Syntaxin 4, VAMP2, and/or VAMP3/cellubrevin are functional target membrane and vesicle SNAP receptors for insulin-stimulated GLUT4 translocation in adipocytes.

Introduction of the cytoplasmic domain of syntaxin 4, using either recombinant vaccinia virus or single-cell microinjection, resulted in an inhibition of insulin-stimulated GLUT4 but not GLUT1 translocation to the plasma membrane. This was specific for syntaxin 4, since neither the expression of syntaxin 3 nor the expression of a syntaxin 4 mutant in which the vesicle-associated membrane protein (VAMP) binding site was deleted had any significant effect. Consistent with the requirement for a functional VAMP binding site, expression of the cytoplasmic domains of VAMP2 or VAMP3/cellubrevin also resulted in an inhibition of insulin-stimulated GLUT4 translocation. In addition, immunoprecipitation of the expressed syntaxin 4 cytoplasmic domain resulted in an insulin-stimulated increase in the coimmunoprecipitation of GLUT4-containing vesicles. Together, these data demonstrate that syntaxin 4, VAMP2, and/or VAMP3/cellubrevin can function as target membrane and vesicle SNAP receptors, respectively, for insulin-responsive GLUT4 translocation to the plasma membrane.     

Glucose deprivation induces Akt-dependent synthesis and incorporation of GLUT1, but not of GLUT4, into the plasma membrane of 3T3-L1 adipocytes

Reduction of the glucose concentration in the culture medium of 3T3-L1 adipose cells below 1.25 mM produces a 4-8-fold stimulation of 2-deoxyglucose uptake which starts after a lag phase of 2 h and is maximal after 10-16 h. In the present study, we employed the 'membrane sheet assay' in order to re-assess the contribution of the transporter isoforms GLUT1 and GLUT4 to this effect. Immunochemical assay of glucose transporters in membranes prepared with the 'sheet assay' revealed that the effect reflected a marked increase of GLUT1 in the plasma membrane with no effect on GLUT4. Glucose deprivation increased the total cellular GLUT1 protein in parallel with the transport activity, whereas GLUT4 was unaltered. The specific PI 3-kinase inhibitor wortmannin inhibited the effect of glucose deprivation on transport activity and also on GLUT1 synthesis. Glucose deprivation produced a moderate, biphasic increase in the activity of the protein kinase Akt/PKB that was inhibitable by wortmannin. When wortmannin was added after stimulation of cells in order to assess the internalization rate of transporters, the effect of insulin was reversed considerably faster (T1/2 = 18 min) than that of glucose deprivation (T1/2 > 60 min). These data are consistent with the conclusion that the effect of glucose deprivation reflects a specific, Akt-dependent de-novo synthesis of GLUT1, and not of GLUT4, and its insertion into a plasma membrane compartment which is distinct from that of the insulin-sensitive GLUT1.     

The t-SNAREs syntaxin4 and SNAP23 but not v-SNARE VAMP2 are indispensable to tether GLUT4 vesicles at the plasma membrane in adipocyte

SNARE proteins (VAMP2, syntaxin4, and SNAP23) have been thought to play a key role in GLUT4 trafficking by mediating the tethering, docking and subsequent fusion of GLUT4-containing vesicles with the plasma membrane. The precise functions of these proteins have remained elusive, however. We have now shown that depletion of the vesicle SNARE (v-SNARE) VAMP2 by RNA interference in 3T3-L1 adipocytes inhibited the fusion of GLUT4 vesicles with the plasma membrane but did not affect tethering of the vesicles to the membrane. In contrast, depletion of the target SNAREs (t-SNAREs) syntaxin4 or SNAP23 resulted in impairment of GLUT4 vesicle tethering to the plasma membrane. Our results indicate that the t-SNAREs syntaxin4 and SNAP23 are indispensable for the tethering of GLUT4 vesicles to the plasma membrane, whereas the v-SNARE VAMP2 is not required for this step but is essential for the subsequent fusion event.     

Protein kinase B stimulates the translocation of GLUT4 but not GLUT1 or transferrin receptors in 3T3-L1 adipocytes by a pathway involving SNAP-23, synaptobrevin-2, and/or cellubrevin.

