Two compartments for insulin-stimulated exocytosis in 3T3-L1 adipocytes defined by endogenous ACRP30 and GLUT4.

Insulin stimulates adipose cells both to secrete proteins and to translocate the GLUT4 glucose transporter from an intracellular compartment to the plasma membrane. We demonstrate that whereas insulin stimulation of 3T3-L1 adipocytes has no effect on secretion of the alpha3 chain of type VI collagen, secretion of the protein hormone adipocyte complement related protein of 30 kD (ACRP30) is markedly enhanced. Like GLUT4, regulated exocytosis of ACRP30 appears to require phosphatidylinositol-3-kinase activity, since insulin-stimulated ACRP30 secretion is blocked by pharmacologic inhibitors of this enzyme. Thus, 3T3-L1 adipocytes possess a regulated secretory compartment containing ACRP30. Whether GLUT4 recycles to such a compartment has been controversial. We present deconvolution immunofluorescence microscopy data demonstrating that the subcellular distributions of ACRP30 and GLUT4 are distinct and nonoverlapping; in contrast, those of GLUT4 and the transferrin receptor overlap. Together with supporting evidence that GLUT4 does not recycle to a secretory compartment via the trans-Golgi network, we conclude that there are at least two compartments that undergo insulin-stimulated exocytosis in 3T3-L1 adipocytes: one for ACRP30 secretion and one for GLUT4 translocation.     

ArPIKfyve-PIKfyve interaction and role in insulin-regulated GLUT4 translocation and glucose transport in 3T3-L1 adipocytes

Insulin activates glucose transport by promoting translocation of the insulin-sensitive fat/muscle-specific glucose transporter GLUT4 from an intracellular storage compartment to the cell surface. Here we report that an optimal insulin effect on glucose uptake in 3T3-L1 adipocytes is dependent upon expression of both PIKfyve, the sole enzyme for PtdIns 3,5-P(2) biosynthesis, and the PIKfyve activator, ArPIKfyve. Small-interfering RNAs that selectively ablated PIKfyve or ArPIKfyve in this cell type depleted the PtdIns 3,5-P(2) pool and reduced insulin-activated glucose uptake to a comparable degree. Combined loss of PIKfyve and ArPIKfyve caused further PtdIns 3,5-P(2) ablation that correlated with greater attenuation in insulin responsiveness. Loss of PIKfyve-ArPIKfyve reduced insulin-stimulated Akt phosphorylation and the cell surface accumulation of GLUT4 or IRAP, but not GLUT1-containing vesicles without affecting overall expression of these proteins. ArPIKfyve and PIKfyve were found to physically associate in 3T3-L1 adipocytes and this was insulin independent. In vitro labeling of membranes isolated from basal or insulin-stimulated 3T3-L1 adipocytes documented substantial insulin-dependent increases of PtdIns 3,5-P(2) production on intracellular membranes. Together, the data demonstrate for the first time a physical association between functionally related PIKfyve and ArPIKfyve in 3T3-L1 adipocytes and indicate that the novel ArPIKfyve-PIKfyve-PtdIns 3,5-P(2) pathway is physiologically linked to insulin-activated GLUT4 translocation and glucose transport.     

Perinuclear localization and insulin responsiveness of GLUT4 requires cytoskeletal integrity in 3T3-L1 adipocytes

The GLUT4 glucose transporter resides mostly in perinuclear membranes in unstimulated 3T3-L1 adipocytes and is acutely translocated to the cell surface in response to insulin. Using a novel method to purify intracellular GLUT4-enriched membranes, we identified by mass spectrometry the intermediate filament protein vimentin and the microtubule protein alpha-tubulin as components of these membranes. Immunoelectron microscopy of the GLUT4-containing membranes also revealed their association with these cytoskeletal proteins. Disruption of intermediate filaments and microtubules in 3T3-L1 adipocytes by microinjection of a vimentin-derived peptide of the helix initiation 1A domain caused marked dispersion of perinuclear GLUT4 to peripheral regions of the cells. Inhibition of the microtubule-based motor dynein by brief cytoplasmic acidification of cultured adipocytes also dispersed perinuclear GLUT4 and inhibited insulin-stimulated GLUT4 translocation to the cell surface. Insulin sensitivity was restored as GLUT4 was again concentrated near the nucleus upon recovery of cells in physiological buffer. These data suggest that GLUT4 trafficking to perinuclear membranes of cultured adipocytes is directed by dynein and is required for optimal GLUT4 regulation by insulin.     

