Syntaxin 4 heterozygous knockout mice develop muscle insulin resistance

To investigate the physiological function of syntaxin 4 in the regulation of GLUT4 vesicle trafficking, we used homologous recombination to generate syntaxin 4–knockout mice. Homozygotic disruption of the syntaxin 4 gene results in early embryonic lethality, whereas heterozygous knockout mice, Syn4^+/–, had normal viability with no significant impairment in growth, development, or reproduction. However, the Syn4^+/– mice manifested impaired glucose tolerance with a 50% reduction in whole-body glucose uptake. This defect was attributed to a 50% reduction in skeletal muscle glucose transport determined by 2-deoxyglucose uptake during hyperinsulinemic-euglycemic clamp procedures. In parallel, insulin-stimulated GLUT4 translocation in skeletal muscle was also significantly reduced in these mice. In contrast, Syn4^+/– mice displayed normal insulin-stimulated glucose uptake and metabolism in adipose tissue and liver. Together, these data demonstrate that syntaxin 4 plays a critical physiological role in insulin-stimulated glucose uptake in skeletal muscle. Furthermore, reduction in syntaxin 4 protein levels in this tissue can account for the impairment in whole-body insulin-stimulated glucose metabolism in this animal model.

It is well known that skeletal muscle and adipose tissue are the major in vivo sites responsible for insulin-stimulated glucose disposal (1–4). In these tissues, the clearance of circulating glucose is dependent on the insulin-stimulated translocation of the GLUT4 glucose transporter protein isoform to the cell surface (5–10). In the basal non–insulin-stimulated state, the majority of the GLUT4 proteins reside in poorly defined intracellular compartments sequestered away from the general membrane recycling system (11–16). However, insulin stimulation generates a complex series of signaling events that ultimately results in a rapid redistribution of these sequestered GLUT4 proteins to the plasma membrane, thereby increasing the rate of glucose uptake. Disruption of any step of this process results in an impairment of GLUT4 translocation and hence insulin resistance, which is an important factor contributing to the development of type 2 diabetes (17–20).

The mechanism of GLUT4 translocation shares several important features with the exocytosis of synaptic vesicles during neurotransmitter release (21–23). In particular, the insulin-stimulated plasma membrane docking and fusion of GLUT4 vesicles appears to require specific interactions between the plasma membrane t-SNARE proteins syntaxin 4 and SNAP23 with the GLUT4 vesicle v-SNARE protein VAMP2 (24–31). For example, previous studies have demonstrated that introduction of either syntaxin 4–specific Ab’s or blocking peptides inhibited insulin-stimulated GLUT4 translocation (26–28, 32). This inhibition was apparently specific because the introduction of a dominant-interfering form of syntaxin 4, but not syntaxin 3, prevented GLUT4 translocation (25). Furthermore, the dominant-interfering function of this truncated syntaxin 4 protein required the presence of the VAMP2-binding site of syntaxin 4. Taken together, these data provide strong evidence that syntaxin 4 is part of the protein machinery required for insulin-stimulated GLUT4 translocation in cultured adipocytes.

In contrast to cultured adipocytes, there have been no in vivo studies on the functional role of syntaxin 4 in skeletal muscle. Therefore, to examine the physiological function of syntaxin 4 in GLUT4 translocation, we used homologous recombination to generate mice with a disruption of the syntaxin 4 gene. In this study, we demonstrate that heterozygotic syntaxin 4–knockout mice, syn4^+/–, develop impaired whole-body insulin-stimulated glucose disposal. The in vivo glucose transport activity in skeletal muscle was decreased by 50% compared with that of wild-type animals, suggesting that syntaxin 4 protein levels play an essential role in insulin-stimulated glucose transport and GLUT4 translocation in skeletal muscle.

