scholarly journals Syntaxin 4 in 3T3-L1 adipocytes: regulation by insulin and participation in insulin-dependent glucose transport.

1996 ◽  
Vol 7 (7) ◽  
pp. 1075-1082 ◽  
Author(s):  
A Volchuk ◽  
Q Wang ◽  
H S Ewart ◽  
Z Liu ◽  
L He ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Glucose Transport ◽  
Glucose Transporter ◽  
Subcellular Fractionation ◽  
Membrane Receptors ◽  
Low Density ◽  
Adipocyte Cell ◽  
Syntaxin 4 ◽  
Insulin Dependent ◽  
Plasma Membrane Receptors

Syntaxins are thought to be membrane receptors that bind proteins of the synaptobrevin/vesicle-associated membrane protein (VAMP) family found on transport vesicles. Recently, we detected synaptobrevin II and cellubrevin on immunopurified vesicles containing the glucose transporter 4 (GLUT4) in insulin-responsive cells. In an effort to identify the plasma membrane receptors for these vesicles, we now examine the expression of syntaxins in the 3T3-L1 adipocyte cell line. Neither syntaxin 1A nor 1B was found, in keeping with the neuronal restriction of these isoforms. In contrast, syntaxins 2 and 4 were readily detectable. By subcellular fractionation and estimation of protein yields, 67% of syntaxin 4 was localized to the plasma membrane, 24% to the low-density microsomes, and 9% to the high-density microsomes. Interestingly, acute insulin treatment decreased the content of syntaxin 4 in low-density microsomes and caused a corresponding gain in the plasma membrane fraction, reminiscent of the recruitment of GLUT4 glucose transporters. In contrast, there was no change in the distribution of syntaxin 2, which was mostly associated in the plasma membrane. A fraction of the intracellular syntaxin 4 was recovered with immunopurified GLUT4-containing vesicles. Moreover, anti-syntaxin 4 antibodies introduced in permeabilized 3T3-L1 adipocytes significantly reduced the insulin-dependent stimulation of glucose transport, in contrast to the introduction of irrelevant immunoglobulin G, which was without consequence. We propose that either the plasma membrane and/or the vesicular syntaxin 4 are involved in docking and/or fusion of GLUT4 vesicles at the cell surface of 3T3-L1 adipocytes.

Additive effect of contractions and insulin on GLUT-4 translocation into the sarcolemma

1994 ◽  
Vol 77 (4) ◽  
pp. 1597-1601 ◽  
Author(s):  
J. Gao ◽  
J. Ren ◽  
E. A. Gulve ◽  
J. O. Holloszy
Keyword(s):  
Plasma Membrane ◽  
Glucose Transport ◽  
Membrane Fraction ◽  
Glucose Transporter ◽  
Subcellular Fractionation ◽  
Muscle Group ◽  
Intracellular Membrane ◽  
Glut 4 ◽  
Incremental Effect ◽  
Glut 4 Translocation

The maximal effects of insulin and muscle contractions on glucose transport are additive. GLUT-4 is the major glucose transporter isoform expressed in skeletal muscle. Muscle contraction and insulin each induce translocation of GLUT-4 from intracellular sites into the plasma membrane. The purpose of this study was to test the hypothesis that the incremental effect of contractions and insulin on glucose transport is mediated by additivity of the maximal effects of these stimuli on GLUT-4 translocation into the sarcolemma. Anesthetized rats were given insulin by intravenous infusion to raise plasma insulin to 2,635 +/- 638 microU/ml. The gastrocnemius-plantaris-soleus group was stimulated to contract via the sciatic nerve by using a protocol that maximally activates glucose transport. After treatment with insulin, contractions, or insulin plus contractions or no treatment, the gastrocnemius-plantaris-soleus muscle group was dissected out and was subjected to subcellular fractionation to separate the plasma membrane and intracellular membrane fractions. Insulin induced a 70% increase and contractions induced a 113% increase in the GLUT-4 content of the plasma membrane fraction. The effects of insulin and contractions were additive, as evidenced by a 185% increase in the GLUT-4 content of the sarcolemmal fraction. This finding provides evidence that the incremental effect of maximally effective insulin and contractile stimuli on glucose transport is mediated by additivity of their effects on GLUT-4 translocation into the sarcolemma.

scholarly journals Septin 7 forms a complex with CD2AP and nephrin and regulates glucose transporter trafficking

