Induction of caveolin during adipogenesis and association of GLUT4 with caveolin-rich vesicles.

Caveolae, also termed plasmalemmal vesicles, are small, flask-shaped, non-clathrin-coated invaginations of the plasma membrane. Caveolin is a principal component of the filaments that make up the striated coat of caveolae. Using caveolin as a marker protein for the organelle, we found that adipose tissue is the single most abundant source of caveolae identified thus far. Caveolin mRNA and protein are strongly induced during differentiation of 3T3-L1 fibroblasts to adipocytes; during adipogenesis there is also a dramatic increase in the complexity of the protein composition of caveolin-rich membrane domains. About 10-15% of the insulin-responsive glucose transporter GLUT4 is found in this caveolin-rich fraction, and immuno-isolated vesicles containing GLUT4 also contain caveolin. However, in non-stimulated adipocytes the majority of caveolin fractionates with the plasma membrane, while most GLUT4 associates with low-density microsomes. Upon addition of insulin to 3T3-L1 adipocytes, there is a significant increase in the amount of GLUT4 associated with caveolin-rich membrane domains, an increase in the amount of caveolin associated with the plasma membrane, and a decrease in the amount of caveolin associated with low-density microsomes. Caveolin does not undergo a change in phosphorylation upon stimulation of 3T3-L1 adipocytes with insulin. However, after treatment with insulin it is associated with a 32-kD phosphorylated protein. Caveolae thus may play an important role in the vesicular transport of GLUT4 to or from the plasma membrane. 3T3-L1 adipocytes offer an attractive system to study the function of caveolae in several cellular trafficking and signaling events.

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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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TC10α Is Required for Insulin-Stimulated Glucose Uptake in Adipocytes

Endocrinology ◽

10.1210/en.2006-1167 ◽

2007 ◽

Vol 148 (1) ◽

pp. 27-33 ◽

Cited By ~ 64

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.

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Potential mechanism of insulin resistance in ageing: impaired insulin-stimulated glucose transport due to a depletion of the intracellular pool of glucose transporters in Fischer rat adipocytes

Journal of Endocrinology ◽

10.1677/joe.0.1260099 ◽

1990 ◽

Vol 126 (1) ◽

pp. 99-107 ◽

Cited By ~ 11

Author(s):

S. Matthaei ◽

H. Benecke ◽

H. H. Klein ◽

A. Hamann ◽

G. Kreymann ◽

...

Keyword(s):

Plasma Membrane ◽

Glucose Transporter ◽

Cytochalasin B ◽

Glucose Transporters ◽

Insulin Binding ◽

Low Density ◽

Intracellular Pool ◽

Subcellular Membrane ◽

Impaired Insulin ◽

In Cells

ABSTRACT To examine the cellular mechanism responsible for impaired insulin action in ageing, we determined various in-vitro parameters involved in the pathogenesis of insulin resistance, i.e. basal and insulin-stimulated [14C]3-O-methylglucose transport (30MG), 125I-labelled insulin binding, activation of insulin receptor kinase (IRKA) in intact cells, and number and subcellular distribution of glucose transporters in subcellular membrane fractions of adipocytes from 6- (FR-6) and 24- (FR-24) month-old Fischer rats. Ageing had no effect on basal 30MG (12±4 vs 13±3 fmol/5 × 104 cells, means ± s.e.m.); in contrast, in FR-24 rats insulin-stimulated 30MG was markedly decreased by 43% when compared with that in FR-6 rats (158±14 vs 90±8 fmol/5 × 104 cells; P < 0·01). Insulin binding to adipocytes from FR-6 rats was 2·40±0·38% compared with 2·28±0·47% in FR-24 (P not significant). Moreover, ageing had no significant effect on IRKA, as determined by insulin-stimulated (0, 1, 4 and 500 ng insulin/ml) 32P-incorporation into histone 2B. In subcellular membrane fractions, low density microsomes and plasma membranes, glucose transporter numbers were determined using [3H]cytochalasin B binding and immunodetection using an antiserum against the C-terminal peptide of the hepatoma-G2-glucose transporter. Cytochalasin B binding revealed that in the basal state the intracellular pool of glucose transporters was depleted in FR-24 by about 39% compared with low density microsomes from FR-6: (48·6±7·2 vs 29·8±5·5 pmol/mg membrane protein; P < 0·01). In consequence, in FR-24 there were fewer glucose transporters available for insulin-induced translocation to the plasma membrane (insulin-treated plasma membrane: 23·9±4·2 (FR-6) vs 14·4±3·1 (FR-24) pmol/mg membrane protein; P < 0·01). These results were confirmed by immunoblotting. In conclusion, (1) maximal insulin-stimulated 30MG was decreased by 43% in cells from FR-24 rats compared with those from FR-6 rats, while basal 30MG was similar in both groups, (2) neither insulin binding nor IRKA were significantly altered in cells from FR-24 rats, and (3) impaired insulin-stimulated 30MG was associated with reduced numbers of glucose transporters in the plasma membrane as a consequence of a depletion of the intracellular pool of glucose transporters in cells from FR-24 rats. Journal of Endocrinology (1990) 126, 99–107

