Vol. 304, Issue 3, 994-1002, March 2003

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

Department of Pharmacology, Miyazaki Medical College, Miyazaki, Japan

Treatment (24 h) of cultured bovine adrenal chromaffin cells with ketoacidosis-related concentrations (3 mM) of acetoacetate (but not -hydroxybutyrate, acetone, and acidic medium) caused a time- and concentration-dependent reduction of cell surface ¹²⁵I-insulin binding by ~38%, with no change in the K_d value. The reduction of ¹²⁵I-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 the trans-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.