Distinct Effects of Ketone Bodies on Down-Regulation of Cell Surface Insulin Receptor and Insulin Receptor Substrate-1 Phosphorylation in Adrenal Chromaffin Cells
- Hiroki Yokoo,
- Tomokazu Saitoh,
- Seiji Shiraishi,
- Toshihiko Yanagita,
- Takashi Sugano,
- Shin-Ichi Minami,
- Hideyuki Kobayashi and
- Akihiko Wada
- Akihiko Wada, Department of Pharmacology, Miyazaki Medical College, Kiyotake, Miyazaki 889-1692, Japan. E-mail: akihiko{at}fc.miyazaki-med.ac.jp
Abstract
Treatment (≧24 h) of cultured bovine adrenal chromaffin cells with ketoacidosis-related concentrations (≧3 mM) of acetoacetate (but not β-hydroxybutyrate, acetone, and acidic medium) caused a time- and concentration-dependent reduction of cell surface125I-insulin binding by ∼38%, with no change in theKd value. The reduction of125I-insulin binding returned to control nontreated level at 24 h after the washout of acetoacetate-treated cells. Acetoacetate did not increase the internalization rate of cell surface insulin receptor (IR), as measured in the presence of brefeldin A, an inhibitor of cell surface vesicular exit from thetrans-Golgi network. Acetoacetate (10 mM for 24 h) lowered cellular levels of the immunoreactive IR precursor molecule (∼190 kDa) and IR by 22 and 28%, respectively. Acetoacetate decreased IR mRNA levels by ∼23% as early as 6 h, producing their maximum plateau reduction at 12 and 24 h. The half-life of IR mRNA was shortened by acetoacetate from 13.6 to 9.5 h. Immunoprecipitation followed by immunoblot analysis revealed that insulin-induced (100 nM for 10 min) tyrosine-phosphorylation of insulin receptor substrate-1 (IRS-1) was attenuated by 56% in acetoacetate-treated cells, with no change in IRS-1 level. These results suggest that chronic treatment with acetoacetate selectively down-regulated the density of cell surface functional IR via lowering IR mRNA levels and IR synthesis, thereby retarding insulin-induced activation of IRS-1.
Footnotes
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This study was supported in part by a grant-in-aid for the 21st Century COE (Centers of Excellence) Program (Life Science) from the Ministry of Education, Culture, Sports, Science and Technology, Japan.
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DOI: 10.1124/jpet.102.044115
- Abbreviations:
- FFA
- free fatty acids
- IR
- insulin receptors
- ER
- endoplasmic reticulum
- IRS-1
- insulin receptor substrate-1
- cPGI2
- 6α-carba-prostaglandin I2
- GAPDH
- glyceraldehyde-3-phosphate-dehydrogenase
- DMSO
- dimethyl sulfoxide
- KRP
- Krebs-Ringer phosphate
- PAGE
- polyacrylamide gel electrophoresis
- SSC
- saline-sodium citrate
- PPAR
- peroxisome proliferator-activated receptors
- kb
- kilobase
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- Received September 5, 2002.
- Accepted November 11, 2002.
- The American Society for Pharmacology and Experimental Therapeutics