2012 ◽  
Vol 23 (17) ◽  
pp. 3370-3379 ◽  
Author(s):  
Anita A. Wasik ◽  
Zydrune Polianskyte-Prause ◽  
Meng-Qiu Dong ◽  
Andrey S. Shaw ◽  
John R. Yates ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Glucose Uptake ◽  
Glucose Transporter ◽  
Subcellular Fractionation ◽  
Actin Organization ◽  
Storage Vesicles ◽  
Uptake Assay ◽  
Syntaxin 4 ◽  
Glomerular Ultrafiltration ◽  
Interfering Rna

Podocytes are insulin-sensitive and take up glucose in response to insulin. This requires nephrin, which interacts with vesicle-associated membrane protein 2 (VAMP2) on GLUT4 storage vesicles (GSVs) and facilitates their fusion with the plasma membrane. In this paper, we show that the filament-forming GTPase septin 7 is expressed in podocytes and associates with CD2-associated protein (CD2AP) and nephrin, both essential for glomerular ultrafiltration. In addition, septin 7 coimmunoprecipitates with VAMP2. Subcellular fractionation of cultured podocytes revealed that septin 7 is found in both cytoplasmic and membrane fractions, and immunofluorescence microscopy showed that septin 7 is expressed in a filamentous pattern and is also found on vesicles and the plasma membrane. The filamentous localization of septin 7 depends on CD2AP and intact actin organization. A 2-deoxy-d-glucose uptake assay indicates that depletion of septin 7 by small interfering RNA or alteration of septin assembly by forchlorfenuron facilitates glucose uptake into cells and further, knockdown of septin 7 increased the interaction of VAMP2 with nephrin and syntaxin 4. The data indicate that septin 7 hinders GSV trafficking and further, the interaction of septin 7 with nephrin in glomeruli suggests that septin 7 may participate in the regulation of glucose transport in podocytes.

scholarly journals Knockout of Syntaxin-4 in 3T3-L1 adipocytes reveals new insight into GLUT4 trafficking and adiponectin secretion

2021 ◽  
Author(s):  
Hannah L. Black ◽  
Rachel Livingstone ◽  
Cynthia C. Mastick ◽  
Mohammed Al Tobi ◽  
Holly Taylor ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Cell Surface ◽  
Glucose Transport ◽  
Metabolic Regulation ◽  
Glucose Transporter ◽  
Ectopic Expression ◽  
Glut4 Translocation ◽  
Syntaxin 4 ◽  
Rate Limiting ◽  
Insight Into

Adipocytes are key to metabolic regulation, exhibiting insulin-stimulated glucose transport which is underpinned by the insulin-stimulated delivery of glucose transporter-4 (GLUT4)- containing vesicles to the plasma membrane where they dock and fuse increasing cell surface GLUT4 levels. Adipocytokines such as adiponectin are secreted via a similar mechanism. We used genome editing to knockout Syntaxin-4 a protein reported to mediate GLUT4-vesicle fusion with the plasma membrane in 3T3-L1 adipocytes. Syntaxin-4 knockout reduced insulin-stimulated glucose transport and adiponectin secretion by ∼50% and reduced GLUT4 levels. Ectopic expression of HA-GLUT4-GFP showed that Syntaxin-4 knockout cells retain significant GLUT4 translocation capacity demonstrating that Syntaxin-4 is dispensable for insulin-stimulated GLUT4 translocation. Analysis of recycling kinetics revealed only a modest reduction in the exocytic rate of GLUT4 in knockout cells, and little effect on endocytosis. These analyses demonstrate that Syntaxin-4 is not always rate limiting for GLUT4 delivery to the cell surface. In sum, we show that Syntaxin-4 knockout results in reduced insulin-stimulated glucose transport, depletion of cellular GLUT4 levels and inhibition of adiponectin secretion but has only modest effects on the translocation capacity of the cells.

