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PubMed1997

Functional studies in 3T3L1 cells support a role for SNARE proteins in insulin stimulation of GLUT4 translocation.

Insulin stimulation of glucose transport in the major insulin-responsive tissues results predominantly from the translocation to the cell surface of a particular glucose transporter isoform, GLUT4, residing normally under basal conditions in intracellular vesicular structures. Recent studies have identified the presence of vesicle-associated membrane protein (VAMP) 2, a protein involved in vesicular trafficking in secretory cell types, in the vesicles of insulin-sensitive cells that contain GLUT4. The plasma membranes of insulin-responsive cells have also been shown to contain syntaxin 4 and the 25 kDa synaptosome-associated protein (SNAP-25), two proteins that form a complex with VAMP 2. The potential functional involvement of VAMP 2, SNAP-25 and syntaxin 4 in the trafficking of GLUT4 was assessed in the present study by determining the effect on GLUT4 translocation of microinjection of toxins that specifically cleave VAMPs or SNAP-25, or microinjection of specific peptides from VAMP 2 and syntaxin 4. Microinjection of tetanus toxin light chain or botulinum D toxin light chain resulted in an 80 and 61% inhibition respectively of insulin stimulation of GLUT4 translocation in 3T3L1 cells assessed using the plasma-membrane lawn assay. Botulinum A toxin light chain, which cleaves SNAP-25, was without effect. Microinjection of an N-terminal VAMP 2 peptide (residues 1-26) inhibited insulin stimulation of GLUT4 translocation by 54%. A syntaxin 4 peptide (residues 106-122) inhibited insulin stimulation of GLUT4 translocation by 40% whereas a syntaxin 1c peptide (residues 226-260) was without effect. These data taken together strongly suggest a role for VAMP 2 in GLUT4 trafficking and also for syntaxin 4. They further indicate that the isoforms of SNAP-25 isolated to date that are sensitive to cleavage by botulinum A toxin light chain do not appear to be involved in GLUT4 translocation.

3T3 Cells↗
PubMed1997

Heterologous expression of rab4 reduces glucose transport and GLUT4 abundance at the cell surface in oocytes.

To evaluate the role of the small rab GTP-binding proteins in glucose transporter trafficking, we have heterologously co-expressed rab4 or rab5 and GLUT4 or GLUT1 glucose transporters in Xenopus oocytes. Co-injection of rab4 and GLUT4 cRNAs resulted in a dose-dependent decrease in glucose transport; this effect was specific for rab4, since co-injection of an inactive rab4 mutant or rab5 cRNA did not have any effect on glucose transport. The effect of rab4 was selective for GLUT4, since no effect was detected in GLUT1-expressing oocytes. The inhibitory effect of rab4 on GLUT4-induced glucose transport was not the result of a change in overall cellular levels of GLUT4 glucose transporters. However, rab4 expression caused a marked decrease in the abundance of GLUT4 transporters present at the cell surface. Finally, rab4 and inhibitors of PtdIns 3-kinase showed additive effects in decreasing glucose transport in GLUT4-expressing oocytes. We conclude that rab4 plays an important role in the regulation of the intracellular GLUT4 trafficking pathway, by contributing to the intracellular retention of GLUT4 through a PtdIns 3-kinase-independent mechanism.

Animals↗
PubMed2001

Lack of in vivo effect of vanadium on GLUT4 translocation in white adipose tissue of streptozotocin-diabetic rats.

