Abstract
BackgroundThe 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. MethodsL6 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.ResultsOur 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 (pGSK3β/GSK3β ratio) in L6 myotubes. These effects were related to the accumulation of sphingosine in palmitate treated myotubes. ConclusionMyriocin 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 GSK3β phosphorylation, glucose uptake and the accumulation of sphingosine.
Highlights
Sphingolipids belong to a group of lipid-derived molecules, comprising a sphingoid base as a backbone to which is attached a single fatty acid (FA) side-chain of varying length and degree of saturation [1,2]
The first and rate-limiting step of de novo synthesis is the condensation of a fatty acyl-CoA, usually palmitoyl-CoA, with serine, which is catalyzed by the enzyme serine palmitoyltransferase (SPT), to form 3-ketosphinganine [1,2,8]
We compared for the first time effects of pharmacological inhibition of SPT and sphingosine kinase (SphK) 1 on palmitate induced insulin resistance in skeletal muscle cells in vitro
Summary
Sphingolipids belong to a group of lipid-derived molecules, comprising a sphingoid base as a backbone to which is attached a single fatty acid (FA) side-chain of varying length and degree of saturation [1,2]. Prolonged inhibition of SphK1 intensified the effect of PA on insulin-stimulated glucose uptake and attenuated further the activity of insulin signaling proteins (pGSK3β/GSK3β ratio) in L6 myotubes. These effects were related to the accumulation of sphingosine in palmitate treated myotubes. 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 GSK3β phosphorylation, glucose uptake and the accumulation of sphingosine
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