| Popis: |
Electroporation of rat adipocytes with guanosine 5′-3-O-(thio)triphosphate (GTPγS) elicited sizable insulin-like increases in glucose transport and GLUT4 translocation. Like insulin, GTPγS 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 GTPγS action, the stable overexpression of Rho in 3T3/L1 adipocytes provoked increases in membrane PI 3-kinase activity. As with insulin treatment, GTPγS 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 GTPγS, as well as insulin, appeared to require Rho, PI 3-kinase, and another downstream kinase, e.g. protein kinase C-ζ (PKC-ζ) and/or protein kinase N (PKN). Whereas insulin activated both PKN and PKC-ζ, GTPγS activated PKN but not PKC-ζ. 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 GTPγS on HA-GLUT4 translocation. Our findings suggest that (a) GTPγS 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-ζ may contribute to increases in glucose transport during insulin, but not GTPγS, action. |