Vanadium treatment, in vivo, corrects the severe hyperglycemia observed in streptozotocin (STZ)-diabetic rats. A number of metabolic effects of vanadium have been demonstrated in vitro and might contribute importantly to normalization of glucose homeostasis. However, many in vitro effects of vanadium occur at concentrations substantially higher than those achieved in vivo. Effects of vanadium on white adipose tissue have been particularly well characterized in vitro. To examine the relationship between in vitro and in vivo actions of vanadium, we examined the effects of vanadium treatment on acute glucose tolerance and adipose tissue GLUT4 control in vivo. In agreement with previous studies, vanadium treatment of STZ-diabetic rats restored normoglycemia with no appreciable restoration of insulin secretion. GLUT4 expression in white adipose tissue was reduced by 22% in STZ-diabetic rats compared with controls. Vanadium treatment did not significantly alter GLUT4 expression in controls, but completely restored normal expression levels in STZ-diabetic rats. In overnight-fasted control animals, GLUT4 translocation to the plasma membrane (PM) was maximally elevated (by 50%) in adipose tissue within 5 to 10 minutes after an intravenous (IV) glucose challenge. No glucose-induced translocation of GLUT4 was detected in diabetic rats, and peak PM GLUT4 content was 40% lower than in controls. Vanadium treatment did not increase peak PM GLUT4 content in either control or diabetic animals in response to a glucose load. Finally, the suppression of whole-body acute glucose tolerance in diabetic animals was only partially normalized by vanadium treatment. We conclude: (1) that concentrations of vanadium effective for maintaining normoglycemia in vivo (typically below 30 micromol/L) promote normal GLUT4 expression, but do not influence the subcellular localization of GLUT4 in white adipose tissue and (2) that in vivo effects of vanadium may not necessarily reflect the actions observed in vitro at supraphysiologic concentrations.

Adipose Tissue↗
PubMed1991

Translocation of the glucose transporter GLUT4 in cardiac myocytes of the rat.

The insulin-regulated glucose transporter GLUT4 was immunolocalized in rat cardiac muscle under conditions of basal and stimulated glucose uptake, achieved by fasting and a combined exercise/insulin stimulus, respectively. In basal myocytes there was very little (less than 1%) GLUT4 in the different domains of the plasma membrane (sarcolemma, intercalated disk, and transverse tubular system). GLUT4 was localized in small tubulo-vesicular elements that occur predominantly near the sarcolemma and the transverse tubular system and in the trans-Golgi region. Upon stimulation approximately 42% of GLUT4 was found in the plasma membrane. Each domain of the plasma membrane contributed equally to this effect. GLUT4-positive, clathrin-coated pits were also present at each cell surface domain. The remainder of the labeling was in tubulo-vesicular elements at the same sites as in basal cells and in the intercalated disk areas. The localization of GLUT4 in cardiac myocytes is essentially the same as in brown adipocytes, skeletal muscle, and white adipocytes. We conclude that increased glucose transport in muscle and fat is accounted for by translocation of GLUT4 from the intracellular tubulo-vesicular elements to the plasma membrane. The labeling of coated pits indicates that in stimulated myocytes, as in adipocytes, GLUT4 recycles constantly between the endosomal compartment and the plasma membrane and that stimulation of the exocytotic rate constant is likely the major mechanism for GLUT4 translocation.

Animals↗
PubMed1993

Regulated expression of an insulin-responsive glucose transporter (GLUT4) minigene in 3T3-L1 adipocytes and transgenic mice.

Preliminary studies showed that up to 7 kb of 5' flanking sequence of the insulin-responsive glucose transporter (GLUT4) gene are insufficient to mediate differentiation-induced reporter gene expression in mouse 3T3-L1 preadipocytes. To locate the regulatory element(s) responsible for this function, a minigene containing the entire GLUT4 gene with substantial 5' and 3' flanking sequence and a short segment of foreign DNA (for transcript identification) was constructed and transfected into mice and 3T3-L1 preadipocytes at relatively low copy number. In transgenic mice the GLUT4 minigene exhibited a pattern of tissue-specific expression similar, but not identical, to that of the endogenous gene. In 3T3-L1 cells expression of minigene mRNA occurred upon differentiation into adipocytes, with kinetics virtually identical to that of endogenous GLUT4 mRNA. In both cultured adipocytes and transgenic mice, the level of expression of the minigene was low relative to that of the endogenous gene. Treatment of minigene-transfected 3T3-L1 adipocytes with 8-bromo-cAMP, which represses transcription of the endogenous GLUT4 gene, also repressed expression of the GLUT4 minigene. However, insulin, which down-regulates transcription of the endogenous GLUT4 gene, failed to normally down-regulate expression of the GLUT4 minigene. These findings indicate that the cis-acting elements required for directing tissue-specific expression (in heart, skeletal muscle, and brown adipose tissue), differentiation-induced activation of transcription, and cAMP-induced repression of transcription are located within the 14-kb GLUT4 minigene. However, the cis elements necessary for maximal tissue-specific expression and for insulin-induced down-regulation of expression are not located in the minigene.

