The SAT2 System A transporter is one of the most highly regulated Na⁺-coupled transport mechanisms for small neutral amino acids (AAs) in mammalian cells and hence one most likely to have an active influence on cellular AA metabolism (Broer, 2002). The transporter is regulated at multiple levels and by many factors, including hormonal and nutritional stimuli that can modulate both its subcellular distribution and cellular expression (Hyde et al. 2001, 2002), and by changes in pH and electrochemical gradients that can alter transporter activity at the plasma membrane (Chaudhry et al. 2002). The sphingolipid ceramide (Cer) has been implicated in the pathogenesis of insulin resistance and apoptosis, because of its widely accepted role in tumour necrosis factor α signalling (Mathias et al. 1998). Since ceramide antagonises insulin action, we sought to determine whether it affects SAT2 function in L6 muscle cells, which is known to be regulated by insulin.

Cer induced a time- and dose-dependent decrease in System A activity, with maximal (~70 %) reduction after 2 h of treatment with 100 µM Cer, whereas an inactive Cer analogue had no effect. This response was associated with a decrease in SAT2 protein in the plasma membrane, although no reduction in total cellular SAT2 content was observed. Protein synthesis and the uptake of leucine and glucose were largely unaffected by Cer, but significant changes in the intracellular AA profile for both System A and non-System A substrates was observed. Anabolic stimuli such as insulin and leucine stimulated System A activity by up to 80 % following a 30 min and 3 h incubation, respectively. System A transport was also enhanced (~2-fold) upon depriving cells of AAs for 4 h. The responses to both insulin and leucine were lost upon preincubation of L6 cells with Cer, whereas the increase in System A activity in response to AA withdrawal was unaffected.

Our results indicate that Cer suppresses transport of AAs via System A by promoting internalisation of SAT2 transporters at the plasma membrane and by desensitising the carrier to the effects of various stimuli. Since Cer levels are elevated in muscle during insulin resistance, the effects of the lipid on SAT2 may have clinical importance in Type II diabetes. Also, since System A is upregulated during cellular proliferation, the effects of pro-apoptotic compounds, such as Cer, on SAT2 may also have a role in regulated cell death.

This work was supported by the BBSRC, MRC and Diabetes UK.