Insulin inhibits the ubiquitin-dependent degrading activity of the 26S proteasome.
A major metabolic effect of insulin is inhibition of cellular proteolysis, but the proteolytic systems involved are unclear. Tissues have multiple proteolytic systems, including the ATP- and ubiquitin-dependent proteasome pathway. The effect of insulin on this pathway was examined in vitro and in cultured cells. Insulin inhibited ATP- and ubiquitin-dependent lysozyme degradation more than 90% by reticulocyte extract, in a dose-dependent manner (IC50 approximately 50 nM). Insulin did not reduce the conjugation of ubiquitin to lysozyme and was not itself ubiquitin-conjugated. In HepG2 cells, insulin increased ubiquitin-conjugate accumulation 80%. The association between the 26S proteasome and an intracellular protease, the insulin-degrading enzyme (IDE), was examined by a purification scheme designed to enrich for the 26S proteasome. Copurification of IDE activity and immunoreactivity with the proteasome were detected through several chromatographic steps. Glycerol gradient analysis revealed cosedimentation of IDE with the 20S proteasome and possibly with the 26S proteasome. The proteasome-associated IDE was displaced when the samples were treated with insulin. These results suggest that insulin regulates protein catabolism, at least in part, by decreasing ubiquitin-mediated proteasomal activity, and provides a new target for insulin action. The displacement of IDE from the proteasome provides a mechanism for this insulin action.
Bennett
R G
RG
Veterans Affairs Medical Center, and the Department of Internal Medicine, University of Nebraska Medical Center, Omaha 68198-3020, USA. rgbennet@unmc.edu
Hamel
F G
FG
Duckworth
W C
WC
D000255:Adenosine Triphosphate / Q000502:physiology; D000818:Animals; D002457:Cell Extracts / Q000494:pharmacology; D003546:Cysteine Endopeptidases / Q000378:metabolism; D006801:Humans; D007328:Insulin / Q000494:pharmacology*; D007339:Insulysin / Q000378:metabolism; D009097:Multienzyme Complexes / Q000378:metabolism; D009113:Muramidase / Q000037:antagonists & inhibitors / Q000378:metabolism; D010447:Peptide Hydrolases / Q000378:metabolism*; D046988:Proteasome Endopeptidase Complex; D011817:Rabbits; D012156:Reticulocytes / Q000737:chemistry; D014407:Tumor Cells, Cultured; D014452:Ubiquitins / Q000502:physiology*
D002457:Cell Extracts; D007328:Insulin; D009097:Multienzyme Complexes; D014452:Ubiquitins; D000255:Adenosine Triphosphate; D009113:Muramidase; D010447:Peptide Hydrolases; D003546:Cysteine Endopeptidases; D007339:Insulysin; D046988:Proteasome Endopeptidase Complex; C061553:ATP dependent 26S protease