Biochemical and Biophysical Research Communications
Syntaxin 16 controls the intracellular sequestration of GLUT4 in 3T3-L1 adipocytes☆
Section snippets
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.
References (17)
- et al.
Molecular basis of insulin stimulated GLUT4 vesicle trafficking: Location! Location! Location!
J. Biol. Chem.
(1999) - et al.
A Leu-Leu sequence is essential for COOH-terminal targeting signal of GLUT4 glucose transporter in fibroblasts
J. Biol. Chem.
(1994) - et al.
Vesicle associated membrane protein-2 (VAMP2) plays a specific role in the insulin-dependent trafficking of the facilitative glucose transporter GLUT4 in 3T3-L1 adipocytes
J. Biol. Chem.
(1998) - et al.
Regulated trafficking of the glucose transporter, Glut4
Nat. Rev. Mol. Cell Biol.
(2002) - et al.
Molecular regulation of GLUT-4 targeting in 3T3-L1 adipocytes
J. Cell Biol.
(1995) - et al.
Mutational analysis of the carboxy-terminal phosphorylation site of GLUT-4 in 3T3-L1 adipocytes
Am. J. Physiol.
(1998) - et al.
Analysis of amino and carboxy terminal GLUT-4 targeting motifs in 3T3-L1 adipocytes using an endosomal ablation technique
Biochemistry
(1998) - et al.
GLUT4 NH2 terminus contains a phenylalanine-based targeting motif that regulates intracellular sequestration
J. Cell Biol.
(1993)
Cited by (43)
Building GLUT4 Vesicles: CHC22 Clathrin's Human Touch
2020, Trends in Cell BiologyThirty sweet years of GLUT4
2019, Journal of Biological ChemistryTUSC5 regulates insulin-mediated adipose tissue glucose uptake by modulation of GLUT4 recycling
2015, Molecular MetabolismExpression of syntaxin 8 in visceral adipose tissue is increased in obese patients with type 2 diabetes and related to markers of insulin resistance and inflammation
2015, Archives of Medical ResearchCitation Excerpt :Data from the present study evidence an increased gene and protein expression of STX8 in VAT of OB-T2D patients showing for the first time that STX8 appears to be implicated in the pathogenesis of T2D. Although the physiological function of STX8 has not been fully elucidated, the involvement of other members of the STX family in the development of insulin resistance has been reported (21,31–33). In this sense, it has been observed that STX4 overexpression increased the insulin-mediated glucose uptake in skeletal muscle (21).
- ☆
Abbreviations: Stx, Syntaxin; TGN, trans Golgi network; MAO, morpholino antisense oligonucleotide; DMEM, Dulbecco’s modified Eagle’s medium; GSV, Glut4-storage vesicles.