GLUT4 Translocation In 3T3-L1 Adipocytes Research Articles

Microtubule integrity is not required for insulin-stimulated GLUT4 translocation in 3T3-L1 adipocytes

Microtubule integrity is not required for insulin-stimulated GLUT4 translocation in 3T3-L1 adipocytes

Substrates of semicarbazide-sensitive amine oxidase co-operate with vanadate to stimulate tyrosine phosphorylation of insulin-receptor-substrate proteins, phosphoinositide 3-kinase activity and GLUT4 translocation in adipose cells

It has been shown that the combination of benzylamine or tyramine and low concentrations of vanadate markedly stimulates glucose transport in rat adipocytes by a mechanism that requires semicarbazide-sensitive amine oxidase (SSAO) activity and H2O2 formation. Here we have further analysed the insulin-like effects of the combination of SSAO substrates and vanadate and we have studied the signal-transduction pathway activated in rat adipocytes. We found that several SSAO substrates (benzylamine, tyramine, methylamine, n-decylamine, histamine, tryptamine or β-phenylethylamine), in combination with low concentrations of vanadate, stimulate glucose transport in isolated rat adipocytes. Furthermore, SSAO substrates together with vanadate stimulated the recruitment of GLUT4 to the cell surface in isolated rat adipocytes. Benzylamine plus vanadate also stimulated glucose transport and GLUT4 translocation in 3T3-L1 adipocytes. Benzylamine or tyramine in combination with vanadate potently stimulated the tyrosine phosphorylation of both insulin receptor substrate (IRS)-1 and IRS-3. In contrast, benzylamine and vanadate caused only a weak stimulation of insulin receptor kinase. Benzylamine or tyramine in combination with vanadate also stimulated phosphoinositide 3-kinase activity; wortmannin abolished the stimulatory effect of benzylamine and vanadate on glucose transport in adipose cells. Furthermore, the administration of benzylamine and vanadate in vivo caused a rapid lowering of plasma glucose levels, which took place in the absence of alterations in plasma insulin. On the basis of these results we propose that SSAO activity regulates glucose transport in adipocytes. SSAO oxidative activity stimulates glucose transport via the translocation of GLUT4 carriers to the cell surface, resulting from a potent tyrosine phosphorylation of IRS-1 and IRS-3 and phosphoinositide 3-kinase activation. Our results also indicate that substrates of SSAO might regulate glucose disposal in vivo.

Substrates of semicarbazide-sensitive amine oxidase co-operate with vanadate to stimulate tyrosine phosphorylation of insulin-receptor-substrate proteins, phosphoinositide 3-kinase activity and GLUT4 translocation in adipose cells

It has been shown that the combination of benzylamine or tyramine and low concentrations of vanadate markedly stimulates glucose transport in rat adipocytes by a mechanism that requires semicarbazide-sensitive amine oxidase (SSAO) activity and H(2)O(2) formation. Here we have further analysed the insulin-like effects of the combination of SSAO substrates and vanadate and we have studied the signal-transduction pathway activated in rat adipocytes. We found that several SSAO substrates (benzylamine, tyramine, methylamine, n-decylamine, histamine, tryptamine or beta-phenylethylamine), in combination with low concentrations of vanadate, stimulate glucose transport in isolated rat adipocytes. Furthermore, SSAO substrates together with vanadate stimulated the recruitment of GLUT4 to the cell surface in isolated rat adipocytes. Benzylamine plus vanadate also stimulated glucose transport and GLUT4 translocation in 3T3-L1 adipocytes. Benzylamine or tyramine in combination with vanadate potently stimulated the tyrosine phosphorylation of both insulin receptor substrate (IRS)-1 and IRS-3. In contrast, benzylamine and vanadate caused only a weak stimulation of insulin receptor kinase. Benzylamine or tyramine in combination with vanadate also stimulated phosphoinositide 3-kinase activity; wortmannin abolished the stimulatory effect of benzylamine and vanadate on glucose transport in adipose cells. Furthermore, the administration of benzylamine and vanadate in vivo caused a rapid lowering of plasma glucose levels, which took place in the absence of alterations in plasma insulin. On the basis of these results we propose that SSAO activity regulates glucose transport in adipocytes. SSAO oxidative activity stimulates glucose transport via the translocation of GLUT4 carriers to the cell surface, resulting from a potent tyrosine phosphorylation of IRS-1 and IRS-3 and phosphoinositide 3-kinase activation. Our results also indicate that substrates of SSAO might regulate glucose disposal in vivo.

