaminoisobutyrate). Ceramide provision resulted in marked inhibition of basal System A activity in a dose- and time-dependent manner (P<0.01 vs. vehicle-treated control after 2 h exposure to 100 µM ceramide; Fig. 1
A, B). An identical protocol caused similar inhibition of the uptake of 500 µM [3H] L-alanine (P<0.01), a physiological substrate for System A, but did not affect basal transport of non-System A substrates, such as L-leucine or the sugar 2-deoxyglucose (Fig. 1C-E
).
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Exposure of L6 cells to 0.75 mM palmitic acid significantly inhibited System A (vehicle 9.31±1.44 pmol/min/mg protein; Palmitate 4.69±1.57 pmol/min/mg protein; P<0.05), whereas palmitoleic acid, a compound that does not increase intracellular ceramide, did not affect System A activity (P>0.05).
Treatment for 30 min with 100 nM insulin increased L6 System A activity by 
40% (P<0.05). In cells pretreated with ceramide, the response of System A to insulin was blocked.
2. Plasma membrane SNAT2 abundance is reduced by ceramide
In L6 myotubes, sodium-dependent neutral amino acid transporter 2 (SNAT2) is the only functionally identified System A carrier expressed. Total expression of SNAT2 in ceramide-treated muscle cells was unchanged after ceramide treatment. However, Western blot analysis of L6 plasma membrane fractions demonstrated a reduction in cell surface SNAT2 abundance after ceramide treatment. The inactive, but structurally related, compound C2-dihydroceramide did not affect SNAT2 localization or System A activity.
3. Divergent signaling mechanisms govern ceramide effects on glucose and amino acid transport
Ceramide inhibits insulin-stimulated muscle glucose transport and attenuates the cell surface recruitment and activation of protein kinase B (PKB). In L6 myotubes, this inhibition can be bypassed after the expression of a membrane-targeted (constitutively active) PKB construct (mPKB). However, although mPKB-expressing L6 cells display elevated (35%, vs. empty vector control) basal System A activity, this cellular manipulation does not counteract the inhibition of System A observed in ceramide treated cells. Tests were performed to pharmacologically characterize the involvement of several other ceramide targets (protein phosphatases and protein kinase C (PKC) isoforms) in System A regulation. This demonstrated that, although PKC isoforms are not involved in mediating the inhibitory effects of ceramide, PKC inhibitors impair the insulin responsiveness of System A.
4. Ceramide depletes the intracellular amino acid pool
SNAT2 is a unidirectional transporter that functions in amino acid influx. HPLC analysis of L6 homogenates was performed to test whether ceramide treatment and the consequential reduction of System A transport activity altered intracellular amino acid abundance. Exposure to 100 µM ceramide for 2 h reduced the intracellular concentration of three of the most abundant cellular amino acids (glutamine, glutamate, and aspartate-reduced, respectively, to 55.5±10.82%, 52.8±10.4%, 56.9±10.89% of control values; P<0.01 in all cases) and reduced the total intracellular amino acid pool by 50%.
5. Diminished mTOR signaling in ceramide-treated cells
The mammalian target of rapamycin (mTOR) controls mRNA translation and is regulated by nutrient availability and hormonal stimuli. Effects of ceramide on mTOR signaling were tested. Ceramide reduced phosphorylation of components of the mTOR pathway (ribosomal protein S6 and its upstream kinase, S6K1) in a time-dependent manner. Ceramide mimicked the cellular response to amino acid deprivation by the mTOR pathway and prevented mTOR signaling in response to amino acid resupply. Control experiments demonstrated that insulin-stimulated mTOR signaling was not impaired by ceramide, although PKB activation was attenuated.
6. Inhibition of protein synthesis by ceramide
Protein synthesis was determined in ceramide-treated L6 myotubes over a 4 h time course (Fig. 2
) to test whether the elevation of intramuscular ceramide may contribute to catabolic muscle wasting. As shown in Fig. 2A
, incubation with ceramide led to progressive inhibition of muscle cell protein synthesis. Ceramide treatment did not affect the distribution of the phenylalanine tracer used (Fig. 2B
), and it is proposed that ceramide-mediated inhibition of protein synthesis occurs due to inhibition of translation factors, including those downstream of mTOR.