An interaction of SNAP-23 and syntaxin 4 on the plasma membrane with vesicle-associated synaptobrevin-2 and/or cellubrevin, known as SNAP (soluble N-ethyl-maleimide-sensitive factor attachment protein) receptors or SNAREs, has been proposed to provide the targeting and/or fusion apparatus for insulin-stimulated translocation of the GLUT4 isoform of glucose transporter to the plasma membrane. By microinjecting 3T3-L1 adipocytes with the Clostridium botulinum toxin B or E, which proteolyzed synaptobrevin-2/cellubrevin and SNAP-23, respectively, we investigated the role of these SNAREs in GLUT4, GLUT1, and transferrin receptor trafficking. As expected, insulin stimulated the translocation of GLUT4, GLUT1, and transferrin receptors to the plasma membrane. By contrast, a constitutively active protein kinase B (PKB-DD) only stimulated a translocation of GLUT4 and not GLUT1 or the transferrin receptor. The GLUT4 response to PKB-DD was abolished by toxins B or E, whereas the insulin-evoked translocation of GLUT4 was inhibited by approximately 65%. These toxins had no significant effect on insulin-stimulated transferrin receptor appearance at the cell surface. Thus, insulin appears to induce GLUT4 translocation via two distinct routes, only one of which involves SNAP-23 and synaptobrevin-2/cellubrevin, and can be mobilized by PKB-DD. The PKB-, SNAP-23-, and synaptobrevin-2/cellubrevin-independent GLUT4 translocation pathway may involve movement through recycling endosomes, together with GLUT1 and transferrin receptors.     

Cytochalasin-D retards sperm incorporation deep into the egg cytoplasm but not membrane fusion with the egg plasma membrane

The fertilization process is impaired when spermatozoa are previously incubated with Cytochalasin-D (Cyt-D). Although this fact reveals the participation of polymerized actin in fertilization, the specific event obstructed by Cyt-D treatment has not been determined. To identify this event, we capacitated guinea pig spermatozoa in minimal capacitating medium with pyruvate and lactate (MCM-PL) with Cyt-D, to inseminate hamster zona pellucida (ZP)-free eggs. Cyt-D (70 microM) decreased F-actin relative concentration in capacitated spermatozoa to a larger extent than in spermatozoa incubated under control conditions. Cyt-D also cancelled the F-actin increase normally observed in acrosome-reacted cells, and decreased the number of these cells with normal F-actin localization at the equatorial zone. Insemination of eggs with Cyt-D treated spermatozoa did not change early fertilization events such as the egg cortical reaction (CR), membranes fusion, and egg F-actin new localization, but clearly retarded, by 16 hr, spermatozoa incorporation deep into the egg cytoplasm, and decondensation of egg metaphase II chromosomes. These results show that actin polymerization is necessary for spermatozoa incorporation deep into the egg cytoplasm, but not for plasma membrane fusion nor egg activation early steps.     

Evidence that a membrane bound lectin mediates fusion of L6 myoblasts.

The external membranes of L₆ myoblasts are shown to posses lectin activity which resides in protein molecules. Thiodigalactoside blocks agglutination of formalinized, trypsin treated rabbit red blood cells caused by the lectin. Thiodigalactoside at a concentration which neither inhibits cell division nor decreases cell yield prevents fusions of myoblasts. It is suggested that this protein which has lectin activity also is an essential participant in the membrane events which cause fusion of myoblasts to form myotubes.     

C2 domain-containing phosphoprotein CDP138 regulates GLUT4 insertion into the plasma membrane

The protein kinase B(β) (Akt2) pathway is known to?mediate insulin-stimulated glucose transport through increasing glucose transporter GLUT4 translocation from intracellular stores to the plasma membrane (PM). Combining quantitative phosphoproteomics with RNAi-based functional analyses, we show that a previously uncharacterized 138?kDa C2 domain-containing phosphoprotein (CDP138) is a substrate for Akt2, and is required for optimal insulin-stimulated glucose transport, GLUT4 translocation, and fusion of GLUT4 vesicles with the PM in live adipocytes. The purified C2 domain is capable of binding Ca(2+) and lipid membranes. CDP138 mutants lacking the Ca(2+)-binding sites in the C2 domain or Akt2 phosphorylation site S197 inhibit insulin-stimulated GLUT4 insertion into the PM, a rate-limiting step of GLUT4 translocation. Interestingly, CDP138 is dynamically associated with the PM and GLUT4-containing vesicles in response to insulin stimulation. Together, these results suggest that CDP138 is a key molecule linking the Akt2 pathway to the regulation of GLUT4 vesicle-PM fusion.     

