title: Effects of ML-9 on insulin stimulation of glucose transport in 3T3-L1 adipocytes.
abstract: Treatment of 3T3-L1 adipocytes with insulin resulted in activation of 2-deoxyglucose transport activity and translocation of glucose transporters (GLUT4 and GLUT1) from the cytoplasmic space to the plasma membrane. ML-9 (a myosin light chain kinase inhibitor) inhibited insulin stimulation of 2-deoxyglucose transport activity by 80% at 100 microM (IC50 = 27 microM) without affecting 2-deoxyglucose transport activity in the basal state. The inhibition was independent of extracellular Ca2+ concentration and almost fully reversible at 40 microM ML-9. ML-9 did not inhibit insulin-stimulated tyrosine phosphorylation of 95-kDa protein in the wheat germ agglutinin-purified preparation and of 95- and 160-kDa proteins in intact cells. However, ML-9 inhibited insulin-induced translocation of both GLUT4 and GLUT1 in a dose-dependent manner. The dose-response curves were similar to those observed for the inhibition of insulin stimulation of 2-deoxyglucose transport activity. Neither insulin nor ML-9 affected the phosphorylation state of both heavy and light chains of myosin. Therefore, it seems likely that ML-9 inhibits the insulin-induced translocation of glucose transporters at a step beyond the insulin receptor kinase activity by a mechanism different from that affecting phosphorylation of the myosin light chain. Phosphorylating activity of microtubule-associated protein 2 and myelin basic protein was stimulated by insulin, and this stimulation was not affected by ML-9. ML-9, however, inhibited the phosphorylating activity in vitro and insulin stimulation of the phosphorylating activity of ribosomal protein S6 in intact cells in a dose-dependent manner similar to that observed for the inhibition of insulin stimulation of glucose transport. These results suggest that mitogen-activated protein kinase may be one of the constituents in intracellular insulin signaling to the glucose transport system.
authors.affiliation: Second Division of Internal Medicine, Kyoto University School of Medicine, Japan.
authors.forename: G
authors.initials: G
authors.lastname: Inoue
authors.forename: H
authors.initials: H
authors.lastname: Kuzuya
authors.forename: T
authors.initials: T
authors.lastname: Hayashi
authors.forename: M
authors.initials: M
authors.lastname: Okamoto
authors.forename: Y
authors.initials: Y
authors.lastname: Yoshimasa
authors.forename: A
authors.initials: A
authors.lastname: Kosaki
authors.forename: S
authors.initials: S
authors.lastname: Kono
authors.forename: M
authors.initials: M
authors.lastname: Okamoto
authors.forename: I
authors.initials: I
authors.lastname: Maeda
authors.forename: M
authors.initials: M
authors.lastname: Kubota
mesh_terms: D016475:3T3 Cells; D000273:Adipose Tissue / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D000818:Animals; D001381:Azepines / Q000494:pharmacology*; D001692:Biological Transport / Q000187:drug effects; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D003847:Deoxyglucose / Q000378:metabolism; D004336:Drug Antagonism; D005947:Glucose / Q000378:metabolism*; D051272:Glucose Transporter Type 1; D051275:Glucose Transporter Type 4; D007328:Insulin / Q000494:pharmacology*; D051379:Mice; D008869:Microtubule-Associated Proteins / Q000378:metabolism; D009004:Monosaccharide Transport Proteins / Q000378:metabolism; D009124:Muscle Proteins; D004676:Myelin Basic Protein / Q000378:metabolism; D009219:Myosin-Light-Chain Kinase / Q000037:antagonists & inhibitors*; D010766:Phosphorylation; D047428:Protein Kinase Inhibitors; D011972:Receptor, Insulin / Q000378:metabolism; D038601:Ribosomal Protein S6; D012269:Ribosomal Proteins / Q000378:metabolism
chemical_list: D001381:Azepines; D051272:Glucose Transporter Type 1; D051275:Glucose Transporter Type 4; D007328:Insulin; D008869:Microtubule-Associated Proteins; D009004:Monosaccharide Transport Proteins; D009124:Muscle Proteins; D004676:Myelin Basic Protein; D047428:Protein Kinase Inhibitors; D038601:Ribosomal Protein S6; D012269:Ribosomal Proteins; C495678:Slc2a1 protein, mouse; C495693:Slc2a4 protein, mouse; C056218:ML 9; D003847:Deoxyglucose; D011972:Receptor, Insulin; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D009219:Myosin-Light-Chain Kinase; D005947:Glucose