3T3 Cells↗
PubMed1996

Insulin-mediated targeting of phosphatidylinositol 3-kinase to GLUT4-containing vesicles.

Phosphatidylinositol (PI) 3-kinase is hypothesized to be a signaling element in the acute redistribution of intracellular GLUT4 glucose transporters to the plasma membrane in response to insulin. However, some receptors activate PI 3-kinase without causing GLUT4 translocation, suggesting specific cellular localization may be critical to this PI 3-kinase function. Consistent with this idea, complexes containing PI 3-kinase bound to insulin receptor substrate 1 (IRS-1) in 3T3-L1 adipocytes are associated with intracellular membranes (Heller-Harrison, R., Morin, M. and Czech, M. (1995) J. Biol. Chem. 270, 24442-24450). We report here that in response to insulin, activated complexes of IRS-1.PI 3-kinase can be immunoprecipitated with anti-IRS-1 antibody from detergent extracts of immunoadsorbed GLUT4-containing vesicles prepared from 3T3-L1 adipocytes. The targeting of PI 3-kinase to rat adipocyte GLUT4-containing vesicles using vesicles prepared by sucrose velocity gradient ultracentrifugation was also demonstrated. Insulin treatment caused a 2.3-fold increase in immunoreactive p85 protein in these GLUT4-containing vesicles while anti-p85 immunoprecipitates of PI 3-kinase activity in GLUT4-containing vesicle extracts increased to a similar extent. HPLC analysis of the GLUT4 vesicle-associated PI 3-kinase activity showed insulin-mediated increases in PI 3-P, PI 3,4-P2, and PI 3,4,5-P3 when PI, PI 4-P, and PI 4,5-P2 were used as substrates. Our data demonstrate that insulin directs the association of PI 3-kinase with GLUT4-containing vesicles in 3T3-L1 and rat adipocytes, consistent with the hypothesis that PI 3-kinase is involved in the insulin-regulated movement of GLUT4 to the plasma membrane.

3T3 Cells↗
PubMed1998

Vesicle-associated membrane protein 2 plays a specific role in the insulin-dependent trafficking of the facilitative glucose transporter GLUT4 in 3T3-L1 adipocytes.

Vesicle-associated membrane protein 2 (VAMP2) has been implicated in the insulin-regulated trafficking of GLUT4 in adipocytes. It has been proposed that VAMP2 co-localizes with GLUT4 in a postendocytic storage compartment (Martin, S., Tellam, J., Livingstone, C., Slot, J. W., Gould, G. W., and James, D. E. (1996) J. Cell Biol. 134, 625-635), suggesting that it may play a role distinct from endosomal v-SNAREs (soluble N-ethylmaleimide-sensitive factor attachment protein receptors) such as cellubrevin that are also expressed in adipocytes. The present study examines the effects of recombinant glutathione S-transferase (GST) fusion proteins encompassing the entire cytoplasmic tails of VAMP1, VAMP2, and cellubrevin on insulin-stimulated GLUT4 translocation in streptolysin O permeabilized 3T3-L1 adipocytes. GST-VAMP2 inhibited insulin-stimulated GLUT4 translocation by approximately 35%, whereas GST-VAMP1 and GST-cellubrevin were without effect. A synthetic peptide corresponding to the unique N terminus of VAMP2 also inhibited insulin-stimulated GLUT4 translocation in a dose-dependent manner. This peptide had no effect on either guanosine 5'-3-O-(thio)triphosphate-stimulated GLUT4 translocation or on insulin-stimulated GLUT1 translocation. These results imply that GLUT4 and GLUT1 may undergo insulin-stimulated translocation to the cell surface from separate intracellular compartments. To confirm this, adipocytes were incubated with a transferrin-horseradish peroxidase conjugate to fill the itinerant endocytic system after which cells were incubated with H2O2 and diaminobenzidine. This treatment completely blocked insulin-stimulated movement of GLUT1, whereas in the case of GLUT4, movement to the surface was delayed but still reached similar levels to that observed in insulin-stimulated control cells after 30 min. These results suggest that the N terminus of VAMP2 plays a unique role in the insulin-dependent recruitment of GLUT4 from its intracellular storage compartment to the cell surface.

