Association of impaired phosphatidylinositol 3-kinase activity in GLUT1-containing vesicles with malinsertion of glucose transporters into the plasma membrane 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 responsible for the defective insulin-stimulated glucose transport in cultured fibroblasts from a patient (VH) with clinical features of Werner syndrome and severe insulin resistance. Thus, in cells derived from VH, the subcellular distribution, structure, functional activity, as well as plasma membrane insertion 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 were characterized. In fibroblasts derived from VH, GLUT1 glucose transporters were overexpressed by 8-fold in plasma membranes (PM) and by 5-fold in high density microsomes, respectively. Exofacial photolabeling revealed that only 14% of the overexpressed PM-GLUT1 transporters were properly inserted into the plasma membrane. The complementary DNA structure of the patient's insulin receptor and 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 associated with a reduced IRS-1-dependent activation of PI 3-kinase by 50.2% and 63.6% after incubation for 5 and 10 min with 100 nmol/L insulin, respectively. Furthermore, immunodetection of small GTP-binding Rab proteins in subcellular membrane fractions indicated a decreased expression of Rab4 in total cellular homogenates as well as in high density microsomes by 70% and 58%, respectively. After preparation of GLUT1-containing vesicles, Rab4 was not 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 fibroblasts. Importantly, the association of the p85 subunit as well as the p85-immunoprecipitable PI 3-kinase activity were markedly reduced in GLUT1-vesicles derived from the patient. In conclusion, impaired PI 3-kinase activity in GLUT1-containing membrane vesicles derived from fibroblasts of VH is associated with a defective docking and/or fusion process of glucose transporters with the plasma membrane and thus might contribute to the molecular defect causing insulin resistance in this patient.
Kausch
C
C
Department of Medicine, University of Tübingen, Germany.
Hamann
A
A
Uphues
I
I
Niendorf
A
A
Müller-Wieland
D
D
Joost
H G
HG
Algenstaedt
P
P
Dreyer
M
M
Rüdiger
H W
HW
Häring
H U
HU
Eckel
J
J
Matthaei
S
S
D000328:Adult; D000345:Affinity Labels; D001386:Azides; D001483:Base Sequence / Q000235:genetics; D002462:Cell Membrane / Q000378:metabolism; D018076:DNA, Complementary / Q000235:genetics; D004187:Disaccharides; D005347:Fibroblasts / Q000378:metabolism*; D051272:Glucose Transporter Type 1; D006027:Glycosides; D006801:Humans; D007333:Insulin Resistance; D007527:Isoenzymes / Q000378:metabolism; D009004:Monosaccharide Transport Proteins / Q000235:genetics / Q000378:metabolism*; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism*; D011437:Propylamines; D012333:RNA, Messenger / Q000378:metabolism; D011972:Receptor, Insulin / Q000235:genetics; D015398:Signal Transduction / Q000502:physiology; D013347:Subcellular Fractions / Q000378:metabolism; D014898:Werner Syndrome / Q000201:enzymology / Q000378:metabolism / Q000503:physiopathology*; D020691:rab GTP-Binding Proteins / Q000378:metabolism
D000345:Affinity Labels; D001386:Azides; D018076:DNA, Complementary; D004187:Disaccharides; D051272:Glucose Transporter Type 1; D006027:Glycosides; D007527:Isoenzymes; D009004:Monosaccharide Transport Proteins; D011437:Propylamines; D012333:RNA, Messenger; C495677:SLC2A1 protein, human; C065412:2-N-(4-(1-azitrifluoroethyl)benzoyl)-1,3-bis-(mannos-4-yloxy)-2-propylamine; D019869:Phosphatidylinositol 3-Kinases; D011972:Receptor, Insulin; D020691:rab GTP-Binding Proteins