618 DIABETES, VOL. 49, APRIL 2000 v- and t-SNARE Protein Expression in Models of Insulin Resistance Normalizatio n of Glycemia by Ro siglitazone Treatment Corrects Overexpression of Cellubrevin, Vesicle-Associated Membrane Protein-2, and Syntaxin 4 in Skeletal Muscle of Zucker Diabetic Fatty Rats Valerie H. Maier, Derek R. Melvin, Carolyn A. Lister, Helen Chapman, Gwyn W. Gould, and Gregory J. Murphy Insulin stimulation of adipose and muscle cells results in the translocation of GLUT4 from an intracellular location to the plasma membrane; this translocation is defective in insulin resistance. Studies have suggested an important role for synaptobrevin and syntaxin homo- logues in this event, particularly the v–soluble N-et hyl- maleimide attachment protein receptors ( SNAREs) cel- lubrevin and vesicle-associated membrane protein-2 ( VAMP-2) and the t-SNARE syntaxin 4, but the expres- sion of these proteins has not been studied in insulin- resistant tissues. Therefore, we examined SNARE pro- tein content in skeletal muscle from Zucker diabetic fatty ( ZDF) rats compared with lean controls and deter- mined the effect of the thiazolidinedione insulin sensi- tizer rosiglitazone on these proteins. GLUT4 levels in skeletal muscle from ZDF rats were similar to those in lean control animals. In contrast, cellubrevin, VA MP -2 , and syntaxin 4 protein levels were elevated (2.8-fold, P = 0.02; 3.7-fold, P = 0.01; and 2.2-fold, P < 0.05, respec- tively) in skeletal muscle from ZDF rats compared with lean controls. Restoration of normoglycemia and normoinsulinemia in ZDF rats with rosiglitazone ( 3 0 μmol/kg) normalized cellubrevin, VAMP-2, and syn- taxin 4 protein to levels approaching those observed in lean control animals. These data show that elevated v- and t-SNARE protein levels are associated with insulin resistance in skeletal muscle and that these increases may be reversed by rosiglitazone treatment concomitant with a restoration of glycemic control. Such increases in SNARE protein levels were not observed in streptozotocin-induced diabetic rats, which suggests that hyperinsulinemia rather than hypergly- cemia may be more important in modulating SNARE protein expression in rodent models of insulin resis- tance. Consistent with this hypothesis, elevated levels of SNARE proteins were also observed in 3T3-L1 adipocytes chronically treated with insulin (500 nmol/l for 24 h). These data argue that SNARE protein levels may be altered in insulin-resistant states and that the levels of these proteins are modulated by agents that increase insulin sensitivity. Moreover, these data demonstrate for the first time altered expression of proteins known to regulate GLUT4 translocation in a model of diabetes. Diabetes 4 9 :6 1 8–625, 2000 T he integral membrane protein GLUT4 is one of a family of membrane proteins responsible for the facilitative diffusion of glucose into mammalian cells (1,2). GLUT4 is expressed predominantly in tissues that exhibit acute insulin-stimulated glucose uptake (brown and white adipocytes, skeletal muscle, and cardio- cytes) and is responsible for the large increase in the rate of glucose transport into these tissues observed in response to insulin (2–4). Unlike other members of the GLUT family, GLUT4 is almost completely intracellularly sequestered under resting or fasting conditions. However, in response to acute insulin elevation or muscle contraction, GLUT4 is rapidly translo cated to the cell surface (2–7). This insulin-reg- ulated mo bilizatio n of GLUT4 is fundamentally impo rtant to the maintenance of blood glucose homeostasis. Defective GLUT4 translo cation is implicated in the etiology of type 2 dia- betes and has been demonstrated in many rodent models of diabetes (4,8–11). Insulin-stimulated GLUT4 translocation represents a highly regulated vectorial delivery of defined intracellular cargo to the plasma membrane. The fidelity of this response is thought to be mediated, at least in part, by the intermedi- acy of soluble N-ethylmaleimide attachment protein receptor (SNARE) proteins. The SNARE hypothesis for membrane trafficking suggests that, for all vesicle trafficking events, a unique vesicle-bound protein (v-SNARE) exists that specifi- cally recognizes and interacts with a cognate t-SNARE local- From the Division of Biochemistry and Molecular Biology (V.H.M., D.R.M., G.W.G.), Institute of Biomedical and Life Sciences, University of Glasgo w, Glas go w, Sco tland; and the Department of Vascular Biology (C.A.L., H.C., G.J.M.), SmithKline Beecham Pharmaceuticals, Harlow, Essex, U.K. Address correspondence and reprint requests to Gwyn W. Gould, PhD, University of Glasgow, Institute of Biomedical and Life Sciences, Division of Biochemistry and Molecular Biology, Davidson Building, Glasgo w, Scot- land, G12 8QQ, U.K. E-mail: g.gould@ bio.gla.ac.uk. Received for publication 30 July 1999 and accepted in revised form 17 December 1999. DMEM, Dulbecco’s modified Eagle’s medium; ECL, enhanced chemi- luminescence; HES, HEPES EDTA sucrose; IRAP, insulin-responsive aminopep- tidase; SNAP-23, synaptosomal protein of 23 kDa; SNARE, soluble N-e thyl- maleimide attachment protein receptor; STZ, streptozotocin; TGN38, tran s Golgi network marker 38; VAMP-2, vesicle-associated membrane protein-2.