Knockout of Syntaxin-4 in 3T3-L1 adipocytes reveals new insight into GLUT4 trafficking and adiponectin secretion

Adipocytes are key to metabolic regulation, exhibiting insulin-stimulated glucose transport which is underpinned by the insulin-stimulated delivery of glucose transporter-4 (GLUT4)- containing vesicles to the plasma membrane where they dock and fuse increasing cell surface GLUT4 levels. Adipocytokines such as adiponectin are secreted via a similar mechanism. We used genome editing to knockout Syntaxin-4 a protein reported to mediate GLUT4-vesicle fusion with the plasma membrane in 3T3-L1 adipocytes. Syntaxin-4 knockout reduced insulin-stimulated glucose transport and adiponectin secretion by ∼50% and reduced GLUT4 levels. Ectopic expression of HA-GLUT4-GFP showed that Syntaxin-4 knockout cells retain significant GLUT4 translocation capacity demonstrating that Syntaxin-4 is dispensable for insulin-stimulated GLUT4 translocation. Analysis of recycling kinetics revealed only a modest reduction in the exocytic rate of GLUT4 in knockout cells, and little effect on endocytosis. These analyses demonstrate that Syntaxin-4 is not always rate limiting for GLUT4 delivery to the cell surface. In sum, we show that Syntaxin-4 knockout results in reduced insulin-stimulated glucose transport, depletion of cellular GLUT4 levels and inhibition of adiponectin secretion but has only modest effects on the translocation capacity of the cells.