The v-SNARE Vti1a Regulates Insulin-stimulated Glucose Transport and Acrp30 Secretion in 3T3-L1 Adipocytes

Abstract

Regulated exocytosis in adipocytes mediates key functions, exemplified by insulin-stimulated secretion of peptides such as adiponectin and recycling of intracellular membranes containing GLUT4 glucose transporters to the cell surface. Using a proteomics approach, the v-SNARE Vti1a (vps10p tail interacting 1a) was identified by mass spectrometry in purified GLUT4-containing membranes. Insulin treatment of 3T3-L1 adipocytes decreased the amounts of both Vti1a and GLUT4 in these membranes, confirming that Vti1a is a component of insulin-sensitive GLUT4-containing vesicles. In the basal state, endogenous Vti1a colocalizes exclusively with perinuclear GLUT4. Although Vti1a has previously been reported to be a v-SNARE localized in the trans-Golgi network, treatment with brefeldin A failed to significantly modify Vti1a or GLUT4 localization while completely dispersing Golgi and transGolgi network marker proteins. Furthermore, depletion of Vti1a protein in cultured adipocytes through small interfering RNAbased gene silencing significantly inhibited both adiponectin secretion and insulin-stimulated deoxyglucose uptake. Taken together, these results suggest that the v-SNARE Vti1a may regulate a step common to both GLUT4 and Acrp30 trafficking in 3T3-L1 adipocytes.Blood glucose homeostasis is maintained by insulin in part through glucose disposal into muscle and fat tissues. This is achieved by means of a redistribution of the facilitative glucose transporter GLUT4 from an internal sequestration compartment to the cell surface where it facilitates glucose uptake into these cells. In the absence of insulin GLUT4 recycles very slowly from internal membranes to the plasma membrane, whereas insulin markedly increases the exocytic rate of these GLUT4-containing vesicles. This results in a net increase of GLUT4 at the plasma membrane. This regulated trafficking pathway for GLUT4 appears to be distinct from that of many other membrane proteins such as the transferrin receptor or the GLUT1 glucose transporter protein.Insulin stimulation of adipocytes increases the abundance of these latter proteins at the cell surface by ϳ2-fold, whereas the amount of GLUT4 is increased between 10 -20-fold (1, 2). Other insulin-regulated exocytic pathways likely operate in adipocytes as well, such as a secretory mechanism for peptides like Acrp30/adiponectin. Like transferrin receptor recycling to the cell surface, Acrp30 secretion is only modestly stimulated by insulin (3). This large difference in insulin sensitivity indicates that the molecular mechanisms of GLUT4 sequestration and trafficking have unique regulatory elements. To understand insulin action on glucose transport it will be necessary to elucidate the nature of GLUT4 sequestration in intracellular membranes, the transit of GLUT4-containing intracellular membranes toward the cell surface, and the fusion of these membranes with the plasma membrane.Although the trafficking of the transferrin receptor and GLUT4 differ in their response to insulin stimulation, it appears that ϳ50% of the ...

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In-depth Analysis of the Adipocyte Proteome by Mass Spectrometry and Bioinformatics

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