Osmotic Shock Stimulates GLUT4 Translocation in 3T3L1 Adipocytes by a Novel Tyrosine Kinase Pathway

Chen, Dong; Elmendorf, Jeffrey S.; Olson, Ann Louise; Xiong, Lei; Earp, H. Shelton; Pessin, Jeffrey E.

doi:10.1074/jbc.272.43.27401

Cited by 141 publications

(160 citation statements)

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“…Thus, its translocation to the plasma membrane (PM) in response to insulin stimulation was assessed. Plasma membrane lawns were prepared as described [17], followed by western blot analysis. As depicted in Figure 2B (left side), insulin induced a 4.89 ± 0.36-fold increase in PM lawn GLUT4 content in control cells.…”

Section: Resultsmentioning

confidence: 99%

“…The homogenate was centrifuged at 1000 g for 3 min, after which supernatant was centrifuged at 245 000 g for 90 min to sediment total membranes. Plasma membranes lawns were prepared as described previously [17]. Cells were washed with PBS and incubated for 1 min with 0.5 mg/ml poly-d-lysine followed by 3 washes with hypotonic buffer (23 mmol/l KCl, 10 mmol/l HEPES, pH 7.5, 1.7 mmol/l MgCl 2 , 1 mmol/l EGTA).…”

Section: Methodsmentioning

confidence: 99%

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Lipoic acid protects against oxidative stress induced impairment in insulin stimulation of protein kinase B and glucose transport in 3T3-L1 adipocytes

et al. 1999

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Oxidative stress has been recently implicated in the pathogenesis of various diseases. Consequently, the potential therapeutic or preventive effects of antioxidative agents has been raised [1]. Both poorly controlled Type I (insulin-dependent) diabetes mellitus as well as Type II (non-insulin-dependent) diabetes mellitus are characterized by reduced capacity of peripheral tissues (i. e. skeletal muscle and adipose tissue) to respond to the metabolic effects of insulin. The causes and cellular mechanisms responsible for this abnormality are still not fully understood despite intense investigative effort. Several lines of evidence suggest that increased oxidative stress occurs in diabetes and could have a role in the development or deterioration of peripheral insulin resistance. These Diabetologia (1999) Abstract Aims/hypothesis. Oxidative stress has been shown to impair insulin-stimulated glucose transporter 4 translocation in 3T3-L1 adipocytes. This study explores the potential of the antioxidant lipoic acid to protect the cells against the induction of insulin resistance when given before exposure to oxidative stress. Methods. 3T3-LI were exposed for 16 h to lipoic acid after which cells were exposed for 2 h to continuous production of H 2 O 2 by adding glucose oxidase to the culture medium. Results. These conditions resulted in a 50±70 % reduction in insulin-stimulated glucose transport activity associated with a decrease in reduced glutathione content from 37.4 ± 3.1 to 26.4 ± 4.9 nmol/mg protein, (p < 0.005). Lipoic acid pretreatment increased insulin-stimulated glucose transport following oxidative stress, reaching 84.8 ± 4.4 % of the control, associated with an increase in reduced glutathione content. Oxidation impaired the 4.89 ± 0.36-fold insulin-stimulated increase in glucose transporter 4 content in plasma membrane lawns of control cells. Lipoic acid pretreatment was, however, associated with preserved insulin-induced glucose transporter 4 translocation in cells exposed to oxidation, yielding 80 % of its content in controls. Although tyrosine phosphorylation patterns were not affected by lipoic acid pretreatment, insulin-stimulated protein kinase B/Akt serine 473 phosphorylation and activity were considerably impaired by oxidation but protected by lipoic acid pretreatment. A protective effect was not observed with either troglitazone, its isolated vitamin E moiety, or with vitamin C. Conclusion/interpretation. This study shows the ability of lipoic acid to provide partial protection against the impaired insulin-stimulated glucose transporter 4 translocation and protein kinase B/Akt activation induced by oxidative stress, potentially by its capacity to maintain intracellular redox state. [Diabetologia (1999) 42: 949±957]