Generation of syntaxin 4–knockout mice. To assess the physiological function of syntaxin 4, we constructed a targeting vector that replaces 3.3 kb of the syntaxin 4 genomic sequence, including the ATG-coding exon, with a neo^R cassette oriented in the opposite direction as the endogenous syntaxin 4 gene (Figure 1, a–c). Of the 96 neomycin-resistant ES cell clones analyzed, five demonstrated the appropriate recombination as confirmed by Southern blot analysis (shown for one clone in Figure 1d). Two of these clones were used to generate chimeric founder mice. Heterozygotic mice from the F₁ generation were identified by PCR analysis (data not shown) and were crossed in order to obtain syntaxin 4–deficient mice. However, of the 186 offspring analyzed, 71 were wild-type and 115 were heterozygous, syn4^+/–, mice, suggesting that homozygotic disruption of the syntaxin 4 gene, syn4^–/–, resulted in embryonic lethality. Subsequent analysis of embryonic development revealed that the syn4^–/– embryos die before embryonic day 7.5.

The relative expression levels of syntaxin 4 protein were next determined by immunoblotting of various mouse tissues (Figure 2). In all the tissues examined, the syntaxin 4 protein was reduced by 36% ± 4.4 in the syn4^+/– mice compared with the wild-type syn4^+/+ littermate controls (Figure 2a). Interestingly, the syntaxin 4–binding regulatory protein, Munc18c, also displayed a 42% ± 2.9 decrease in all tissues, with the exception of the heart (Figure 2a). Although we do not know the molecular basis for reduction of Munc18c protein in the syn4^+/– mice, these data suggest the presence of a coordinate regulatory mechanism. However, the parallel decrease in syntaxin 4 and Munc18c was not shared by the other syntaxin 4–binding SNARE protein, SNAP23, and synaptobrevin 2 (syb2), also known as VAMP2 (Figure 2a). SNAP23 associates with syntaxin 4 as part of the plasma membrane–t-SNARE complex, and synaptobrevin 2 is a GLUT4 vesicle v-SNARE protein (27, 28, 36–38). As expected, GLUT4 displayed its normal tissue-specific expression pattern being restricted to heart, skeletal muscle, and fat (Figure 2b). Similar to SNAP23 and synaptobrevin 2, there was no significant difference in the levels of GLUT4 protein expression in the syn4^+/– mice compared with the control syn4^+/+ mice. Finally, the insulin-responsive aminopeptidase (IRAP) is an ubiquitously expressed protein (39–41). In the heart, skeletal muscle, and fat, IRAP is localized to the same intracellular compartments as the GLUT4 protein and undergoes an identical pattern of insulin-stimulated translocation (39–44). In any case, the expression of IRAP was not significantly affected in syn4^+/– mice in these tissues (Figure 2b). Thus, the syn4^+/– mice appear to have a selective and coordinate decrease of the syntaxin 4 and Munc18c proteins without any change on other SNARE or GLUT4 vesicle-containing proteins.

Figure 2

The syn4^+/– mice have reduced tissue expression of syntaxin 4 and Munc18c proteins. Tissue extracts from heart, hindquarter skeletal muscle (Skm), liver, brain, lung, spleen, pancreas (Panc), and epididymal adipose depots (Fat) were isolated from the syn4^+/– and control syn4^+/+ mice as described in Methods. The samples were resolved by SDS-PAGE and immunoblotted with Ab’s for (a) syntaxin 4 (syn4), Munc18c, SNAP23, and synaptobrevin 2 (Syb2), or (b) GLUT4 and IRAP. These are representative immunoblots performed independently four times with four separate pairs of syn4^+/+ and syn4^+/– mice.

Impaired clearance of circulating glucose in the heterozygous syntaxin 4–knockout mice. Previous studies have indicated an important involvement of syntaxin 4 in the docking/fusion of GLUT4-containing vesicles in adipose and muscle cell lines in culture (24–29, 45). Therefore, we next determined the ability of these mice to respond to a glucose challenge (Figure 3). After a single intraperitoneal bolus injection of glucose, circulating glucose levels maximally increased at 60 minutes and slowly declined over the next 90 minutes. Compared with the wild-type littermate controls, the syn4^+/– mice displayed a considerably greater glucose excursion. These data directly demonstrate that the syn4^+/– mice have a reduced ability to clear glucose from the peripheral circulation. Consistent with this finding, fasting serum insulin levels in the syn4^+/– mice are higher than those in the wild-type controls (64 ± 9 pM vs. 43 ± 9 pM). However, other metabolic parameters assessed, including serum triglyceride, free fatty acids, and cholesterol were not significantly different between the wild-type and syn4^+/– mice in either the fed or fasted state (data not shown).

