Mikłosz Agnieszka, Łukaszuk Bartłomiej, Baranowski Marcin, Górski Jan, Chabowski Adrian
Department of Physiology, Medical University of Bialystok, Bialystok, Poland.
PLoS One. 2013 Dec 23;8(12):e85547. doi: 10.1371/journal.pone.0085547. eCollection 2013.
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.
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.
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 (pGSK3β/GSK3β ratio) in L6 myotubes. These effects were related to the accumulation of sphingosine in palmitate treated myotubes.
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 GSK3β phosphorylation, glucose uptake and the accumulation of sphingosine.
本研究的目的是检测短期（2小时）和长期（18小时）抑制丝氨酸棕榈酰转移酶（SPT）和鞘氨醇激酶1（SphK1）对棕榈酸酯（PA）诱导的L6肌管胰岛素抵抗的影响。
L6肌管用PA与myriocin（SPT抑制剂）或PA与Ski II（SphK1抑制剂）同时处理不同时间段（2小时和18小时）。使用放射性同位素测量胰岛素刺激的葡萄糖摄取。使用蛋白质免疫印迹分析确定胰岛素信号蛋白的表达。通过高效液相色谱法估计细胞内鞘脂含量[鞘氨醇（SFA）、神经酰胺（CER）、鞘氨醇（SFO）、1-磷酸鞘氨醇（S1P）]。
我们的结果显示，myriocin对SPT的短期和长期抑制均足以防止神经酰胺积累，并同时逆转棕榈酸酯诱导的胰岛素刺激的葡萄糖转运抑制。相反，长期抑制SphK1增强了PA对胰岛素刺激的葡萄糖摄取的影响，并进一步减弱了L6肌管中胰岛素信号蛋白的活性（pGSK3β/GSK3β比值）。这些效应与棕榈酸酯处理的肌管中鞘氨醇的积累有关。
与SKII（一种特异性SphK1抑制剂）相比，无论SPT抑制时间长短，myriocin在恢复L6肌细胞中棕榈酸酯诱导的胰岛素抵抗方面更有效。观察到的胰岛素信号蛋白变化与特定鞘脂的含量有关，即与神经酰胺的减少有关。有趣的是，SphK1的失活增强了PA诱导的L6肌管胰岛素抵抗的效应，这与胰岛素刺激的PKB和GSK3β磷酸化、葡萄糖摄取的进一步抑制以及鞘氨醇的积累有关。