3T3 Cells↗
PubMed1999

Endothelin-1-induced GLUT4 translocation is mediated via Galpha(q/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.

3T3 Cells↗
PubMed2000

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.

3T3 Cells↗
PubMed2000

Munc18c regulates insulin-stimulated glut4 translocation to the transverse tubules in skeletal muscle.

To examine the intracellular trafficking and translocation of GLUT4 in skeletal muscle, we have generated transgenic mouse lines that specifically express a GLUT4-EGFP (enhanced green fluorescent protein) fusion protein under the control of the human skeletal muscle actin promoter. These transgenic mice displayed EGFP fluorescence restricted to skeletal muscle and increased glucose tolerance characteristic of enhanced insulin sensitivity. The GLUT4-EGFP protein localized to the same intracellular compartment as the endogenous GLUT4 protein and underwent insulin- and exercise-stimulated translocation to both the sarcolemma and transverse-tubule membranes. Consistent with previous studies in adipocytes, overexpression of the syntaxin 4-binding Munc18c isoform, but not the related Munc18b isoform, in vivo specifically inhibited insulin-stimulated GLUT4-EGFP translocation. Surprisingly, however, Munc18c inhibited GLUT4 translocation to the transverse-tubule membrane without affecting translocation to the sarcolemma membrane. The ability of Munc18c to block GLUT4-EGFP translocation to the transverse-tubule membrane but not the sarcolemma membrane was consistent with substantially reduced levels of syntaxin 4 in the transverse-tubule membrane. Together, these data demonstrate that Munc18c specifically functions in the compartmentalized translocation of GLUT4 to the transverse-tubules in skeletal muscle. In addition, these results underscore the utility of this transgenic model to directly visualize GLUT4 translocation in skeletal muscle.

Animals↗
PubMed2005

Free fatty acids repress the GLUT4 gene expression in cardiac muscle via novel response elements.

Hyperlipidemia (HL) impairs cardiac glucose homeostasis, but the molecular mechanisms involved are yet unclear. We examined HL-regulated GLUT4 and peroxisome proliferator-activated receptor (PPAR) gamma gene expression in human cardiac muscle. Compared with control patients, GLUT4 protein levels were 30% lower in human cardiac muscle biopsies from patients with HL and/or type 2 diabetes mellitus, whereas GLUT4 mRNA levels were unchanged. PPARgamma mRNA levels were 30-50% lower in patients with HL and/or diabetes mellitus type 2 than in controls. Reporter studies in H9C2 cardiomyotubes showed that HL in vitro, induced by high levels of arachidonic (AA) stearic, linoleic, and oleic acids (24 h, 200 mum) repressed transcription from the GLUT4 promoter; AA also repressed transcription from the PPARgamma1 and PPARgamma2 promoters. Co-expression of PPARgamma2 repressed GLUT4 promoter activity, and the addition of AA further enhanced this effect. 5'-Deletion analysis revealed three GLUT4 promoter regions that accounted for AA-mediated effects: two repression-mediating sequences at -443/-423 bp and -222/-197 bp, the deletion of either or both of which led to a partial derepression of promoter activity, and a third derepression-mediating sequence at -612/-587 bp that was required for sustaining this derepression effect. Electromobility shift assay further shows that AA enhanced binding to two of the three regions of cardiac nuclear protein(s), the nature of which is still unknown. We propose that HL, exhibited as a high free fatty acid level, modulates GLUT4 gene expression in cardiac muscle via a complex mechanism that includes: (a) binding of AA mediator proteins to three newly identified response elements on the GLUT4 promoter gene and (b) repression of GLUT4 and the PPARgamma genes by AA.

