Insulin-mediated Targeting of Phosphatidylinositol 3-Kinase to GLUT4-containing Vesicles

Abstract

Phosphatidylinositol (PI) 3-kinase is hypothesized to be a signaling element in the acute redistribution of intracellular GLUT4 glucose transporters to the plasma membrane in response to insulin. However, some receptors activate PI 3-kinase without causing GLUT4 translocation, suggesting specific cellular localization may be critical to this PI 3-kinase function. Consistent with this idea, complexes containing PI 3-kinase bound to insulin receptor substrate 1 (IRS-1) in 3T3-L1 adipocytes are associated with intracellular membranes (Heller-Harrison, R., Morin, M. and Czech, M. (1995) J. Biol. Chem. 270, 24442-24450). We report here that in response to insulin, activated complexes of IRS-1⅐PI 3-kinase can be immunoprecipitated with anti-IRS-1 antibody from detergent extracts of immunoadsorbed GLUT4-containing vesicles prepared from 3T3-L1 adipocytes. The targeting of PI 3-kinase to rat adipocyte GLUT4-containing vesicles using vesicles prepared by sucrose velocity gradient ultracentrifugation was also demonstrated. Insulin treatment caused a 2.3-fold increase in immunoreactive p85 protein in these GLUT4-containing vesicles while anti-p85 immunoprecipitates of PI 3-kinase activity in GLUT4-containing vesicle extracts increased to a similar extent. HPLC analysis of the GLUT4 vesicle-associated PI 3-kinase activity showed insulin-mediated increases in PI 3-P, PI 3,4-P 2 , and PI 3,4,5-P 3 when PI, PI 4-P, and PI 4,5-P 2 were used as substrates. Our data demonstrate that insulin directs the association of PI 3-kinase with GLUT4-containing vesicles in 3T3-L1 and rat adipocytes, consistent with the hypothesis that PI 3-kinase is involved in the insulin-regulated movement of GLUT4 to the plasma membrane.

GLUT4 vesicle dynamics in living 3T3 L1 adipocytes visualized with green-fluorescent protein

“…Our data suggest that the primary site of insulin action must be to release GLUT4 vesicles from their tether, but it remains to be established whether insulin also acts at the translocation and fusion steps. This hypothesis is consistent with recent data suggesting that insulin-stimulated GLUT4 translocation may require targeting of PtdIns 3-kinase to the high-density microsomal fraction [23][24][25], and that additional PtdIns 3-kinase-independent events are also required [26]. Future work must be directed at resolving the identity of the intracellular site of attachment of GLUT4 vesicles, and determining whether PtdIns 3-kinase plays a role in the release of these vesicles, allowing them to translocate to the plasma membrane.…”

Cardiac insulin resistance is associated with an impaired recruitment of phosphatidylinositol 3-kinase to GLUT4 vesicles

“…1 The common mechanism of Pl 3-kinase action is at present unclear, but observations that PDGF and YMXM peptides stimulate the total cellular Pl 3-kinase but not the glucose transport activities in 3T3-L1 cells are best explained by a speci®c insulin-dependent targeting of Pl 3-kinase to an intracellular site which is associated with the lowdensity microsomal fraction of cells. This hypothesis is supported by the work of Heller-Harrison et al, 2 who reported that GLUT4 vesicles isolated from 3T3-L1 cells contain Pl 3-kinase that is precipitable by an IRS-1 antibody. The purpose of the present investigation was to correlate the impaired insulin action in obesity to the translocation of cardiac GLUT4 with special attention to the Pl 3-kinase.…”

Suppression of insulin‐stimulated phosphatidylinositol 3‐kinase activity by the β3‐adrenoceptor agonist CL316243 in rat adipocytes

“…Furthermore, insulin increased an immunoreactive tyrosine-phosphorylated p85 protein, and the increase in the phospho-p85 protein was suppressed by CL in the presence of ADA. Heller-Harrison et al [31] demonstrated that insulin directs the association of PI 3-kinase with GLUT 4-containing vesicles in rat adipocytes. Our results suggest that the inhibitory effect of CL on insulin-stimulated 2-DG uptake is mediated via cAMP-dependent mechanisms by which the appearance of PI 3-kinase on the cell surface, tyrosine-phosphorylation of p85, and subsequent GLUT 4 translocation to the plasma membrane fractions were inhibited.…”