Insulin Suppresses Ubiquitination via the Deubiquitinating Enzyme Ubiquitin-Specific Protease 14, Independent of Proteasome Activity in H4IIEC3 Hepatocytes

Abstract

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.

Complexity in Hepatic Insulin Resistance – Unraveling the Role of Ubiquitin-Specific Protease 14 in Protein Homeostasis of Metabolic Transcription Factors

“…proves the levels of insulin-induced phosphorylation of Akt (Otoda et al, 2013), demonstrating that reducing ER stress that is caused by inhibition of the 26S proteasome can improve hepatic insulin signaling in vitro. Kamoshita et al (2022) reveal a significant negative correlation of hepatic USP14 mRNA expression in 21 individuals with and 11 without type 2 diabetes with glucose clearance rate under hyperglycemic clamp conditions (Fig. 1).…”

“…1). The 26S proteasome, however, is not significantly impacted by insulin treatment or cotreatment with IU1 in any of the three cell lines tested (Kamoshita et al, 2022) (Fig. 1).…”

“…As such, the authors sought to identify the mechanisms regulating the balance of proteasome activity and the number of ubiquitinated proteins under the control of insulin signaling and USP14. Using native PAGE assays, the authors demonstrate that 2 hours of insulin treatment reduces the accumulation of ubiquitinated proteins in rat H4IIEC3 hepatocytes and in human HepG2 hepatoma cells, which is restored upon inactivation of USP14 with IU1 (Kamoshita et al, 2022) (Fig. 1).…”

“…Next, the authors sought to determine if insulin-activated USP14 also reduces ubiquitinated proteins by enhancing proteasome activity or by modulating ER stress. In-gel assays reveal that inhibition of USP14 activity with IU1 does not prevent insulin-mediated increases in 20S proteasome activity in both rat H4IIEC3 hepatocytes and human HepG2 cells (Kamoshita et al, 2022) (Fig. 1).…”

“…1). Interestingly, the response to insulin and IU1 cotreatment is not maintained in murine Hepa 1-6 cells, where ubiquitinated protein levels are unchanged by insulin treatment (Kamoshita et al, 2022). Next, the authors sought to determine if insulin-activated USP14 also reduces ubiquitinated proteins by enhancing proteasome activity or by modulating ER stress.…”