scholarly journals Actin filaments participate in the relocalization of phosphatidylinositol3-kinase to glucose transporter-containing compartments and in the stimulation of glucose uptake in 3T3-L1 adipocytes

1998 ◽  
Vol 331 (3) ◽  
pp. 917-928 ◽  
Author(s):  
Qinghua WANG ◽  
Philip J. BILAN ◽  
Theodoros TSAKIRIDIS ◽  
Aleksander HINEK ◽  
Amira KLIP
Keyword(s):  
Plasma Membrane ◽  
Glucose Uptake ◽  
Actin Filament ◽  
Actin Filaments ◽  
Glucose Transporter ◽  
Kinase Activity ◽  
Glucose Transporters ◽  
Cytochalasin D ◽  
Insulin Dependent ◽  
Stimulation Of

Insulin stimulates the rate of glucose uptake into muscle and adipose cells by translocation of glucose transporters from an intracellular storage pool to the plasma membrane. This event requires the prior activation of phosphatidylinositol 3-kinase (PI 3-kinase). Here we report that insulin causes an increase in wortmannin-sensitive PI 3-kinase activity and a gain in the enzyme's regulatory and catalytic subunits p85α and p110β (but not p110α) in the intracellular compartments containing glucose transporters. The hormone also caused a marked reorganization of actin filaments, which was prevented by cytochalasin D. Cytochalasin D also decreased significantly the insulin-dependent association of PI 3-kinase activity and the levels of insulin receptor substrate (IRS)-1, p85α and p110β with immunopurified GLUT4-containing compartments. In contrast, the drug did not alter the insulin-induced tyrosine phosphorylation of IRS-1, the association of PI 3-kinase with IRS-1, or the stimulation of PI 3-kinase by insulin in anti-(IRS-1) or anti-p85 immunoprecipitates from whole cell lysates. Cytochalasin D, and the chemically unrelated latrunculin B, which also inhibits actin filament reassembly, prevented the insulin stimulation of glucose transport by approx. 50%. Cytochalasin D decreased by about one-half the insulin-dependent translocation to the plasma membrane of the GLUT1 and GLUT4 glucose transporters. The results suggest that the existence of intact actin filament is correlated with the full recruitment of glucose transporters by insulin. The underlying function of the actin filaments might be to facilitate the insulin-mediated association of the p85–p110 PI 3-kinase with glucose-transporter-containing compartments.

Stimulation of glucose transport by thyroid hormone in 3T3-L1 adipocytes: increased abundance of GLUT1 and GLUT4 glucose transporter proteins

2000 ◽  
Vol 164 (2) ◽  
pp. 187-195 ◽  
Author(s):  
R Romero ◽  
B Casanova ◽  
N Pulido ◽  
AI Suarez ◽  
E Rodriguez ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Glucose Uptake ◽  
Glucose Transport ◽  
Plasma Membranes ◽  
Glucose Transporter ◽  
Fold Increase ◽  
Glucose Transporters ◽  
Insulin Binding ◽  
Total Protein Content ◽  
Glucose Transporter Proteins

In 3T3-L1 adipocytes we have examined the effect of tri-iodothyronine (T(3)) on glucose transport, total protein content and subcellular distribution of GLUT1 and GLUT4 glucose transporters. Cells incubated in T(3)-depleted serum were used as controls. Cells treated with T(3) (50 nM) for three days had a 3.6-fold increase in glucose uptake (P<0.05), and also presented a higher insulin sensitivity, without changes in insulin binding. The two glucose carriers, GLUT1 and GLUT4, increased by 87% (P<0.05) and 90% (P<0. 05), respectively, in cells treated with T(3). Under non-insulin-stimulated conditions, plasma membrane fractions obtained from cells exposed to T(3) were enriched with both GLUT1 (3. 29+/-0.69 vs 1.20+/-0.29 arbitrary units (A.U.)/5 microg protein, P<0.05) and GLUT4 (3.50+/-1.16 vs 0.82+/-0.28 A.U./5 microg protein, P<0.03). The incubation of cells with insulin produced the translocation of both glucose transporters to plasma membranes, and again cells treated with T(3) presented a higher amount of GLUT1 and GLUT4 in the plasma membrane fractions (P<0.05 and P<0.03 respectively). These data indicate that T(3) has a direct stimulatory effect on glucose transport in 3T3-L1 adipocytes due to an increase in GLUT1 and GLUT4, and by favouring their partitioning to plasma membranes. The effect of T(3) on glucose uptake induced by insulin can also be explained by the high expression of both glucose transporters.