Figure 3

GTT on the heterozygous syntaxin 4–mutant mice. D-glucose (20% solution, 2 g/kg of body weight) was injected intraperitoneally into 16-week-old mice fasted for 12 hours. Blood glucose was measured at 0, 30, 60, 90, and 120 minutes after injection using a glucometer as described under Methods. Data shown are the average ± SD from 12 syn4^+/– and 12 syn4^+/+ littermate controls.

The decreased clearance of plasma glucose could result from either an impairment of insulin secretion, increased hepatic glucose output, or peripheral tissue insulin resistance. To gain detailed insight into glucose metabolism in syn4^+/– mice, we next examined the metabolism of glucose during a hyperinsulinemic-euglycemic clamp procedure (Figure 4). The glucose infusion rate necessary to maintain euglycemia (110 mg/dl) in the presence of a constant infusion of insulin (2.5 mU/kg/min) was nearly twofold lower in syn4^+/– mice than in the wild-type littermate controls (Figure 4a). Accordingly, the insulin-stimulated whole-body glucose-disposal rate was decreased by nearly 50% in syn4^+/– mice (Figure 4b). This was accompanied by significant decreases in whole-body glycolysis (Figure 4b). In contrast, whole-body glycogen synthesis (Figure 4b) and hepatic glucose output (Figure 5) were unaffected in these mice. Since whole-body glycogen synthesis in mice primarily reflects liver glycogen synthesis, these data strongly suggest that the decreased whole-body glucose clearance was not due to altered liver function.

Figure 4

Whole-body metabolic parameters during hyperinsulinemic-euglycemic clamp procedures in awake mice. (a) Steady-state glucose infusion rate, obtained from averaged rates of 90–120 minutes of hyperinsulinemic-euglycemic clamp procedures in the wild-type (open bar) and heterozygous syntaxin 4–knockout mice (filled bar). (b) Insulin-stimulated whole-body glucose transport, glycolysis, and glycogen synthesis in vivo in the wild-type mice (open bars) and heterozygous syntaxin 4–knockout mice (filled bars). Data shown are the average ± SD of three independent experiments; P < 0.05 versus wild-type mice by unpaired Student’s t test.

Figure 5

HGP during hyperinsulinemic-euglycemic clamp procedure in awake mice. Basal and insulin-stimulated rates of HGP in the wild-type (open bars) and heterozygous syntaxin 4–knockout mice (filled bars). Data shown are the average ± SD of three independent experiments, P < 0.05 versus wild-type mice.

Impaired skeletal muscle glucose transport in the heterozygous syntaxin 4–knockout mice. Having eliminated a defect in liver function, we next examined the ability of muscle and adipose tissue to undergo insulin-stimulated glucose uptake. Analysis of 2DG uptake into skeletal muscle during the clamp procedure demonstrated a 50% decrease in the syn4^+/– mice compared with the control mice (Figure 6). Since glucose uptake under these conditions is rate limiting for glycolysis, there was a corresponding twofold decrease in muscle glycolytic rate. Despite the very low rate of muscle glycogen synthesis, there was also an apparent decrease in the rate of skeletal muscle glycogen synthesis.

Figure 6

Skeletal muscle glucose transport and metabolism during hyperinsulinemic-euglycemic clamp procedures in awake mice. Insulin-stimulated glucose transport, glycolysis, and glycogen synthesis in skeletal muscle (gastrocnemius) in the wild-type mice (open bars) and heterozygous syntaxin 4–knockout mice (filled bars). Data shown are the average ± SD of three independent experiments, P < 0.05 versus wild-type mice.

