Cullin neddylation inhibitor attenuates hyperglycemia by enhancing hepatic insulin signaling through insulin receptor substrate stabilization

Hepatic insulin resistance is a hallmark feature of nonalcoholic fatty liver disease and type-2 diabetes and significantly contributes to systemic insulin resistance. Abnormal activation of nutrient and stress-sensing kinases leads to serine/threonine phosphorylation of insulin receptor substrate (IRS) and subsequent IRS proteasome degradation, which is a key underlying cause of hepatic insulin resistance. Recently, members of the cullin-RING E3 ligases (CRLs) have emerged as mediators of IRS protein turnover, but the pathophysiological roles and therapeutic implications of this cellular signaling regulation is largely unknown. CRLs are activated upon cullin neddylation, a process of covalent conjugation of a ubiquitin-like protein called Nedd8 to a cullin scaffold. Here, we report that pharmacological inhibition of cullin neddylation by MLN4924 (Pevonedistat) rapidly decreases hepatic glucose production and attenuates hyperglycemia in mice. Mechanistically, neddylation inhibition delays CRL-mediated IRS protein turnover to prolong insulin action in hepatocytes. In vitro knockdown of either cullin 1 or cullin 3, but not other cullin members, attenuates insulin-induced IRS protein degradation and enhances cellular insulin signaling activation. In contrast, in vivo knockdown of liver cullin 3, but not cullin 1, stabilizes hepatic IRS and decreases blood glucose, which recapitulates the effect of MLN4924 treatment. In summary, these findings suggest that pharmacological inhibition of cullin neddylation represents a therapeutic approach for improving hepatic insulin signaling and lowering blood glucose.

A Cullin-RING E3 ligase (CRL) is a multiprotein complex generally consisting of a cullin protein, a RING E3 ligase, and a substrate receptor that recognizes specific substrates for ubiquitination and proteasomal degradation (1). Each of the cullin member proteins serves as the scaffold of a functionally distinct CRL complex. Mammalian cells express a large number of substrate receptors in a tissue-specific manner, which further determines the substrate specificity of a unique CRL complex. CRLs are activated upon cullin neddylation, a process of covalent conjugation of a ubiquitin-like protein called Nedd8 to a conserved lysine on a cullin protein (1). Neddylation is mediated by a set of specialized Nedd8 E1, E2, and E3 enzymes that sequentially transfer Nedd8 to a cullin protein. Unlike protein ubiquitination, current knowledge supports that cullin proteins are the predominant neddylation targets in mammalian cells (2). Recently, CRLs have emerged as attractive targets for drug development (3). In the last 10 y, CRLs have drawn major attention in cancer research owing to CRL regulation of oncogenes and tumor suppressors (4). The Nedd8-activating E1 enzyme (NAE1) is the only known Nedd8 E1 enzyme (1). MLN4924 (Pevonedistat) is the first in-class small molecule inhibitor of NAE1 and has entered Phase-I/II clinical trials for various cancer treatments (5). In contrast, the translational potential of targeting cullin neddylation for treating other diseases is still largely unknown.Hepatic insulin resistance is a hallmark pathogenic feature of fatty liver disease and type-2 diabetes, and decreased hepatocellular insulin receptor substrate (IRS) is a key underlying cause (6, 7). Following insulin activation, IRS signaling is feedback inactivated by mechanistic target of rapamycin (mTOR)-mediated serine/threonine phosphorylation and subsequent IRS proteosome degradation (8, 9). Although this feedback mechanism prevents sustained insulin signaling activation in cells, overactivation of this desensitization mechanism by mTOR and other stress/nutrient kinases significantly contributes to hepatic insulin resistance in fatty livers (8, 10). However, the broad cellular functions of these kinases present a major obstacle in targeting them to prevent insulin resistance (11). Serine/threonine-phosphorylated IRS proteins are destined for ubiquitylation and proteasome degradation (9, 12), but targeting the proteasome pathway is unlikely a viable approach due to the perceived broad biological impact. Interestingly, studies of mouse embryonic fibroblasts have revealed that CRLs are involved in mediating IRS1 protein turnover (13, 14). These findings prompted us to ask two intriguing questions: First, are CRLs involved in regulating hepatic insulin signaling, and if yes, which CRL may mediate such an effect in hepatocytes? Second, can CRLs serve as potentially new therapeutic targets for modulating insulin signaling and glucose homeostasis in fatty liver disease in vivo? Here, we report findings that neddylation inhibition effectively enhances hepatic insulin signaling by delaying CRL-mediated IRS turnover, resulting in attenuated hyperglycemia in obese mice.