scholarly journals Methyl-beta-cyclodextrin stimulates glucose uptake in Clone 9 cells: a possible role for lipid rafts

2004 ◽  
Vol 378 (2) ◽  
pp. 343-351 ◽  
Author(s):  
Kay BARNES ◽  
Jean C. INGRAM ◽  
Matthew D. M. BENNETT ◽  
Gordon W. STEWART ◽  
Stephen A. BALDWIN
Keyword(s):  
Plasma Membrane ◽  
Protein Kinase ◽  
Glucose Uptake ◽  
Glucose Transporter ◽  
Gradient Centrifugation ◽  
Metabolic Stress ◽  
Mitogen Activated Protein Kinase ◽  
Cholesterol Depletion ◽  
Lipid Environment ◽  
Stimulation Of

An acute increase in the Vmax for glucose uptake occurs in many mammalian cell types after exposure to osmotic or metabolic stress. In the rat epithelial Clone 9 cell line, the glucose transporter isoform GLUT1 is responsible for this enhanced uptake. Although stimulation of transport in these cells is known to result from the unmasking of ‘cryptic’ exofacial permeant-binding sites in GLUT1 molecules resident in the plasma membrane, the mechanism of such unmasking remains unclear. One possibility involves changes in the lipid environment of the transporter: reconstitution experiments have shown that transport activity in vitro is acutely sensitive to the phospholipid and cholesterol composition of the membrane. In the current study we found that treatment of Clone 9 cells with methyl-β-cyclodextrin, which removed >80% of the cell cholesterol, led to a 3.5-fold increase in the Vmax for 3-O-methyl-d-glucose transport while having little effect on the Km. In contrast to the metabolic stress induced by inhibition of oxidative phosphorylation, cholesterol depletion led neither to depletion of cellular ATP nor stimulation of AMP-activated protein kinase. Similarly, it did not result in stimulation of members of the stress- and mitogen-activated protein kinase families. In unstressed, cholesterol-replete cells, a substantial proportion of GLUT1 in detergent lysates co-fractionated with the lipid-raft proteins caveolin and stomatin on density-gradient centrifugation. Immunocytochemistry also revealed the presence of GLUT1-enriched domains, some of which co-localized with stomatin, in the plasma membrane. Both techniques revealed that the abundance of such putative GLUT1-containing domains was decreased not only by cholesterol depletion but also in cells subjected to metabolic stress. Taken together, these data suggest that a change in the lipid environment of GLUT1, possibly associated with its re-distribution between different microdomains of the plasma membrane, could play a role in its activation in response to stress.

scholarly journals TC10α Is Required for Insulin-Stimulated Glucose Uptake in Adipocytes

Endocrinology ◽  
2007 ◽  
Vol 148 (1) ◽  
pp. 27-33 ◽  
Author(s):  
Louise Chang ◽  
Shian-Huey Chiang ◽  
Alan R. Saltiel
Keyword(s):  
Plasma Membrane ◽  
Growth Factor ◽  
Glucose Uptake ◽  
Glucose Transporter ◽  
Platelet Derived Growth Factor ◽  
Phosphatidylinositol 3 Kinase ◽  
Independent Manner ◽  
Rho Family ◽  
Glucose Transporter Glut4 ◽  
Stimulation Of

Previous studies have suggested that activation of the Rho family member GTPase TC10 is necessary but not sufficient for the stimulation of glucose transport by insulin. We show here that endogenous TC10α is rapidly activated in response to insulin in 3T3L1 adipocytes in a phosphatidylinositol 3-kinase-independent manner, whereas platelet-derived growth factor was without effect. Knockdown of TC10α but not TC10β by RNA interference inhibited insulin-stimulated glucose uptake as well as the translocation of the insulin-sensitive glucose transporter GLUT4 from intracellular sites to the plasma membrane. In contrast, loss of TC10α had no effect on the stimulation of Akt by insulin. Additionally, knockdown of TC10α inhibited insulin-stimulated translocation of its effector CIP4. These data indicate that TC10α is specifically required for insulin-stimulated glucose uptake in adipocytes.