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.

scholarly journals Increased intracellular sequestration of the insulin-regulated aminopeptidase upon differentiation of 3T3-L1 cells

1998 ◽  
Vol 330 (2) ◽  
pp. 1003-1008 ◽  
Author(s):  
A. Stuart ROSS ◽  
R. Susanna KELLER ◽  
E. Gustav LIENHARD
Keyword(s):  
Plasma Membrane ◽  
Cell Surface ◽  
Glucose Transporter ◽  
Fold Increase ◽  
Membrane Receptors ◽  
Cell Surface Biotinylation ◽  
A Cell ◽  
Plasma Membrane Receptors ◽  
Glucose Transporter Glut4 ◽  
Intracellular Sequestration

In fat and muscle cells, the glucose transporter GLUT4 is sequestered in an intracellular compartment under basal conditions and redistributes markedly to the plasma membrane in response to insulin. Recently, we characterized a membrane aminopeptidase, designated IRAP (insulin-regulated aminopeptidase), that colocalizes with intracellular GLUT4 and similarly redistributes markedly to the plasma membrane in response to insulin in adipocytes. In contrast to GLUT4, IRAP is also expressed in 3T3-L1 fibroblasts, and this finding provided an opportunity to compare its subcellular distribution in fibroblasts and adipocytes. The relative amount of IRAP at the cell surface was measured by a cell surface biotinylation method. The portion of total IRAP at the cell surface in unstimulated adipocytes was 30% of that in unstimulated fibroblasts. Upon insulin treatment the portion of IRAP at the cell surface was the same in fibroblasts and adipocytes, and was increased 1.8-fold in fibroblasts and 8-fold in adipocytes. A similar analysis of the distribution of the transferrin receptor (TfR), the paradigm for recycling plasma membrane receptors, revealed that the portions of the TfR at the cell surface in both the basal and insulin-treated states were almost unchanged upon differentiation, and that insulin caused an increase of about 1.6-fold in the amount of TfR at the cell surface. These results show that enhanced intracellular sequestration of IRAP occurs during adipogenesis, and that this effect underlies the larger insulin-elicited fold increase of IRAP at the cell surface in adipocytes.

Exercise training increases GLUT-4 protein in rat adipose cells

1993 ◽  
Vol 264 (6) ◽  
pp. E882-E889 ◽  
Author(s):  
M. F. Hirshman ◽  
L. J. Goodyear ◽  
E. D. Horton ◽  
L. J. Wardzala ◽  
E. S. Horton
Keyword(s):  
Plasma Membrane ◽  
Exercise Training ◽  
Glucose Transport ◽  
Glucose Transporter ◽  
Low Density ◽  
Basal Cells ◽  
Glut 1 ◽  
Glut 4 ◽  
Cell Plasma ◽  
Adipose Cells

The relative abundance and subcellular distribution of the GLUT-1 and GLUT-4 glucose transporter isoforms were determined in basal and insulin-stimulated adipose cells from wheel cage exercise-trained rats and compared with both age-matched sedentary controls and young cell size-matched sedentary controls. Exercise training increased total estimated GLUT-4 by 67 and 54% compared with age-matched and young controls, respectively. Total estimated GLUT-1 per cell was not significantly different among the three groups. Expressed per cell, plasma membrane GLUT-4 protein in basal adipose cells from exercise-trained and age-matched control rats was 2.5-fold greater than in young controls (P < 0.05) and was associated with higher basal rates of glucose transport in these cells (P < 0.02). In insulin-stimulated cells, plasma membrane GLUT-4 was 67% greater in the exercise-trained animals than young controls (P < 0.01), and 31% greater than in age-matched controls. Rates of glucose transport were correspondingly higher. In basal cells, low-density microsomal GLUT-4 from exercise-trained rats was approximately twofold greater than from age-matched controls and young controls. With insulin stimulation, GLUT-4 in low-density microsomes decreased to similar levels in all groups. We conclude that the total amount of GLUT-4 protein, but not GLUT-1, is increased in adipose cells by exercise training and that this increase in GLUT-4 is due primarily to an increase in intracellular GLUT-4.(ABSTRACT TRUNCATED AT 250 WORDS)

scholarly journals Plasma membrane glycosphingolipid signaling: a turning point

2021 ◽  
Author(s):  
Elena Chiricozzi
Keyword(s):  
Plasma Membrane ◽  
Chemical Synthesis ◽  
Bioinformatic Analysis ◽  
Membrane Receptors ◽  
Outer Side ◽  
Neuronal Homeostasis ◽  
Extracellular Stimuli ◽  
Conceptual Shift ◽  
Plasma Membrane Receptors ◽  
Key Events

AbstractPlasma membrane interaction is highly recognized as an essential step to start the intracellular events in response to extracellular stimuli. The ways in which these interactions take place are less clear and detailed. Over the last decade my research has focused on developing the understanding of the glycosphingolipids-protein interaction that occurs at cell surface. By using chemical synthesis and biochemical approaches we have characterized some fundamental interactions that are key events both in the immune response and in the maintenance of neuronal homeostasis. In particular, for the first time it has been demonstrated that a glycolipid, present on the outer side of the membrane, the long-chain lactosylceramide, is able to directly modulate a cytosolic protein. But the real conceptual change was the demonstration that the GM1 oligosaccharide chain is able, alone, to replicate numerous functions of GM1 ganglioside and to directly interact with plasma membrane receptors by activating specific cellular signaling. In this conceptual shift, the development and application of multidisciplinary techniques in the field of biochemistry, from chemical synthesis to bioinformatic analysis, as well as discussions with several national and international colleagues have played a key role.

