Nuciferine blocks MIB2-mediated CARD6 polyubiquitination and degradation in the amelioration of high fructose-induced liver lipid accumulation

Abstract

Dietary alkaloid nuciferine isolated from the leaves of Nelumbo nucifera can ameliorate dyslipidemia and liver lipid accumulation, but the underlying mechanism remains unclear. Caspase recruitment domain protein family member 6 (CARD6) is suggested to play an important role in metabolic diseases. This study aimed to investigate the role and the upstream regulator of CARD6 in high fructose-induced liver lipid accumulation and whether and how the anti-lipid accumulation effect of nuciferine was related to CARD6. Herein, we found that high fructose decreased CARD6 expression and increased ASK1 and JNK1/2 phosphorylation in rat livers and hepatocytes, which were attenuated by nuciferine. Furthermore, after the transfection with HA-CARD6, CARD6 siRNA and MIB2 siRNA, the data showed that CARD6 overexpression blocked high fructose-induced upregulation of ASK1 and JNK1/2 phosphorylation as well as lipid accumulation in hepatocytes. CARD6 siRNA reversed the amelioration of nuciferine to high fructose-induced upregulation of ASK1 and JNK1/2 phosphorylation in hepatocyte lipid accumulation. Mechanistically, high fructose upregulated MIB2 expression by interacting with CARD6 and promoting K48-linked CARD6 polyubiquitination and degradation in high fructose-stimulated hepatocytes which were explored by immunoblotting, immunofluorescence, and immunoprecipitation. However, MIB2 siRNA reversed high fructose-induced downregulation of CARD6 and lipid accumulation in hepatocytes. Notably, nuciferine reduced MIB2 expression and thus decreased K48-linked CARD6 polyubiquitination and degradation in the amelioration of high fructose-induced lipid accumulation in hepatocytes. These results suggested that nuciferine exhibited a protective effect against high fructose-induced liver lipid accumulation through blocking MIB2-mediated CARD6 polyubiquitination and degradation.