Role of EHD1 and EHBP1 in perinuclear sorting and insulin-regulated GLUT4 recycling in 3T3-L1 adipocytes

Insulin stimulates glucose transport in muscle and adipose tissues by recruiting intracellular membrane vesicles containing the glucose transporter GLUT4 to the plasma membrane. The mechanisms involved in the biogenesis of these vesicles and their translocation to the cell surface are poorly understood. Here, we report that an Eps15 homology (EH) domain-containing protein, EHD1, controls the normal perinuclear localization of GLUT4-containing membranes and is required for insulin-stimulated recycling of these membranes in cultured adipocytes. EHD1 is a member of a family of four closely related proteins (EHD1, EHD2, EHD3, and EHD4), which also contain a P-loop near the N terminus and a central coiled-coil domain. Analysis of cultured adipocytes stained with anti-GLUT4, anti-EHD1, and anti-EHD2 antibodies revealed that EHD1, but not EHD2, partially co-localizes with perinuclear GLUT4. Expression of a dominant-negative construct of EHD1 missing the EH domain (DeltaEH-EHD1) markedly enlarged endosomes, dispersed perinuclear GLUT4-containing membranes throughout the cytoplasm, and inhibited GLUT4 translocation to the plasma membranes of 3T3-L1 adipocytes stimulated with insulin. Similarly, small interfering RNA-mediated depletion of endogenous EHD1 protein also markedly dispersed perinuclear GLUT4 in cultured adipocytes. Moreover, EHD1 is shown to interact through its EH domain with the protein EHBP1, which is also required for insulin-stimulated GLUT4 movements and hexose transport. In contrast, disruption of EHD2 function was without effect on GLUT4 localization or translocation to the plasma membrane. Taken together, these results show that EHD1 and EHBP1, but not EHD2, are required for perinuclear localization of GLUT4 and reveal that loss of EHBP1 disrupts insulin-regulated GLUT4 recycling in cultured adipocytes.     

A role for phospholipase D in GLUT4 glucose transporter translocation

Based on recent studies showing that phospholipase D (PLD)1 is associated with intracellular membranes and promotes membrane budding from the trans-Golgi, we tested its possible role in the membrane trafficking of GLUT4 glucose transporters. Using immunofluorescence confocal microscopy, expressed Myc epitope-tagged PLD1 was found to associate with intracellular vesicular structures by a mechanism that requires its N-terminal pleckstrin homology domain. Partial co-localization with expressed GLUT4 fused to green fluorescent protein in both 3T3-L1 adipocytes and Chinese hamster ovary cells was evident. Furthermore, microinjection of purified PLD into cultured adipocytes markedly potentiated the effect of a submaximal concentration of insulin to stimulate GLUT4 translocation to cell surface membranes. Insulin stimulated PLD activity in cells expressing high levels of insulin receptors but no such insulin effect was detected in 3T3-L1 adipocytes. Taken together, these results are consistent with the hypothesis that PLD1 associated with GLUT4-containing membranes acts in a constitutive manner to promote the mechanism of GLUT4 translocation by insulin.     

A role for kinesin in insulin-stimulated GLUT4 glucose transporter translocation in 3T3-L1 adipocytes

Insulin regulates glucose uptake in adipocytes and muscle by stimulating the movement of sequestered glucose transporter 4 (GLUT4) proteins from intracellular membranes to the cell surface. Here we report that optimal insulin-mediated GLUT4 translocation is dependent upon both microtubule and actin-based cytoskeletal structures in cultured adipocytes. Depolymerization of microtubules and F-actin in 3T3-L1 adipocytes causes the dispersion of perinuclear GLUT4-containing membranes and abolishes insulin action on GLUT4 movements to the plasma membrane. Furthermore, heterologous expression in 3T3-L1 adipocytes of the microtubule-binding protein hTau40, which impairs kinesin motors that move toward the plus ends of microtubules, markedly delayed the appearance of GLUT4 at the plasma membrane in response to insulin. The hTau40 protein had no detectable effect on microtubule structure or perinuclear GLUT4 localization under these conditions. These results are consistent with the hypothesis that both the actin and microtubule-based cytoskeleton, as well as a kinesin motor, direct the translocation of GLUT4 to the plasma membrane in response to insulin.     

