Role Of FoxO In Stress-induced Lipid Metabolism Disorer

Stress systems play a crucial role in maintaining homoeostasis to adapt to the environmental demands imposed by change. However, strong and long-lasting stress stimuli could disturbances in the internal environment, thereby increasing the risk of various health problems. In modern society, which is characterized by a rapid pace of life, individuals are continuously confronted with an increasing number of stressful stimuli, such as emotional stimuli and social stress. It had been validated by clinical trials that chronic stress from work and low quality of life may be the risk factor of obesity and metabolism syndrome.Forkhead proteins, and FoxO1 in particular, play a significant role in regulating whole body energy metabolism. Glucose homeostasis is achieved by adjusting endogenous glucose production as well as glucose uptake by peripheral tissues in response to insulin. In the fasted state, the liver is primarily responsible for maintaining glucose levels, with FoxO playing a key role in promoting the expression of gluconeogenic enzymes. Recent studies showed that FoxO is not only take part in glucose metabolism, but also in lipid metabolism.Non-alcoholic fatty liver disease (NAFLD), characterized by the accumulation of large droplets of triglyceride within hepatocytes in the absence of chronic alcohol consumption, is a leading cause of hepatic dysfunction. NAFLD represents a wide spectrum of diseases, ranging from simple steatosis, through steatosis with inflammation (non-alcoholic steatohepatitis, NASH) to cirrhosis. Al-though simple hepatic steatosis is a slowly developed and asymptomatic disease, the next stage NASH is more likely to cause progressive cirrhosis, hepatocellular carcinoma and increased mortality. The prevalence of NAFLD has increased dramatically over the last three decades; at nearly 15-30% in the general population in western countries and approximate 15% in big cities of China. Despite its high prevalence, factors leading to progression from NAFLD to NASH remain obscure and there is no pharmacological agent approved for the treatment of NAFLD.Since chronic stress could lead to systematic inflammation, we assume that chronic stress may take part in the process of NAFLD. Yet the relationship between chronic stress and NAFLD has not been clarified. Thus, the overall objective of this study was to demonstrate whether chronic stress could lead to NAFLD.FoxO1 is highly expressed in NAFLD patients compared with normal ones. At the same time, these patients had elevated lipid related gene expression, suggesting that FoxO1 might involve in NAFLD development. Furthermore, FoxO1 regulates HSL expression in adipocyte, promoting lipolysis. Qu et al. also found that FoxO1 played an important role in macrophage inflammatory factor IL-1βsecretion. Our studies also demonstrated lipolysis and elevated IL-1β in chronic stress-induced NAFLD. Thus we assume that FoxO1 might take part in this process.Results:1. Chronic stress could lead to food intake and body weight decrease. To investigate whether chronic stress is linked to NAFLD progression, we made a 12 weeks stress protocol for C57BL/6 mice in order to lead to chronic stress status. During 12 weeks, the food intake and body weight of control group were steadily increased, while stress group didn’t show increased food intake and body weight gain. At the end of the time, both body weight and food intake gain was significantly lower in stress group.2. Chronic stress promote adipose lipolysis, thus increase free fatty acid input and adipocytokines. The stress mice exhibited remarkable reduction of visceral mass compared to control ones, and HE staining showed that adipocytes of stress group were significantly smaller than control ones. Consistence with these results, Serum FFA was significantly elevated in stress group. Adipose adipokine levels were examined after 12 weeks stress protocol. The results turned out that although the amount of visceral adipose was decreased, a variety of inflammatory factors of visceral adipose were elevated in stress mice. IL-6 and IL-1β were significantly increased. While others like TNF-a and IL-18 were not changed. Combined with cytokines, a lot of chemokines such as macrophage inflammatory protein 2 (MIP-2), monocyte chemoattractant protein-1 (MCP-1) and PAI-1, related to inflammatory cells gathering were elevated too.3. Chronic stress treated mice had steatosis and increased inflammatory lesions. Although stress group had less food intake and weight gain, their hepatic index (HI, the ratio of liver weight to body weight×100), which usually reflects the fatty liver status, was significantly elevated. Serum TC and TG showed increasing tendency but had no statistical significance. Quantitative analysis showed a significant increase in hepatic TG and TC content in stressed mice.4. Chronic stress increase inflammation factors in both liver and serum. To figure out which cytokines take part in NASH, we detected the cytokines closely related to NASH in both liver and serum. For IL-6 and TNF-a, stress mice had both higher serum and liver levels than control ones. For IL-1β and MCP-1, serum levels were higher in mice given chronic stress compared with controls. For IL-18, the two groups showed same level in serum and liver after the stress treatment.5. Chronic stress induced steatosis is mainly due to increased liver TG synthesis. Fasn and SCD1, two TG synthesis genes, were significantly elevated in stress group.6. In chronic stress model, FoxO1 mRNA and protein level were increased. Liver FoxO1 total mRNA level and protein were significantly higher in 12 weeks stress mice compared with control ones. Besides the change of amount, the activity of FoxO1 was elevated at chronic stress state. To figure out whether FoxO1 protein activity was changed in this model, we separated nuclear protein and applied western blot. The result shows that nuclear FoxO1 protein was more than control group. Chronic stress might through IRS2-Akt-FoxO1 pathway to activate FoxO1.Liver IRS2 mRNA expression was significantly decreased in stress group and downstream p-Akt/Akt was decreased as well.7. Fox06 transgenic mice had hypercholesterolemia. Compared with wild type mice, Fox06 transgenic mice showed significantly higher FoxO6 gene and protein expression in liver. FoxO6 transgenic mice had normal body weight, while serum cholesterol significantly elevated.8. FoxO6 induced hypercholesterolemia is due to LDLR decrease. Liver LDL receptor (LDLR) mRNA and protein level were reduced in FoxO6 transgenic mice. The regulation factors such as SREBP2 and PCSK9 didn’t change in this model. In Fox06-adv infected primary hepatocytes, LDLR mRNA and protein level were down regulated. Component with in vivo results, SREBP2 expression didn’t change in vitro.9. FoxO6 block SREBP2 binds on LDLR promoter to decrease LDLR expression. We designed the luciferase plasmid including LDLR promoter region to develop FoxO6 regulation of LDLR. Results showed that SREBP2 significantly increased LDLR expression in HepG2 cells. This effect could be inhibited after adding in FoxO6 virus. We used Chip assay to further investigate the way of FoxO6 regulation on LDLR. Results shows that, SREBP2 binding ability was blocked after adding in FoxO6-adv.