Syntaxin 16 controls the intracellular sequestration of GLUT4 in 3T3-L1 adipocytes

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Abstract

The regulated delivery of Glut4-containing vesicles to the plasma membrane is a specialised example of regulated membrane trafficking. Present models favour the transporter trafficking through two inter-related endosomal cycles. The first is the proto-typical endosomal system. This is a fast trafficking event that, in the absence of insulin, serves to internalise Glut4 from the plasma membrane. Once in this pathway, Glut4 is further sorted into a slowly recycling pathway that operates between recycling endosomes, the trans Golgi network, and a population of vesicles often referred to as Glut4-storage vesicles. Little is known about the molecules that regulate these distinct sorting steps. Here, we have studied the role of Stx16 in Glut4 trafficking. Using two independent strategies, we show that Stx16 plays a crucial role in Glut4 traffic in 3T3-L1 adipocytes. Over-expression of a mutant form of Stx16 devoid of a transmembrane anchor was found to significantly slow the reversal of insulin-stimulated glucose transport. Depletion of Stx16 using antisense approaches profoundly reduced insulin-stimulated glucose transport but was without effect on cell surface transferrin receptor levels, and also reduced the extent of Glut4 translocation to the plasma membrane in response to insulin. These data support a model in which Stx16 is crucial in the sorting of Glut4 from the fast cycling to the slow cycling intracellular trafficking pathways in adipocytes.

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Materials and methods

Materials. Porcine insulin was from Drs. Svend Harelund and Jens Dangaard (Novo Nordisk, Baagsvaerd, Denmark). Stx16 cDNA was generously provided by Harald Stenmark (Radium Hospital, Oslo, Norway), and antibodies specific for Stx16 were from Wanjin Hong (Institute of Molecular Cell Biology, Singapore) or Synaptic Systems GmbH (Goettingen, Germany), who also supplied monoclonal Stx6. All other reagents were as described in [13].

Cell culture. 3T3-L1 fibroblasts were grown and differentiated into

Over-expression of Stx16-cyto perturbs Glut4 traffic

In order to determine the role of Stx16 in Glut4 traffic, we set out to produce a mutant version of Stx16, devoid of its trans-membrane domain as a putative poison protein (hereafter referred to as Stx16-cyto). This approach has been successfully employed to inhibit SNARE-mediated transport events in a variety of cell types (see, for example [13], [15], [17]). Accordingly, we generated a recombinant adenovirus to drive the expression of Stx16-cyto at high levels in adipocytes. Fig. 1A shows

Discussion

Of central importance to the insulin-regulated trafficking of Glut4 is the sequestration of the transporter intracellularly in the absence of insulin. In the absence of insulin, greater than 95% of Glut4 localises intracellularly, but upon insulin stimulation, a significant fraction of the transporter is mobilised to the cell surface, resulting in a dramatic increase in insulin-stimulated glucose transport [1]. Recent studies support a model in which the intracellular Glut4 itinerary involves

Acknowledgments

We thank Harald Stenmark for the Stx16 cDNA and Mike Czech for help with the electroporation methodology. This work was supported by grants from The Wellcome Trust (studentship to K.M.P.; Research Leave Award to G.W.G.), The Medical Research Council (Fellowship to S.C.M.M.), and The Royal College of Physicians and Surgeons, Glasgow (to S.C.M.M.). N.J.B. is a Prize Fellow of the Lister Institute of Preventive Medicine.

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Abbreviations: Stx, Syntaxin; TGN, trans Golgi network; MAO, morpholino antisense oligonucleotide; DMEM, Dulbecco’s modified Eagle’s medium; GSV, Glut4-storage vesicles.

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