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Golgin-160 Is Required for the Golgi Membrane Sorting of the Insulin-responsive Glucose Transporter GLUT4 in Adipocytes

Molecular Biology of the Cell ◽

10.1091/mbc.e06-05-0386 ◽

2006 ◽

Vol 17 (12) ◽

pp. 5346-5355 ◽

Cited By ~ 44

Author(s):

Dumaine Williams ◽

Stuart W. Hicks ◽

Carolyn E. Machamer ◽

Jeffrey E. Pessin

Keyword(s):

Plasma Membrane ◽

Membrane Trafficking ◽

Glucose Transporter ◽

General Distribution ◽

Amino Terminal ◽

Vesicular Stomatitis ◽

Glucose Transporter Glut4 ◽

Golgi Network ◽

Interfering Rna ◽

Regulated Trafficking

The peripheral Golgi protein golgin-160 is induced during 3T3L1 adipogenesis and is primarily localized to the Golgi cisternae distinct from the trans-Golgi network (TGN) in a general distribution similar to p115. Small interfering RNA (siRNA)-mediated reduction in golgin-160 protein resulted in an increase accumulation of the insulin-responsive amino peptidase (IRAP) and the insulin-regulated glucose transporter (GLUT4) at the plasma membrane concomitant with enhanced glucose uptake in the basal state. The redistribution of GLUT4 was rescued by expression of a siRNA-resistant golgin-160 cDNA. The basal state accumulation of plasma membrane GLUT4 occurred due to an increased rate of exocytosis without any significant effect on the rate of endocytosis. This GLUT4 trafficking to the plasma membrane in the absence of golgin-160 was independent of TGN/Golgi sorting, because it was no longer inhibited by the expression of a dominant-interfering Golgi-localized, γ-ear–containing ARF-binding protein mutant and displayed reduced binding to the lectin wheat germ agglutinin. Moreover, expression of the amino terminal head domain (amino acids 1–393) had no significant effect on the distribution or insulin-regulated trafficking of GLUT4 or IRAP. In contrast, expression of carboxyl α helical region (393–1498) inhibited insulin-stimulated GLUT4 and IRAP translocation, but it had no effect on the sorting of constitutive membrane trafficking proteins, the transferrin receptor, or vesicular stomatitis virus G protein. Together, these data demonstrate that golgin-160 plays an important role in directing insulin-regulated trafficking proteins toward the insulin-responsive compartment in adipocytes.

