Protein phosphatase-2C alpha as a positive regulator of insulin sensitivity through direct activation of phosphatidylinositol 3-kinase in 3T3-L1 adipocytes.
During differentiation, expression of protein phosphatase-2Calpha (PP2Calpha) is increased in 3T3-L1 adipocytes. To elucidate the role of PP2Calpha in insulin signaling, we overexpressed wild-type (WT) PP2Calpha by adenovirus-mediated gene transfer in 3T3-L1 adipocytes. Overexpression of PP2Calpha-WT enhanced the insulin sensitivity of glucose uptake without any changes in the early steps of insulin signaling. Infection with adenovirus 5 expressing PP2Calpha-WT increased phosphatidylinositol 3-kinase (PI3K) activities in the immunoprecipitate using antibody against the p85 or p110 subunit under both basal and insulin-stimulated conditions, followed by activation of downstream steps in the PI3K pathway, such as phosphorylation of Akt, glycogen synthase kinase-3, and atypical protein kinase C. In contrast, overexpression of the phosphatase-defective mutant PP2Calpha(R174G) did not produce such effects. Furthermore, overexpression of PP2Calpha-WT (but not PP2Calpha(R174G)) decreased the (32)P-labeled phosphorylation state as well as the gel mobility shift of the p85 subunit, suggesting that dephosphorylation of the p85 subunit by PP2Calpha activation might stimulate PI3K catalytic activity. Moreover, knockdown of PP2Calpha by transfection of small interfering RNA led to a significant decrease in Akt phosphorylation. In addition, microinjection of anti-PP2Calpha antibody or PP2Calpha small interfering RNA led to decreased insulin-stimulated GLUT4 translocation. In conclusion, PP2Calpha is a new positive regulator of insulin sensitivity that acts through a direct activation of PI3K in 3T3-L1 adipocytes.
Yoshizaki
Takeshi
T
Department of Medicine, Shiga University of Medical Science, Otsu, Shiga 520-2192, Japan.
Maegawa
Hiroshi
H
Egawa
Katsuya
K
Ugi
Satoshi
S
Nishio
Yoshihiko
Y
Imamura
Takeshi
T
Kobayashi
Takayasu
T
Tamura
Shinri
S
Olefsky
Jerrold M
JM
Kashiwagi
Atsunori
A
D041721:3T3-L1 Cells; D000256:Adenoviridae / Q000235:genetics / Q000378:metabolism; D017667:Adipocytes / Q000378:metabolism*; D000818:Animals; D015152:Blotting, Northern; D015153:Blotting, Western; D002454:Cell Differentiation; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D005347:Fibroblasts / Q000378:metabolism; D005947:Glucose / Q000493:pharmacokinetics; D051275:Glucose Transporter Type 4; D006801:Humans; D007328:Insulin / Q000378:metabolism*; D051379:Mice; D008954:Models, Biological; D009004:Monosaccharide Transport Proteins / Q000378:metabolism; D009124:Muscle Proteins; D009154:Mutation; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism*; D010749:Phosphoprotein Phosphatases / Q000378:metabolism / Q000502:physiology*; D010766:Phosphorylation; D011233:Precipitin Tests; D000071636:Protein Phosphatase 2C; D021381:Protein Transport; D034741:RNA, Small Interfering / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D015398:Signal Transduction; D013997:Time Factors; D014162:Transfection
D051275:Glucose Transporter Type 4; D007328:Insulin; D009004:Monosaccharide Transport Proteins; D009124:Muscle Proteins; D034741:RNA, Small Interfering; D011994:Recombinant Proteins; C495692:SLC2A4 protein, human; C495693:Slc2a4 protein, mouse; D019869:Phosphatidylinositol 3-Kinases; C000607456:PPM1A protein, human; C000607457:PPM1B protein, human; C000607460:PPM1G protein, human; D010749:Phosphoprotein Phosphatases; C000607466:Ppm1a protein, mouse; D000071636:Protein Phosphatase 2C; D005947:Glucose