The primary mechanism for insulin-stimulated glucose uptake in skeletal muscle is the translocation of intracellular sequestered GLUT4 protein to the cell-surface membrane. This can be assessed by subcellular fractionation of mouse hindquarter skeletal muscle followed by GLUT4 immunoblotting (34, 46–49). In wild type syn4^+/+ mice, insulin stimulated a small increase in the amount of surface membrane–distributed GLUT4 protein (Figure 7a, lanes 1 and 2). In contrast, there was no discernible change in the amount of GLUT4 protein in the syn4^+/– mice (Figure 7a, lanes 3 and 4). Although the insulin-stimulated translocation of GLUT4 is difficult to detect in mouse skeletal muscle due to contamination of the cell-surface membrane fraction, the decrease in the intracellular fractions are readily observed in the syn4^+/+ mice (Figure 7b). Consistent with the lack of insulin-stimulated appearance of GLUT4 at the cell-surface membrane fraction in the syn4^+/– mice, there was also a marked reduction in the ability of insulin to decrease the intracellular content of GLUT4 protein (Figure 7b). Thus, these data demonstrated that the syn4^+/– mice have reduced insulin-stimulated glucose uptake due to a reduction in the cell-surface translocation of the GLUT4 protein.

Figure 7

Insulin stimulation results in the skeletal muscle surface-membrane translocation of GLUT4 in wild-type syn4^+/+ but not heterozygotic syn4^+/– mice. Syn4^+/+ and syn4^+/– mice were fasted overnight and either left untreated or stimulated with insulin as described in Methods. The hindquarter skeletal muscle was dissected and subjected to differential and sucrose velocity centrifugation as described under Methods. The surface-membrane fraction (a), as well as the intracellular-membrane fractions (b), were immunoblotted with the GLUT4 Ab. These are representative immunoblots performed from three different independent pairs of wild-type syn4^+/+ and knockout syn4^+/– mice.

In contrast to the inhibition of glucose uptake and GLUT4 translocation in skeletal muscle, there was no significant difference in glucose uptake into either white or brown adipose tissue during the euglycemic clamp procedure (Figure 8a). This was confirmed in measurements of insulin-stimulated glucose transport in isolated primary adipocytes from syn4^+/– mice (Figure 8b). As observed for the in vivo glucose transport, insulin-stimulated glucose uptake in isolated primary adipocytes was not significantly different in syn4^+/– and wild-type cells. Together these data indicated that the insulin resistance observed in vivo specifically reflects decreased insulin-stimulated glucose uptake into skeletal muscle of the syn4^+/– mice.

Figure 8

Glucose transport in adipose tissues and isolated primary adipocytes. (a) In vivo glucose transport in white and brown adipose tissues during hyperinsulinemic-euglycemic clamp procedures. (b) 2DG uptake in isolated primary white adipocytes from 12-week-old wild-type mice (open bars) or heterozygous syntaxin 4–knockout mice (filled bars). Data shown are the average ± SD of three independent experiments.

It is well established that insulin stimulation enhances glucose uptake primarily in striated muscle and adipose tissue by recruiting the intracellular compartmentalized GLUT4 protein to the cell-surface membrane (1–4). Previous in vitro studies have suggested that syntaxin 4 functions as a plasma membrane t-SNARE during insulin-stimulated GLUT4 translocation (24–29, 45). However, to examine the function of syntaxin 4 in a physiological context, we generated syntaxin 4–knockout mice by homologous recombination. Our data clearly demonstrated that heterozygous syntaxin 4–mutant mice have an approximate 40% loss of syntaxin 4 protein and develop insulin resistance, as manifested by significant decreases in rates of whole-body glucose disposal and glycolysis. The reduction in syntaxin 4 protein levels correlated directly with a decrease in insulin-stimulated skeletal muscle glucose uptake without any significant effect on the basal rate of glucose transport. Importantly, the decrease in insulin-stimulated skeletal muscle glucose uptake occurred in parallel with a reduction in GLUT4 translocation. Thus, the inability of insulin to induce GLUT4 translocation in skeletal muscle of the syn4^+/– mice was directly reflected in an impairment of glucose uptake and glucose disposal.

