Overexpression of SH2-containing inositol phosphatase 2 results in negative regulation of insulin-induced metabolic actions in 3T3-L1 adipocytes via its 5'-phosphatase catalytic activity.
Phosphatidylinositol (PI) 3-kinase plays an important role in various metabolic actions of insulin including glucose uptake and glycogen synthesis. Although PI 3-kinase primarily functions as a lipid kinase which preferentially phosphorylates the D-3 position of phospholipids, the effect of hydrolysis of the key PI 3-kinase product PI 3,4,5-triphosphate [PI(3,4,5)P3] on these biological responses is unknown. We recently cloned rat SH2-containing inositol phosphatase 2 (SHIP2) cDNA which possesses the 5'-phosphatase activity to hydrolyze PI(3,4,5)P3 to PI 3,4-bisphosphate [PI(3,4)P2] and which is mainly expressed in the target tissues of insulin. To study the role of SHIP2 in insulin signaling, wild-type SHIP2 (WT-SHIP2) and 5'-phosphatase-defective SHIP2 (Delta IP-SHIP2) were overexpressed in 3T3-L1 adipocytes by means of adenovirus-mediated gene transfer. Early events of insulin signaling including insulin-induced tyrosine phosphorylation of the insulin receptor beta subunit and IRS-1, IRS-1 association with the p85 subunit, and PI 3-kinase activity were not affected by expression of either WT-SHIP2 or Delta IP-SHIP2. Because WT-SHIP2 possesses the 5'-phosphatase catalytic region, its overexpression marked by decreased insulin-induced PI(3,4,5)P3 production, as expected. In contrast, the amount of PI(3,4,5)P3 was increased by the expression of Delta IP-SHIP2, indicating that Delta IP-SHIP2 functions in a dominant-negative manner in 3T3-L1 adipocytes. Both PI(3,4,5)P3 and PI(3,4)P2 were known to possibly activate downstream targets Akt and protein kinase C lambda in vitro. Importantly, expression of WT-SHIP2 inhibited insulin-induced activation of Akt and protein kinase C lambda, whereas these activations were increased by expression of Delta IP-SHIP2 in vivo. Consistent with the regulation of downstream molecules of PI 3-kinase, insulin-induced 2-deoxyglucose uptake and Glut4 translocation were decreased by expression of WT-SHIP2 and increased by expression of Delta IP-SHIP2. In addition, insulin-induced phosphorylation of GSK-3beta and activation of PP1 followed by activation of glycogen synthase and glycogen synthesis were decreased by expression of WT-SHIP2 and increased by the expression of Delta IP-SHIP2. These results indicate that SHIP2 negatively regulates metabolic signaling of insulin via the 5'-phosphatase activity and that PI(3,4,5)P3 rather than PI(3,4)P2 is important for in vivo regulation of insulin-induced activation of downstream molecules of PI 3-kinase leading to glucose uptake and glycogen synthesis.
First Department of Internal Medicine, Toyama Medical and Pharmaceutical University, Sugitani, Japan.
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T
Wada
T
T
Sasaoka
M
M
Funaki
H
H
Hori
S
S
Murakami
M
M
Ishiki
T
T
Haruta
T
T
Asano
W
W
Ogawa
H
H
Ishihara
M
M
Kobayashi
D016475:3T3 Cells; D000256:Adenoviridae / Q000235:genetics; D017667:Adipocytes / Q000201:enzymology*; D000818:Animals; D029681:Arabidopsis Proteins; D015153:Blotting, Western; D002384:Catalysis; D002851:Chromatography, High Pressure Liquid; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000378:metabolism; D003847:Deoxyglucose / Q000493:pharmacokinetics; D018014:Gene Transfer Techniques; D005799:Genes, Dominant; D051272:Glucose Transporter Type 1; D051275:Glucose Transporter Type 4; D006003:Glycogen / Q000096:biosynthesis; D006006:Glycogen Synthase / Q000378:metabolism; D007328:Insulin / Q000378:metabolism / Q000494:pharmacology; D055504:Insulin Receptor Substrate Proteins; D007527:Isoenzymes; D007763:Lac Operon; D051379:Mice; D018613:Microscopy, Confocal; D008957:Models, Genetic; D009004:Monosaccharide Transport Proteins / Q000378:metabolism; D009124:Muscle Proteins; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D000072183:Phosphatidylinositol-3,4,5-Trisphosphate 5-Phosphatases; D010716:Phosphatidylinositols / Q000378:metabolism; D010750:Phosphoproteins / Q000378:metabolism; D010744:Phosphoric Monoester Hydrolases / Q000096:biosynthesis* / Q000737:chemistry / Q000502:physiology*; D010766:Phosphorylation; D010940:Plant Proteins / Q000378:metabolism; D015221:Potassium Channels / Q000378:metabolism; D011233:Precipitin Tests; D011493:Protein Kinase C / Q000378:metabolism; D051381:Rats; D015398:Signal Transduction; D013997:Time Factors; D014162:Transfection; D018909:src Homology Domains; D000099293:Protein Kinase C-lambda
D029681:Arabidopsis Proteins; D018076:DNA, Complementary; D003847:Deoxyglucose; D051272:Glucose Transporter Type 1; D051275:Glucose Transporter Type 4; D006003:Glycogen; D006006:Glycogen Synthase; D007328:Insulin; D055504:Insulin Receptor Substrate Proteins; D007527:Isoenzymes; D009004:Monosaccharide Transport Proteins; D009124:Muscle Proteins; D019869:Phosphatidylinositol 3-Kinases; D000072183:Phosphatidylinositol-3,4,5-Trisphosphate 5-Phosphatases; D010716:Phosphatidylinositols; D010750:Phosphoproteins; D010744:Phosphoric Monoester Hydrolases; D010940:Plant Proteins; D015221:Potassium Channels; D011493:Protein Kinase C; D000099293:Protein Kinase C-lambda; C526295:Irs1 protein, mouse; C526296:Irs1 protein, rat; C495693:Slc2a4 protein, mouse; C495694:Slc2a4 protein, rat; C118303:phosphoinositide-3,4,5-triphosphate; C118301:phosphoinositide-3,4-bisphosphate; C074500:AKT1 protein, Arabidopsis; C098706:INPPL1 protein, human