Aged↗
PubMed2001

Insulin induces translocation of glucose transporter GLUT4 to plasma membrane caveolae in adipocytes.

Insulin-stimulated glucose uptake in muscle and adipose tissue is the result of translocation of insulin-regulated glucose transporters (GLUT4) from intracellular vesicles to the plasma membrane. Here we report that GLUT4 in the plasma membrane of 3T3-L1 adipocytes were located predominantly in caveolae invaginations: by immunogold electron microscopy of plasma membranes, 88% of GLUT4 were localized to caveolae structures and this distribution within the plasma membrane was not affected by insulin. By immunofluorescence microscopy, a major part of GLUT 4 was colocalized with caveolin. The total amount of GLUT4 in the plasma membrane increased 2.2-fold in response to insulin as determined by immunogold electron or immunofluorescence microscopy. GLUT4 were enriched in caveolae fractions isolated without detergents from plasma membranes of rat adipocytes. In these fractions, GLUT4 were largely confined to caveolin-containing membranes of the caveolae preparation isolated from insulin-stimulated cells, determined by electron microscopy. Insulin increased the amount of GLUT4 2.7-fold in this caveolae fraction. Caveolae were purified further by immunoisolation with antibodies against caveolin. The amount of GLUT4 increased to the same extent in the immunopurified caveolae as in the cruder caveolae fractions from insulin-stimulated cells. We conclude that insulin induces translocation of GLUT4 to caveolae.

3T3 Cells↗
PubMed2004

Insulin stimulates the entry of GLUT4 into the endosomal recycling pathway by a quantal mechanism.

The insulin-sensitive glucose transporter GLUT4 mediates the uptake of glucose into adipocytes and muscle cells. In this study we have used a novel 96-well plate fluorescence assay to study the kinetics of GLUT4 trafficking in 3T3-L1 adipocytes. We have found evidence for a graded release mechanism whereby GLUT4 is released into the plasma membrane recycling system in a nonkinetic manner as follows: the kinetics of appearance of GLUT4 at the plasma membrane is independent of the insulin concentration; a large proportion of GLUT4 molecules do not participate in plasma membrane recycling in the absence of insulin; and with increasing insulin there is an incremental increase in the total number of GLUT4 molecules participating in the recycling pathway rather than simply an increased rate of recycling. We propose a model whereby GLUT4 is stored in a compartment that is disengaged from the plasma membrane recycling system in the basal state. In response to insulin, GLUT4 is quantally released from this compartment in a pulsatile manner, leaving some sequestered from the recycling pathway even in conditions of excess insulin. Once disengaged from this location we suggest that in the continuous presence of insulin this quanta of GLUT4 continuously recycles to the plasma membrane, possibly via non-endosomal carriers that are formed at the perinuclear region.

3T3-L1 Cells↗
PubMed1995

Roles of 1-phosphatidylinositol 3-kinase and ras in regulating translocation of GLUT4 in transfected rat adipose cells.

Insulin stimulates glucose transport in insulin target tissues by recruiting glucose transporters (primarily GLUT4) from an intracellular compartment to the cell surface. Previous studies have demonstrated that insulin receptor tyrosine kinase activity and subsequent phosphorylation of insulin receptor substrate 1 (IRS-1) contribute to mediating the effect of insulin on glucose transport. We have now investigated the roles of 1-phosphatidylinositol 3-kinase (PI 3-kinase) and ras, two signaling proteins located downstream from tyrosine phosphorylation. Rat adipose cells were cotransfected with expression vectors that allowed transient expression of epitope-tagged GLUT4 and the other genes of interest. Overexpression of a mutant p85 regulatory subunit of PI 3-kinase lacking the ability to bind and activate the p110 catalytic subunit exerted a dominant negative effect to inhibit insulin-stimulated translocation of epitope-tagged GLUT4 to the cell surface. In addition, treatment of control cells with wortmannin (an inhibitor of PI 3-kinase) abolished the ability of insulin to recruit epitope-tagged GLUT4 to the cell surface. Thus, our data suggest that PI 3-kinase plays an essential role in insulin-stimulated GLUT4 recruitment in insulin target tissues. In contrast, over-expression of a constitutively active mutant of ras (L61-ras) resulted in high levels of cell surface GLUT4 in the absence of insulin that were comparable to levels seen in control cells treated with a maximally stimulating dose of insulin. However, wortmannin treatment of cells overexpressing L61-ras resulted in only a small decrease in the amount of cell surface GLUT4 compared with that of the same cells in the absence of wortmannin. Therefore, while activated ras is sufficient to recruit GLUT4 to the cell surface, it does so by a different mechanism that is probably not involved in the mechanism by which insulin stimulates GLUT4 translocation in physiological target tissues.

