Standaert M, Bandyopadhyay G, Galloway L, Ono Y, Mukai H, Farese R
J. A. Haley Veterans Hospital Research Service, University of South Florida College of Medicine, Tampa, Florida 33612, USA.
J Biol Chem. 1998 Mar 27;273(13):7470-7. doi: 10.1074/jbc.273.13.7470.
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.
用5'-3-O-(硫代)三磷酸鸟苷（GTPγS）对大鼠脂肪细胞进行电穿孔可引发葡萄糖转运和GLUT4转位出现可观的胰岛素样增加。与胰岛素一样，GTPγS可激活大鼠脂肪细胞膜磷脂酰肌醇（PI）3-激酶，但与胰岛素不同的是，这种激活被肉毒杆菌C3转移酶阻断，这表明需要小G蛋白RhoA。同样表明Rho可能在GTPγS作用期间在PI 3-激酶上游发挥作用的是，Rho在3T3/L1脂肪细胞中的稳定过表达导致膜PI 3-激酶活性增加。与胰岛素处理一样，C3转移酶、渥曼青霉素、LY294002和RO 31-8220可阻断GTPγS对大鼠脂肪细胞葡萄糖转运的刺激；因此，GTPγS以及胰岛素对葡萄糖转运的激活似乎需要Rho、PI 3-激酶和另一种下游激酶，例如蛋白激酶C-ζ（PKC-ζ）和/或蛋白激酶N（PKN）。胰岛素可激活PKN和PKC-ζ，而GTPγS可激活PKN但不能激活PKC-ζ。在3T3/L1细胞的转染研究中，野生型Rho和PKN的稳定表达可激活葡萄糖转运，而Rho和PKN的显性负性形式可抑制胰岛素刺激的葡萄糖转运。在大鼠脂肪细胞的转染研究中，野生型和组成型Rho以及野生型PKN的瞬时表达可促使血凝素（HA）标记的GLUT4向质膜转位增加；相反，Rho和PKN的显性负性形式的瞬时表达可抑制胰岛素和GTPγS对HA-GLUT4转位的作用。我们的研究结果表明：（a）GTPγS和胰岛素激活Rho、PI 3-激酶和PKN，尽管机制不同；（b）这些信号物质中的每一种似乎都是葡萄糖转运增加所必需的，并且可能对此有贡献；（c）PKC-ζ可能在胰岛素作用期间对葡萄糖转运增加有贡献，但在GTPγS作用期间则不然。