The insulin resistance observed in these mice was specific to skeletal muscle because liver glucose metabolism and adipose tissue glucose uptake was unaffected. In hindsight, the apparent lack of effect in liver was not surprising, because syntaxin 4 appears to function primarily in the regulation of GLUT4 translocation. Furthermore, since liver does not express the GLUT4 protein and is not a regulator site for insulin-stimulated glucose uptake, syntaxin 4 probably plays other roles in this tissue unrelated to glucose metabolism.

In contrast, several reports have confirmed that in adipocytes (primary isolated or in tissue culture), syntaxin 4 functions as a specific t-SNARE for GLUT4 translocation (24–27, 29, 45). We were therefore very surprised that we did not detect any significant change in adipose tissue glucose uptake. However, this may be due to the fact that syntaxin 4 is abundantly expressed in the plasma membrane of adipocytes (data not shown). As such, the amount of cell-surface syntaxin 4 in adipocytes may provide a sufficient reservoir so that even with a 40% reduction there is still an excess of syntaxin 4 protein. Recently, a similar phenomenon has been observed in the case of the specific syntaxin 4–binding protein Munc18c (50). In this case, overexpression of Munc18c inhibited insulin-stimulated GLUT4 translocation to the transverse tubules but not the sarcolemma membrane of skeletal muscle. This was apparently due to a relatively higher level of syntaxin 4 at the sarcolemma compared with the transverse tubules. Under these conditions, the overexpressed Munc18c protein was able to saturate the transverse-tubule syntaxin 4 protein, but not the sarcolemma syntaxin 4. Thus, we would expect that the skeletal muscle transverse-tubule membrane would be more sensitive to perturbation of SNARE protein stoichiometry than either the sarcolemma membrane or the plasma membrane in adipocytes. This hypothesis is also consistent with the transverse-tubule membrane accounting for a greater extent of insulin-stimulated GLUT4 translocation and glucose uptake than the sarcolemma membrane in skeletal muscle (46, 51).

In summary, our generation and characterization of the syn4^+/– mice clearly support a critical role of syntaxin 4 in insulin-stimulated glucose transport. The syn4^+/– mice represent the first mouse model for the study of SNARE proteins in GLUT4 translocation and glucose transport. Although homozygotic disruption of the syntaxin 4 gene results in early embryonic lethality, rescuing of these animals using a regulated promoter driving syntaxin 4 expression or the development of a tissue-specific knockout will allow more detailed analysis of t-SNARE function in the regulation of GLUT4 translocation in vivo.

We would like to thank Ahmir Kahn and Diana Boeglin for their excellent technical assistance. This work was supported by research grants DK-25295 and DK-55811 from the NIH.

Chunmei Yang and Kenneth J. Coker’s present address is: Lexicon Genetics Inc., The Woodlands, Texas, USA.

Chunmei Yang and Kenneth J. Coker contributed equally to this work.