Temporal separation of insulin-stimulated GLUT4/IRAP vesicle plasma membrane docking and fusion in 3T3L1 adipocytes

Examination of the time and temperature dependence of insulin-stimulated GLUT4/IRAP-containing vesicle trafficking demonstrated an approximate 7-fold increase in the half-time for plasma membrane translocation at 23 degrees C (t((1)/(2)) = approximately 30 min) compared with 37 degrees C (t((1)/(2)) = approximately 4 min) without a significant change in the extent of either GLUT4 or IRAP translocation. Localization of the endogenous GLUT4 and expressed GLUT4-enhanced green fluorescent protein fusion protein in intact 3T3L1 adipocytes demonstrated that at 23 degrees C there was a time-dependent accumulation of discrete GLUT4-containing vesicles adjacent to the inner face of the cell surface membrane but that was not contiguous and/or physically incorporated into the plasma membrane. Together, these data demonstrate that the temperature-dependent decrease in the rate of GLUT4 and IRAP translocation results from a reduction in GLUT4/IRAP-containing vesicle fusion and not trafficking or docking to the plasma membrane.     

Interleukin-4 mediates STAT6 activation in 3T3-L1 preadipocytes but not adipocytes

STAT6 is abundantly expressed in 3T3-L1 preadipocytes and adipocytes but activating ligands are not well defined. In this report, we provide evidence that interleukin 4 (IL-4) induced JAK2-mediated STAT6 tyrosine phosphorylation and DNA binding in 3T3-L1 preadipocytes but not in 3T3-L1 adipocytes. Loss of IL-4-mediated STAT6 tyrosine phosphorylation occurred 2 days after preadipocytes were induced to differentiate into adipocytes but when cells remained phenotypically preadipocytes. 3T3-L1 adipocytes were still responsive to IL-4 through tyrosine phosphorylation of other cellular proteins. We conclude that IL-4 signals through STAT6 in 3T3-L1 preadipocytes but not in 3T3-L1 adipocytes. This differentiation-dependent loss of STAT6 activation may be critical for distinct biological effects of IL-4 in 3T3-L1 preadipocytes and adipocytes.     

Detection of insulin-regulated GLUT4-translocation by the insertion of a protein C epitope in L6 myoblasts

Insulin stimulates glucose transport by translocation of the membrane glucose transporter GLUT4 from intracellular vesicles to the plasma membrane. GLUT4 is highly expressed in adipose tissue and skeletal muscle. We have constructed a cDNA containing the human GLUT4 inserted by a 12 amino acid protein C epitope in the first extracellular (exofacial) domain of the human GLUT4 (GLUT4-PC). Stable expression of GLUT4-PC in L6 myoblasts (L6-GLUT4-PC) was confirmed in immunofluorescence using monoclonal antibodies against protein C. The protein C staining yielded labeling in perinuclear vesicles strongly co-localizing with GLUT4 detected with antibodies directed against the endofacial part of GLUT4. The L6-GLUT4-PC cells were further characterized in a direct cell-based enzyme-linked immunosorbent assay by the use of beta-galactosidase. Cell surface binding of monoclonal protein C antibodies was detected with beta-galactosidase-conjugated secondary antibodies and chlorophenolred-beta-D-galactopyranoside (CPRG) as substrate in 2% paraformaldehyde fixed cells. In this assay, stimulation with insulin created a rapidly detectable recruitment of GLUT4-PC to the cell surface. This cell-based enzyme-linked immunosorbent GLUT4 assay was shown to be comparable with that of previously reported radioactive assays.     

