State Key Laboratory of Pathogenesis, Prevention and Treatment of High Incidence Diseases in Central Asia, Department of Endocrinology, The First Affiliated Hospital of Xinjiang Medical University, Urumqi 830000, China.
Department of Endocrinology and Metabolism, Zhongshan Hospital and Fudan Institute for Metabolic Diseases, Fudan University, Shanghai 200032, China.
Exp Biol Med (Maywood). 2023 Jan;248(1):44-51. doi: 10.1177/15353702221131886. Epub 2022 Nov 20.
Disturbed insulin receptor (InsR) trafficking is associated with impaired insulin signaling and the development of diabetes. Sphingosine kinase (SphK), including SphK1 and SphK2, is a key enzyme of sphingolipid metabolism, which has been implicated in the regulation of membrane trafficking. More recently, we have reported that SphK2 is a key regulator of hepatic insulin signaling and glucose homeostasis. However, the role of SphK in InsR trafficking is still undefined. Huh7 cells were treated with specific SphK1 and SphK2 inhibitors or SphK1- and SphK2-specific small interfering RNA (siRNA) in the presence or absence of insulin. Flow cytometry and immunofluorescence assays were carried out to investigate the role of SphK in InsR trafficking. InsR endocytosis, recycling, and insulin signaling were analyzed. Inhibition of SphK2, but not SphK1, by either specific pharmaceutic inhibitors or siRNA, significantly suppressed InsR endocytosis and recycling following insulin stimulation. Consequently, the insulin-stimulated Akt activation was significantly attenuated by SphK2 inhibition in hepatocytes. Moreover, the effect of SphK2 on InsR trafficking was mediated via the clathrin-dependent mechanism. Thus, our results show that SphK2 is able to regulate InsR trafficking. These findings suggest that SphK2 may impinge on hepatic insulin signaling by regulating InsR trafficking, providing further mechanistic evidence that SphK2 could serve as a potential intervention target against insulin resistance and T2D (type 2 diabetes).
胰岛素受体（InsR）的转运异常与胰岛素信号受损和糖尿病的发生有关。鞘氨醇激酶（SphK），包括 SphK1 和 SphK2，是鞘脂代谢的关键酶，已被证实参与膜转运的调节。最近，我们报道了 SphK2 是肝胰岛素信号和葡萄糖稳态的关键调节因子。然而，SphK 在 InsR 转运中的作用仍未确定。在存在或不存在胰岛素的情况下，用特定的 SphK1 和 SphK2 抑制剂或 SphK1 和 SphK2 特异性小干扰 RNA（siRNA）处理 Huh7 细胞。通过流式细胞术和免疫荧光分析来研究 SphK 在 InsR 转运中的作用。分析 InsR 内吞作用、循环和胰岛素信号。无论是通过特定的药物抑制剂还是 siRNA 抑制 SphK2，而不是 SphK1，均显著抑制胰岛素刺激后的 InsR 内吞作用和循环。因此，SphK2 抑制显著减弱了肝细胞中胰岛素刺激的 Akt 激活。此外，SphK2 对 InsR 转运的影响是通过网格蛋白依赖性机制介导的。因此，我们的结果表明 SphK2 能够调节 InsR 转运。这些发现表明，SphK2 可能通过调节 InsR 转运来影响肝胰岛素信号，为 SphK2 可能作为针对胰岛素抵抗和 T2D（2 型糖尿病）的潜在干预靶点提供了进一步的机制证据。