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Specialized sorting of GLUT4 and its recruitment to the cell surface are independently regulated by distinct Rabs

Molecular Biology of the Cell ◽

10.1091/mbc.e13-02-0103 ◽

2013 ◽

Vol 24 (16) ◽

pp. 2544-2557 ◽

Cited By ~ 51

Author(s):

L. Amanda Sadacca ◽

Joanne Bruno ◽

Jennifer Wen ◽

Wenyong Xiong ◽

Timothy E. McGraw

Keyword(s):

Plasma Membrane ◽

Glucose Uptake ◽

Glucose Transporter ◽

Receptor Activation ◽

Glut4 Translocation ◽

Insulin Stimulation ◽

Transport Vesicles ◽

Glucose Transporter Glut4

Adipocyte glucose uptake in response to insulin is essential for physiological glucose homeostasis: stimulation of adipocytes with insulin results in insertion of the glucose transporter GLUT4 into the plasma membrane and subsequent glucose uptake. Here we establish that RAB10 and RAB14 are key regulators of GLUT4 trafficking that function at independent, sequential steps of GLUT4 translocation. RAB14 functions upstream of RAB10 in the sorting of GLUT4 to the specialized transport vesicles that ferry GLUT4 to the plasma membrane. RAB10 and its GTPase-activating protein (GAP) AS160 comprise the principal signaling module downstream of insulin receptor activation that regulates the accumulation of GLUT4 transport vesicles at the plasma membrane. Although both RAB10 and RAB14 are regulated by the GAP activity of AS160 in vitro, only RAB10 is under the control of AS160 in vivo. Insulin regulation of the pool of RAB10 required for GLUT4 translocation occurs through regulation of AS160, since activation of RAB10 by DENND4C, its GTP exchange factor, does not require insulin stimulation.

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CD44 Selectively Associates With Active Src Family Protein Tyrosine Kinases Lck and Fyn in Glycosphingolipid-Rich Plasma Membrane Domains of Human Peripheral Blood Lymphocytes

Blood ◽

10.1182/blood.v91.10.3901.3901_3901_3908 ◽

1998 ◽

Vol 91 (10) ◽

pp. 3901-3908 ◽

Cited By ~ 3

Author(s):

Subburaj Ilangumaran ◽

Anne Briol ◽

Daniel C. Hoessli

Keyword(s):

Plasma Membrane ◽

T Lymphocytes ◽

Peripheral Blood ◽

Tyrosine Kinases ◽

Human Peripheral Blood ◽

Membrane Microdomains ◽

Low Density ◽

Membrane Domains ◽

Protein Tyrosine ◽

Plasma Membrane Microdomains

CD44 is the major cell surface receptor for the extracellular matrix glycosaminoglycan hyaluronan and is implicated in a variety of biological events that include embryonic morphogenesis, lymphocyte recirculation, inflammation, and tumor metastasis. CD44 delivers activation signals to T lymphocytes, B lymphocytes, natural killer cells, polymorphonuclear leukocytes, and macrophages by stimulating protein tyrosine phosphorylation and calcium influx. The mechanism of signal transduction via CD44 remains undefined, although CD44 was shown to physically associate with intracellular protein tyrosine kinase Lck in T lymphocytes. In the present report, we show that a significant proportion of CD44 in human peripheral blood T lymphocytes and endothelial cells is associated with low-density plasma membrane fractions that represent specialized plasma membrane domains enriched in glycosphingolipids and glycosylphosphatidylinositol (GPI)-anchored proteins. CD44 and the GPI-anchored CD59 do not appear to directly interact in the low-density membrane fractions. In human peripheral blood T lymphocytes, 20% to 30% of the Src family protein tyrosine kinases, Lck and Fyn, are recovered from these fractions. CD44-associated protein kinase activity was selectively recovered from the low-density membrane fractions, corresponding to glycosphingolipid-rich plasma membrane microdomains. Reprecipitation of the in vitro phosphorylated proteins showed that CD44 associates not only with Lck but also with Fyn kinase in these membrane domains. Our results suggest that cellular stimulation via CD44 may proceed through the signaling machinery of glycosphingolipid-enriched plasma membrane microdomains and, hence, depend on the functional integrity of such domains.

