Insulin Stimulation of GLUT4 Translocation

Recent studies indicate that insulin stimulation of glucose transporter (GLUT)4 translocation requires at least two distinct insulin receptor–mediated signals: one leading to the activation of phosphatidylinositol 3 (PI-3) kinase and the other to the activation of the small GTP binding protein TC10. We now demonstrate that TC10 is processed through the secretory membrane trafficking system and localizes to caveolin-enriched lipid raft microdomains. Although insulin activated the wild-type TC10 protein and a TC10/H-Ras chimera that were targeted to lipid raft microdomains, it was unable to activate a TC10/K-Ras chimera that was directed to the nonlipid raft domains. Similarly, only the lipid raft–localized TC10/ H-Ras chimera inhibited GLUT4 translocation, whereas the TC10/K-Ras chimera showed no significant inhibitory activity. Furthermore, disruption of lipid raft microdomains by expression of a dominant-interfering caveolin 3 mutant (Cav3/DGV) inhibited the insulin stimulation of GLUT4 translocation and TC10 lipid raft localization and activation without affecting PI-3 kinase signaling. These data demonstrate that the insulin stimulation of GLUT4 translocation in adipocytes requires the spatial separation and distinct compartmentalization of the PI-3 kinase and TC10 signaling pathways.

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

Biochemical Journal ◽

10.1042/bj3240217 ◽

1997 ◽

Vol 324 (1) ◽

pp. 217-224 ◽

Cited By ~ 33

Author(s):

S. Lance MACAULAY ◽

Dean R. HEWISH ◽

Keith H. GOUGH ◽

Violet STOICHEVSKA ◽

Susan F. MACPHERSON ◽

...

Keyword(s):

Light Chain ◽

Plasma Membranes ◽

Glucose Transporter ◽

Cell Types ◽

Glut4 Translocation ◽

Insulin Stimulation ◽

Functional Studies ◽

Botulinum A Toxin ◽

Syntaxin 4 ◽

Stimulation Of

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.

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Substrate specificity and effect on GLUT4 translocation of the Rab GTPase-activating protein Tbc1d1

Biochemical Journal ◽

10.1042/bj20061798 ◽

2007 ◽

Vol 403 (2) ◽

pp. 353-358 ◽

Cited By ~ 131

Author(s):

William G. Roach ◽

Jose A. Chavez ◽

Cristinel P. Mîinea ◽

Gustav E. Lienhard

Keyword(s):

Plasma Membrane ◽

Glucose Transporter ◽

Insulin Treatment ◽

Ectopic Expression ◽

Rab Gtpase ◽

Glut4 Translocation ◽

Gtpase Activating Protein ◽

Insulin Stimulation ◽

Glucose Transporter Glut4 ◽

Stimulation Of

Insulin stimulation of the trafficking of the glucose transporter GLUT4 to the plasma membrane is controlled in part by the phosphorylation of the Rab GAP (GTPase-activating protein) AS160 (also known as Tbc1d4). Considerable evidence indicates that the phosphorylation of this protein by Akt (protein kinase B) leads to suppression of its GAP activity and results in the elevation of the GTP form of a critical Rab. The present study examines a similar Rab GAP, Tbc1d1, about which very little is known. We found that the Rab specificity of the Tbc1d1 GAP domain is identical with that of AS160. Ectopic expression of Tbc1d1 in 3T3-L1 adipocytes blocked insulin-stimulated GLUT4 translocation to the plasma membrane, whereas a point mutant with an inactive GAP domain had no effect. Insulin treatment led to the phosphorylation of Tbc1d1 on an Akt site that is conserved between Tbc1d1 and AS160. These results show that Tbc1d1 regulates GLUT4 translocation through its GAP activity, and is a likely Akt substrate. An allele of Tbc1d1 in which Arg125 is replaced by tryptophan has very recently been implicated in susceptibility to obesity by genetic analysis. We found that this form of Tbc1d1 also inhibited GLUT4 translocation and that this effect also required a functional GAP domain.

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Botulinum E Toxin Light Chain Does Not Cleave SNAP-23 and Only Partially Impairs Insulin Stimulation of GLUT4 Translocation in 3T3-L1 Cells

Biochemical and Biophysical Research Communications ◽

10.1006/bbrc.1997.7143 ◽

1997 ◽

Vol 237 (2) ◽

pp. 388-393 ◽

Cited By ~ 19

Author(s):

S.Lance Macaulay ◽

Shane Rea ◽

Keith H. Gough ◽

Colin W. Ward ◽

David E. James

Keyword(s):

Light Chain ◽

Glut4 Translocation ◽

Insulin Stimulation ◽

Stimulation Of

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Regulation of glucose transporters by insulin and extracellular glucose in C2C12 myotubes

AJP Endocrinology and Metabolism ◽

10.1152/ajpendo.00194.2006 ◽

2006 ◽

Vol 291 (4) ◽

pp. E817-E828 ◽

Cited By ~ 69

Author(s):

Taku Nedachi ◽

Makoto Kanzaki

Keyword(s):

