Phosphoinositide 3-Kinase Is Required for Insulin-Induced but Not for Growth Hormone- or Hyperosmolarity-Induced Glucose Uptake in 3T3-L1 Adipocytes

Abstract

The(1) regulatory mechanism of glucose uptake in 3T3-L1 adipocytes was investigated with the use of recombinant adenovirus vectors encoding various dominant negative proteins. Infection with a virus encoding a mutant regulatory subunit of phosphoinositide (PI) 3-kinase that does not bind the 110-kDa catalytic subunit (delta p85) inhibited the insulin-induced increase in PI 3-kinase activity co-precipitated by antibodies to phosphotyrosine and glucose uptake in a virus dose-dependent manner. Overexpression of a dominant negative RAS mutant in which Asp57 is replaced with tyrosine (RAS57Y) or of a dominant negative SOS mutant that lacks guanine nucleotide exchange activity (delta SOS) abolished the insulin-induced increase in mitogen-activated protein kinase activity, but had no effect on PI 3-kinase activity or glucose uptake. Although GH and hyperosmolarity attributable to 300 mM sorbitol each promoted glucose uptake and translocation of glucose transporter (GLUT)4 to an extent comparable to that of insulin, these stimuli triggered little or no association of PI 3-kinase activity with tyrosine-phosphorylated proteins. Overexpression of delta p85 or treatment of cells with wortmannin, an inhibitor of PI 3-kinase activity, had no effect on glucose uptake or translocation of GLUT4 stimulated by GH or hyperosmolarity. Moreover, overexpression of delta SOS or RAC17N also did not affect the increase in glucose uptake induced by these stimuli. A serine/threonine kinase Akt, a constitutively active mutant of which was previously shown to stimulate glucose uptake, is activated by insulin, GH, and hyperosmolarity to approximately 4-fold, approximately 2.1-fold, and approximately 2.3-fold over basal level, respectively. These results suggest that insulin-induced but neither GH- or hyperosmolarity-induced glucose uptake is PI 3-kinase-dependent, and neither RAS nor RAC is required for glucose uptake induced by these stimuli in 3T3-L1 adipocytes.

Role of MAPK Phosphatase-1 (MKP-1) in Adipocyte Differentiation

“…Antibodies to aP2 (14) and troglitazone were kindly provided by D. Bernlohr (University of Minnesota, Minneapolis) and Sankyo Pharmaceutical Co. (Tokyo, Japan), respectively. The kinase activity of immunoprecipitates prepared with antibodies to p42/ p44 MAPK (␣C92) was assayed with myelin basic protein as substrate as described previously (15).…”

Akt2 Phosphorylates Ezrin to Trigger NHE3 Translocation and Activation

“…For example, GLUT4 is translocated from intracellular pools to the adipocyte plasma membrane by Akt-and p38 MAP kinase-dependent mechanisms after insulin stimulation (53)(54)(55). In these cells, PI3 kinase mediates Akt activation (56,57), but PI3 kinase-independent events are also required (58). These include activation of the small GTPase TC10, whose activity within lipid rafts regulates cortical actin (59) and is essential for docking and fusion of GLUT4-containing vesicles with the plasma membrane (60,61).…”

Reciprocal regulation of MelCAM and AKT in human melanoma

“…Adenoviral vectors Myr-AKT and Dp85 were kindly provided by Dr Masahiro Oka (Sakaue et al, 1997;Kotani et al, 1999;Oka et al, 2000). Control adenoviral vector containing the LacZ cDNA (lacZ/Ad5) was purchased from Vector Core at the Institute for Human Gene Therapy of the University of Pennsylvania (Philadelphia, PA, USA).…”