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Stimulation of glucose transport in skeletal muscle by hypoxia

Journal of Applied Physiology ◽

10.1152/jappl.1991.70.4.1593 ◽

1991 ◽

Vol 70 (4) ◽

pp. 1593-1600 ◽

Cited By ~ 203

Author(s):

G. D. Cartee ◽

A. G. Douen ◽

T. Ramlal ◽

A. Klip ◽

J. O. Holloszy

Keyword(s):

Skeletal Muscle ◽

Plasma Membrane ◽

Muscle Contraction ◽

Glucose Transport ◽

Glucose Transporter ◽

Cytochalasin B ◽

Sugar Transport ◽

Transport Activity ◽

Hindlimb Muscles ◽

Stimulation Of

Hypoxia caused a progressive cytochalasin B-inhibitable increase in the rate of 3-O-methylglucose transport in rat epitrochlearis muscles to a level approximately six-fold above basal. Muscle ATP concentration was well maintained during hypoxia, and increased glucose transport activity was still present after 15 min of reoxygenation despite repletion of phosphocreatine. However, the increase in glucose transport activity completely reversed during a 180-min-long recovery in oxygenated medium. In perfused rat hindlimb muscles, hypoxia caused an increase in glucose transporters in the plasma membrane, suggesting that glucose transporter translocation plays a role in the stimulation of glucose transport by hypoxia. The maximal effects of hypoxia and insulin on glucose transport activity were additive, whereas the effects of exercise and hypoxia were not, providing evidence suggesting that hypoxia and exercise stimulate glucose transport by the same mechanism. Caffeine, at a concentration too low to cause muscle contraction or an increase in glucose transport by itself, markedly potentiated the effect of a submaximal hypoxic stimulus on sugar transport. Dantrolene significantly inhibited the hypoxia-induced increase in 3-O-methylglucose transport. These effects of caffeine and dantrolene suggest that Ca2+ plays a role in the stimulation of glucose transport by hypoxia.

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Phosphorylation of the adipose/muscle-type glucose transporter (GLUT4) and its relationship to glucose transport activity

Biochemical Journal ◽

10.1042/bj2850223 ◽

1992 ◽

Vol 285 (1) ◽

pp. 223-228 ◽

Cited By ~ 11

Author(s):

A Schürmann ◽

G Mieskes ◽

H G Joost

Keyword(s):

Protein Kinase ◽

Glucose Transport ◽

Plasma Membranes ◽

Glucose Transporter ◽

Low Density ◽

Transport Activity ◽

Muscle Type ◽

Kinase C ◽

Glucose Transporter Glut4

The effects of protein phosphorylation and dephosphorylation on glucose transport activity reconstituted from adipocyte membrane fractions and its relationship to the phosphorylation state of the adipose/muscle-type glucose transporter (GLUT4) were studied. In vitro phosphorylation of membranes in the presence of ATP and protein kinase A produced a stimulation of the reconstituted glucose transport activity in plasma membranes and low-density microsomes (51% and 65% stimulation respectively), provided that the cells had been treated with insulin prior to isolation of the membranes. Conversely, treatment of membrane fractions with alkaline phosphatase produced an inhibition of reconstituted transport activity. However, in vitro phosphorylation catalysed by protein kinase C failed to alter reconstituted glucose transport activity in membrane fractions from both basal and insulin-treated cells. In experiments run under identical conditions, the phosphorylation state of GLUT4 was investigated by immunoprecipitation of glucose transporters from membrane fractions incubated with [32P]ATP and protein kinases A and C. Protein kinase C stimulated a marked phosphate incorporation into GLUT4 in both plasma membranes and low-density microsomes. Protein kinase A, in contrast to its effect on reconstituted glucose transport activity, produced a much smaller phosphorylation of the GLUT4 in plasma membranes than in low-density microsomes. The present data suggest that glucose transport activity can be modified by protein phosphorylation via an insulin-dependent mechanism. However, the phosphorylation of the GLUT4 itself was not correlated with changes in its reconstituted transport activity.

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Phosphatidylinositol 3-kinase acts at an intracellular membrane site to enhance GLUT4 exocytosis in 3T3-L1 cells

Biochemical Journal ◽

10.1042/bj3130125 ◽

1996 ◽

Vol 313 (1) ◽

pp. 125-131 ◽

Cited By ~ 68

Author(s):

Jing YANG ◽

James F. CLARKE ◽

Catriona J. ESTER ◽

Paul W. YOUNG ◽

Masato KASUGA ◽

...