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Lipoic acid protects against oxidative stress induced impairment in insulin stimulation of protein kinase B and glucose transport in 3T3-L1 adipocytes

et al. 1999

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“…Materials-The GLUT4 and syntaxin 4 antibodies were isolated as described previously (10,29). The Munc18c polyclonal antibody was obtained by immunization of a carboxyl-terminal synthetic peptide as described previously (13).…”

Section: Methodsmentioning

confidence: 99%

Regulation of Insulin-stimulated GLUT4 Translocation by Munc18c in 3T3L1 Adipocytes

Thurmond¹,

Ceresa²,

Okada

et al. 1998

Journal of Biological Chemistry

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216

237

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Insulin stimulates glucose transporter (GLUT) 4 vesicle translocation from intracellular storage sites to the plasma membrane in 3T3L1 adipocytes through a VAMP2-and syntaxin 4-dependent mechanism. We have observed that Munc18c, a mammalian homolog of the yeast syntaxin-binding protein n-Sec1p, competed for the binding of VAMP2 to syntaxin 4. Consistent with an inhibitory function for Munc18c, expression of Munc18c, but not the related Munc18b isoform, prevented the insulin stimulation of GLUT4 and IRAP/vp165 translocation to the plasma membrane without any significant effect on GLUT1 trafficking. As expected, overexpressed Munc18c was found to co-immunoprecipitate with syntaxin 4 in the basal state. However, these complexes were found to dissociate upon insulin stimulation. Furthermore, endogenous Munc18c was predominantly localized to the plasma membrane and its distribution was not altered by insulin stimulation. Although expression of enhanced green fluorescent protein-Munc18c primarily resulted in a dispersed cytosolic distribution, co-expression with syntaxin 4 resulted in increased localization to the plasma membrane. Together, these data suggest that Munc18c inhibits the docking/fusion of GLUT4-containing vesicles by blocking the binding of VAMP2 to syntaxin 4. Insulin relieves this inhibition by inducing the dissociation of Munc18c from syntaxin 4 and by sequestering Munc18c to an alternative plasma membrane binding site.The binding of insulin to its heterotetrameric integral-membrane receptor activates its intracellular tyrosine kinase domain and thereby triggers a signaling cascade resulting in the translocation and fusion of intracellular GLUT4 1 isoform-containing vesicles to the plasma membrane (1-3). Although most cell types also constitutively express the GLUT1 isoform at the cell surface, the insulin-stimulated increase in plasma membrane-associated GLUT4 protein accounts for the majority of post-prandial glucose disposal in both muscle and adipose tissue (4).The insulin-stimulated translocation of these GLUT4-containing vesicles has several features in common with the regulated exocytosis pathway of synaptic vesicle trafficking in neurotransmitter release (5). The machinery involved in the regulation of synaptic vesicle priming/docking/fusion entails the pairing of protein complexes in the vesicle compartment (v-SNAREs, for vesicle SNAP receptors) with their cognate receptor complexes at the target membrane (t-SNAREs, for target membrane SNAP receptors). Recently, several of the vand t-SNARE proteins have been identified that specifically participate in the insulin-regulated docking and fusion of GLUT4 vesicles with the adipocyte plasma membrane. GLUT4 vesicles co-purify with both the VAMP2 and VAMP3 v-SNARE isoforms and specific proteolytic cleavage of VAMP2, expression of a dominant-interfering VAMP2 mutant or inhibitory peptides impairs insulin-stimulated GLUT4 translocation (6 -11). In addition, transferrin-horseradish peroxidase ablation of recycling endosomes resulted in a selective loss ...