1. Mueckler, M. Facilitative glucose transporters. Eur J Biochem 1994. 219:713-725.
   View this article via: PubMed CrossRef Google Scholar
2. Zorzano, A, Fandos, C, Palacin, M. Role of plasma membrane transporters in muscle metabolism. Biochem J 2000. 349:667-688.
   View this article via: PubMed Google Scholar
3. Goodyear, LJ, Kahn, BB. Exercise, glucose transport, and insulin sensitivity. Annu Rev Med 1998. 49:235-261.
   View this article via: PubMed CrossRef Google Scholar
4. Galuska, D, Ryder, J, Kawano, Y, Charron, MJ, Zierath, JR. Insulin signaling and glucose transport in insulin resistant skeletal muscle. Special reference to GLUT4 transgenic and GLUT4 knockout mice. Adv Exp Med Biol 1998. 441:73-85.
   View this article via: PubMed Google Scholar
5. Charron, MJ, Brosius (III), FC, Alper, SL, Lodish, HF. A glucose transport protein expressed predominately in insulin-responsive tissues. Proc Natl Acad Sci USA 1989. 86:2535-2539.
   View this article via: PubMed CrossRef Google Scholar
6. Birnbaum, MJ. Identification of a novel gene encoding an insulin-responsive glucose transporter protein. Cell 1989. 57:305-315.
   View this article via: PubMed CrossRef Google Scholar
7. James, DE, Brown, R, Navarro, J, Pilch, PF. Insulin-regulatable tissues express a unique insulin-sensitive glucose transport protein. Nature 1988. 333:183-185.
   View this article via: PubMed CrossRef Google Scholar
8. James, DE, Strube, M, Mueckler, M. Molecular cloning and characterization of an insulin-regulatable glucose transporter. Nature 1989. 338:83-87.
   View this article via: PubMed CrossRef Google Scholar
9. Kaestner, KH, et al. Sequences, tissue distribution, and differential expression of mRNA for a putative insulin-responsive glucose transporter in mouse 3T3-L1 adipocytes. Proc Natl Acad Sci USA 1989. 86:3150-3154.
   View this article via: PubMed CrossRef Google Scholar
10. Fukumoto, H, et al. Cloning and characterization of the major insulin-responsive glucose transporter expressed in human skeletal muscle and other insulin-responsive tissues. J Biol Chem 1989. 264:7776-7779.
    View this article via: PubMed Google Scholar
11. Rodnick, KJ, et al. Immunocytochemical and biochemical studies of GLUT4 in rat skeletal muscle. J Biol Chem 1992. 267:6278-6285.
    View this article via: PubMed Google Scholar
12. Jhun, BH, Rampal, AL, Liu, H, Lachaal, M, Jung, CY. Effects of insulin on steady state kinetics of GLUT4 subcellular distribution in rat adipocytes. J Biol Chem 1992. 268:17710-17715.
13. Slot, JW, Geuze, HJ, Gigengack, S, Lienhard, GE, James, DE. Immuno-localization of the insulin regulatable glucose transporter in brown adipose tissue of the rat. J Cell Biol 1991. 113:123-135.
    View this article via: PubMed CrossRef Google Scholar
14. Slot, JW, Geuze, HJ, Gigengack, S, James, DE, Lienhard, GE. Translocation of the glucose transporter GLUT4 in cardiac myocytes of the rat. Proc Natl Acad Sci USA 1991. 88:7815-7819.
    View this article via: PubMed CrossRef Google Scholar
15. Satoh, S, et al. Use of bismannose photolabel to elucidate insulin-regulated GLUT4 subcellular trafficking kinetics in rat adipose cells. Evidence that exocytosis is a critical site of hormone action. J Biol Chem 1993. 268:17820-17829.
    View this article via: PubMed Google Scholar
16. Yang, J, Holman, GD. Comparison of GLUT4 and GLUT1 subcellular trafficking in basal and insulin-stimulated 3T3-L1 cells. J Biol Chem 1993. 268:4600-4603.
    View this article via: PubMed Google Scholar
17. Kahn, BB. Facilitative glucose transporters: regulatory mechanisms and dysregulation in diabetes. J Clin Invest 1992. 89:1367-1374.
    View this article via: JCI PubMed CrossRef Google Scholar
18. Mueckler, M. The molecular biology of glucose transport: relevance to insulin resistance and non-insulin-dependent diabetes mellitus. J Diabetes Complications 1993. 7:130-141.
    View this article via: PubMed CrossRef Google Scholar