Multiple plasma membrane receptors but not NPC1L1 mediate high-affinity, ezetimibe-sensitive cholesterol uptake into the intestinal brush border membrane

We compared cholesterol uptake into brush border membrane vesicles (BBMV) made from the small intestines of either wild-type or Niemann-Pick C1-like 1 (NPC1L1) knockout mice to elucidate the contribution of NPC1L1 to facilitated uptake; this uptake involves cholesterol transport from lipid donor particles into the BBM of enterocytes. The lack of NPC1L1 in the BBM of the knockout mice had no effect on the rate of cholesterol uptake. It follows that NPC1L1 cannot be the putative high-affinity, ezetimibe-sensitive cholesterol transporter in the brush border membrane (BBM) as has been proposed by others. The following findings substantiate this conclusion: (I) NPC1L1 is not a brush border membrane protein but very likely localized to intracellular membranes; (II) the cholesterol absorption inhibitor ezetimibe and its analogues reduce cholesterol uptake to the same extent in wild-type and NPC1L1 knockout mouse BBMV. These findings indicate that the prevailing belief that NPC1L1 facilitates intestinal cholesterol uptake into the BBM and its interaction with ezetimibe is responsible for the inhibition of this process can no longer be sustained.     

Overexpression of Vesicle-associated Membrane Protein (VAMP) 3, but Not VAMP2, Protects Glucose Transporter (GLUT) 4 Protein Translocation in an in Vitro Model of Cardiac Insulin Resistance

Cardiac glucose utilization is regulated by reversible translocation of the glucose transporter GLUT4 from intracellular stores to the plasma membrane. During the onset of diet-induced insulin resistance, elevated lipid levels in the circulation interfere with insulin-stimulated GLUT4 translocation, leading to impaired glucose utilization. Recently, we identified vesicle-associated membrane protein (VAMP) 2 and 3 to be required for insulin- and contraction-stimulated GLUT4 translocation, respectively, in cardiomyocytes. Here, we investigated whether overexpression of VAMP2 and/or VAMP3 could protect insulin-stimulated GLUT4 translocation under conditions of insulin resistance. HL-1 atrial cardiomyocytes transiently overexpressing either VAMP2 or VAMP3 were cultured for 16 h with elevated concentrations of palmitate and insulin. Upon subsequent acute stimulation with insulin, we measured GLUT4 translocation, plasmalemmal presence of the fatty acid transporter CD36, and myocellular lipid accumulation. Overexpression of VAMP3, but not VAMP2, completely prevented lipid-induced inhibition of insulin-stimulated GLUT4 translocation. Furthermore, the plasmalemmal presence of CD36 and intracellular lipid levels remained normal in cells overexpressing VAMP3. However, insulin signaling was not retained, indicating an effect of VAMP3 overexpression downstream of PKB/Akt. Furthermore, we revealed that endogenous VAMP3 is bound by the contraction-activated protein kinase D (PKD), and contraction and VAMP3 overexpression protect insulin-stimulated GLUT4 translocation via a common mechanism. These observations indicate that PKD activates GLUT4 translocation via a VAMP3-dependent trafficking step, which pathway might be valuable to rescue constrained glucose utilization in the insulin-resistant heart.     

Insulin-sensitive targeting of the GLUT4 glucose transporter in L6 myoblasts is conferred by its COOH-terminal cytoplasmic tail

