Ubiquitin-proteasome dysfunction contributes to obesity-related metabolic disorders such as diabetes and fatty liver disease. However, the regulation of ubiquitin-proteasome activity by insulin remains to be elucidated.Here, we show that prolonged insulin stimulation activates proteasome function even though it reduces the ubiquitinated proteins in H4IIEC3 hepatocytes. Looking for a pathway by which insulin inhibits ubiquitination, we found that hepatic expression of ubiquitin-specific protease 14 (USP14) was upregulated in the liver of patients with insulin resistance. Indeed, the USP14-specific inhibitor IU1 canceled the insulin-mediated reduction of ubiquitinated proteins.Furthermore, insulin induced endoplasmic reticulum (ER) stress, which was canceled by IU1, suggesting that USP14 activity is involved in insulin-induced ER stress. Co-stimulation with insulin and IU1 for 2 h upregulated the nuclear translocation of the lipogenic transcription factor, sterol regulatory element binding protein-1c (SREBP-1c), upregulated the expression of the lipogenic gene, fatty acid synthase (Fasn), and repressed the gluconeogenic genes.In conclusion, insulin activates proteasome function even though it inhibits protein ubiquitination by activating USP14 in hepatocytes. USP14 activation by insulin inhibits mature SREBP-1c while upregulating ER stress and the expression of genes involved in gluconeogenesis. Further understanding mechanisms underlying the USP14 activation and its pleiotropic effects may lead to therapeutic development for obesity-associated metabolic disorders such as diabetes and fatty liver disease. SIGNIFICANCE STATEMENTThis study shows that insulin stimulation inhibits ubiquitination by activating USP14, independent of its effect on proteasome activity in hepatocytes. USP14 also downregulates the nuclear translocation of the lipogenic transcription factor SREBP-1c and upregulates the expression of genes involved in gluconeogenesis. Since USP14 is upregulated in the liver of insulin-resistant patients, understanding mechanisms underlying the USP14 activation and its pleiotropic effects will help develop treatments for metabolic disorders such as diabetes and fatty liver.

NK1, a splicing variant of hepatocyte growth factor (HGF), binds to and activates Met receptor by forming an NK1 dimer and 2:2 complex with Met. Although the structural mechanism underlying Met activation by HGF remains incompletely resolved, it has been proposed that the NK1 dimer structure participates in this activation. We investigated the NK1 dimer interface’s role in Met activation by HGF. Because N127, V140, and K144 are closely involved in the head-to-tail NK1 dimer formation, mutant NK1 proteins with replacement of these residues by alanine were prepared. In Met tyrosine phosphorylation assays, N127-NK1, V140-NK1, and K144-NK1 showed 8.3%, 23.8%, and 52.2% activity, respectively, compared with wild-type NK1. Although wild-type NK1 promoted cell migration and scattering, N127-NK1, V140-NK1, and K144-NK1 hardly or marginally promoted them, indicating loss of activity of these mutant NK1 proteins to activate Met. In contrast, mutant HGFs (N127-HGF, V140-HGF, and K144-HGF) with the same amino acid replacements as in NK1 induced Met tyrosine phosphorylation and biological responses at levels comparable to those of wild-type HGF. These results indicate that the structural basis responsible for NK1-dependent Met dimer formation and activation differs from, or is at least distinguishable from, the structural basis responsible for HGF-dependent Met activation.