Adipocytes↗
PubMed2002

GLUT4-containing vesicles are released from membranes by phospholipase D cleavage of a GPI anchor.

We have previously developed a cell-free assay from rat skeletal muscle that displayed in vitro glucose transporter 4 (GLUT4) transfer from large to small membrane structures by the addition of a cytosolic protein fraction. By combining protein fractionation and the in vitro GLUT4 transfer assay, we have purified a glycosylphosphatidylinositol (GPI) phospholipase D (PLD) that induces transfer of GLUT4 from small to large membranes. The in vitro GLUT4 transfer was activated and inhibited by suramin and 1,10-phenanthroline (an activator and an inhibitor of GPI-PLD activity, respectively). Furthermore, upon purification of the GLUT4 transporter protein, the protein displayed an elution profile in which the molecular mass was related to the charge, suggesting the presence or absence of phosphate. Second, by photoaffinity labeling of the purified GLUT4 with 3-(trifluoromethyl)-3-(m-[(125)I]iodopenyl)diazirine, both labeled phosphatidylethanolamine and fatty acids (constituents of a GPI link) were recovered. Third, by using phase transition of Triton X-114, the purified GLUT4 was found to be partly detergent resistant, which is a known characteristic of GPI-linked proteins. Fourth, the purified GLUT4 protein was recognized by an antibody raised specifically against GPI links. In conclusion, GLUT4-containing vesicles may be released from a membrane compartment by action of a GPI-PLD.

Blotting, Western↗
PubMed2005

Activators of AMP-activated protein kinase enhance GLUT4 translocation and its glucose transport activity in 3T3-L1 adipocytes.

To determine whether the increase in glucose uptake following AMP-activated protein kinase (AMPK) activation in adipocytes is mediated by accelerated GLUT4 translocation into plasma membrane, we constructed a chimera between GLUT4 and enhanced green fluorescent protein (GLUT4-eGFP) and transferred its cDNA into the nucleus of 3T3-L1 adipocytes. Then, the dynamics of GLUT4-eGFP translocation were visualized in living cells by means of laser scanning confocal microscopy. It was revealed that the stimulation with 5-aminoimidazole-4-carboxamide-1-beta-D-ribofuranoside (AICAR) and 2,4-dinitrophenol (DNP), known activators of AMPK, promptly accelerates its translocation within 4 min, as was found in the case of insulin stimulation. The insulin-induced GLUT4 translocation was markedly inhibited after addition of wortmannin (P < 0.01). However, the GLUT4 translocation through AMPK activators AICAR and DNP was not affected by wortmannin. Insulin- and AMPK-activated translocation of GLUT4 was not inhibited by SB-203580, an inhibitor of p38 mitogen-activated protein kinase (MAPK). Glucose uptake was significantly increased after addition of AMPK activators AICAR and DNP (P < 0.05). AMPK- and insulin-stimulated glucose uptake were similarly suppressed by wortmannin (P < 0.05-0.01). In addition, SB-203580 also significantly prevented the enhancement of glucose uptake induced by AMPK and insulin (P < 0.05). These results suggest that AMPK-activated GLUT4 translocation in 3T3-L1 adipocytes is mediated through the insulin-signaling pathway distal to the site of activated phosphatidylinositol 3-kinase or through a signaling system distinct from that activated by insulin. On the other hand, the increase of glucose uptake dependent on AMPK activators AICAR and DNP would be additionally due to enhancement of the intrinsic activity in translocated GLUT4 protein, possibly through a p38 MAPK-dependent mechanism.

