Research On The Key Role And Mechanisms Of Insulin Signaling In Alcoholic Liver Injury

【Background】Alcoholic liver injury has seriously threatened to the health of crowd with drinking and also becomes as serious problem to public health worldwide. It is generally believed that oxidative stress, inflammation, apoptosis, mitochondrial dysfunction and the toxicity of acetaldehyde are all closely related with the pathogenesis of alcoholic liver injury.Many studies have found that excessive ethanol consumption could lead to the damnification of insulin signaling. However, the effects of insulin signaling on the susceptibility of alcoholic liver injury are not well understood until now. Previous literatures suggest that insulin, as an important endocrine hormone regulating glucose homeostasis, possesses various biological effects including maintaining mitochondrial function, anti-apoptosis, anti-inflammatory, anti-oxidative and so on. Recently research in our laboratory also confirmed that insulin signal plays an important role in the regulation of intracellular redox balance via Nrf-2 dependent antioxidase activation. The liver is also one of the insulin sensitive target organs. These phenomenones suggest that insulin signaling activation might be an important endogenous protective mechanism againstalcoholic liver injury. The most typical manifestation of insulin signaling dysfunction is insulin resistance and many researches indicated that insulin resistance can cause the disorder of redox imbalance in many tissues and organs including the liver. We hypothesized that insulin signaling dysfunction in drinkers may play an important promoting role in the occurrence and development of alcoholic liver injury. According to the above research progress and our prophase work, we propose the following scientific problems: What are the effects and relative molecular mechanisms of insulin signaling activation on alcoholic liver injury? What are the effects and relative molecular mechanisms of insulin signaling dysfunction on alcoholic liver injury? The answers of these above questions will undoubtedly help to elucidate the key role of insulin signaling in the pathogenesis of alcoholic liver injury, and it also will provide a new insight for the prevention and treatment of alcoholic liver injury. Based on above, it will help to preliminarily reveal the different sensitivity on drinking harm between population with different insulin level or different state of insulin sensitivity, it also will help to provide theoretical and experimental foundation for preventing and treating alcoholic liver injury in targeted people(such as type 1 diabetes and type 2 diabetes mellitus patients).【Aims】1. To investigate the effects and underlying mechanisms of insulin signaling activating on alcoholic liver injury.2. To investigate the effects and underlying mechanisms of insulin resistance on alcoholic liver injury.3. To clarify the changes of oxidative stress, mitochondrial dysfunction, apoptosis,inflammation, lipid metabolism disorder in the effects of insulin signaling on alcoholic liver injury.4. To elucidate the role of Nrf-2 relative antioxidant defense system, alcohol metabolizing enzyme represented by CYP2E1, NO generation mediated by i NOS, and serum Adiponectin level in the effects of insulin signaling on alcoholic liver injury.【Methods】1. The vitro experiment about the effects of insulin signaling activation on alcoholic liver injury: L02 cells were incubated with 50 n M insulin for 4 h and interval blank treatment for 4 h, and then exposed to high dose of ethanol. Cell viability detection by MTT, ALT and AST activities detections in culture medium by kits, and morphological observation were used to evaluate cell injury. Cell apoptosis detection by Annexin V-FITC/ PI apoptosis detection kit, Bax/Bcl-2 protein expressions by Western Blotting,Caspase-3 activity detection by kit were used to used to evaluate apoptosis. MMP detection by Rhodamine 123, Mitochondrial ROS detection by Mito SOX, open of m PTP detection by kit, ATP detection by kit, and Rate of mitochondrial oxygen consumption detection by Clark oxygen consumption instrument were used to evaluate mitochondrial function. Intracellular ROS detections by DCFH-DA and DHE, MDA and GSSG detections by corresponding kits were used to evaluate oxidative stress. GSH level and GSH/GSSG ratio detections by corresponding kits, SOD and GR activities detections by corresponding kits, the detection of Nrf-2 protein expressions by Western Blotting were used to evaluate antioxidant defense system. The CYP2E1 activity detection by kit,protein expressions of ADH,ALDH,CYP2E1 detection by Western Blotting, CYP2E1 m RNA expression detection by RT-PCR were used to evaluate the changes of ethanol metabolism enzymes. The detections of phosphorylation protein expressions of Akt and Erk and the using of PI3 K inhibitor wortmannin and ERK inhibitor U0126 were used to investigate the role of PI3K/Akt and Erk signaling in the effects of insulin signaling activation.2. The vivo experiment about the effects of insulin signaling activation on alcoholic liver injury: Mice were given insulin by intraperitoneal injection to activate the insulin signaling 4h-prior and then following to ethanol insult twice a day for 7 days. After the experiment,the body weight, the values of liver weight/body weight, blood glucose were detected. Liver histological alterations by H-E Stain, ALT and AST activities in serum detections by corresponding kits were used to evaluate liver injury. Adiponectin level detection in serum by ELISA was used to investigate the role of serum Adiponectin in thecirculatory system. Hepatic MPO activity detection by kit, hepatic m RNA expressions of TNF-α and IL-6 by RT-PCR, TNF-α level in serum by ELISA were used to evaluate inflammation. Hepatic caspases-3 activity detection by kit, protein expressions of hepatic Bcl-2 and Bax detection by Western Blotting were used to evaluate apoptosis.Mitochondrial viability detection by MTT, ATP detection in liver