The Trimeric GTP-binding Protein (Gq/G11) α Subunit Is Required for Insulin-stimulated GLUT4 Translocation in 3T3L1 Adipocytes

To investigate the potential role of trimeric GTP-binding proteins regulating GLUT4 translocation in adipocytes, wild type and constitutively active G(q) (G(q)/Q209L), G(i) (G(i)/Q205L), and G(s) (G(s)/Q227L) alpha subunit mutants were expressed in 3T3L1 adipocytes. Although expression of neither the wild type nor G(i)/Q205L and G(s)/Q227L alpha subunit mutants had any effect on the basal or insulin-stimulated translocation of a co-expressed GLUT4-enhanced green fluorescent protein (EGFP) fusion protein, expression of G(q)/Q209L resulted in GLUT4-EGFP translocation in the absence of insulin. In contrast, microinjection of an inhibitory G(q)/G(11) alpha subunit-specific antibody but not a G(i) or G(s) alpha subunit antibody prevented insulin-stimulated endogenous GLUT4 translocation. Consistent with a required role for GTP-bound G(q)/G(11), expression of the regulators of G protein signaling (RGS4 and RGS16) also attenuated insulin-stimulated GLUT4-EGFP translocation. To assess the relationship between G(q)/G(11) function with the phosphatidylinositol 3-kinase dependent pathway, expression of a dominant-interfering p85 regulatory subunit, as well as wortmannin treatment inhibited insulin-stimulated but not G(q)/Q209L-stimulated GLUT4-EGFP translocation. Furthermore, G(q)/Q209L did not induce the in vivo accumulation of phosphatidylinositol-3,4,5-trisphosphate (PIP(3)), whereas expression of the RGS proteins did not prevent the insulin-stimulated accumulation of PIP(3). Together, these data demonstrate that insulin stimulation of GLUT4 translocation requires at least two independent signal transduction pathways, one mediated through the phosphatidylinositol 3-kinase and another through the trimeric GTP-binding proteins G(q) and/or G(11).

Munc18c function is required for insulin-stimulated plasma membrane fusion of GLUT4 and insulin-responsive amino peptidase storage vesicles.

To examine the functional role of the interaction between Munc18c and syntaxin 4 in the regulation of GLUT4 translocation in 3T3L1 adipocytes, we assessed the effects of introducing three different peptide fragments (20 to 24 amino acids) of Munc18c from evolutionarily conserved regions of the Sec1 protein family predicted to be solvent exposed. One peptide, termed 18c/pep3, inhibited the binding of full-length Munc18c to syntaxin 4, whereas expression of the other two peptides had no effect. In parallel, microinjection of 18c/pep3 but not a control peptide inhibited the insulin-stimulated translocation of endogenous GLUT4 and insulin-responsive amino peptidase (IRAP) to the plasma membrane. In addition, expression of 18c/pep3 prevented the insulin-stimulated fusion of endogenous and enhanced green fluorescent protein epitope-tagged GLUT4- and IRAP-containing vesicles into the plasma membrane, as assessed by intact cell immunofluorescence. However, unlike the pattern of inhibition seen with full-length Munc18c expression, cells expressing 18c/pep3 displayed discrete clusters of GLUT4 abd IRAP storage vesicles at the cell surface which were not contiguous with the plasma membrane. Together, these data suggest that the interaction between Munc18c and syntaxin 4 is required for the integration of GLUT4 and IRAP storage vesicles into the plasma membrane but is not necessary for the insulin-stimulated trafficking to and association with the cell surface.

Endothelin-1-induced GLUT4 Translocation Is Mediated via Gαq/11 Protein and Phosphatidylinositol 3-Kinase in 3T3-L1 Adipocytes