A Highlights from MBoC Selection: Characterization of VAMP isoforms in 3T3-L1 adipocytes: implications for GLUT4 trafficking

The fusion of GLUT4-containing vesicles with the plasma membrane of adipocytes is a key facet of insulin action. This process is mediated by the formation of functional soluble N-ethylmaleimide–sensitive factor attachment protein receptor (SNARE) complexes between the plasma membrane t-SNARE complex and the vesicle v-SNARE or VAMP. The t-SNARE complex consists of Syntaxin4 and SNAP23, and whereas many studies identify VAMP2 as the v-SNARE, others suggest that either VAMP3 or VAMP8 may also fulfil this role. Here we characterized the levels of expression, distribution, and association of all the VAMPs expressed in 3T3-L1 adipocytes to provide the first systematic analysis of all members of this protein family for any cell type. Despite our finding that all VAMP isoforms form SDS-resistant SNARE complexes with Syntaxin4/SNAP23 in vitro, a combination of levels of expression (which vary by >30-fold), subcellular distribution, and coimmunoprecipitation analyses lead us to propose that VAMP2 is the major v-SNARE involved in GLUT4 trafficking to the surface of 3T3-L1 adipocytes.     

Role of SNAP23 in insulin-induced translocation of GLUT4 in 3T3-L1 adipocytes. Mediation of complex formation between syntaxin4 and VAMP2

Both syntaxin4 and VAMP2 are implicated in insulin regulation of glucose transporter-4 (GLUT4) trafficking in adipocytes as target (t) soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNARE) and vesicle (v)-SNARE proteins, respectively, which mediate fusion of GLUT4-containing vesicles with the plasma membrane. Synaptosome-associated 23-kDa protein (SNAP23) is a widely expressed isoform of SNAP25, the principal t-SNARE of neuronal cells, and colocalizes with syntaxin4 in the plasma membrane of 3T3-L1 adipocytes. In the present study, two SNAP23 mutants, SNAP23-DeltaC8 (amino acids 1 to 202) and SNAP23-DeltaC49 (amino acids 1 to 161), were generated to determine whether SNAP23 is required for insulin-induced translocation of GLUT4 to the plasma membrane in 3T3-L1 adipocytes. Wild-type SNAP23 (SNAP23-WT) promoted the interaction between syntaxin4 and VAMP2 both in vitro and in vivo. Although SNAP23-DeltaC49 bound to neither syntaxin4 nor VAMP2, the SNAP23-DeltaC8 mutant bound to syntaxin4 but not to VAMP2. In addition, although SNAP23-DeltaC8 bound to syntaxin4, it did not mediate the interaction between syntaxin4 and VAMP2. Moreover, overexpression of SNAP23-DeltaC8 in 3T3-L1 adipocytes by adenovirus-mediated gene transfer inhibited insulin-induced translocation of GLUT4 but not that of GLUT1. In contrast, overexpression of neither SNAP23-WT nor SNAP23-DeltaC49 in 3T3-L1 adipocytes affected the translocation of GLUT4 or GLUT1. Together, these results demonstrate that SNAP23 contributes to insulin-dependent trafficking of GLUT4 to the plasma membrane in 3T3-L1 adipocytes by mediating the interaction between t-SNARE (syntaxin4) and v-SNARE (VAMP2).     