homogenate by kit and the rate of mitochondrial oxygen consumption detection by oxygen consumption instrument were used to evaluate mitochondrial function. Hepatic ROS accumulation by DHE staining, hepatic MDA and GSSG levels and mitochondrial MDA level detections by kits were used to evaluate hepatic oxidative stress. Hepatic GSH level and the value of GSH/GSSG detection by kits, the protein expressions of Nrf-2 and related antioxidant enzymes by Western Blotting, and the activities of PRX-1, CAT, SOD-1, SOD-2, GR,GCLC and GPX-1 by corresponding kits were used to evaluate hepatic antioxidant defense system. The CYP2E1 m RNA expressions detections by RT-PCR, ADH, ALDH,CYP2E1 activities and protein expressions detections were used to evaluate the changes of hepatic ethanol metabolism pathway. At the end, hepatic triglyceride detection by kit and SRBEP-1c m RNA and protein expression detections were used to observe the change of hepatic steatosis.3. The vitro experiment about the effects of insulin signaling dysfunction on alcoholic liver injury: L02 cells were incubated with 5μM dexamethasone for 24 h to induce IR,insulin-stimulated phosphorylation of Akt and cellular glucose uptake were used to identify insulin resistance. We observed the effects of insulin resistance on alcohol induced cell viability loss, apoptosis, mitochondrial dysfunction and oxidative stress,while the changes of antioxidant defense system and CYP2E1 were also observed. The main experimental methods are consistent with that of vitro experiment(1) about insulin signaling activation.4. The vivo experiment about the effects of insulin signaling dysfunction on alcoholic liver injury: Mice were given high fat diet with t BHP for 30 days to induce IR and then following to ethanol insult twice a day for 7 days. Fasting glucose and insulin level detections by kits, and IPGTT/IPITT were used to indentify IR. We observed the effects ofinsulin resistance on alcohol induced liver injury, apoptosis, mitochondrial dysfunction,oxidative stress and hepatic steatosis, while the changes of antioxidant defense system and ethanol metabolism enzymes were also detected. The main experimental methods are consistent with that of vivo experiment(2) about insulin signaling activation.【Results】1. Insulin signaling activation by insulin pre-treatment could exert significantly inhibitory effects on ethanol-induced hepatocellular viability injury, apoptosis,mitochondrial dysfunction, and oxidative stress. Insulin pre-treatment enhanced antioxidant defense system via Nrf-2 activation and inhibited CYP2E1 activation in cells exposed to ethanol. PI3 K and Erk inhibitors could abolish the protective effects exerted by insulin pre-treatment to a certain extent.2. Insulin signaling activation by insulin pre-treatment inhibited ethanol-induced the decrease of liver weight/body weight and the decrease of blood glucose, it also alleviated liver injury, inflammation, apoptosis, mitochondrial dysfunction and oxidative stress in mice exposed to ethanol. Insulin pre-administration enhanced the capacity of antioxidant defense system via Nrf-2 activation, meanwhile it also inhibited ethanol-induced hepatic CYP2E1 and i NOS activation, and the decrease of serum Adiponectin level. But insulin pre-treatment also promoted the hepatic SREBP-1 activation and the increase of hepatic triglyceride in mice exposed to ethanol.3. Dexamethasone treatment successfully induced insulin resistance in L02 cells, it also increase the intracellular ROS level. Insulin resistance promoted ethanol-induced hepatocellular viability injury, apoptosis, mitochondrial dysfunction, and oxidative stress.Meanwhile we found that insulin resistance weakened the antioxidant defense system via Nrf-2 inactivation, and it also further enhanced CYP2E1 activation in cells exposed to ethanol.4. High fat diet and t BHP administration successfully induced insulin resistance in mice. IR promoted ethanol-induced the decrease of body weight and the increase of liver weight/body weight. Insulin resistance also enhanced liver injury, inflammation, apoptosis,mitochondrial dysfunction and oxidative stress in mice exposed to ethanol. Insulinresistance could weaken the capacity of antioxidant defense system via Nrf-2 inactivation,meanwhile insulin resistance further promoted the hepatic CYP2E1 and i NOS activation,and the decrease of serum Adiponectin level in mice exposed to ethanol. And insulin resistance promoted ethanol-induced hepatic SREBP-1 activation and the increase of hepatic triglyceride.【Conclusions】1. Insulin signaling activation exerted protective effects against oxidative liver injury through anti-oxidant, anti-inflammatory, anti-apoptosis and the maintaining mitochondrial function, but meanwhile it also deteriorated hepatic lipid metabolism disorder. These results suggested that insulin signaling activation might be an important endogenous protective mechanism against alcoholic oxidative liver injury, but also acted as an important factor to promote alcoholic fatty liver, being manifested by “double-edged sword” effect.2. The protective mechanisms of insulin signaling activation on alcoholic liver oxidative damage might closely related to the enhance of antioxidant defense system function dependent on Nrf-2 activation, the inhibition of CYP2E1 and i NOS, and increase of serum adiponectin level. The mechanism of insulin signaling activation promoting alcoholic fatty liver might be closely related to the activation of hepatic SREBP-1c.3. Insulin resistance promoted alcoholic liver injury through pro-oxidative,pro-inflammatory, pro-apoptosis and the deterioration of mitochondrial function,suggesting that insulin signaling dysfunction may be an important risk factor for alcoholic liver injury.4. The mechanisms of insulin resistance promoting alcoholic liver injury might closely related to the weakening of antioxidant defense system function dependent on Nrf-2 activation, the activation of CYP2E1 and i NOS, the decrease of serum adiponectin level, and the activation of hepatic SREBP-1c.