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1.
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. 相似文献
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Identification and partial purification of the insulin-responsive glucose transporter from 3T3-L1 adipocytes 总被引:3,自引:0,他引:3
D W Schroer S C Frost R A Kohanski M D Lane G E Lienhard 《Biochimica et biophysica acta》1986,885(3):317-326
The glucose transporter in 3T3-L1 adipocytes has been identified as a polypeptide of average Mr 51000 by means of its reaction with antibodies raised against the purified human erythrocyte glucose transporter and by photolabeling with [3H]cytochalasin B. The finding that the antibodies immunoprecipitated the photolabeled polypeptide demonstrated that both methods detected the same polypeptide. The 3T3-L1 adipocyte glucose transporter has been partially purified. The main steps in the purification procedure were the preparation of salt-washed cellular membranes, Triton X-100 solubilization, and immunoaffinity chromatography on affinity-purified antibodies against the human erythrocyte transporter. A simple method of affinity purification of these antibodies, which consists of adsorption from serum onto protein-depleted erythrocyte membranes and release with acid, and an assay for the 3T3-L1 adipocyte transporter polypeptide, which employs immunoblotting, have been developed. 相似文献
3.
Cytoplasmic Ca2+ during differentiation of 3T3-L1 adipocytes. Effect of insulin and relation to glucose transport 总被引:1,自引:0,他引:1
The cytoplasmic concentration of ionized Ca2+ [( Ca2+]i) was determined in 3T3-L1 cells during their differentiation from fibroblasts to adipocytes, suspended and loaded with the fluorescent Ca2+ indicators quin2 or indo-1. In undifferentiated fibroblasts, as well as in differentiated adipocytes up to day 9, [Ca2+]i was steady around 170 nM, and it increased significantly only in old adipocytes (day 12). During differentiation, stimulation of glucose uptake by insulin increased from a few percent to severalfold. Stimulation of uptake was already apparent after 10 min of addition of the hormone, and 10 nM insulin produced maximal stimulation in 30 min. Insulin (10(-6) M) added to quin2- or indo-1-loaded, suspended adipocytes had no detectable effect on [Ca2+]i for at least 10 min. In contrast, addition of the general anesthetic halothane increased [Ca2+]i from 172 to 251 nM in 3 min. In EGTA solution, the Ca2+ ionophore ionomycin elicited release of Ca2+ from intracellular stores that resulted in a transient increase in [Ca2+]i. A smaller but measurable Ca2+ release from intracellular stores (increasing [Ca2+]i by 20 nM) resulted upon addition of 20 micrograms/ml phosphatidic acid. In contrast, insulin did not produce any detectable release of Ca2+ from intracellular stores. Incubation of 3T3-L1 adipocytes with insulin in the presence of EGTA (the latter in excess over the Ca2+ concentration of the medium) did not prevent the stimulation of hexose uptake by the hormone, indicating that extracellular Ca2+ does not play a role in the insulin response. Furthermore, incubation of cells with quin2/AM in EGTA medium during exposure to insulin did not prevent stimulation of hexose uptake. Under these conditions it is demonstrated that intracellular quin2 suffices to chelate cytoplasmic Ca2+ even if releasable Ca2+ from intracellular stores were to pour into the cytoplasm. Thus, quin2 effectively lowers [Ca2+]i without impairing insulin action. It is concluded that insulin does not produce changes in [Ca2+]i and that chelating intracellular Ca2+ does not prevent stimulation of hexose uptake by insulin. These results suggest that it is unlikely that changes in [Ca2+]i may play a role in the transduction of information in insulin stimulation of glucose uptake in 3T3-L1 adipocytes. 相似文献
4.
Isolation of vesicles containing insulin-responsive, intracellular glucose transporters from 3T3-L1 adipocytes 总被引:7,自引:0,他引:7
Insulin stimulation of glucose transport in fat and muscle cells occurs, at least in part, by the translocation of glucose transporters from intracellular membranes to the plasma membrane. In this report, we describe the isolation and partial characterization of vesicles containing translocatable intracellular transporters from 3T3-L1 adipocytes. The glucose transporter content of light microsomes in a 44,000 X g cell supernatant was found to decrease by 50% in response to insulin treatment of the adipocytes. A procedure was developed for the purification of transporter-containing vesicles from this supernatant by immunoadsorption onto Staphylococcus aureus cells coated with anti-transporter antibodies. The vesicles are about 50 nm in diameter and have a distinct polypeptide composition. After insulin treatment the number of transporter-containing vesicles decreased by about 50%, as determined both by microscopic analysis of vesicle number and by the relative abundance of vesicle polypeptides. 相似文献
5.