Endothelin-1 (ET-1) can stimulate insulin-responsive glucose transporter (GLUT4) translocation in 3T3-L1 adipocytes (Wu-Wong, J. R., Berg, C. E., Wang, J., Chiou, W. J., and Fissel, B. (1999) J. Biol. Chem. 274, 8103-8110), and in the current study, we have evaluated the signaling pathway leading to this response. First, we inhibited endogenous Galpha(q/11) function by single-cell microinjection using anti-Galpha(q/11) antibody or RGS2 protein (a GTPase activating protein for Galpha(q)) followed by immunostaining to quantitate GLUT4 translocation in 3T3-L1 adipocytes. ET-1-stimulated GLUT4 translocation was markedly decreased by 70 or 75% by microinjection of Galpha(q/11) antibody or RGS2 protein, respectively. Pretreatment of cells with the Galpha(i) inhibitor (pertussis toxin) or microinjection of a Gbetagamma inhibitor (glutathione S-transferase-beta-adrenergic receptor kinase (GST-BARK)) did not inhibit ET-1-induced GLUT4 translocation, indicating that Galpha(q/11 )mediates ET-1 signaling to GLUT4 translocation. Next, we found that ET-1-induced GLUT4 translocation was inhibited by the phosphatidylinositol (PI) 3-kinase inhibitors wortmannin or LY294002, but not by the phospholipase C inhibitor U-73122. ET-1 stimulated the PI 3-kinase activity of the p110alpha subunit (5.5-fold), and microinjection of anti-p110alpha or PKC-lambda antibodies inhibited ET-stimulated GLUT4 translocation. Finally, we found that Galpha(q/11) formed immunocomplexes with the type-A endothelin receptor and the 110alpha subunit of PI 3-kinase and that ET-1 stimulation enhances tyrosine phosphorylation of Galpha(q/11). These results indicate that: 1) ET-1 signaling to GLUT4 translocation is dependent upon Galpha(q/11) and PI 3-kinase; and 2) Galpha(q/11) can transmit signals from the ET(A) receptor to the p110alpha subunit of PI 3-kinase, as does insulin, subsequently leading to GLUT4 translocation.

G alpha-q/11 protein plays a key role in insulin-induced glucose transport in 3T3-L1 adipocytes.

We evaluated the role of the G alpha-q (Galphaq) subunit of heterotrimeric G proteins in the insulin signaling pathway leading to GLUT4 translocation. We inhibited endogenous Galphaq function by single cell microinjection of anti-Galphaq/11 antibody or RGS2 protein (a GAP protein for Galphaq), followed by immunostaining to assess GLUT4 translocation in 3T3-L1 adipocytes. Galphaq/11 antibody and RGS2 inhibited insulin-induced GLUT4 translocation by 60 or 75%, respectively, indicating that activated Galphaq is important for insulin-induced glucose transport. We then assessed the effect of overexpressing wild-type Galphaq (WT-Galphaq) or a constitutively active Galphaq mutant (Q209L-Galphaq) by using an adenovirus expression vector. In the basal state, Q209L-Galphaq expression stimulated 2-deoxy-D-glucose uptake and GLUT4 translocation to 70% of the maximal insulin effect. This effect of Q209L-Galphaq was inhibited by wortmannin, suggesting that it is phosphatidylinositol 3-kinase (PI3-kinase) dependent. We further show that Q209L-Galphaq stimulates PI3-kinase activity in p110alpha and p110gamma immunoprecipitates by 3- and 8-fold, respectively, whereas insulin stimulates this activity mostly in p110alpha by 10-fold. Nevertheless, only microinjection of anti-p110alpha (and not p110gamma) antibody inhibited both insulin- and Q209L-Galphaq-induced GLUT4 translocation, suggesting that the metabolic effects induced by Q209L-Galphaq are dependent on the p110alpha subunit of PI3-kinase. In summary, (i) Galphaq appears to play a necessary role in insulin-stimulated glucose transport, (ii) Galphaq action in the insulin signaling pathway is upstream of and dependent upon PI3-kinase, and (iii) Galphaq can transmit signals from the insulin receptor to the p110alpha subunit of PI3-kinase, which leads to GLUT4 translocation.

Arrest of endosome acidification by bafilomycin A1 mimics insulin action on GLUT4 translocation in 3T3-L1 adipocytes

Arrest of endosome acidification by bafilomycin A1 mimics insulin action on GLUT4 translocation in 3T3-L1 adipocytes

Arrest of endosome acidification by bafilomycin A1 mimics insulin action on GLUT4 translocation in 3T3-L1 adipocytes