An angiotensin II AT1 receptor antagonist, telmisartan augments glucose uptake and GLUT4 protein expression in 3T3-L1 adipocytes

Evidence has accumulated that some of the angiotensin II AT1 receptor antagonists have insulin-sensitizing property. We thus examined the effect of telmisartan on insulin action using 3T3-L1 adipocytes. With standard differentiation inducers, a higher dose of telmisartan effectively facilitated differentiation of 3T3-L1 preadipocytes. Treatment of both differentiating adipocytes and fully differentiated adipocytes with telmisartan caused a dose-dependent increase in mRNA levels for PPARgamma target genes such as aP2 and adiponectin. By contrast, telmisartan attenuated 11beta-hydroxysteroid dehydrogenase type 1 mRNA level in differentiated adipocytes. Of note, we demonstrated for the first time that telmisartan augmented GLUT4 protein expression and 2-deoxy glucose uptake both in basal and insulin-stimulated state of adipocytes, which may contribute, at least partly, to its insulin-sensitizing ability.     

Golgi-localized, gamma-ear-containing, Arf-binding protein adaptors mediate insulin-responsive trafficking of glucose transporter 4 in 3T3-L1 adipocytes

Small glucose transporter 4 (Glut4)-containing vesicles represent the major insulin-responsive compartment in fat and skeletal muscle cells. The molecular mechanism of their biogenesis is not yet elucidated. Here, we studied the role of the newly discovered family of monomeric adaptor proteins, GGA (Golgi-localized, gamma-ear-containing, Arf-binding proteins), in the formation of small Glut4 vesicles and acquisition of insulin responsiveness in 3T3-L1 adipocytes. In these cells, all three GGA isoforms are expressed throughout the differentiation process. In particular, GGA2 is primarily present in trans-Golgi network and endosomes where it demonstrates a significant colocalization with the recycling pool of Glut4. Using the techniques of immunoadsorption as well as glutathione-S-transferase pull-down assay we found that Glut4 vesicles (but not Glut4 per se) interact with GGA via the Vps-27, Hrs, and STAM (VHS) domain. Moreover, a dominant negative GGA mutant inhibits formation of Glut4 vesicles in vitro. To study a possible role of GGA in Glut4 traffic in the living cell, we stably expressed a dominant negative GGA mutant in 3T3-L1 adipocytes. Formation of small insulin-responsive Glut4-containing vesicles and insulin-stimulated glucose uptake in these cells were markedly impaired. Thus, GGA adaptors participate in the formation of the insulin-responsive vesicular compartment from the intracellular donor membranes both in vivo and in vitro.     

Apelin stimulates glucose uptake through the PI3K/Akt pathway and improves insulin resistance in 3T3-L1 adipocytes

Apelin, a cytokine mainly secreted by adipocytes, is closely related with insulin resistance. The underlying molecular mechanisms of how apelin affects insulin resistance, however, are poorly understood. This study aimed to investigate the effect of apelin on glucose metabolism and insulin resistance in 3T3-L1 adipocytes. After 10 ng/ml TNF-α treatment for 24 h, insulin-stimulated glucose uptake was reduced by 47% in 3T3-L1 adipocytes. Apelin treatment improved glucose uptake in a time- and dose-dependent manner. Treatment of 1,000 nM apelin for 60 min maximally augmented glucose uptake in insulin-resistant 3T3-L1 adipocytes. Furthermore, apelin pre-incubation also increased adipocytes' insulin-stimulated glucose uptake, and PI3K/Akt pathway were involved in these effects. In addition, immunocytochemistry staining and western blotting analysis indicated that apelin could increase glucose transporter 4 translocation from the cytoplasm to the plasma membrane. Apelin also increased the anti-inflammatory adipokine adiponectin mRNA expression while reducing that of pro-inflammatory adipokine interleukin-6 in insulin-resistant 3T3-L1 adipocytes. These results suggest that apelin stimulates glucose uptake through the PI3K/Akt pathway, promotes GLUT4 translocation from the cytoplasm to the plasma membrane, and modulates inflammatory responses in insulin-resistant 3T3-L1 adipocytes.     