Differentiating 3T3-L1 cells exhibit a dramatic increase in the rate of insulin-stimulated glucose transport during their conversion from proliferating fibroblasts to nonproliferating adipocytes. On day 3 of 3T3-L1 cell differentiation, basal glucose transport and cell surface transferrin binding are markedly diminished. This occurs concomitant with the formation of a distinct insulin-responsive vesicular pool of intracellular glucose transporter 1 (GLUT1) and transferrin receptors as assessed by sucrose velocity gradients. The intracellular distribution of the insulin-responsive aminopeptidase is first readily detectable on day 3, and its gradient profile and response to insulin at this time are identical to that of GLUT1. With further time of differentiation, GLUT4 is expressed and targeted to the same insulin-responsive vesicles as the other three proteins. Our data are consistent with the notion that a distinct insulin-sensitive vesicular cargo compartment forms early during fat call differentiation and its formation precedes GLUT4 expression. The development of this compartment may result from the differentiation-dependent inhibition of constitutive GLUT1 and transferrin receptor trafficking such that there is a large increase in, or the new formation of, a population of postendosomal, insulin-responsive vesicles. 相似文献
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Differential regulation of secretory compartments containing the insulin-responsive glucose transporter 4 in 3T3-L1 adipocytes 总被引:8,自引:0,他引:8
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Millar CA Shewan A Hickson GR James DE Gould GW 《Molecular biology of the cell》1999,10(11):3675-3688
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. 相似文献
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We used nigericin, a K+/H+ exchanger, to test whether glucose transport in 3T3-L1 adipocytes was modulated by changes in intracellular pH. Our results showed that nigericin increased basal but decreased insulin-stimulated glucose uptake in a time- and dose-dependent manner. Whereas the basal translocation of GLUT1 was enhanced, insulin-stimulated GLUT4 translocation was inhibited by nigericin. On the other hand, the total amount of neither transporter protein was altered. The finding that insulin-stimulated phosphoinositide 3-kinase (PI 3-kinase) activity was not affected by nigericin implies that nigericin exerted its inhibition at a step downstream of PI 3-kinase activation. At maximal dose, nigericin rapidly lowered cytosolic pH to 6.7; however, this effect was transient and cytosolic pH was back to normal in 20 min. Removal of nigericin from the incubation medium after 20 min abolished its enhancing effect on basal but had little influence on its inhibition of insulin-stimulated glucose transport. Moreover, lowering cytosolic pH to 6.7 with an exogenously added HCl solution had no effect on glucose transport. Taken together, it appears that nigericin may inhibit insulin-stimulated glucose transport mainly by interfering with GLUT4 translocation, probably by a mechanism not related to changes in cytosolic pH. 相似文献
11.
