Expression of a Dominant Interfering Dynamin Mutant in 3T3L1 Adipocytes Inhibits GLUT4 Endocytosis without Affecting Insulin Signaling
ABSTRACT To examine the role of clathrin-coated vesicle endocytosis in insulin receptor signaling and GLUT4 trafficking, we used recombinant
adenovirus to express a dominant interfering mutant of dynamin (K44A/dynamin) in 3T3L1 adipocytes. Functional expression of
K44A/dynamin, as measured by inhibition of transferrin receptor internalization, did not affect insulin-stimulated insulin
receptor autophosphorylation, Shc tyrosine phosphorylation, or mitogen-activated protein kinase activation. Although the tyrosine
phosphorylation of insulin receptor substrate-1 was slightly reduced, correlating with a 25% decrease in insulin receptor
substrate-1-associated phosphatidylinositol 3-kinase activity, insulin-stimulated Akt kinase activation was unaffected. In
contrast, expression of K44A/dynamin resulted in the cell-surface accumulation of GLUT4 under basal conditions and an inhibition
of GLUT4 endocytosis without affecting insulin-stimulated GLUT4 exocytosis. These data demonstrate that disruption of clathrin-mediated
endocytosis does not significantly perturb insulin receptor signal transduction pathways. Furthermore, K44A/dynamin expression
causes an accumulation of GLUT4 at the cell surface, suggesting that GLUT4 vesicles exist in at least two distinct intracellular
compartments, one that undergoes continuous recycling and a second that is responsive to insulin.
- SourceAvailable from: PubMed Central
[Show abstract] [Hide abstract]
- "Insulin-stimulated transfected adipocytes were chilled to 4 ° C and incubated with the myc monoclonal antibody for 1 h to label GLUT4 at the plasma membrane. Cells were then washed to remove insulin and excess myc antibody as described previously ( Kao et al., 1998 ). The cells were placed at 37 ° C and incubated for various times to allow the myc antibody – bound GLUT4 to internalize. "
ABSTRACT: The functional trafficking steps used by soluble NSF attachment protein receptor (SNARE) proteins have been difficult to establish because of substantial overlap in subcellular localization and because in vitro SNARE-dependent binding and fusion reactions can be promiscuous. Therefore, to functionally identify the site of action of the vesicle-associated membrane protein (VAMP) family of R-SNAREs, we have taken advantage of the temporal requirements of adipocyte biosynthetic sorting of a dual-tagged GLUT4 reporter (myc-GLUT4-GFP) coupled with small interfering RNA gene silencing. Using this approach, we confirm the requirement of VAMP2 and VAMP7 for insulin and osmotic shock trafficking from the vesicle storage sites, respectively, and fusion with the plasma membrane. Moreover, we identify a requirement for VAMP4 for the initial biosynthetic entry of GLUT4 from the Golgi apparatus into the insulin-responsive vesicle compartment, VAMP8, for plasma membrane endocytosis and VAMP2 for sorting to the specialized insulin-responsive compartment after plasma membrane endocytosis.The Journal of Cell Biology 02/2008; 180(2):375-87. DOI:10.1083/jcb.200709108 · 9.69 Impact Factor
[Show abstract] [Hide abstract]
- "Basal golgin-160 knockdown cells and insulin-stimulated control adipocytes were chilled and incubated with the myc monoclonal antibody for 1 h at 4°C to label the GLUT4 at the plasma membrane. Cells were then washed to remove insulin and excess myc antibody as described previously (Kao et al., 1998). The cells were placed at 37°C and incubated for various times to allow myc antibody-bound GLUT4 to internalize. "
ABSTRACT: The peripheral Golgi protein golgin-160 is induced during 3T3L1 adipogenesis and is primarily localized to the Golgi cisternae distinct from the trans-Golgi network (TGN) in a general distribution similar to p115. Small interfering RNA (siRNA)-mediated reduction in golgin-160 protein resulted in an increase accumulation of the insulin-responsive amino peptidase (IRAP) and the insulin-regulated glucose transporter (GLUT4) at the plasma membrane concomitant with enhanced glucose uptake in the basal state. The redistribution of GLUT4 was rescued by expression of a siRNA-resistant golgin-160 cDNA. The basal state accumulation of plasma membrane GLUT4 occurred due to an increased rate of exocytosis without any significant effect on the rate of endocytosis. This GLUT4 trafficking to the plasma membrane in the absence of golgin-160 was independent of TGN/Golgi sorting, because it was no longer inhibited by the expression of a dominant-interfering Golgi-localized, gamma-ear-containing ARF-binding protein mutant and displayed reduced binding to the lectin wheat germ agglutinin. Moreover, expression of the amino terminal head domain (amino acids 1-393) had no significant effect on the distribution or insulin-regulated trafficking of GLUT4 or IRAP. In contrast, expression of carboxyl alpha helical region (393-1498) inhibited insulin-stimulated GLUT4 and IRAP translocation, but it had no effect on the sorting of constitutive membrane trafficking proteins, the transferrin receptor, or vesicular stomatitis virus G protein. Together, these data demonstrate that golgin-160 plays an important role in directing insulin-regulated trafficking proteins toward the insulin-responsive compartment in adipocytes.Molecular Biology of the Cell 01/2007; 17(12):5346-55. DOI:10.1091/mbc.E06-05-0386 · 4.55 Impact Factor
[Show abstract] [Hide abstract]
- "In order to investigate whether HMIT internalization was dependent on dynamin-mediated endocytosis, we infected HMIT-expressing cells with the GTPase-deficient dominantnegative form of dynamin (dynaminK44A) (Ceresa et al, 1998; Kao et al, 1998). Under basal conditions, cell surface expression of HMIT could be clearly detected in cells expressing dynaminK44A but not in control cells. "
ABSTRACT: Phosphoinositides, synthesized from myo-inositol, play a critical role in the development of growth cones and in synaptic activity. As neurons cannot synthesize inositol, they take it up from the extracellular milieu. Here, we demonstrate that, in brain and PC12 cells, the recently identified H(+)/myo-inositol symporter HMIT is present in intracellular vesicles that are distinct from synaptic and dense-core vesicles. We further show that HMIT can be triggered to appear on the cell surface following cell depolarization, activation of protein kinase C or increased intracellular calcium concentrations. HMIT cell surface expression takes place preferentially in regions of nerve growth and at varicosities and leads to increased myo-inositol uptake. The symporter is then endocytosed in a dynamin-dependent manner and becomes available for a subsequent cycle of stimulated exocytosis. HMIT is thus expressed in a vesicular compartment involved in activity-dependent regulation of myo-inositol uptake in neurons. This may be essential for sustained signaling and vesicular traffic activities in growth cones and at synapses.The EMBO Journal 03/2004; 23(3):531-40. DOI:10.1038/sj.emboj.7600072 · 10.75 Impact Factor