The GLUT4 glucose transporter appears to be targeted to a unique insulin-sensitive intracellular membrane compartment in fat and muscle cells. Insulin stimulates glucose transport in these cell types by mediating the partial redistribution of GLUT4 from this intracellular compartment to the plasma membrane. The structural basis for the unique targeting behavior of GLUT4 was investigated in the insulin-sensitive L6 myoblast cell line. Analysis of immunogold-labeled cells of independent clonal lines by electron microscopy indicated that 51-53% of GLUT1 was present in the plasma membrane in the basal state. Insulin did not significantly affect this distribution. In contrast, only 4.2- 6.1% of GLUT4 was present in the plasma membrane of basal L6 cells and insulin increased this percentage by 3.7-6.1-fold. Under basal conditions and after insulin treatment, GLUT4 was detected in tubulovesicular structures, often clustered near Golgi stacks, and in endosome-like vesicles. Analysis of 25 chimeric transporters consisting of reciprocal domains of GLUT1 and GLUT4 by confocal immunofluorescence microscopy indicated that only the final 25 amino acids of the COOH- terminal cytoplasmic tail of GLUT4 were both necessary and sufficient for the targeting pattern observed for GLUT4. A dileucine motif present in the COOH-terminal tail of GLUT4 was found to be necessary, but not sufficient, for intracellular targeting. Contrary to previous studies, the NH2 terminus of GLUT4 did not affect the subcellular distribution of chimeras. Analysis of a chimera containing the COOH-terminal tail of GLUT4 by immunogold electron microscopy indicated that its subcellular distribution in basal cells was very similar to that of wild-type GLUT4 and that its content in the plasma membrane increased 6.8-10.5-fold in the presence of insulin. Furthermore, only the chimera containing the COOH terminus of GLUT4 enhanced insulin responsive 2-deoxyglucose uptake. GLUT1 and two other chimeras lacking the COOH terminus of GLUT4 were studied by immunogold electron microscopy and did not demonstrate insulin-mediated changes in subcellular distribution. The NH2-terminal cytoplasmic tail of GLUT4 did not confer intracellular sequestration and did not cause altered subcellular distribution in the presence of insulin. Intracellular targeting of one chimera to non-insulin- sensitive compartments was also observed. We conclude that the COOH terminus of GLUT4 is both necessary and sufficient to confer insulin- sensitive subcellular targeting of chimeric glucose transporters in L6 myoblasts.     

Akt2 regulates Rac1 activity in the insulin-dependent signaling pathway leading to GLUT4 translocation to the plasma membrane in skeletal muscle cells

The small GTPase Rac1 plays a pivotal role in insulin-stimulated glucose uptake in skeletal muscle, which is mediated by GLUT4 translocation to the plasma membrane. However, regulatory mechanisms for Rac1 and its role in the signaling pathway composed of phosphoinositide 3-kinase and the serine/threonine kinase Akt remain obscure. Here, we investigate the role of Akt in the regulation of Rac1 in myocytes. Insulin-induced, but not constitutively activated Rac1-induced, GLUT4 translocation was suppressed by Akt inhibitor IV. Insulin-induced Rac1 activation, on the other hand, was completely inhibited by this inhibitor. Constitutively activated phosphoinositide 3-kinase induced Rac1 activation and GLUT4 translocation. This GLUT4 translocation was almost completely suppressed by Rac1 knockdown. Furthermore, constitutively activated phosphoinositide 3-kinase-induced, but not constitutively activated Rac1-induced, GLUT4 translocation was suppressed by Akt2 knockdown. Finally, insulin-induced Rac1 activation was indeed inhibited by Akt2 knockdown. Together, these results reveal a novel regulatory mechanism involving Akt2 for insulin-dependent Rac1 activation.     

PI 4,5-P2 stimulates glucose transport activity of GLUT4 in the plasma membrane of 3T3-L1 adipocytes

Insulin-stimulated glucose uptake through GLUT4 plays a pivotal role in maintaining normal blood glucose levels. Glucose transport through GLUT4 requires both GLUT4 translocation to the plasma membrane and GLUT4 activation at the plasma membrane. Here we report that a cell-permeable phosphoinositide-binding peptide, which induces GLUT4 translocation without activation, sequestered PI 4,5-P2 in the plasma membrane from its binding partners. Restoring PI 4,5-P2 to the plasma membrane after the peptide treatment increased glucose uptake. No additional glucose transporters were recruited to the plasma membrane, suggesting that the increased glucose uptake was attributable to GLUT4 activation. Cells overexpressing phosphatidylinositol-4-phosphate 5-kinase treated with the peptide followed by its removal exhibited a higher level of glucose transport than cells stimulated with a submaximal level of insulin. However, only cells treated with submaximal insulin exhibited translocation of the PH-domains of the general receptor for phosphoinositides (GRP1) to the plasma membrane. Thus, PI 4,5-P2, but not PI 3,4,5-P3 converted from PI 4,5-P2, induced GLUT4 activation. Inhibiting F-actin remodeling after the peptide treatment significantly impaired GLUT4 activation induced either by PI 4,5-P2 or by insulin. These results suggest that PI 4,5-P2 in the plasma membrane acts as a second messenger to activate GLUT4, possibly through F-actin remodeling.     

