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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.

ABSTRACT: 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 p85alpha and p110beta (but not p110alpha) 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, p85alpha and p110beta 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.

SUBMITTER: Wang Q

PROVIDER: S-EPMC1219436 | biostudies-other | 1998 May

REPOSITORIES: biostudies-other

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Project description:The translocation of a unique facilitative glucose transporter isoform (GLUT4) from an intracellular site to the plasma membrane accounts for the large insulin-dependent increase in glucose transport observed in muscle and adipose tissue. The intracellular location of GLUT4 in the basal state and the pathway by which it reaches the cell surface upon insulin stimulation are unclear. Here, we have examined the colocalization of GLUT4 with the transferrin receptor, a protein which is known to recycle through the endosomal system. Using an anti-GLUT4 monoclonal antibody we immunoisolated a vesicular fraction from an intracellular membrane fraction of 3T3-L1 adipocytes that contained > 90% of the immunoreactive GLUT4 found in this fraction, but only 40% of the transferrin receptor (TfR). These results suggest only a limited degree of colocalization of these proteins. Using a technique to cross-link and render insoluble ("ablate') intracellular compartments containing the TfR by means of a transferrin-horseradish peroxidase conjugate (Tf-HRP), we further examined the relationship between the endosomal recycling pathway and the intracellular compartment containing GLUT4 in these cells. Incubation of non-stimulated cells with Tf-HRP for 3 h at 37 degrees C resulted in quantitative ablation of the intracellular TfR, GLUT1 and mannose-6-phosphate receptor and a shift in the density of Rab5-positive membranes. In contrast, only 40% of intracellular GLUT4 was ablated under the same conditions. Ablation was specific for the endosomal system as there was no significant ablation of either TGN38 or lgp120, which are markers for the trans Golgi reticulum and lysosomes respectively. Subcellular fractionation analysis revealed that most of the ablated pools of GLUT4 and TfR were found in the intracellular membrane fraction. The extent of ablation of GLUT4 from the intracellular fraction was unchanged in cells which were insulin-stimulated prior to ablation, whereas GLUT1 exhibited increased ablation in insulin-stimulated cells. Pretreatment of adipocytes with okadaic acid, an inhibitor of Type-I and -IIa phosphatases, increased GLUT4 ablation in the presence of insulin, consistent with okadaic acid increasing the internalization of GLUT4 from the plasma membrane under these conditions. Using a combination of subcellular fractionation, vesicle immunoadsorption and compartment ablation using the Tf-HRP conjugate we have been able to resolve overlapping but distinct intracellular distributions of the TfR and GLUT4 in adipocytes. At least three separate compartments were identified: TfR-positive/GLUT4-negative. TfR-negative/GLUT4-positive, and TfR-positive/GLUT4-positive, as defined by the relative abundance of these two markers. We propose that the TfR-negative/GLUT4-positive compartment, which contains approximately 60% of the intracellular GLUT4, represents a specialized intracellular compartment that is withdrawn from the endosomal system. The biosynthesis and characteristics of this compartment may be fundamental to the unique insulin regulation of GLUT4.

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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.

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