Deletion Of Nrf2Aggravate Inhepatic Insulin Resistance In Mice Fed With High Fat Diet

Non-alcoholic fatty liver disease (NAFLD), ranging from simple steatosis tosteatohepatitis(NASH) which can progress to liver cirrhosis and hepatocellular cancer，isthe most common form of chronic liver disease all over the world. But the exactpathological mechanism is still not so clear.Because of its strong association with obesity, type2diabetes mellitus, hypertensionand hypertriglyceridaemia, NAFLD is universally considered as the hepatic manifestationof metabolic syndrome. It is generally accepted that hepatic insulin resistance is a keypathophysiological hallmark in NAFLD and might play a role in the progression fromsimple fatty liver to NASH by promoting the development of steatosis and the subsequentcell injury and inflammation. So a better understanding on the underlying mechanismshow hepatic insulin resistance develops is pivotal to devise rational treatments that could prevent the progression of NAFLD to advanced forms.One of the mechanisms proposed to account for the development of insulin resistanceafter the administration of a high fat diet is ROS upregulation in the liver. Markers of lipidperoxidation and oxidative DNA damage, such as hepatic content of both HNE and8-hydroxydeoxygnanosine are correlated with the severity of insulin resistance. Indeed,ROS and lipid peroxidation products act together to trigger the development of insulinresistance through phosphorylation of proteins of the insulin signaling cascade.In addition, recent evidence suggest that part of the role of oxidative stress on insulinresistance could contribute to secondary inflammation state. Furthermore, it is notewortythat these two events are intertwined and a vicious cycle can take place between them.One hand, ROS and products of lipid peroxidation can activate IKK/NF-kappaB signalpathway, which induce the synthesis of several proinflammatory cytokines includingTNFalpha and IL-6, leading to insulin resistance via paracrine effects. On the other hand,those cytokines, especially TNFalpha and IL-6, can induce mitochoudrial dysfunction bypromoting ROS and RNS production, which contributes significantly to the progression ofinsulin resistance and NASH. So it is reasonable to speculate that cutting off the loop willbe an attractive therapeutic target to inhibit the development of insulin resistance and theprogression of NAFLD to NASH.Nrf2is a key transcription factor that regulates many kinds of antioxidant genes anddetoxifying enzymes against oxidative stress and electronic stress. Under normal condition,Nrf2is sequestered in the cytosol by Kelch-like Ech-associated protein (Keap1). But whenchallenged with oxidative stress or electronic stress, Nrf2dissociates with Keap1, thentranslocates into nucleus, and promotes the expression of a number of antioxidant genessuch as GCLC,Nqo1and Gsta1. It was showed that MCD or HFD could induce moreaggravated oxidative stress injury and inflammation in Nrf2-null mice than in WT mice.On the contrary, such damages can be attenuated through elevating the level of Nrf2by itsinducer or genenic disruption of Keap1.Our previous study also showed that activation ofNrf2by curcumin could significantly attenuate the oxidative stress injury in hepatocyteand hepatic stellate cells induced by GO. ObjectiveTo investigate the influence of oxidation stress on nuclear translocation of Nrf2inmouse hepatic insulin resistance model induced by high fat diet. Furthermore, wehypothesis that deletion of Nrf2markedly accelerates the onset and progression of hepaticinsulin resistance due to the more serious oxidative stress and inflammation. It willprovide a new experimental and theoretical basis for the prevention and treatment ofNASH.Methods1．Male8week-old WT and Nrf2-null mices on ICR background were randomlydivided into four groups after adopt to the lab condition for1week and were free access toirradiated hight-fat diet(10%lard,2%cholesterol,0.5%bile salt and87.5%base forage)or a control diet for8weeks(n=5). Body weight were measured weekly. At the end of theexperiment,liver weight were measured. After that, the changes of pathology of liver wereobserved. Then the levels of liver TG, hepatic methane dicarboxylic aldehyde (MDA) andglutathione (GSH) were examined by spectrophotometric method. The expression of liverNrf2and Nqo1were determined by western-blot.2．