In insulin-sensitive fat and muscle cells, the major glucose transporter GLUT4 is constitutively sequestered in endosomal tubulovesicular membranes, and moves to the cell surface in response to insulin. While sequence information within GLUT4 appears to be responsible for its constitutive intracellular sequestration, the regulatory elements and mechanisms that enable this protein to achieve its unique sorting pattern under basal and insulin-stimulated conditions are poorly understood. We show here that arrest of endosome acidification in insulin-sensitive 3T3-L1 adipocytes by bafilomycin A1, a specific inhibitor of the vacuolar proton pump, results in the rapid and dose-dependent translocation of GLUT4 from the cell interior to the membrane surface; the effects of maximally stimulatory concentrations of bafilomycin A1 (400-800 nM) were equivalent to 50-65% of the effects of acute insulin treatment. Like insulin, bafilomycin A1 induced the redistribution of GLUT1 and Rab4, but not that of other proteins whose membrane localization has been shown to be insulin-insensitive. Studies to address the mechanism of this effect demonstrated that neither autophosphorylation nor internalization of the insulin receptor was altered by bafilomycin A1 treatment. Bafilomycin-induced GLUT4 translocation was not blocked by cell pretreatment with wortmannin. Taken together, these data indicate that arrest of endosome acidification mimics insulin action on GLUT4 and GLUT1 translocation by a mechanism distal to insulin receptor and phosphatidylinositol 3-kinase activation, and suggest an important role for endosomal pH in the membrane dynamics of the glucose transporters.

Prolonged oxidative stress impairs insulin-induced GLUT4 translocation in 3T3-L1 adipocytes.

Prolonged exposure of 3T3-L1 adipocytes to micromolar concentrations of H2O2 results in an impaired response to the acute metabolic effects of insulin. In this study, we further characterized the mechanisms by which oxidative stress impairs insulin stimulation of glucose transport activity. Although insulin induced a 2.5-fold increase in plasma membrane GLUT4 content and a 50% reduction in its abundance in the low-density microsomal (LDM) fraction in control cells, oxidation completely prevented these responses. The net effect of insulin on 2-deoxyglucose uptake activity was reduced in oxidized cells and could be attributed to GLUT1 translocation. Insulin stimulation of insulin receptor substrate (IRS) 1 tyrosine phosphorylation and the association of IRS-1 with phosphatidylinositol (PI) 3-kinase were not impaired by oxidative stress. However, a 1.9-fold increase in the LDM content of the p85 subunit of PI 3-kinase after insulin stimulation was observed in control, but not in oxidized, cells. Moreover, although insulin induced an increase in IRS-1-associated PI 3-kinase activity in the LDM in control cells, this effect was prevented by oxidation. These findings suggest that prolonged low-grade oxidative stress impairs insulin-stimulated GLUT4 translocation, potentially by interfering with compartment-specific activation of PI 3-kinase.

The small guanosine triphosphate-binding protein Rab4 is involved in insulin-induced GLUT4 translocation and actin filament rearrangement in 3T3-L1 cells.

Insulin's stimulation of glucose transport involves the translocation of vesicles containing the glucose transporter GLUT4 to the plasma membrane. Small GTP-binding proteins have been implicated in the regulation of vesicular traffic. We studied the effects of microinjection of wild-type Rab4 glutathione S-transferase fusion protein (WT Rab4), a GTP-binding defective mutant (Rab4 N121I), a guanosine triphosphatase-defective mutant (Rab4 Q67L), and a Rab4 antibody on insulin-induced GLUT4 translocation in 3T3-L1 adipocytes. Microinjection of Rab4 N121I and Rab4 antibodies had no effect on basal GLUT4 staining, but inhibited insulin-induced GLUT4 translocation by 50% compared with that in control IgG-injected cells. WT Rab4 and Rab4 Q67L microinjection had no effect on either basal or insulin-induced GLUT4 translocation. Premixing and coinjection of the Rab4 antibody with WT Rab4 almost completely abolished its inhibitory effect on insulin-induced GLUT4 translocation. In contrast, microinjection of an antibody directed against the highly conserved region of Rab3 proteins had no effect on insulin-induced GLUT4. These results point to a direct role of Rab4 in insulin-induced GLUT4 translocation, and that this effect is dependent on nucleotide binding to the protein. We also studied the effect of microinjection of the same proteins on insulin-induced actin filament rearrangement (membrane ruffling) in the same cell line. Microinjection of Rab4 N121I and Rab4 antibodies inhibited insulin-induced membrane ruffling by 40%, whereas WT Rab4 or a Rab3 antibody injection had no effect on cytoskeletal rearrangement. In summary, 1) Rab4 is a necessary component of the insulin/GLUT4 translocation signaling pathway; 2) the function of Rab4 in this pathway requires GTP binding; 3) Rab4 also participates in the process of insulin-induced membrane ruffling; and 4) Rab3 proteins do not seem to be involved in these processes.