A study of the SNARE protein syntaxin 16 and its role in the intracellular trafficking of glucose transporter GLUT4 in 3T3-L1 adipocytes

In muscle and adipose tissue insulin promotes glucose uptake by stimulating the translocation of the glucose transporter GLUT4 from an intracellular location to the plasma membrane. This exocytic delivery of GLUT4 to the plasma membrane is fundamental for the maintenance of blood glucose homeostasis. Individuals with insulin resistance or type II diabetes exhibit a blunted ability of insulin to stimulate GLUT4 translocation, and, as a result, a decreased rate of insulin stimulated glucose transport. To design effective therapies we require a clear understanding of the molecular basis of intracellular GLUT4 trafficking. Recent studies support a model in which the intracellular GLUT4 itinerary involves two intracellular cycles. In the basal state, GLUT4 recycles between the PM and endosomes in the fast recycling endosomal system. However, unique sequences within GLUT4 cause a large population to be sorted away from the endosomal system, into a slowly recycling intracellular pathway between endosomes and the TGN. This pathway involves the sorting of GLUT4 into unique storage vesicles, (GLUT4 storage vesicles, GSVs) which are mobilised in response to insulin, and are responsible for the majority of GLUT4 that is delivered to the cell surface in response to insulin. The intracellular sorting of GLUT4 is therefore essential to direct GLUT4 into the insulin-responsive compartment and to effectively sequester GLUT4 from the cell surface in the basal state. However, the molecules responsible for regulating this intracellular sorting pathway remain largely unknown. Previous studies have implicated the TGN localised t-SNARE, STX16, in GLUT4 trafficking in 3T3-L1 adipocytes. STX16 is a phosphoprotein in these cells and phosphorylation is decreased in response to acute insulin stimulation. This study aimed to elucidate the role, if any, of STX16 in GLUT4 trafficking, and to determine whether phosphorylation was responsible for the regulation of this process. In this study, experiments to identify the exact site of insulin-regulated phosphorylation were carried out in HEK 293 cells and suggest that STX16 phosphorylation may be specific to adipocytes. Immunoprecipitation studies revealed the existence of a STX16 containing SNARE complex in 3T3-L1 adipocytes with the t-SNAREs STX6 and Vtila; however, this SNARE complex does not appear to be regulated by insulin. These studies also suggest that STX16 does not bind to its cognate SM protein, mVps45, when part of this SNARE complex, either in the presence or absence of insulin. Recombinant adenoviruses were generated to express wild-type STX16 and STX16 mutants in 3T3-L1 adipocytes to assess their effects on glucose transport. Expression of these STX16 mutants proved problematic due to a property inherent in STX16, which requires stabilisation by its cognate SM protein, mVps45. It appears that STX16 expressed in excess of mVps45 is subject to proteasomal degradation, since expression could be dramatically increased when proteasome function was inhibited. One mutant, STX16cyt, constituted the dominant negative cytosolic domain of STX16 and was expressed in adipocytes to perturb endogenous STX16 function. Expression of STX16cyt significantly slowed the endocytic retrieval of GLUT4 following insulin- withdrawal. In contrast, expression of STX16cyt had no effect on the secretion of an adipocyte-derived adipokine, ACRP30. In a complementary approach, endogenous STX16 was knocked down by > 90 % using a specific Morpholino Antisense Oligonucleotide. 3T3-L1 adipocytes depleted of STX16 in this way showed a significant decrease in insulin- stimulated glucose transport. This corresponded with ~ 30 % decrease in cellular GLUT4 levels. These data were rationalised within a model in which STX16 functions to sequester GLUT4 from the PM in the basal state in a step which involves the intracellular sorting of GLUT4 from the fast cycling endosomal system into the slowly recycling intracellular pool of which GSVs are part. In cells depleted of STX16, this pathway is blocked and GLUT4 appears to be directed into a degradative pathway.