The glucose transporter 4-regulating protein TUG is essential for highly insulin-responsive glucose uptake in 3T3-L1 adipocytes

Insulin stimulates glucose uptake in fat and muscle by redistributing GLUT4 glucose transporters from intracellular membranes to the cell surface. We previously proposed that, in 3T3-L1 adipocytes, TUG retains GLUT4 within unstimulated cells and insulin mobilizes this retained GLUT4 by stimulating its dissociation from TUG. Yet the relative importance of this action in the overall control of glucose uptake remains uncertain. Here we report that transient, small interfering RNA-mediated depletion of TUG causes GLUT4 translocation and enhances glucose uptake in unstimulated 3T3-L1 adipocytes, similar to insulin. Stable TUG depletion or expression of a dominant negative fragment likewise stimulates GLUT4 redistribution and glucose uptake, and insulin causes a 2-fold further increase. Microscopy shows that TUG governs the accumulation of GLUT4 in perinuclear membranes distinct from endosomes and indicates that it is this pool of GLUT4 that is mobilized by TUG disruption. Interestingly, in addition to translocating GLUT4 and enhancing glucose uptake, TUG disruption appears to accelerate the degradation of GLUT4 in lysosomes. Finally, we find that TUG binds directly and specifically to a large intracellular loop in GLUT4. Together, these findings demonstrate that TUG is required to retain GLUT4 intracellularly in 3T3-L1 adipocytes in the absence of insulin and further implicate the insulin-stimulated dissociation of TUG and GLUT4 as an important action by which insulin stimulates glucose uptake.     

Differential regulation of NHE1 phosphorylation and glucose uptake by inhibitors of the ERK pathway and p90RSK in 3T3-L1 adipocytes

Insulin stimulates trafficking of GLUT4 to the cell surface for glucose uptake into target cells, and phosphorylation of Ser703 of the Na⁺/H⁺ exchanger NHE1, which activates proton efflux. The latter has been proposed to facilitate optimal glucose uptake into cardiomyocytes. We found that the insulin-stimulated phosphorylation of Ser703 of NHE1 is mediated by p90RSK but not directly coupled to glucose uptake in 3T3-L1 adipocytes in the short-term. Inhibiting Erk1/2 activation prevented NHE1 phosphorylation but not glucose uptake in 3T3-L1 adipocytes. In contrast, both NHE1 phosphorylation and insulin-stimulated uptake of glucose into 3T3-L1 adipocytes were blocked by inhibitors of the N-terminal kinase domain of p90RSK, namely BI-D1870 and SL0101, but not the FMK inhibitor of the C-terminal kinase domain of p90RSK, though in our hands FMK did not inhibit p90RSK in 3T3-L1 adipocytes. Further experiments were consistent with phosphorylation of AS160 by PKB/Akt mediating insulin-stimulated trafficking of GLUT4 to the plasma membrane. BI-D1870 and SL0101 however, inhibited glucose uptake without blocking GLUT4 translocation. While BI-D1870 partially inhibited insulin-stimulated PKB activation in these cells, this only partially inhibited AS160 phosphorylation and did not block GLUT4 trafficking, suggesting that p90RSK might regulate glucose transport after GLUT4 translocation. Moreover, BI-D1870 also prevented PMA-induced glucose transport in 3T3-L1 adipocytes further suggesting a role for p90RSK in regulating uptake of glucose into the cells. Kinetic experiments are consistent with SL0101 being a direct competitor of 2-deoxyglucose entry into cells, and this compound might also inhibit uptake of glucose into cells via inhibiting p90RSK, as revealed by comparison with the inactive form of the inhibitor. Taken together, we propose that BI-D1870 and SL0101 might exert their inhibitory effects on glucose uptake in 3T3-L1 adipocytes at least partially through a p90RSK dependent step after GLUT4 becomes associated with the plasma membrane.     

