Omega-3 polyunsaturated fatty acids promote SNAREs mediated GLUT4 vesicle docking and fusion.

Glucose homeostasis imbalance and insulin resistance (IR) are major contributors to the incidence of type 2 diabetes. Omega-3 polyunsaturated fatty acids (PUFAs) are key ingredients for maintaining cellular functions and improving insulin sensitivity. However, how omega-3 PUFAs modulate the dynamic process of glucose transport at the cellular level remains unclear. Here we unraveled eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) may regulate the glucose transporter 4 (GLUT4) vesicle trafficking in both normal and IR adipocytes. Both omega-3 PUFAs significantly increase glucose consumption within a range of 10-32% in the basal state. Furthermore, both EPA (200 µM) and DHA (100 µM) may significantly promote the serine/threonine protein kinase (Akt) phosphorylation by 70% and 40% in the physiological state of adipocytes, respectively. Both omega-3 PUFAs significantly advanced the Akt phosphorylation in a dose-dependent way and showed a ∼2-fold increase at the dose of 200 µM in the IR pathological state. However, they could not up-regulate the expression of GLUT4 and insulin-regulated aminopeptidase protein. We further revealed that both omega-3 PUFAs dynamically promote insulin-stimulated GLUT4 vesicle translocation and soluble N-ethylmaleimide-sensitive factor attachment protein receptor mediated vesicle docking and fusion to the plasma membrane via specifically modulating the expression of vesicle-associated membrane protein 2. Understanding the mechanisms by which omega-3 PUFAs modulate cellular metabolism and IR in peripheral tissues may provide novel insights into the potential impact of omega-3 PUFAs on the metabolic function and the management of IR.

Dual Function of PI(4,5)P2 in Insulin-Regulated Exocytic Trafficking of GLUT4 in Adipocytes.

Phosphoinositides are important signaling molecules involved in the regulation of vesicular trafficking. It has been implicated that phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] is involved in insulin-regulated GLUT4 translocation in adipocytes. However, it remains unclear where and how PI(4,5)P2 regulates discrete steps of GLUT4 vesicle translocation in adipocytes, especially on the exocytic arm of regulation. Here, we employed optogenetic tools to acutely control the PI(4,5)P2 metabolism in living cells. By combination of TIRFM imaging, we were able to monitor the temporal-spatial-dependent PI(4,5)P2 regulation on discrete steps of GLUT4 translocation in adipocytes. We found that the plasma membrane localized PI(4,5)P2 is crucial for proper insulin signaling propagation and for insulin-stimulated GLUT4 vesicle translocation in 3T3-L1 adipocytes. Global depletion of PI(4,5)P2 on the cell surface blunted insulin-stimulated Akt phosphorylation and abolished insulin effects in promotion of the docking and fusion of GLUT4 vesicle with the plasma membrane. Furthermore, by development of a novel optogenetic module to selectively modulate PI(4,5)P2 levels on the GLUT4 vesicle docking site, we identified an important regulatory role of PI(4,5)P2 in controlling of vesicle docking process. Local depletion of PI(4,5)P2 at the vesicle docking site promoted GLUT4 vesicle undocking, diminished insulin-stimulated GLUT4 vesicle docking and fusion, but without perturbation of insulin signaling propagation in adipocytes. Our results provide strong evidence that cell surface PI(4,5)P2 plays two distinct functions on regulation of the exocytic trafficking of GLUT4 in adipocytes. PI(4,5)P2 not only regulates the proper activation of insulin signaling in general but also controls GLUT4 vesicle docking process at the vesicle-membrane contact sites.

Sec3 knockdown inhibits TGF-ß induced epithelial-mesenchymal transition through the down-regulation of Akt phosphorylation in A549 cells.

The exocyst, an evolutionarily conserved octomeric protein complex, has been demonstrated as an essential component for vesicle tethering during cell exocytosis, and participates in various physiological processes in the cell. Although subunits of the exocyst complex have been reported to be involved in the regulation of TGF-ß induced cancer cell migration and epithelial-mesenchymal transition (EMT), the potential function of Sec3 in these regulated processes remains unclear. Here, we show that Sec3 knockdown abolishes TGF-ß stimulated A549 lung cancer cell migration in vitro and causes defects in the regulated EMT process. In addition, we find that depletion of Sec3 significantly inhibits TGF-ß stimulated Akt phosphorylation in A549 cells, whereas the increase of Smad2 phosphorylation is unaffected. Furthermore, replenishment of an RNAi-resistant form of Sec3 is shown to restore the defects of TGF-ß induced cell migration, EMT and Akt signaling activation. In summary, our study provides evidence that Sec3 is involved in TGF-ß induced cell migration and EMT processes, presumably through the regulation of PI3K/Akt signaling activation in A549 cancer cells.

Spatiotemporal Regulators for Insulin-Stimulated GLUT4 Vesicle Exocytosis.

Insulin increases glucose uptake and storage in muscle and adipose cells, which is accomplished through the mobilization of intracellular GLUT4 storage vesicles (GSVs) to the cell surface upon stimulation. Importantly, the dysfunction of insulin-regulated GLUT4 trafficking is strongly linked with peripheral insulin resistance and type 2 diabetes in human. The insulin signaling pathway, key signaling molecules involved, and precise trafficking itinerary of GSVs are largely identified. Understanding the interaction between insulin signaling molecules and key regulatory proteins that are involved in spatiotemporal regulation of GLUT4 vesicle exocytosis is of great importance to explain the pathogenesis of diabetes and may provide new potential therapeutic targets.