Urotensin II-induced insulin resistance is mediated by NADPH oxidase-derived reactive oxygen species in HepG2 cells.

AIM

To investigated the effects of urotensin II (UII) on hepatic insulin resistance in HepG2 cells and the potential mechanisms involved.

METHODS

Human hepatoma HepG2 cells were cultured with or without exogenous UII for 24 h, in the presence or absence of 100 nmol/L insulin for the last 30 min. Glucose levels were detected by the glucose-oxidase method and glycogen synthesis was analyzed by glycogen colorimetric/fluorometric assay. Reactive oxygen species (ROS) levels were detected with a multimode reader using a 2',7'-dichlorofluorescein diacetate probe. The protein expression and phosphorylation levels of c-Jun N-terminal kinase (JNK), insulin signal essential molecules such as insulin receptor substrate -1 (IRS-1), protein kinase B (Akt), glycogen synthase kinase-3β (GSK-3β), and glucose transporter-2 (Glut 2), and NADPH oxidase subunits such as gp91(phox), p67(phox), p47(phox), p40(phox), and p22(phox) were evaluated by Western blot.

RESULTS

Exposure to 100 nmol/L UII reduced the insulin-induced glucose consumption (P < 0.05) and glycogen content (P < 0.01) in HepG2 cells compared with cells without UII. UII also abolished insulin-stimulated protein expression (P < 0.01) and phosphorylation of IRS-1 (P < 0.05), associated with down-regulation of Akt (P < 0.05) and GSK-3β (P < 0.05) phosphorylation levels, and the expression of Glut 2 (P < 0.001), indicating an insulin-resistance state in HepG2 cells. Furthermore, UII enhanced the phosphorylation of JNK (P < 0.05), while the activity of JNK, insulin signaling, such as total protein of IRS-1 (P < 0.001), phosphorylation of IRS-1 (P < 0.001) and GSK-3β (P < 0.05), and glycogen synthesis (P < 0.001) could be reversed by pretreatment with the JNK inhibitor SP600125. Besides, UII markedly improved ROS generation (P < 0.05) and NADPH oxidase subunit expression (P < 0.05). However, the antioxidant/NADPH oxidase inhibitor apocynin could decrease UII-induced ROS production (P < 0.05), JNK phosphorylation (P < 0.05), and insulin resistance (P < 0.05) in HepG2 cells.

CONCLUSION

UII induces insulin resistance, and this can be reversed by JNK inhibitor SP600125 and antioxidant/NADPH oxidase inhibitor apocynin targeting the insulin signaling pathway in HepG2 cells.