Pioglitazone acutely stimulates adiponectin secretion from mouse and human adipocytes via activation of the phosphatidylinositol 3'-kinase

AIMS: Thiazolidinediones increase circulating adiponectin. We have previously demonstrated the involvement of the phosphatidylinositol 3-kinase (PI3K) signaling pathway in insulin-stimulated adiponectin secretion. We therefore investigated the effects of the thiazolidinedione pioglitazone on acute adiponectin secretion, and the involvement of the PI3K signaling pathway in this action. MAIN METHODS: We treated murine 3T3-L1 and human primary adipocytes with 1-10 uM pioglitazone for 2 h, +/-PI3K inhibition by Wortmannin (WT). Secreted adiponectin was measured by Western blot. PI3K activity following 15-minute treatments with 1-10 uM pioglitazone was measured by thin layer chromatography. Pioglitazone's effect on adiponectin synthesis and on secretion of newly synthesized adiponectin was studied in 3T3-L1 adipocytes using a pulse-chase technique. KEY FINDINGS: Pioglitazone was found to increase adiponectin secretion and PI3K activity in a dose-dependent manner from 3T3-L1 and human adipocytes. In 3T3-L1 adipocytes, 10 uM pioglitazone increased adiponectin secretion by 84+/-14% (p<0.0001) at 2 h. Similarly, in human adipocytes there was a 56+/-18% (p<0.02) increase in secretion. WT blocked the pioglitazone effect and decreased adiponectin secretion at 2 h (47% of pioglitazone treated, p<0.006). Pioglitazone increased PI3K activity in a dose-dependent manner in both 3T3-L1 (1.7 vs. 2.7-fold increase over control at 2 uM vs. 10 uM dose, p=0.02) and human adipocytes. SIGNIFICANCE: Our data show that pioglitazone acutely stimulates adiponectin secretion from both 3T3-L1 and human adipocytes. This acute effect of pioglitazone is PI3K-dependent.     

Evidence that erythroid-type glucose transporter intrinsic activity is modulated by cadmium treatment of mouse 3T3-L1 cells

Previous studies suggest that regulation of hexose uptake in Chinese hamster ovary fibroblasts can occur by alterations in glucose transporter intrinsic activity without changes in cell surface transporter number (Harrison, S. A., Buxton, J. M., Helgerson, A. L., MacDonald, R. G., Chlapowski, F. J., Carruthers, A., and Czech, M. P. (1990) J. Biol. Chem. 265, 5793-5801). We tested this hypothesis using 3T3-L1 fibroblasts and adipocytes which exhibit 5-6-fold increases in 2-deoxyglucose or 3-O-methylglucose uptake when exposed to low micromolar concentrations of cadmium for 18 h. Cadmium treatment decreased the apparent Km of 3T3-L1 fibroblasts for 3-O-methylglucose influx from approximately 28 to 9 mM and increased the apparent Vmax by 2-3-fold. These fibroblasts lack the skeletal muscle/adipocyte-type (GLUT4) transporter and showed only a small increase in total cellular immunoreactive HepG2 type (GLUT1) transporter in response to cadmium. Furthermore, cell surface GLUT1 levels did not change in 3T3-L1 fibroblasts exposed to cadmium, as assessed by the binding to intact cells of an antibody which recognizes an extracellular GLUT1 epitope. Insulin enhanced 2-deoxyglucose uptake 2-fold in 3T3-L1 fibroblasts, but did not further stimulate cadmium-activated transport rates. In contrast, insulin stimulated hexose transport 15-fold in 3T3-L1 adipocytes, which express both GLUT1 and GLUT4 proteins, and this effect was fully additive with the 5-fold effect of cadmium. Cadmium had little or no effect on immunoreactive GLUT1 or GLUT4 in isolated 3T3-L1 adipocyte plasma membranes. In contrast, insulin action led to marked recruitment (3-fold) of GLUT4 to the plasma membrane fraction in adipocytes treated with or without cadmium. Taken together, these data are consistent with the hypothesis that cadmium-activated sugar uptake is catalyzed by GLUT1, whereas insulin-stimulated sugar uptake is catalyzed predominantly by GLUT4 in 3T3-L1 adipocytes. Furthermore, the data suggest that the GLUT1 transporter can undergo significant increases in intrinsic catalytic activity in response to cadmium treatment of 3T3-L1 fibroblasts and adipocytes.     