Skeletal Muscle ◽

Glucose Uptake ◽

Glucose Transport ◽

Glucose Transporter ◽

C2c12 Myotubes ◽

Glut4 Translocation ◽

Insulin Stimulation ◽

Extracellular Glucose ◽

Insulin Dependent ◽

Stimulation Of

It is well established that insulin stimulation of glucose uptake in skeletal muscle cells is mediated through translocation of GLUT4 from intracellular storage sites to the cell surface. However, the established skeletal muscle cell lines, with the exception of L6 myocytes, reportedly show minimal insulin-dependent glucose uptake and GLUT4 translocation. Using C2C12 myocytes expressing exofacial-Myc-GLUT4-enhanced cyan fluorescent protein, we herein show that differentiated C2C12 myotubes are equipped with basic GLUT4 translocation machinery that can be activated by insulin stimulation (∼3-fold increase as assessed by anti-Myc antibody uptake and immunostaining assay). However, this insulin stimulation of GLUT4 translocation was difficult to demonstrate with a conventional 2-deoxyglucose uptake assay because of markedly elevated basal glucose uptake via other glucose transporter(s). Intriguingly, the basal glucose transport activity in C2C12 myotubes appeared to be acutely suppressed within 5 min by preincubation with a pathophysiologically high level of extracellular glucose (25 mM). In contrast, this activity was augmented by acute glucose deprivation via an unidentified mechanism that is independent of GLUT4 translocation but is dependent on phosphatidylinositol 3-kinase activity. Taken together, these findings indicate that regulation of the facilitative glucose transport system in differentiated C2C12 myotubes can be achieved through surprisingly acute glucose-dependent modulation of the activity of glucose transporter(s), which apparently contributes to obscuring the insulin augmentation of glucose uptake elicited by GLUT4 translocation. We herein also describe several methods of monitoring insulin-dependent glucose uptake in C2C12 myotubes and propose this cell line to be a useful model for analyzing GLUT4 translocation in skeletal muscle.

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Insulin Stimulation of Amino Acid Uptake in Rat Diaphragm

Journal of Biological Chemistry ◽

10.1016/s0021-9258(18)93519-2 ◽

1968 ◽

Vol 243 (8) ◽

pp. 1846-1853 ◽

Cited By ~ 5

Author(s):

L J Elsas ◽

I Albrecht ◽

L E Rosenberg

Keyword(s):

Amino Acid ◽

Amino Acid Uptake ◽

Acid Uptake ◽

Rat Diaphragm ◽

Insulin Stimulation ◽

Stimulation Of

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Insulin stimulation of glucose transport in adipose cells. An energy-dependent process

Biochemistry ◽

10.1021/bi00625a019 ◽

1977 ◽

Vol 16 (6) ◽

pp. 1151-1158 ◽

Cited By ~ 52

Author(s):

Visvanathan Chandramouli ◽

Marianne Milligan ◽

James R. Carter

Keyword(s):

Glucose Transport ◽

Insulin Stimulation ◽

Dependent Process ◽

Energy Dependent ◽

Adipose Cells ◽

Stimulation Of

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Effects of ML-9 on insulin stimulation of glucose transport in 3T3-L1 adipocytes.

Journal of Biological Chemistry ◽

10.1016/s0021-9258(18)53529-8 ◽

1993 ◽

Vol 268 (7) ◽

pp. 5272-5278 ◽

Cited By ~ 1

Author(s):

G. Inoue ◽

H. Kuzuya ◽

T. Hayashi ◽

M. Okamoto ◽

Y. Yoshimasa ◽

...

Keyword(s):

Glucose Transport ◽

Insulin Stimulation ◽

Stimulation Of

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Immunoelectron microscopic evidence that GLUT4 translocation explains the stimulation of glucose transport in isolated rat white adipose cells

Journal of Cell Science ◽

10.1242/jcs.113.23.4203 ◽

2000 ◽

Vol 113 (23) ◽

pp. 4203-4210 ◽

Cited By ~ 1

Author(s):

D. Malide ◽

G. Ramm ◽

S.W. Cushman ◽

J.W. Slot

Keyword(s):

Adipose Tissue ◽

Glucose Transport ◽

Morphological Evidence ◽

Glut4 Translocation ◽

Transport Activity ◽

Brown Adipose ◽

Quantitative Immunoelectron Microscopy ◽

Adipose Cells ◽

Isolated Rat ◽

Stimulation Of

We used an improved cryosectioning technique in combination with quantitative immunoelectron microscopy to study GLUT4 compartments in isolated rat white adipose cells. We provide clear evidence that in unstimulated cells most of the GLUT4 localizes intracellularly to tubulovesicular structures clustered near small stacks of Golgi and endosomes, or scattered throughout the cytoplasm. This localization is entirely consistent with that originally described in brown adipose tissue, strongly suggesting that the GLUT4 compartments in white and brown adipose cells are morphologically similar. Furthermore, insulin induces parallel increases (with similar magnitudes) in glucose transport activity, approximately 16-fold, and cell-surface GLUT4, approximately 12-fold. Concomitantly, insulin decreases GLUT4 equally from all intracellular locations, in agreement with the concept that the entire cellular GLUT4 pool contributes to insulin-stimulated exocytosis. In the insulin-stimulated state, GLUT4 molecules are not randomly distributed on the plasma membrane, but neither are they enriched in caveolae. Importantly, the total number of GLUT4 C-terminal epitopes detected by the immuno-gold method is not significantly different between basal and insulin-stimulated cells, thus arguing directly against a reported insulin-induced unmasking effect. These results provide strong morphological evidence (1) that GLUT4 compartments are similar in all insulin-sensitive cells and (2) for the concept that GLUT4 translocation almost fully accounts for the increase in glucose transport in response to insulin.

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