title: Comparative effects of GTPgammaS and insulin on the activation of Rho, phosphatidylinositol 3-kinase, and protein kinase N in rat adipocytes. Relationship to glucose transport.
abstract: Electroporation of rat adipocytes with guanosine 5'-3-O-(thio)triphosphate (GTPgammaS) elicited sizable insulin-like increases in glucose transport and GLUT4 translocation. Like insulin, GTPgammaS activated membrane phosphatidylinositol (PI) 3-kinase in rat adipocytes, but, unlike insulin, this activation was blocked by Clostridium botulinum C3 transferase, suggesting a requirement for the small G-protein, RhoA. Also suggesting that Rho may operate upstream of PI 3-kinase during GTPgammaS action, the stable overexpression of Rho in 3T3/L1 adipocytes provoked increases in membrane PI 3-kinase activity. As with insulin treatment, GTPgammaS stimulation of glucose transport in rat adipocytes was blocked by C3 transferase, wortmannin, LY294002, and RO 31-8220; accordingly, the activation of glucose transport by GTPgammaS, as well as insulin, appeared to require Rho, PI 3-kinase, and another downstream kinase, e.g. protein kinase C-zeta (PKC-zeta) and/or protein kinase N (PKN). Whereas insulin activated both PKN and PKC-zeta, GTPgammaS activated PKN but not PKC-zeta. In transfection studies in 3T3/L1 cells, stable expression of wild-type Rho and PKN activated glucose transport, and dominant-negative forms of Rho and PKN inhibited insulin-stimulated glucose transport. In transfection studies in rat adipocytes, transient expression of wild-type and constitutive Rho and wild-type PKN provoked increases in the translocation of hemagglutinin (HA)-tagged GLUT4 to the plasma membrane; in contrast, transient expression of dominant-negative forms of Rho and PKN inhibited the effects of both insulin and GTPgammaS on HA-GLUT4 translocation. Our findings suggest that (a) GTPgammaS and insulin activate Rho, PI 3-kinase, and PKN, albeit by different mechanisms; (b) each of these signaling substances appears to be required for, and may contribute to, increases in glucose transport; and (c) PKC-zeta may contribute to increases in glucose transport during insulin, but not GTPgammaS, action.
authors.affiliation: J. A. Haley Veterans Hospital Research Service, University of South Florida College of Medicine, Tampa, Florida 33612, USA.
authors.forename: M
authors.initials: M
authors.lastname: Standaert
authors.forename: G
authors.initials: G
authors.lastname: Bandyopadhyay
authors.forename: L
authors.initials: L
authors.lastname: Galloway
authors.forename: Y
authors.initials: Y
authors.lastname: Ono
authors.forename: H
authors.initials: H
authors.lastname: Mukai
authors.forename: R
authors.initials: R
authors.lastname: Farese
mesh_terms: D036002:ADP Ribose Transferases / Q000378:metabolism; D017667:Adipocytes / Q000187:drug effects / Q000201:enzymology*; D000730:Androstadienes / Q000494:pharmacology; D000818:Animals; D001692:Biological Transport; D001905:Botulinum Toxins; D002867:Chromones / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D019204:GTP-Binding Proteins / Q000378:metabolism*; D005947:Glucose / Q000378:metabolism*; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000494:pharmacology*; D007211:Indoles / Q000494:pharmacology; D007328:Insulin / Q000494:pharmacology*; D008297:Male; D009025:Morpholines / Q000494:pharmacology; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism*; D011493:Protein Kinase C / Q000378:metabolism*; D051381:Rats; D017207:Rats, Sprague-Dawley; D000077191:Wortmannin; D020741:rho GTP-Binding Proteins
chemical_list: D000730:Androstadienes; D002867:Chromones; D004791:Enzyme Inhibitors; D007211:Indoles; D007328:Insulin; D009025:Morpholines; C085911:2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D036002:ADP Ribose Transferases; C063007:exoenzyme C3, Clostridium botulinum; D019869:Phosphatidylinositol 3-Kinases; C061570:protein kinase N; D011493:Protein Kinase C; D001905:Botulinum Toxins; D019204:GTP-Binding Proteins; D020741:rho GTP-Binding Proteins; D005947:Glucose; C064758:Ro 31-8220; D000077191:Wortmannin