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CONCLUSIONS
Results from our study demonstrate that, in skeletal muscle cells, ceramide has profound effects on amino acid transport and protein synthesis in addition to its known effects on insulin-stimulated glucose deposition. Muscle ceramide is generated after cytokine signaling and via metabolism of saturated fatty acids; increased levels may contribute to insulin resistance in this tissue. Ceramide levels are elevated in catabolic and insulin-resistant muscle, but a connection between muscle ceramide levels and muscle protein metabolism remains untested. Given the inhibitory effects of ceramide on protein synthesis and amino acid transport presented in this article, it is conceivable that ceramide has a protein catabolic function in vivo.
Our current model for regulation of muscle System A activity is presented in Fig. 3
. In unstimulated cells (Fig. 3A
), cell surface SNAT2 carriers transport extracellular amino acids to the intracellular pool, where they may be used for protein synthesis and maintain mTOR signaling. Elevated ceramide levels stimulate internalization of SNAT2 (Fig. 3B
), thus reducing System A activity; this may contribute to diminution of the amino acid pool. Ceramide reduces protein synthesis and signaling by the mTOR pathway, and we propose that a causal link may exist between the reduced intracellular amino acid availability and these phenomena. Nonetheless, the possibility exists that ceramide may inhibit mTOR signaling either downstream or independent of amino acid sensing pathways (indicated by dotted line in Fig. 3B
). Insulin (Fig. 3C
) stimulates System A and glucose transport, both of which are attenuated by ceramide. However, distinct pathways are implicated, since the effect of ceramide on glucose transport, but not System A, can be bypassed after the constitutive activation of PKB.
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Amino acids are potent signaling molecules in skeletal muscle, where they influence a variety of catabolic and anabolic processes. Our group recently suggested an intracellular location for amino acid sensor(s) upstream of the mTOR pathway. The present results are in agreement with this proposition, since ceramide reduces both intracellular amino acid abundance and signaling by mTOR, without change to the extracellular amino acid availability. To our knowledge this is the first demonstration of cross talk between lipid (ceramide) and amino acid (mTOR) sensitive signaling pathways. The possibility that ceramide may inhibit mTOR signaling at a locus downstream of the putative amino acid sensors exists. However, the ability of insulin to stimulate mTOR was maintained in the presence of ceramide – demonstrating that the core mTOR pathway remains intact regardless of ceramide content. Intriguingly, PKB has been implicated in mTOR activation in transformed cells and overexpression studies. The PKB substrate TSC2 may contribute to this effect. PKB signaling was attenuated by ceramide in our system, but TSC2 phosphorylation was maintained, suggestive of insulin-sensitive PKB-independent routes to mTOR activation.
Subcellular redistribution of the SNAT2 transporter is suggested to account for System A activation in response to insulin (in muscle) and hepatectomy (in liver). Our data provide the first demonstration of controlled System A down-regulation through translocation events. The signaling pathways and molecular machinery involved in SNAT2 redistribution are poorly characterized, in contrast to GLUT4. However, the results suggest that divergence in these processes exists, while highlighting (through pharmacological means) potential SNAT2 regulatory pathways. The implication of PKC signaling in System A regulation is of particular interest as dysregulation of PKC isoforms has been demonstrated in insulin-resistant and diabetic muscle.
FOOTNOTES
To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.04-2284fje;
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), 10 µM C2-ceramide (
), 100 µM C2-ceramide (
), or 5 µg/mL cycloheximide (•). B) Incorporation of extracellular Phe into the intracellular free pool (TCA nonprecipitable) is presented as the ratio of TCA supernatant-associated radioactivity (CPMi) to the radioactivity associated with 1 mL [3H]-Phe labeled 