C2C12 myocytes lack an insulin-responsive vesicular compartment despite dexamethasone-induced GLUT4 expression

2002 ◽  
Vol 283 (3) ◽  
pp. E514-E524 ◽  
Author(s):  
Lori L. Tortorella ◽  
Paul F. Pilch
Keyword(s):  
Glucose Transporter ◽  
Fold Increase ◽  
Snare Proteins ◽  
Vesicular Traffic ◽  
Glut4 Expression ◽  
Protein Receptor ◽  
Syntaxin 4 ◽  
Insulin Dependent ◽  
Glucose Transporter Glut4 ◽  
Vesicular Compartment

Insulin regulates the uptake of glucose into skeletal muscle and adipocytes by redistributing the tissue-specific glucose transporter GLUT4 from intracellular vesicles to the cell surface. To date, GLUT4 is the only protein involved in insulin-regulated vesicular traffic that has this tissue distribution, thus raising the possibility that its expression alone may allow formation of an insulin-responsive vesicular compartment. We show here that treatment of differentiating C2C12myoblasts with dexamethasone, acting via the glucocorticoid receptor, causes a ≥10-fold increase in GLUT4 expression but results in no significant change in insulin-stimulated glucose transport. Signaling from the insulin receptor to its target, Akt2, and expression of the soluble N-ethylmaleimide-sensitive factor-attachment protein receptor, or SNARE, proteins syntaxin 4 and vesicle-associated membrane protein are normal in dexamethasone-treated C2C12 cells. However, these cells show no insulin-dependent trafficking of the insulin-responsive aminopeptidase or the transferrin receptor, respective markers for intracellular GLUT4-rich compartments and endosomes that are insulin responsive in mature muscle and adipose cells. Therefore, these data support the hypothesis that GLUT4 expression by itself is insufficient to establish an insulin-sensitive vesicular compartment.

scholarly journals Expression and compartmentalization of caveolin in adipose cells: coordinate regulation with and structural segregation from GLUT4.

1995 ◽  
Vol 129 (4) ◽  
pp. 999-1006 ◽  
Author(s):  
K V Kandror ◽  
J M Stephens ◽  
P F Pilch
Keyword(s):  
Cell Surface ◽  
Glucose Transporter ◽  
Buoyant Density ◽  
Fat Cells ◽  
Structural Protein ◽  
Coordinate Regulation ◽  
Rat Adipocytes ◽  
Transport Vesicles ◽  
Adipocyte Cell ◽  
Insulin Dependent

Native rat adipocytes and the mouse adipocyte cell line, 3T3-L1, possess transport vesicles of apparently uniform composition and size which translocate the tissue-specific glucose transporter isoform, GLUT4, from an intracellular pool to the cell surface in an insulin-sensitive fashion. Caveolin, the presumed structural protein of caveolae, has also been proposed to function in vesicular transport. Thus, we studied the expression and subcellular distribution of caveolin in adipocytes. We found that rat fat cells express the highest level of caveolin protein of any tissue studied, and caveolin is also expressed at high levels in cardiac muscle, another tissue possessing insulin responsive GLUT4 translocation. Both proteins are absent from 3T3-L1 fibroblasts and undergo a dramatic coordinate increase in expression upon differentiation of these cells into adipocytes. However, unlike GLUT4 in rat adipocytes not exposed to insulin, the majority of caveolin is present in the plasma membrane. In native rat adipocytes, intracellular GLUT4 and caveolin reside in vesicles practically indistinguishable by their size and buoyant density in sucrose gradients, and both proteins show insulin-dependent translocation to the cell surface. However, by immunoadsorption of GLUT4-containing vesicles with anti-GLUT4 antibody, we show that these vesicles have no detectable caveolin, and therefore, this protein is present in a distinct vesicle population. Thus, caveolin has no direct structural relation to the organization of the intracellular glucose transporting machinery in fat cells.

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