Differential localization of glucose transporter isoforms in non-polarized mammalian cells: distribution of GLUT1 but not GLUT3 to detergent-resistant membrane domains

The hexose transporter family, which mediates a facilitated uptake in mammalian cells, consists of more than 10 members containing 12 membrane-spanning segments with a single N-glycosylation site. However, it remains unknown how these isoforms are functionally organized in the membrane domains. In this report, we describe a differential distribution of the glucose transporter isoforms GLUT1 and GLUT3 to detergent-resistant membrane domains (DRMs) in non-polarized mammalian cells. Whereas more than 80% of cellular proteins containing GLUT3 in HeLa cell lines was solubilized by a non-ionic detergent (either Triton X-100 or Lubrol WX) at 4 degrees C, GLUT1 remained insoluble together with the DRM-associated proteins, such as caveolin-1 and intestinal alkaline phosphatase (IAP). These DRM-associated proteins and the ganglioside GM1 were shown to float to the upper fractions when Triton X-100-solubilized cell extracts were centrifuged on a density gradient. In contrast, GLUT3 as well as most soluble proteins remained in the lower layers. Furthermore, perturbations of DRMs due to depletion of cholesterol by methyl-beta-cyclodextrin (m beta CD) rendered GLUT1 soluble in Triton X-100. Immunostaining patterns for these isoforms detected by confocal laser scanning microscopy in a living cell were also distinctive. These results suggest that in non-polarized mammalian cells, GLUT1 can be organized into a raft-like DRM domain but GLUT3 may distribute to fluid membrane domains. This differential distribution may occur irrespective of the N-glycosylation state or cell type.     

Decorin modulates collagen I-stimulated, but not fibronectin-stimulated, migration of C2C12 myoblasts

Extracellular matrix factors, specifically fibronectin and collagen I, are essential for structural support during muscle regeneration. Decorin has been identified as an anti-fibrotic agent with binding sites located on both fibronectin and collagen I. Upon injury, activated myoblasts are required to migrate through the extracellular matrix factors deposited by the myofibroblasts to facilitate skeletal muscle regeneration. In this study we looked at the effects decorin on fibronectin- and collagen I-stimulated myoblast migration. Dose response studies demonstrated 10 μg/ml, 5 μg/ml and 25 μg/ml as the optimal stimulatory concentrations of decorin (1.2 fold increase), fibronectin (3.5 fold increase) and collagen I (2.4 fold increase), when compared with control respectively. A synergistic effect was identified when decorin and collagen I were added in combination; this effect was not evident when decorin was added with fibronectin. The effects of these factors on the ROCK signalling pathway were also analyzed. ROCK-2 was identified as the key Rho-activated kinase isoform involved in migration, due to its higher expression levels and localisation to focal points within migrating C2C12 myoblasts. Decorin and collagen I in combination stimulated an increase in the number of ROCK-2 localized focal points when compared with control, decorin and collagen I added separately. Fibronectin did not show any increase in ROCK-2 focal points when compared with control. These results show for the first time that decorin can modify collagen I-stimulated, but not fibronectin-stimulated myoblast migration in vitro. Furthermore, the synergistic, rather than additive, effect observed suggests a direct modification of collagen I signalling by decorin mediated, at least in part, by ROCK-2 rather than ROCK-1.     

Bi-directional transport of GLUT4 vesicles near the plasma membrane of primary rat adipocytes

Insulin stimulates glucose uptake into adipocytes by mobilizing intracellular membrane vesicles containing GLUT4 proteins to the plasma membrane. Here we applied time-lapse total internal reflection fluorescence microscopy to study moving parameters and characters of exogenously expressed GLUT4 vesicles in basal, insulin and nocodazole treated primary rat adipocytes. Our results showed that microtubules were essential for long-range transport of GLUT4 vesicles but not obligatory for GLUT4 distribution in rat adipocytes. Insulin reduced the mobility of the vesicles, made them tethered/docked to the PM and finally had constitutive exocytosis. Moreover, long-range bi-directional movements of GLUT4 vesicles were visualized for the first time by TIRFM. It is likely that there are interactions between insulin signaling and microtubules, to regulating GLUT4 translocation in rat adipocytes.