Arsenite stimulated glucose transport in 3T3-L1 adipocytes involves both Glut4 translocation and p38 MAPK activity. 总被引:2,自引:0,他引:2
Merlijn Bazuine D Margriet Ouwens Daan S Gomes de Mesquita J Antonie Maassen 《European journal of biochemistry》2003,270(19):3891-3903
The protein-modifying agent arsenite stimulates glucose uptake in 3T3-L1 adipocytes. In the current study we have analysed the signalling pathways that contribute to this response. By subcellular fractionation we observed that arsenite, like insulin, induces translocation of the GLUT1 and GLUT4 glucose transporters from the low-density membrane fraction to the plasma membrane. Arsenite did not activate early steps of the insulin receptor (IR)-signalling pathway and the response was insensitive to inhibition of phosphatidylinositol-3'-kinase (PI-3') kinase by wortmannin. These findings indicate that the 'classical' IR-IR substrate-PI-3' kinase pathway, that is essential for insulin-induced GLUT4 translocation, is not activated by arsenite. However, arsenite-treatment did induce tyrosine-phosphorylation of c-Cbl. Furthermore, treatment of the cells with the tyrosine kinase inhibitor, tyrphostin A25, abolished arsenite-induced glucose uptake, suggesting that the induction of a tyrosine kinase by arsenite is essential for glucose uptake. Both arsenite and insulin-induced glucose uptake were inhibited partially by the p38 MAP kinase inhibitor, SB203580. This compound had no effect on the magnitude of translocation of glucose transporters indicating that the level of glucose transport is determined by additional factors. Arsenite- and insulin-induced glucose uptake responded in a remarkably similar dose-dependent fashion to a range of pharmacological- and peptide-inhibitors for atypical PKC-lambda, a downstream target of PI-3' kinase signalling in insulin-induced glucose uptake. These data show that in 3T3-L1 adipocytes both arsenite- and insulin-induced signalling pathways project towards a similar cellular response, namely GLUT1 and GLUT4 translocation and glucose uptake. This response to arsenite is not functionally linked to early steps of the IR-IRS-PI-3' kinase pathway, but does coincide with c-Cbl phosphorylation, basal levels of PKC-lambda activity and p38 MAPK activation. 相似文献
12.
In this note, we present a detailed procedure for highly effective and reproducible 3T3-L1 cell differentiation. Due to their potential to differentiate from fibroblasts to adipocytes, 3T3-L1 cells are widely used for studying adipogenesis and the biochemistry of adipocytes. However, using different kits and protocols published so far, we were not able to obtain full differentiation of the currently available American Type Culture Collection (ATCC) 3T3-L1 cell lots. Using rosiglitazone (2 μM) as an additional prodifferentiative agent, we achieved apparently complete differentiation of 3T3-L1 cells within 10 to 12 days that persisted for at least up to cell culture passage 10. 相似文献
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Kitamura A Nishizuka M Tominaga K Tsuchiya T Nishihara T Imagawa M 《Biochemical and biophysical research communications》2001,280(2):435-439
PCR and antisense oligodeoxy-nucleotide (ODN) blocking were used to identify a calcium (Ca) channel in rabbit proximal tubule (PT) cells. The subcloned Ca channel is identical to the rabbit cardiac Ca channel (alpha(1)) except a 33 base deletion at the fourth S3-S4 linker in PT cells. Anti-sense ODN treatment (18 h) inhibited 73 and 44% of Ca influxes induced by hypoosmotic stress (220 Osm) and by 1-oleoyl-2-acetyl-sn-glycerol (5 microM), respectively. The results indicate that the subcloned channel is a spliced variant of the cardiac Ca channel and that it plays a critical role in regulation of Ca signaling in these cells. 相似文献
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Kovacic PB Chowdhury HH Velebit J Kreft M Jensen J Zorec R 《The Journal of biological chemistry》2011,286(15):13370-13381
Cytosolic glucose concentration reflects the balance between glucose entry across the plasma membrane and cytosolic glucose utilization. In adipocytes, glucose utilization is considered very rapid, meaning that every glucose molecule entering the cytoplasm is quickly phosphorylated. Thus, the cytosolic free glucose concentration is considered to be negligible; however, it was never measured directly. In the present study, we monitored cytosolic glucose dynamics in 3T3-L1 fibroblasts and adipocytes by expressing a fluorescence resonance energy transfer (FRET)-based glucose nanosensor: fluorescent indicator protein FLIPglu-600μ. Specifically, we monitored cytosolic glucose responses by varying transmembrane glucose concentration gradient. The changes in cytosolic glucose concentration were detected in only 56% of 3T3-L1 fibroblasts and in 14% of 3T3-L1 adipocytes. In adipocytes, the resting cytosolic glucose concentration was reduced in comparison with the one recorded in fibroblasts. Membrane permeabilization increased cytosolic glucose concentration in adipocytes, and glycolytic inhibitor iodoacetate failed to increase cytosolic glucose concentration, indicating low adipocyte permeability for glucose at rest. We also examined the effects of insulin and adrenaline. Insulin significantly increased cytosolic glucose concentration in adipocytes by a factor of 3.6; however, we recorded no effect on delta ratio (ΔR) in fibroblasts. Adrenaline increased cytosolic glucose concentration in fibroblasts but not in adipocytes. However, in adipocytes in insulin-stimulated conditions, glucose clearance was significantly faster following adrenaline addition in comparison with controls (p < 0.001). Together, these results demonstrate that during differentiation, adipocytes develop more efficient mechanisms for maintaining low cytosolic glucose concentration, predominantly with reduced membrane permeability for glucose. 相似文献
17.
