title: Regulation of glucose transport by ROCK1 differs from that of ROCK2 and is controlled by actin polymerization.
abstract: A role of Rho-associated coiled-coil-containing protein kinase (ROCK)1 in regulating whole-body glucose homeostasis has been reported. However, cell-autonomous effects of ROCK1 on insulin-dependent glucose transport in adipocytes and muscle cells have not been elucidated. To determine the specific role of ROCK1 in glucose transport directly, ROCK1 expression in 3T3-L1 adipocytes and L6 myoblasts was biologically modulated. Here, we show that small interfering RNA-mediated ROCK1 depletion decreased insulin-induced glucose transport in adipocytes and myoblasts, whereas adenovirus-mediated ROCK1 expression increased this in a dose-dependent manner, indicating that ROCK1 is permissive for glucose transport. Inhibition of ROCK1 also impaired glucose transporter 4 translocation in 3T3-L1 adipocytes. Importantly, the ED₅₀ of insulin for adipocyte glucose transport was reduced when ROCK1 was expressed, leading to hypersensitivity to insulin. These effects are dependent on actin cytoskeleton remodeling, because inhibitors of actin polymerization significantly decreased ROCK1's effect to promote insulin-stimulated glucose transport. Unlike ROCK2, ROCK1 binding to insulin receptor substrate (IRS)-1 was not detected by immunoprecipitation, although cell fractionation demonstrated both ROCK isoforms localize with IRS-1 in low-density microsomes. Moreover, insulin's ability to increase IRS-1 tyrosine 612 and serine 632/635 phosphorylation was attenuated by ROCK1 suppression. Replacing IRS-1 serine 632/635 with alanine reduced insulin-stimulated phosphatidylinositol 3-kinase activation and glucose transport in 3T3-L1 adipocytes, indicating that phosphorylation of these serine residues of IRS-1, which are substrates of the ROCK2 isoform in vitro, are crucial for maximal stimulation of glucose transport by insulin. Our studies identify ROCK1 as an important positive regulator of insulin action on glucose transport in adipocytes and muscle cells.
authors.affiliation: Beth Israel Deaconess Medical Center, 330 Brookline Avenue, CLS-736, Boston, Massachusetts 02216, USA.
authors.forename: Kwang-Hoon
authors.initials: KH
authors.lastname: Chun
authors.forename: Kazushi
authors.initials: K
authors.lastname: Araki
authors.forename: Yuna
authors.initials: Y
authors.lastname: Jee
authors.forename: Dae-Ho
authors.initials: DH
authors.lastname: Lee
authors.forename: Byung-Chul
authors.initials: BC
authors.lastname: Oh
authors.forename: Hu
authors.initials: H
authors.lastname: Huang
authors.forename: Kyong Soo
authors.initials: KS
authors.lastname: Park
authors.forename: Sam W
authors.initials: SW
authors.lastname: Lee
authors.forename: Janice M
authors.initials: JM
authors.lastname: Zabolotny
authors.forename: Young-Bum
authors.initials: YB
authors.lastname: Kim
mesh_terms: D041721:3T3-L1 Cells; D000199:Actins / Q000378:metabolism*; D017667:Adipocytes / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D000818:Animals; D001692:Biological Transport / Q000502:physiology; D002478:Cells, Cultured; D005947:Glucose / Q000378:metabolism*; D051275:Glucose Transporter Type 4 / Q000378:metabolism; D066298:In Vitro Techniques; D007328:Insulin / Q000494:pharmacology; D055504:Insulin Receptor Substrate Proteins / Q000378:metabolism; D051379:Mice; D023421:Models, Animal; D032448:Myoblasts, Skeletal / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D058105:Polymerization; D054460:rho-Associated Kinases / Q000378:metabolism*
chemical_list: D000199:Actins; D051275:Glucose Transporter Type 4; D007328:Insulin; D055504:Insulin Receptor Substrate Proteins; C526295:Irs1 protein, mouse; D019869:Phosphatidylinositol 3-Kinases; C515854:Rock1 protein, mouse; C515855:Rock2 protein, mouse; D054460:rho-Associated Kinases; D005947:Glucose