Male8week-old WT and Nrf2-null mices on ICR background were randomlydivided into four groups after adopt to the lab condition for1week and were free access toirradiated hight-fat diet(10%lard,2%cholesterol,0.5%bile salt and87.5%base forage)or a control diet for8weeks(n=5). Body weight were measured weekly. At the end of theexperiment, iPGTT was performed before sacrifice. After that, the changes of pathology ofliver were observed. Then the levels of blood glucose, hepatic methane dicarboxylicaldehyde (MDA) and glutathione (GSH) were examined by spectrophotometric method.The levels hepatic TNFα and IL-6were examined by ELISA and RT-PCR. The expressionof liver NF-κB, IRS-1, Akt, p-GSK-3β and p-FoXO1were determined by western-blot.Results1．After feeding the diet for8weeks, weight gain in both WT and Nrf2-null miceincreased significantly compared with weight gain after intake of control diet. And the tendency to be higher in Nrf2-null mice, although there were no significantly differenceswhen compared to WT intake of HFD. Also the liver weight increased significantly both inWT and Nrf2-null mice after feeding HFD for8weeks in compared with control diet, butno significant differences were noted between WT and Nrf2-null mice. In line with this,WT and Nrf2-null on HFD showed a comparable increase in hepatic TG at8weeks.Moreover, the magnitudes were significantly higher in Nrf2-null mice than in WT mice.2．After start the HF diet for8weeks, both WT and Nrf2-null mice showed moremicrovesicular lipid accumulation in livers compared with the normal diet groups. Asexpected, the Nrf2-null mice exhibited increases in infiltration of inflammatory cells in thelivers, compared with the findings in the livers of the WT mice intake of HFD3．The level of liver MDA increased significantly both in WT and Nrf2-null miceafter feeding HFD for8weeks in compared with control diet. Furthermore, themagnitudes were significantly greater in Nrf2-null mice than in WT mice. In contrast,feeding HFD for8weeks significantly reduced the concentration of liver GSH comparedwith intake of control diet and the tendency was to be more obervious in Nrf2-null mouse.Feeding HFD for8weeks significantly increased the expression of nuclear Nrf2protein inWT mouse liver, wherase no Nrf2expression were noted in Nrf2-null mouse liver.Furthermore, the basal protein levels of the typical Nrf2-regulated genes, Nqo1, weresignificantly lower in the livers of Nrf2-null mice than in WT mice fed the control diet andwere significantly induced by HFD feeding in liver of WT mouse.4．8weeks HFD fed resulted in a notable increase of FGB both in WT mice andNrf2-null compared with control. Moreover, the increase in Nrf2-null mice were moresignificant than that in WT mices. Also,8weeks HFD fed leaded to markedly increase ofthe blood glucose of WT and Nrf2-null mices following i.p. glucose load at both baselineand after15min,60min and90mmin. Additionally, the area under the curve (AUC), whichis a measure of impaired glucose tolerance, also significantly increased in iPGTT in WTand Nrf2-null mices. Further,the magnitude of the above increase were both greater in theNrf2-null mice than those in the WT mice. 5．After start the HF diet for8weeks, there were no significant changes of totalIRS-1and total Akt, but tyrosine phosphorylation of IRS-1and the phosphorylation of Aktwere significantly reduced after the intake of HFD for8weeks. Also, the phosphorylationof GSK-3β and FoXO1were significantly reduced. Moreover, the above decreasestended to be more significant in Nrf2-null mice indicating that HFD feeding induced moreserious hepatic insulin resistance in nrf2-null mice.6．The mRNA levels of TNFα and IL-6were significantly higher in the livers ofNrf2-null mice than in those of WT mice at8weeks after the start of HFD feeding.Furthermore, the protein levels of TNFα and IL-6were correlated with the mRNA levels.As expected, the nucleus expression of NF-κB was significantly upregulated both in WTand Nrf2-null mice liver at8weeks after the start of HFD feeding and the upregulationwas more significant in Nrf2-null mice liver than in WT mice liver.ConclusionThe study shows that oxidative stress induced by high fat diet can activate the nucleartranslocation of Nrf2to develop its biological effects. Meanwhile, deletion of Nrf2significantly exacerbate intrahepatic insulin resistance through decreasing intracellar GSHlevel and aggravatting the degress of oxidative stress, promotting the translocation ofNF-κB and the release of inflammatory factors. The findings imply that the translocationof Nrf2could be a target for the prevention and treatment of NASH.