19. Garvey, WT, Hardin, D, Juhaszova, M, Dominguez, JH. Effects of diabetes on myocardial glucose transport system in rats: implications for diabetic cardiomyopathy. Am J Physiol 1993. 264:H837-H844.
    View this article via: PubMed Google Scholar
20. James, DE, Piper, RC. Insulin resistance, diabetes, and the insulin-regulated trafficking of GLUT-4. J Cell Biol 1994. 126:1123-1126.
    View this article via: PubMed CrossRef Google Scholar
21. Rea, S, James, DE. Moving GLUT4: the biogenesis and trafficking of GLUT4 storage vesicles. Diabetes 1997. 46:1667-1677.
    View this article via: PubMed CrossRef Google Scholar
22. Pessin, JE, Thurmond, DC, Elmendorf, JS, Coker, KJ, Okada, S. Molecular basis of insulin-stimulated GLUT4 vesicle trafficking. Location! location! location! J Biol Chem 1999. 274:2593-2596.
    View this article via: PubMed CrossRef Google Scholar
23. St-Denis, SF, Cushman, SW. Role of SNARE’s in the Glut4 translocation response to insulin in adipose cells and muscle. J Basic Clin Physiol Pharmacol 1998. 9:153-165.
    View this article via: PubMed Google Scholar
24. Cheatham, B, et al. Insulin-stimulated translocation of GLUT4 glucose transporters requires SNARE-complex proteins. Proc Natl Acad Sci USA 1996. 93:15169-15173.
    View this article via: PubMed CrossRef Google Scholar
25. Olson, AL, Knight, JB, Pessin, JE. Syntaxin 4, VAMP2, and/or VAMP3/cellubrevin are functional target membrane and vesicle SNAP receptors for insulin-stimulated GLUT4 translocation in adipocytes. Mol Cell Biol 1997. 17:2425-2435.
    View this article via: PubMed Google Scholar
26. Volchuk, A, et al. Syntaxin 4 in 3T3-L1 adipocytes: regulation by insulin and participation in insulin-dependent glucose transport. Mol Biol Cell 1996. 7:1075-1082.
    View this article via: PubMed Google Scholar
27. Martin, LB, Shewan, A, Millar, CA, Gould, GW, James, DE. Vesicle-associated membrane protein 2 plays a specific role in the insulin-dependent trafficking of the facilitative glucose transporter GLUT4 in 3T3-L1 adipocytes. J Biol Chem 1998. 273:1444-1452.
    View this article via: PubMed CrossRef Google Scholar
28. Rea, S, et al. Syndet, an adipocyte target SNARE involved in the insulin-induced translocation of GLUT4 to the cell surface. J Biol Chem 1998. 273:18784-18792.
    View this article via: PubMed CrossRef Google Scholar
29. Macaulay, SL, et al. Functional studies in 3T3L1 cells support a role for SNARE proteins in insulin stimulation of GLUT4 translocation. Biochem J 1997. 324:217-224.
    View this article via: PubMed Google Scholar
30. Kawanishi, M, et al. Role of SNAP23 in insulin-induced translocation of GLUT4 in 3T3-L1 adipocytes. Mediation of complex formation between syntaxin4 and VAMP2. J Biol Chem 2000. 275:8240-8247.
    View this article via: PubMed CrossRef Google Scholar
31. Randhawa, V, et al. VAMP2, but not VAMP3/cellubrevin, mediates insulin-dependent incorporation of GLUT4 into the plasma membrane of L6 myoblasts. Mol Biol Cell 2000. 11:2403-2407.
    View this article via: PubMed Google Scholar
32. Tellam, JT, et al. Characterization of Munc-18c and syntaxin-4 in 3T3-L1 adipocytes. Putative role in insulin-dependent movement of GLUT-4. J Biol Chem 1997. 272:6179-6186.
    View this article via: PubMed CrossRef Google Scholar
33. Thurmond, DC, et al. Regulation of insulin-stimulated GLUT4 translocation by munc18c in 3T3L1 adipocytes. J Biol Chem 1998. 273:33876-33883.
    View this article via: PubMed CrossRef Google Scholar
34. Zhou, M, et al. Insulin-dependent protein trafficking in skeletal muscle cells. Am J Physiol 1998. 275:E187-E196.
    View this article via: PubMed Google Scholar
35. Enrique-Tarancon, G, et al. Role of semicarbazide-sensitive amine oxidase on glucose transport and GLUT4 recruitment to the cell surface in adipose cells. J Biol Chem 1998. 273:8025-8032.
    View this article via: PubMed CrossRef Google Scholar