Whitehead JP Molero JC Clark S Martin S Meneilly G James DE 《The Journal of biological chemistry》2001,276(30):27816-27824
We have examined the requirement for Ca2+ in the signaling and trafficking pathways involved in insulin-stimulated glucose uptake in 3T3-L1 adipocytes. Chelation of intracellular Ca2+, using 1,2-bis (o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetra (acetoxy- methyl) ester (BAPTA-AM), resulted in >95% inhibition of insulin-stimulated glucose uptake. The calmodulin antagonist, W13, inhibited insulin-stimulated glucose uptake by 60%. Both BAPTA-AM and W13 inhibited Akt phosphorylation by 70-75%. However, analysis of insulin-dose response curves indicated that this inhibition was not sufficient to explain the effects of BAPTA-AM and W13 on glucose uptake. BAPTA-AM inhibited insulin-stimulated translocation of GLUT4 by 50%, as determined by plasma membrane lawn assay and subcellular fractionation. In contrast, the insulin-stimulated appearance of HA-tagged GLUT4 at the cell surface, as measured by surface binding, was blocked by BAPTA-AM. While the ionophores or ionomycin prevented the inhibition of Akt phosphorylation and GLUT4 translocation by BAPTA-AM, they did not overcome the inhibition of glucose transport. Moreover, glucose uptake of cells pretreated with insulin followed by rapid cooling to 4 degrees C, to promote cell surface expression of GLUT4 and prevent subsequent endocytosis, was inhibited specifically by BAPTA-AM. This indicates that inhibition of glucose uptake by BAPTA-AM is independent of both trafficking and signal transduction. These data indicate that Ca2+ is involved in at least two different steps of the insulin-dependent recruitment of GLUT4 to the plasma membrane. One involves the translocation step. The second involves the fusion of GLUT4 vesicles with the plasma membrane. These data are consistent with the hypothesis that Ca2+/calmodulin plays a fundamental role in eukaryotic vesicle docking and fusion. Finally, BAPTA-AM may inhibit the activity of the facilitative transporters by binding directly to the transporter itself. 相似文献
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Evidence that erythroid-type glucose transporter intrinsic activity is modulated by cadmium treatment of mouse 3T3-L1 cells 总被引:6,自引:0,他引:6
S A Harrison J M Buxton B M Clancy M P Czech 《The Journal of biological chemistry》1991,266(29):19438-19449
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. 相似文献
20.
Jang MK Kim CH Seong JK Jung MH 《Biochemical and biophysical research communications》2012,418(1):38-43
The global spread of highly pathogenic avian influenza A H5N1 viruses raises concerns about more widespread infection in the human population. Pre-pandemic vaccine for H5N1 clade 1 influenza viruses has been produced from the A/Viet Nam/1194/2004 strain (VN1194), but recent prevalent avian H5N1 viruses have been categorized into the clade 2 strains, which are antigenically distinct from the pre-pandemic vaccine. To understand the antigenicity of H5N1 hemagglutinin (HA), we produced a neutralizing monoclonal antibody (mAb12-1G6) using the pre-pandemic vaccine. Analysis with chimeric and point mutant HAs revealed that mAb12-1G6 bound to the loop (amino acid positions 140-145) corresponding to an antigenic site A in the H3 HA. mAb12-1G6 failed to bind to the mutant VN1194 HA when only 3 residues were substituted with the corresponding residues of the clade 2.1.3.2 A/Indonesia/5/05 strain (amino acid substitutions at positions Q142L, K144S, and S145P), suggesting that these amino acids are critical for binding of mAb12-1G6. Escape mutants of VN1194 selected with mAb12-1G6 carried a S145P mutation. Interestingly, mAb12-1G6 cross-neutralized clade 1 and clade 2.2.1 but not clade 2.1.3.2 or clade 2.3.4 of the H5N1 virus. We discuss the cross-reactivity, based on the amino acid sequence of the epitope. 相似文献