| Diabetes mellitus is caused by absolute or relative insufficient insulin and hyperglycemia is one of the main characteristics of diabetes mellitus. Free fatty acids (FFA) are critical correlation between lipid metabolism disorder and insulin resistance or hyperinsulinemia. Increased FFA originated from obesity contributes to insulin action and glucose metabolism. It is important to insulin resistance's pathology. Liver is one of the target organs of insulin. The insulin resistance of liver plays a critical role in the generation and development of type 2 diabetes. It is declared that oxidative stress induced from high FFA is the crucial cause of insulin resistance of liver. Oxidative stress is caused by an imbalance between the production of reactive oxygen species (ROS) or reactive nitrogen species (RNS) and a biological system's ability to readily detoxify the reactive intermediates or easily repair the resulting damage. The potential sources of ROS include NADPH oxidase (NOX), xanthine oxidase, cytochrome p-450, uncoupled endothelial nitric oxide synthase and lipoxygenase. NOX is considered as the main source of ROS in liver. NOX2, the complex enzyme NOX composed of five subunits including cytomembrane components (p22phox and NOX2) and cytosolic components (p47phox, p67phox and Rac), was firstly found in phagocyte cells. NOX2, which has potential binding sites of NADPH, heme and FAD, and p22phox are regulated by p47phox and Rac. Seven members of NOX family including NOX1, NOX2 (formerly named gp91PHOX), NOX3, NOX4, NOX5, Duox1 (Dual oxidase 1) and Duox2 have been identified in different cells. It is considered that NOX3 is the main NOX expressed in liver. Although some experiments indicated that FFA play an important role during the generation of insulin resistance originated from FFA, its molecular mechanism is unclear. In order to investigate the role of ROS derived by NOX3 in insulin resistance induced by FFA, first of all, human liver cell line HepG2 was treated with different concentrations of palmitate(0.15-0.35mmol/L), and ROS level was detected by flow cytometry. Comparing with the control group, treatment of palmitate could lead to excessive production of ROS in HepG2 in dependent way on concentration and time. However, pre-incubation of HepG2 with DPI, the NOX inhibitor and Rotenone, but not Oxypurinol or L-NAME could inhibit the generation of ROS, indicating that NOX is the main source of FFA-induced ROS generation in HepG2. Further, the expression of NOX subunits was investigated by Western blot. The results showed that NOX3 and sub-units p22PHOX, p47PHOX, p67PHOX, Rac-1 were expressed in HepG2, but only the expression of NOX3 and p47PHOX were significantly up-regulated after treatment of HepG2 with 0.25 mmol/L palmitate. p47PHOX was translocated to cell membrane from cytoplasm to mediate the activation of NOX3 and the generation of ROS. We tested the glycogen in HepG2 and quantity of glucose in the medium to evaluate insulin resistance in HepG2. It was found that palmitate could stimulate the reduction of glycogen in HepG2 and elevation of glucose level in medium and induce insulin resistance in HepG2. Moreover, lost of function was carried out to observe effects of ROS derived from NOX3 during insulin resistance. The NOX3 was knocked down by transfection of HepG2 with NOX3-siRNA. The results showed that down-regulation of NOX3 significantly reduced the level of ROS and prevented HepG2 from insulin resistance. It means that NOX3 is involved in insulin resistance induced by FFA. Furthermore, to study the mechanisms of insulin resistance generation induced by FFA. After 0.25mmol/L palmitate treatment, the levels of phosphorylated p38MAPK,PTEN,JNK,IRS1 were elevated, however, the levels of phosphorylated AKT,GSK,FOXO1 were decreased with up-regulated the expression of NOX3 and production of ROS. These pointed out that FFA-induced oxidative stress originated by NOX resulted in insulin resistance in HepG2. p38MAPK/PTEN,JNK and PI-3K/AKT pathways were involved in insulin resistance in HepG2. We used db/db mice as an insulin resistance model to further certificate the mechanisms of role of NOX3-derived ROS in insulin resistance of liver induced by FFA. The oil red O staining for lipofuchsin showed the lipid storage in liver of db/db mice. Compared with normal mice, serum FFA and GLU levels in db/db mice were elevated. Serum MDA level and the levels of ROS and NOX3 in liver were considered as evaluation target, showing that serum MDA and the levels of ROS and NOX3 in liver were significantly elevated in db/db mice. Another result that the levels of plasma insulin and glycosylated hemoglobin in db/db mice were much higher than that in control group means that insulin resistance occurred in db/db mice. To further investigate the insulin resistance in liver of db/db mice, we tested glycogen in liver. The results showed that glycogen of liver in db/db mice is much lower than that in normal group, indicating that db/db mice were in the insulin resistance state. Same as the results from vitro, p38MAPK/PTEN,JNK and PI-3K/AKT pathways were involved in the insulin resistance in liver.In summary, our results provide the molecular evidence that NOX3-derived ROS could play an important role in oxidative stress and insulin resistance in liver induced by high concentration of FFA. Specifically inhibiting NOX activity may be a novel target preventing insulin resistance and diabetes.
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