title: 7-hydroxystaurosporine (UCN-01) inhibition of Akt Thr308 but not Ser473 phosphorylation: a basis for decreased insulin-stimulated glucose transport.
abstract: 7-hydroxystaurosporine (UCN-01) infused for 72 hours by continuous i.v. infusion induced insulin resistance during phase I clinical trials. To understand the mechanism for this observation, we examined the effect of UCN-01 on insulin-stimulated glucose transport activity with 3-O-methylglucose in isolated rat adipose cells. UCN-01 inhibits glucose transport activity in a dose-dependent manner at all insulin concentrations. At the clinically relevant concentration of 0.25 mumol/L UCN-01, glucose transport is inhibited 66, 29, and 26% at insulin concentrations of 10, 50, and 100,000 (100K) microunits/mL respectively, thus shifting the dose-response curve to the right. Increasing concentrations of UCN-01 up to 2.5 mumol/L progressively shift the insulin dose-response curve even further. As Akt is known to mediate in part action initiated at the insulin receptor, we also studied the effect of UCN-01 on Akt activation in whole-cell homogenates of these cells. Decreased glucose transport activity directly parallels decreased Akt Thr308 phosphorylation in both an insulin and UCN-01 dose-dependent manner, whereas Akt Ser473 phosphorylation is inhibited only at the lowest insulin concentration, and then, only modestly. UCN-01 also inhibits insulin-induced Thr308 but not Ser473 phosphorylation of Akt associated with the plasma membranes and low-density microsomes and inhibits translocation of GLUT4 from low-density microsomes to plasma membranes as expected from the glucose transport activity measurements. These data suggest that UCN-01 induces clinical insulin resistance by blocking Akt activation and subsequent GLUT4 translocation in response to insulin, and this effect appears to occur by inhibiting Thr308 phosphorylation even in the face of almost completely unaffected Ser473 phosphorylation.
authors.affiliation: Developmental Therapeutics Program, National Cancer Institute, and Experimental Diabetes, National Institutes of Diabetes, Digestive and Kidney Diseases, NIH, Bethesda, Maryland, USA. kondapakas@ncifcrf.gov
authors.forename: Sudhir B
authors.initials: SB
authors.lastname: Kondapaka
authors.forename: Maryjane
authors.initials: M
authors.lastname: Zarnowski
authors.forename: Dena R
authors.initials: DR
authors.lastname: Yver
authors.forename: Edward A
authors.initials: EA
authors.lastname: Sausville
authors.forename: Samuel W
authors.initials: SW
authors.lastname: Cushman
mesh_terms: D000273:Adipose Tissue / Q000166:cytology / Q000378:metabolism; D000818:Animals; D000970:Antineoplastic Agents / Q000494:pharmacology*; D001692:Biological Transport; D015153:Blotting, Western; D002462:Cell Membrane / Q000378:metabolism; D003600:Cytosol / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004789:Enzyme Activation; D005947:Glucose / Q000378:metabolism*; D051275:Glucose Transporter Type 4; D015151:Immunoblotting; D007328:Insulin / Q000378:metabolism* / Q000494:pharmacology; D008297:Male; D008861:Microsomes / Q000378:metabolism; D009004:Monosaccharide Transport Proteins / Q000378:metabolism; D009124:Muscle Proteins / Q000378:metabolism; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D011518:Proto-Oncogene Proteins / Q000378:metabolism*; D051057:Proto-Oncogene Proteins c-akt; D051381:Rats; D012694:Serine / Q000737:chemistry*; D019311:Staurosporine / Q000031:analogs & derivatives* / Q000494:pharmacology*; D013347:Subcellular Fractions / Q000378:metabolism; D013912:Threonine / Q000737:chemistry*
chemical_list: D000970:Antineoplastic Agents; D051275:Glucose Transporter Type 4; D007328:Insulin; D009004:Monosaccharide Transport Proteins; D009124:Muscle Proteins; D011518:Proto-Oncogene Proteins; C495694:Slc2a4 protein, rat; D013912:Threonine; D012694:Serine; C054852:7-hydroxystaurosporine; C494920:Akt1 protein, rat; D017346:Protein Serine-Threonine Kinases; D051057:Proto-Oncogene Proteins c-akt; D019311:Staurosporine; D005947:Glucose