Association of impaired phosphatidylinositol 3-kinase activity in GLUT1-containing vesicles with malinsertion of glucose transporters into the plasmamembrane of fibroblasts from a patient with severe insulin resistance and clinical features of Werner syndrome

The purpose of this study was to examine the molecular mechanism responsibl e for the defective insulin-stimulated glucose transport in cultured fibrob lasts from a patient (VH) with clinical features of Werner syndrome and sev ere insulin resistance. Thus, in cells derived from VH, the subcellular dis tribution, structure, functional activity, as well as plasma membrane inser tion of GLUT1 glucose transporters were analyzed. Furthermore, the insulin signal transduction pathway leading to activation of phosphatidylinositol ( PI) 3-kinase as well as components of GLUT1-containing membrane vesicles we re characterized. In fibroblasts derived from VH, GLUT1 glucose transporters were overexpress ed by 8-fold in plasma membranes (PM) and by 5-fold in high density microso mes, respectively, Exofacial photolabeling revealed that only 14% of the ov erexpressed PM-GLUT1 transporters were properly inserted into the plasma me mbrane. The complementary DNA structure of the patient's insulin receptor a nd the GLUT1 glucose transporter, the intrinsic activity of plasma membrane glucose transporters, the tyrosine phosphorylation, as well as the protein expression of insulin receptor substrate-1/2 and p85 alpha/beta- and p110 alpha/beta-subunits of PI 3-kinase were normal. However, insulin-stimulated association of the p85 subunit of PI 3-kinase was defective in fibroblasts derived from VH compared to those from controls, and this defect was assoc iated with a reduced IRS-l-dependent activation of PI 3-kinase by 50.2% and 63.6% after incubation for 5 and 10 min with 100 nmol/L insulin, respectiv ely. Furthermore, immunodetection of small GTP-binding Rab proteins in subc ellular membrane fractions indicated a decreased expression of Rab4 in tota l cellular homogenates as well as in high density microsomes by 70% and 58% , respectively. After preparation of GLUT1-containing vesicles, Rab4 was no t detected to be a component of these vesicles. Analysis of the PI 3-kinase in GLUT1-containing membrane vesicles revealed insulin-dependent targeting of the p85 subunit to the vesicles immunoadsorbed from VH and control fibr oblasts. Importantly, the association of the p85 subunit as well as the p85 -immunoprecipitable PI 3-kinase activity were markedly reduced in GLUT1-ves icles derived from the patient. In conclusion, impaired PL 3-kinase activity in GLUT1-containing membrane v esicles derived from fibroblasts of VH is associated with a defective docki ng and/or fusion process of glucose transporters with the plasma membrane a nd thus might contribute to the molecular defect causing insulin resistance in this patient.