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“…Plasma Membrane Sheet Preparation-Plasma membrane sheets were prepared from 3T3L1 adipocytes as described (24). For 2-deoxyglucose (2DG) treatment of intact 3T3L1 adipocytes, cells were serum starved and placed into glucose-free Dulbecco's modified Eagle's medium containing 5 mM pyruvate and 5 mM glutamine with or without 5 mM 2DG for 5 min before insulin stimulation.…”

Section: Methodsmentioning

confidence: 99%

Aldolase Mediates the Association of F-actin with the Insulin-responsive Glucose Transporter GLUT4

Kao¹,

Noda²,

Johnson³

et al. 1999

Journal of Biological Chemistry

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112

102

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To identify potential proteins interacting with the insulin-responsive glucose transporter (GLUT4), we generated fusion proteins of glutathione S-transferase (GST) and the final 30 amino acids from GLUT4 (GST-G4) or GLUT1 (GST-G1). Incubation of these carboxylterminal fusion proteins with adipocyte cell extracts revealed a specific interaction of GLUT4 with fructose 1,6-bisphosphate aldolase. In the presence of aldolase, GST-G4 but not GST-G1 was able to co-pellet with filamentous (F)-actin. This interaction was prevented by incubation with the aldolase substrates, fructose 1,6-bisphosphate or glyceraldehyde 3-phosphate. Immunofluorescence confocal microscopy demonstrated a significant co-localization of aldolase and GLUT4 in intact 3T3L1 adipocytes, which decreased following insulin stimulation. Introduction into permeabilized 3T3L1 adipocytes of fructose 1,6-bisphosphate or the metabolic inhibitor 2-deoxyglucose, two agents that disrupt the interaction between aldolase and actin, inhibited insulin-stimulated GLUT4 exocytosis without affecting GLUT4 endocytosis. Furthermore, microinjection of an aldolase-specific antibody also inhibited insulin-stimulated GLUT4 translocation. These data suggest that aldolase functions as a scaffolding protein for GLUT4 and that glucose metabolism may provide a negative feedback signal for the regulation of glucose transport by insulin.The insulin-responsive glucose transporter GLUT4 is expressed primarily in adipose tissue, skeletal, and cardiac muscle (1-4). Under basal conditions, GLUT4 slowly recycles between poorly defined intracellular compartments and the plasma membrane with the vast majority sequestered in these intracellular storage sites. Insulin stimulates a large increase in the rate of GLUT4 exocytosis concomitant with a smaller decrease in the rate of GLUT4 endocytosis (5-7). The overall insulin-induced changes in GLUT4 trafficking kinetics result in a 10 -20-fold increase in the number of cell surface GLUT4 proteins that accounts for the majority of insulin-stimulated increases in glucose transport activity (8,9).Recently, several laboratories have begun to examine the subcellular distribution of GLUT4 to identify the mechanism responsible for the intracellular sequestration of the GLUT4 protein. Steady-state and kinetic analysis of various expressed GLUT4 chimeric proteins have indicated that both the aminoand carboxyl-terminal domains are important in GLUT4 internalization from the plasma membrane (10 -15). In particular, the carboxyl-terminal dileucine motif (SLL) was found to substantially alter GLUT4 trafficking kinetics and steady-state localization (16,17). Although a specific GLUT4 sequence responsible for intracellular localization has yet to be identified, the presence of a carboxyl-terminal retention signal is consistent with the observation that expression of the GLUT4 carboxyl-terminal domain results in the translocation of the endogenous GLUT4 protein to the plasma membrane (18). In addition, the presence of a GLUT4 carboxyl-terminal binding protein...

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Osmotic Shock Stimulates GLUT4 Translocation in 3T3L1 Adipocytes by a Novel Tyrosine Kinase Pathway

Cited by 141 publications

References 90 publications

Lipoic acid protects against oxidative stress induced impairment in insulin stimulation of protein kinase B and glucose transport in 3T3-L1 adipocytes

Lipoic acid protects against oxidative stress induced impairment in insulin stimulation of protein kinase B and glucose transport in 3T3-L1 adipocytes

Regulation of Insulin-stimulated GLUT4 Translocation by Munc18c in 3T3L1 Adipocytes

Aldolase Mediates the Association of F-actin with the Insulin-responsive Glucose Transporter GLUT4

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