2,4-Dinitrophenol↗
PubMed2005

Regulation of muscle GLUT4 enhancer factor and myocyte enhancer factor 2 by AMP-activated protein kinase.

As the primary glucose transporter in skeletal muscle, GLUT4 is an important factor in the regulation of blood glucose. We previously reported that stimulation of AMP-activated protein kinase (AMPK) with 5-aminoimidazole-4-carboxamide-1-beta-d-ribofuranoside (AICAR) increased GLUT4 expression in muscle. GLUT4 enhancer factor (GEF) and myocyte enhancer factor 2 (MEF2) have been shown to be important for normal GLUT4 expression because deletion or truncation of the consensus sequences on the promoter causes depressed GLUT4 mRNA expression. This led to the current study to investigate possible roles for GEF and MEF2 in mediating the activation of GLUT4 gene transcription in response to AMPK. Here we show that, although AMPK does not appear to phosphorylate MEF2A, AMPK directly phosphorylates the GEF protein in vitro. MEF2 and GEF are activated in response to AMPK as we observed translocation of both to the nucleus after AICAR treatment. Nuclear MEF2 protein content was increased after 2 h, and GEF protein was increased in the nucleus 1 and 2 h post-AICAR treatment. Last, GEF and MEF2 increase in binding to the GLUT4 promoter within 2 h after AICAR treatment. Thus we conclude that GEF and MEF2 mediate the AMPK-induced increase in transcription of skeletal muscle GLUT4. AMPK can phosphorylate GEF and in response to AICAR, GEF, and MEF2 translocate to the nucleus and have increased binding to the GLUT4 promoter.

AMP-Activated Protein Kinases↗
PubMed1992

Expression of GLUT1 and GLUT4 glucose transporters in skeletal muscle of humans with insulin-dependent diabetes mellitus: regulatory effects of metabolic factors.

Insulin-dependent diabetes mellitus (IDDM) is associated with insulin deficiency and insulin-resistant glucose uptake in skeletal muscle. To investigate the molecular mechanisms for this insulin resistance, we examined the expression of GLUT1 and GLUT4, glucose transporter genes in vastus lateralis muscle from 20 IDDM subjects and 10 nondiabetic controls. Both groups had a mean age of 34 yr and were nonobese. Fasting free plasma insulin levels were similar in control and IDDM subjects but hemoglobin A1c (HbA1c), fasting plasma glucose and free fatty acid levels were significantly higher in IDDM subjects. Euglycemic clamp studies over a range of insulin concentrations in these IDDM subjects previously showed both decreased insulin sensitivity and decreased maximally insulin stimulated glucose utilization. In this study, Northern blotting of muscle ribonucleic acid (RNA) revealed a single 3.0-3.5 kb transcript for both GLUT1 and GLUT4 with no change in messenger RNA (mRNA) size or abundance with IDDM. In IDDM subjects, GLUT1 mRNA levels correlated positively with HbA1c whereas GLUT4 mRNA levels correlated negatively with fasting plasma glucose but not with HbA1c. Neither mRNA correlated with fasting plasma insulin or free fatty acid levels or with daily insulin dose. Immunoblotting of total muscle membranes for GLUT4 showed a single band of mol mass of approximately 45 kilodaltons with no change in size or abundance with IDDM. There was no significant correlation between GLUT4 polypeptide levels and HbA1c, fasting plasma glucose, insulin, or free fatty acids, daily insulin dose, duration of diabetes, or subject age but in IDDM subjects GLUT4 protein levels correlated negatively with body mass index. Thus, impaired expression of glucose transporters in muscle is not essential for the pathogenesis of insulin-resistant glucose uptake in IDDM. No direct regulatory role of chronic glycemic control or plasma insulin levels on GLUT4 expression is evident. In contrast, recent ambient glucose levels may affect levels of GLUT4 mRNA but not GLUT4 protein, suggesting important posttranscriptional regulation of this protein. Since glucose transport has been shown to be rate limiting for glucose utilization in muscle in IDDM, these results suggest impaired translocation or activation of glucose transporters in IDDM.

Actins↗