Differential regulation of secretory compartments containing the insulin-responsive glucose transporter 4 in 3T3-L1 adipocytes

Insulin and guanosine-5'-O-(3-thiotriphosphate) (GTPgammaS) both stimulate glucose transport and translocation of the insulin-responsive glucose transporter 4 (GLUT4) to the plasma membrane in adipocytes. Previous studies suggest that these effects may be mediated by different mechanisms. In this study we have tested the hypothesis that these agonists recruit GLUT4 by distinct trafficking mechanisms, possibly involving mobilization of distinct intracellular compartments. We show that ablation of the endosomal system using transferrin-HRP causes a modest inhibition ( approximately 30%) of insulin-stimulated GLUT4 translocation. In contrast, the GTPgammaS response was significantly attenuated ( approximately 85%) under the same conditions. Introduction of a GST fusion protein encompassing the cytosolic tail of the v-SNARE cellubrevin inhibited GTPgammaS-stimulated GLUT4 translocation by approximately 40% but had no effect on the insulin response. Conversely, a fusion protein encompassing the cytosolic tail of vesicle-associated membrane protein-2 had no significant effect on GTPgammaS-stimulated GLUT4 translocation but inhibited the insulin response by approximately 40%. GTPgammaS- and insulin-stimulated GLUT1 translocation were both partially inhibited by GST-cellubrevin ( approximately 50%) but not by GST-vesicle-associated membrane protein-2. Incubation of streptolysin O-permeabilized 3T3-L1 adipocytes with GTPgammaS caused a marked accumulation of Rab4 and Rab5 at the cell surface, whereas other Rab proteins (Rab7 and Rab11) were unaffected. These data are consistent with the localization of GLUT4 to two distinct intracellular compartments from which it can move to the cell surface independently using distinct sets of trafficking molecules.     

Thyroid hormone promotes insulin-induced glucose uptake by enhancing Akt phosphorylation and VAMP2 translocation in 3T3-L1 adipocytes

The purpose of this study was to test a hypothesis that T3 promotes glucose uptake via enhancing insulin-induced Akt phosphorylation and VAMP2 translocation in 3T3-L1 adipocytes. T3 significantly enhanced insulin-induced phosphorylation of Akt, cytoplasma to cell membrane translocations of vesicle-associated membrane protein 2 (VAMP2) and glucose transporter 4 (GLUT4), and glucose uptake in adipocytes. Akt inhibitor X abolished the promoting effects of T3, suggesting that Akt activation is essential for T3 to enhance these insulin-induced events in adipocytes. Knockdown of VAMP2 using siRNA abrogated the effects of T3 on insulin-induced GLUT4 translocation and glucose uptake, suggesting that VAMP2 is an important mediator of these processes. These data suggest that T3 may promote glucose uptake via enhancing insulin-induced phosphorylation of Akt and subsequent translocations of VAMP2 and GLUT4 in 3T3-L1 adipocytes. Akt phosphorylation is necessary for the promoting effects of T3 on insulin-stimulated VAMP2 translocation. Further, VAMP2 is essential for T3 to increase insulin-stimulated translocation of GLUT4 and subsequent uptake of glucose in adipocytes.     

Quantification of SNARE protein levels in 3T3-L1 adipocytes: implications for insulin-stimulated glucose transport

