Program in Cell Biology, The Hospital for Sick Children, Toronto, ON M5G 1X8, Canada Department of Biochemistry, University of Toronto, Toronto, ON M5S 1A8, Canada.
Program in Cell Biology, The Hospital for Sick Children, Toronto, ON M5G 1X8, Canada Department of Biochemistry, University of Toronto, Toronto, ON M5S 1A8, Canada
Biol Open. 2014 Apr 4;3(5):314-25. doi: 10.1242/bio.20147898.
GLUT4 constitutively recycles between the plasma membrane and intracellular depots. Insulin shifts this dynamic equilibrium towards the plasma membrane by recruiting GLUT4 to the plasma membrane from insulin-responsive vesicles. Muscle is the primary site for dietary glucose deposition; however, how GLUT4 sorts into insulin-responsive vesicles, and if and how insulin resistance affects this process, is unknown. In L6 myoblasts stably expressing myc-tagged GLUT4, we analyzed the intracellular itinerary of GLUT4 as it internalizes from the cell surface and examined if such sorting is perturbed by C2-ceramide, a lipid metabolite causing insulin resistance. Surface-labeled GLUT4myc that internalized for 30 min accumulated in a Syntaxin-6 (Stx6)- and Stx16-positive perinuclear sub-compartment devoid of furin or internalized transferrin, and displayed insulin-responsive re-exocytosis. C2-ceramide dispersed the Stx6-positive sub-compartment and prevented insulin-responsive re-exocytosis of internalized GLUT4myc, even under conditions not affecting insulin-stimulated signaling towards Akt. Microtubule disruption with nocodazole prevented pre-internalized GLUT4myc from reaching the Stx6-positive perinuclear sub-compartment and from undergoing insulin-responsive exocytosis. Removing nocodazole allowed both parameters to recover, suggesting that the Stx6-positive perinuclear sub-compartment was required for GLUT4 insulin-responsiveness. Accordingly, Stx6 knockdown inhibited by ∼50% the ability of internalized GLUT4myc to undergo insulin-responsive re-exocytosis without altering its overall perinuclear accumulation. We propose that Stx6 defines the insulin-responsive compartment in muscle cells. Our data are consistent with a model where ceramide could cause insulin resistance by altering intracellular GLUT4 sorting.
GLUT4 在内质网和质膜之间持续循环。胰岛素通过从胰岛素应答小泡中募集 GLUT4 到质膜来改变这种动态平衡。肌肉是膳食葡萄糖沉积的主要部位；然而，GLUT4 如何分拣到胰岛素应答小泡中，以及胰岛素抵抗是否会影响这一过程，目前尚不清楚。在稳定表达 myc 标记 GLUT4 的 L6 成肌细胞中，我们分析了 GLUT4 从细胞表面内化的细胞内途径，并研究了这种分拣是否会受到 C2-神经酰胺的干扰，C2-神经酰胺是一种导致胰岛素抵抗的脂质代谢物。表面标记的 GLUT4myc 在 30 分钟内内化，在 Syntaxin-6（Stx6）和 Stx16 阳性的核周亚区室中积累，该亚区室不含 furin 或内化的转铁蛋白，并且显示出胰岛素应答的再胞吐作用。C2-神经酰胺分散了 Stx6 阳性亚区室，并阻止了内化的 GLUT4myc 的胰岛素应答再胞吐作用，即使在不影响 Akt 信号转导的情况下也是如此。用诺考达唑破坏微管阻止了内化的 GLUT4myc 到达 Stx6 阳性核周亚区室并进行胰岛素应答的胞吐作用。去除诺考达唑后，这两个参数都可以恢复，这表明 Stx6 阳性核周亚区室是 GLUT4 胰岛素反应性所必需的。因此，Stx6 敲低抑制了内化的 GLUT4myc 进行胰岛素应答再胞吐的能力约 50%，而不改变其整体核周积累。我们提出 Stx6 定义了肌肉细胞中的胰岛素应答区室。我们的数据与一种模型一致，即神经酰胺可能通过改变细胞内 GLUT4 分拣而导致胰岛素抵抗。