SNAP23 promotes insulin-dependent glucose uptake in 3T3-L1 adipocytes: possible interaction with cytoskeleton

1999 ◽  
Vol 276 (5) ◽  
pp. C1108-C1114 ◽  
Author(s):  
Leonard J. Foster ◽  
Karen Yaworsky ◽  
William S. Trimble ◽  
Amira Klip
Keyword(s):  
Plasma Membrane ◽  
Glucose Uptake ◽  
Glucose Transporter ◽  
Carboxy Terminus ◽  
Insulin Effect ◽  
Permeabilized Cells ◽  
Glut 4 ◽  
Syntaxin 4 ◽  
Insulin Dependent ◽  
Cytoskeletal Elements

The acute stimulation of glucose uptake by insulin in fat and muscle cells is primarily the result of translocation of facilitative glucose transporter 4 (GLUT-4) from an internal compartment to the plasma membrane. Here, we investigate the role of SNAP23 (a 23-kDa molecule resembling the 25-kDa synaptosome associated protein) in GLUT-4 translocation and glucose uptake in 3T3-L1 adipocytes. Microinjection of a polyclonal antibody directed to the carboxy terminus of SNAP23 inhibited GLUT-4 incorporation into the membrane in response to insulin, whereas microinjection of full-length recombinant SNAP23 enhanced the insulin effect. Introduction of recombinant SNAP23 into chemically permeabilized cells also enhanced insulin-stimulated glucose transport. These results indicate that SNAP23 is required for insulin-dependent, functional incorporation of GLUT-4 into the plasma membrane and that the carboxy terminus of the protein is essential for this process. SNAP23 is therefore likely to be a fusion catalyst along with syntaxin-4 and vesicle-associated membrane protein (VAMP)-2. Furthermore, the endogenous content of SNAP23 appears to be limiting for insulin-dependent GLUT-4 exposure at the cell surface. A measurable fraction of SNAP23 was sedimented with cytoskeletal elements when extracted with Triton X-100, unlike VAMP-2 and syntaxin-4, which were exclusively soluble in detergent. We hypothesize that SNAP23 and its interaction with the cytoskeleton may be targets for regulation of GLUT-4 traffic.

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

2006 ◽  
Vol 291 (4) ◽  
pp. E817-E828 ◽  
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.

Actin filaments regulate epithelial Na+ channel activity

1991 ◽  
Vol 261 (5) ◽  
pp. C882-C888 ◽  
Author(s):  
H. F. Cantiello ◽  
J. L. Stow ◽  
A. G. Prat ◽  
D. A. Ausiello
Keyword(s):  
Actin Filament ◽  
Actin Filaments ◽  
Functional Role ◽  
Cytochalasin D ◽  
Actin Binding ◽  
Na Channel ◽  
Na Channels ◽  
Channel Activity ◽  
Cortical Actin ◽  
Excised Patches

The functional role of the cytoskeleton in the control of ion channel activity is unknown. In the present study, immunocolocalization of Na+ channels with specific antibodies and fluorescein isothiocyanate-phalloidin to stain the cortical cytoskeleton indicates that actin is always present in close proximity to apical Na+ channels in A6 cells. The patch-clamp technique was used to assess the effect of cortical actin networks on apical Na+ channels in these A6 epithelial cells. The actin filament disrupter, cytochalasin D (5 micrograms/ml), induced Na+ channel activity in cell-attached patches within 5 min of addition. Cytochalasin D also induced and/or increased Na+ channel activity in 90% of excised patches tested within 2 min. Addition of short actin filaments (greater than 5 microM) to excised patches also induced channel activity. This effect was enhanced by addition of ATP and/or cytochalasin D. The effect of actin on Na+ channel activity was reversed by addition of the G actin-binding protein DNase I or completely prevented by treatment of the excised patches with this enzyme. Addition of the actin-binding protein, filamin, reversibly inhibited both spontaneous and actin-induced Na+ channels. Thus actin filament networks, achieved by either depolymerizing endogenous actin filaments by treatment with cytochalasin D, the addition of exogenous short actin filaments plus ATP, or actin plus cytochalasin D, regulate apical Na+ channel activity. This conclusion was supported by the observation that the addition of short actin filaments in the form of actin-gelsolin complexes in molar ratios less than 8:1 was also effective in activating Na+ channels. We have thus demonstrated a functional role for the cortical actin network in the regulation of epithelial Na+ channels that may complement a structural role for membrane protein targetting and assembly.

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