Insulin-stimulates glucose transport in peripheral tissues by stimulating the movement ('translocation') of a pool of intracellular vesicles containing the glucose transporter Glut4 to the cell surface. The fusion of these vesicles with the plasma membrane results in a large increase in the numbers of Glut4 molecules at the cell surface and a concomitant enhancement of glucose uptake. It is well established that proteins of the VAMP- (synaptobrevin) and syntaxin-families play a fundamental role in the insulin-stimulated fusion of Glut4-containing vesicles with the plasma membrane. Studies have identified key roles for vesicle associated membrane protein-2 (VAMP2) and syntaxin-4 in this event, and more recently have also implicated SNAP-23 and Munc18c in this process. In this study, we have quantified the absolute levels of expression of these proteins in murine 3T3-L1 adipocytes, with the objective of determining the stoichiometry of these proteins both relative to each other and also in comparison with previous estimates of Glut4 levels within these cells. To achieve this, we performed quantitative immunoblot analysis of these proteins in 3T3-L1 membranes compared to known amounts of purified recombinant proteins. Such analyses suggest that in 3T3-L1 adipocytes there are approximately 374,000 copies of syntaxin 4, 1.15 x 10(6) copies of SNAP23, 495,000 copies of VAMP2, 4.3 x 10(6) copies of cellubrevin and 452,000 copies of Munc18c per cell, compared to previous estimates of 280,000 copies of Glut4. Thus, the main SNARE proteins involved in insulin-stimulated Glut4 exocytosis (syntaxin 4 and VAMP2) are expressed in approximately equimolar amounts in adipocytes, whereas by contrast the endosomal v-SNARE cellubrevin is present at approximately 10-fold higher levels and the t-SNARE SNAP-23 is also present in an approximately 3-fold molar excess. The implications of this quantification for the mechanism of insulin-stimulated Glut4 translocation are discussed.     

An inhibitor of p38 mitogen-activated protein kinase prevents insulin-stimulated glucose transport but not glucose transporter translocation in 3T3-L1 adipocytes and L6 myotubes

The precise mechanisms underlying insulin-stimulated glucose transport still require investigation. Here we assessed the effect of SB203580, an inhibitor of the p38 MAP kinase family, on insulin-stimulated glucose transport in 3T3-L1 adipocytes and L6 myotubes. We found that SB203580, but not its inactive analogue (SB202474), prevented insulin-stimulated glucose transport in both cell types with an IC50 similar to that for inhibition of p38 MAP kinase (0.6 microM). Basal glucose uptake was not affected. Moreover, SB203580 added only during the transport assay did not inhibit basal or insulin-stimulated transport. SB203580 did not inhibit insulin-stimulated translocation of the glucose transporters GLUT1 or GLUT4 in 3T3-L1 adipocytes as assessed by immunoblotting of subcellular fractions or by immunofluorescence of membrane lawns. L6 muscle cells expressing GLUT4 tagged on an extracellular domain with a Myc epitope (GLUT4myc) were used to assess the functional insertion of GLUT4 into the plasma membrane. SB203580 did not affect the insulin-induced gain in GLUT4myc exposure at the cell surface but largely reduced the stimulation of glucose uptake. SB203580 had no effect on insulin-dependent insulin receptor substrate-1 phosphorylation, association of the p85 subunit of phosphatidylinositol 3-kinase with insulin receptor substrate-1, nor on phosphatidylinositol 3-kinase, Akt1, Akt2, or Akt3 activities in 3T3-L1 adipocytes. In conclusion, in the presence of SB203580, insulin caused normal translocation and cell surface membrane insertion of glucose transporters without stimulating glucose transport. We propose that insulin stimulates two independent signals contributing to stimulation of glucose transport: phosphatidylinositol 3-kinase leads to glucose transporter translocation and a pathway involving p38 MAP kinase leads to activation of the recruited glucose transporter at the membrane.     

Glucosamine-induced insulin resistance is coupled to O-linked glycosylation of Munc18c

Evidence suggests that glucosamine inhibits distal components regulating insulin-stimulated GLUT4 translocation to the plasma membrane. Here we assessed whether key membrane docking and fusion events were targeted. Consistent with a plasma membrane-localized effect, 3T3-L1 adipocytes exposed to glucosamine displayed an increase in cell-surface O-linked glycosylation and a simultaneously impaired mobilization of GLUT4 by insulin. Analysis of syntaxin 4 and SNAP23, plasma membrane-localized target receptor proteins (t-SNAREs) for the GLUT4 vesicle, showed that they were not cell-surface targets of O-linked glycosylation. However, the syntaxin 4 binding protein, Munc18c, was targeted by O-linked glycosylation. This occurred concomitantly with a block in insulin-stimulated association of syntaxin 4 with its cognate GLUT4 vesicle receptor protein (v-SNARE), VAMP2. In conclusion, our data suggest that the mechanism by which glucosamine inhibits insulin-stimulated GLUT4 translocation involves modification of Munc18c.