Keyword(s):

Plasma Membrane ◽

Insulin Receptor ◽

Time Course ◽

Glucose Transporter ◽

Kinase Activity ◽

Insulin Receptor Substrate ◽

Low Density ◽

Intracellular Membrane ◽

Phosphatidylinositol 3 Kinase ◽

Phosphatidylinositol 3

Glucose transporters (GLUTs) are continuously recycled in 3T3-L1 cells and so insulin, through its action on phosphatidylinositol 3-kinase (PI 3-kinase), could potentially alter the distribution of these transporters by enhancing retention in the plasma membrane or acting intracellularly to increase exocytosis, either by stimulating a budding or a docking and fusion process. To examine the site of involvement of PI 3-kinase in the glucose transporter recycling pathway, we have determined the kinetics of recycling under conditions in which the PI 3-kinase activity is inhibited by wortmannin. Wortmannin addition to fully insulin-stimulated cells induces a net reduction of glucose transport activity with a time course that is consistent with a major effect on the return of internalized transporters to the plasma membrane. The exocytosis of GLUT1 and GLUT4 is reduced to very low levels in wortmannin-treated cells (≈ 0.009 min-1), but the endocytosis of these isoforms is not markedly perturbed and the rate constants are approx. 10-fold higher than for exocytosis (0.099 and 0.165 min-1, respectively). The slow reduction in basal activity following treatment with wortmannin is consistent with a wortmannin effect on constitutive recycling as well as insulin-regulated exocytosis. PI 3-kinase activity that is precipitated by anti-phosphotyrosine, anti-[insulin receptor substrate 1 (IRS1)] and anti-α-p85 antibodies show the same level of insulin-stimulated activity, ≈ 0.5 pmol/20 min per dish of 3T3-L1 cells. Since the activities precipitated by all three antibodies are similar, it seems unlikely that a second insulin receptor substrate, IRS2, contributes significantly to the insulin signalling observed in 3T3-L1 cells. To examine whether insulin targets PI 3-kinase to intracellular membranes we have carried out subcellular fractionation studies. These suggest that nearly all the insulin-stimulated PI 3-kinase activity is located on intracellular, low-density, membranes. In addition, the association of PI 3-kinase with IRS1 appears to partially deplete the cytoplasm of α-p85-precipitatable activity, suggesting that IRS1 may redistribute PI 3-kinase from the cytoplasm to the low-density microsome membranes. Taken together, the trafficking kinetic and PI 3-kinase distribution studies suggest an intracellular membrane site of action of the enzyme in enhancing glucose transporter exocytosis.

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Control of glucose transport by GLUT1: Regulated secretion in an unexpected environment

Bioscience Reports ◽

10.1007/bf01204347 ◽

1995 ◽

Vol 15 (6) ◽

pp. 427-443 ◽

Cited By ~ 4

Author(s):

Christopher C. Widnell

Keyword(s):

Plasma Membrane ◽

Glucose Transport ◽

Glucose Transporter ◽

Standard Medium ◽

Cytoplasmic Vesicles ◽

Infected Cells ◽

Vesicular Stomatitis ◽

Cellular Stresses ◽

Specific Membrane ◽

Stimulation Of

Studies designed to elucidate the mechanism of regulation of the GLUT1 isoform of the glucose transporter in response to a variety of cellular stresses are reviewed. Using ts mutants of vesicular stomatitis virus, it was shown that the viral L gene was responsible for the stimulation of glucose transport in infected cells. Immunofluorescence of GLUT1 demonstrated that the increase in glucose transport was the consequence of a translocation of the transporter from a reservoir in cytoplasmic vesicles to the plasma membrane. When cells were cycled between deficient and standard medium, the change in glucose transport rates was paralleled by a cycling of the transporter between the plasma membrane and the cytoplasmic vesicles. The redistribution of GLUT1 was not a consequence of a general redistribution of recycling plasma membrane proteins. Instead, the findings focus attention on the regulated exocytosis of specific membrane constituents in cells that, until recently, were not thought to exhibit this capacity.

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