36. Ralston, E, Beushausen, S, Ploug, T. Expression of the synaptic vesicle proteins VAMPs/synaptobrevins 1 and 2 in non-neural tissues. J Biol Chem 1994. 269:15403-15406.
    View this article via: PubMed Google Scholar
37. Jagadish, MN, et al. Insulin-responsive tissues contain the core complex protein SNAP-25 (synaptosomal-associated protein 25) A and B isoforms in addition to syntaxin 4 and synaptobrevins 1 and 2. Biochem J 1996. 317:945-954.
    View this article via: PubMed Google Scholar
38. Martin, S, et al. The glucose transporter (GLUT-4) and vesicle-associated membrane protein-2 (VAMP-2) are segregated from recycling endosomes in insulin-sensitive cells. J Cell Biol 1996. 134:625-635.
    View this article via: PubMed CrossRef Google Scholar
39. Kandror, KV, Pilch, PF. gp160, a tissue-specific marker for insulin-activated glucose transport. Proc Natl Acad Sci USA 1994. 91:8017-8021.
    View this article via: PubMed CrossRef Google Scholar
40. Kandror, K, Pilch, PF. Identification and isolation of glycoproteins that translocate to the cell surface from GLUT4-enriched vesicles in an insulin-dependent fashion. J Biol Chem 1994. 269:138-142.
    View this article via: PubMed Google Scholar
41. Keller, SR, Scott, HM, Mastick, CC, Aebersold, R, Lienhard, GE. Cloning and characterization of a novel insulin-regulated membrane aminopeptidase from Glut4 vesicles. J Biol Chem 1995. 270:23612-23618.
    View this article via: PubMed CrossRef Google Scholar
42. Kandror, KV, Yu, L, Pilch, PF. The major protein of GLUT4-containing vesicles, gp160, has aminopeptidase activity. J Biol Chem 1994. 269:30777-30780.
    View this article via: PubMed Google Scholar
43. Martin, S, et al. The glucose transporter GLUT4 and the aminopeptidase vp165 colocalise in tubulo-vesicular elements in adipocytes and cardiomyocytes. J Cell Sci 1997. 110:2281-2291.
    View this article via: PubMed Google Scholar
44. Ross, SA, Herbst, JJ, Keller, SR, Lienhard, GE. Trafficking kinetics of the insulin-regulated membrane aminopeptidase in 3T3-L1 adipocytes. Biochem Biophys Res Commun 1997. 239:247-251.
    View this article via: PubMed CrossRef Google Scholar
45. Timmers, KI, et al. Identification of SNAP receptors in rat adipose cell membrane fractions and in SNARE complexes co-immunoprecipitated with epitope-tagged N-ethylmaleimide-sensitive fusion protein. Biochem J 1996. 320:429-436.
    View this article via: PubMed Google Scholar
46. Marette, A, Burdett, E, Douen, A, Vranic, M, Klip, A. Insulin induces the translocation of GLUT4 from a unique intracellular organelle to transverse tubules in rat skeletal muscle. Diabetes 1992. 41:1562-1569.
    View this article via: PubMed CrossRef Google Scholar
47. Ploug, T, van Deurs, B, Ai, H, Cushman, SW, Ralston, E. Analysis of GLUT4 distribution in whole skeletal muscle fibers: identification of distinct storage compartments that are recruited by insulin and muscle contractions. J Cell Biol 1998. 142:1429-1446.
    View this article via: PubMed CrossRef Google Scholar
48. Munoz, P, Rosemblatt, M, Testar, X, Palacin, M, Zorzano, A. Isolation and characterization of distinct domains of sarcolemma and T-tubules from rat skeletal muscle. Biochem J 1995. 307:273-280.
    View this article via: PubMed Google Scholar
49. Goodyear, LJ, Hirshman, MF, Valyou, PM, Horton, ES. Glucose transporter number, function, and subcellular distribution in rat skeletal muscle after exercise training. Diabetes 1992. 41:1091-1099.
    View this article via: PubMed CrossRef Google Scholar
50. Khan, AH, et al. Munc18c regulates insulin-stimulated GLUT4 translocation to the transverse tubules in skeletal muscle. J Biol Chem 2001. 276:4063-4069.
    View this article via: PubMed CrossRef Google Scholar
51. Munoz, P, et al. The T-tubule is a cell-surface target for insulin-regulated recycling of membrane proteins in skeletal muscle. Biochem J 1995. 312:393-400.
    View this article via: PubMed Google Scholar