Cytoskeletal reorganization induced by insulin: involvement of Grb2/Ash, Ras and phosphatidylinositol 3-kinase signalling.
BACKGROUND
Cytoskeletal reorganization is important for a wide variety of insulin-mediated biological actions, including cell growth, migration and metabolism, but the intracellular signalling pathways leading to insulin-induced cytoskeletal reorganization have largely been unknown. We therefore investigated the involvement of Grb2/Ash-Ras and phosphatidylinositol (PI) 3-kinase in the insulin-induced morphological changes in fibroblasts over-expressing human insulin receptors (HIRcB cells).


RESULTS
Insulin, as well as 12-O-tetradecanoylphorbol-13-acetate (TPA) and 8-bromo-cAMP, induced a unique morphological change associated with actin cytoskeletal reorganization characterized by the disruption of actin stress fibres and thicker actin bundle formation. Microinjection of an anti-Grb2/Ash antibody, but not control IgG, inhibited the insulin-induced actin reorganization, whereas the TPA- and 8-bromo-cAMP-induced morphological changes were not inhibited by microinjection of the anti-Grb2/Ash antibody. In addition, microinjection of dominant negative ras p21 protein, but not the heat-treated protein, inhibited insulin-induced cytoskeletal reorganization. Microinjection of activated p21ras protein resulted in very similar cytoskeletal reorganization with actin bundle formation in the cytoplasm. The PI3-kinase inhibitor wortmannin inhibited insulin-induced cytoskeletal reorganization, but not the TPA- nor 8-bromo-cAMP-induced reorganization. Interestingly, wortmannin also inhibited the activated p21ras-induced morphological change.


CONCLUSIONS
We concluded that Grb2/Ash-Ras activation and probably Ras-associated PI3-kinase activation are involved in the insulin-induced morphological change.
Tobe
Kazuyuki
K
Department of Internal Medicine, Graduate School of Medicine, University of Tokyo, Hongo, Bunkyo-ku, Tokyo 113-8655, Japan.
Asai
Shohji
S
Matuoka
Koozi
K
Yamamoto
Tadashi
T
Chida
Kazuhiro
K
Kaburagi
Yasushi
Y
Akanuma
Yasuo
Y
Kuroki
Toshio
T
Takenawa
Tadaomi
T
Kimura
Satoshi
S
Nagai
Ryozo
R
Kadowaki
Takashi
T
D015124:8-Bromo Cyclic Adenosine Monophosphate / Q000494:pharmacology; D000199:Actins / Q000502:physiology; D048868:Adaptor Proteins, Signal Transducing; D000730:Androstadienes / Q000494:pharmacology; D000818:Animals; D002460:Cell Line; D003599:Cytoskeleton / Q000187:drug effects / Q000502:physiology*; D004789:Enzyme Activation; D051380:GRB2 Adaptor Protein; D006801:Humans; D007328:Insulin / Q000494:pharmacology*; D007329:Insulin Antagonists / Q000494:pharmacology; D015689:Oncogene Protein p21(ras); D010703:Phorbol Esters / Q000494:pharmacology; D019869:Phosphatidylinositol 3-Kinases / Q000502:physiology*; D011506:Proteins / Q000276:immunology / Q000502:physiology*; D051381:Rats; D015398:Signal Transduction / Q000502:physiology*; D013314:Stress, Mechanical; D000077191:Wortmannin; D018631:ras Proteins / Q000502:physiology*
D000199:Actins; D048868:Adaptor Proteins, Signal Transducing; D000730:Androstadienes; D051380:GRB2 Adaptor Protein; C495948:GRB2 protein, human; C495950:Grb2 protein, rat; D007328:Insulin; D007329:Insulin Antagonists; D010703:Phorbol Esters; D011506:Proteins; D015124:8-Bromo Cyclic Adenosine Monophosphate; D019869:Phosphatidylinositol 3-Kinases; D015689:Oncogene Protein p21(ras); D018631:ras Proteins; D000077191:Wortmannin