Effects of Inhibition of Serine Palmitoyltransferase (SPT) and Sphingosine Kinase 1 (SphK1) on Palmitate Induced Insulin Resistance in L6 Myotubes

Background: The objective of this study was to examine the effects of short (2 h) and prolonged (18 h) inhibition of serine palmitoyltransferase (SPT) and sphingosine kinase 1 (SphK1) on palmitate (PA) induced insulin resistance in L6 myotubes. Methods: L6 myotubes were treated simultaneously with either PA and myriocin (SPT inhibitor) or PA and Ski II (SphK1inhibitor) for different time periods (2 h and 18 h). Insulin stimulated glucose uptake was measured using radioactive isotope. Expression of insulin signaling proteins was determined using Western blot analyses. Intracellular sphingolipids content [sphinganine (SFA), ceramide (CER), sphingosine (SFO), sphingosine-1-phosphate (S1P)] were estimated by HPLC. Results: Our results revealed that both short and prolonged time of inhibition of SPT by myriocin was sufficient to prevent ceramide accumulation and simultaneously reverse palmitate induced inhibition of insulin-stimulated glucose transport. In contrast, prolonged inhibition of SphK1 intensified the effect of PA on insulin-stimulated glucose uptake and attenuated further the activity of insulin signaling proteins (pGSK3 beta/GSK3 beta ratio) in L6 myotubes. These effects were related to the accumulation of sphingosine in palmitate treated myotubes. Conclusion: Myriocin is more effective in restoration of palmitate induced insulin resistance in L6 myocytes, despite of the time of SPT inhibition, comparing to SKII (a specific SphK1 inhibitor). Observed changes in insulin signaling proteins were related to the content of specific sphingolipids, namely to the reduction of ceramide. Interestingly, inactivation of SphK1 augmented the effect of PA induced insulin resistance in L6 myotubes, which was associated with further inhibition of insulin stimulated PKB and GSK3 beta phosphorylation, glucose uptake and the accumulation of sphingosine.