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The effects of insulin pre-administration in mice exposed to ethanol: alleviating hepatic oxidative injury through anti-oxidative, anti-apoptotic activities and deteriorating hepatic steatosis through SRBEP-1c activation.

Liu J, Wang X, Peng Z, Zhang T, Wu H, Yu W, Kong D, Liu Y, Bai H, Liu R, Zhang X, Hai C - Int. J. Biol. Sci. (2015)

Bottom Line: Previous studies have showed that insulin, a classic regulator of glucose metabolism, has significant anti-oxidative function and plays an important role in maintaining the redox balance.First, we found insulin pre-administration alleviated acute ethanol exposure-induced liver injury and inflammation reflected by the decrease of serum AST and ALT activities, the improvement of pathological alteration and the inhibition of TNF-α and IL-6 expressions.Our study provided novel insight about the effects and mechanisms of insulin on ethanol-induced liver injury.

View Article: PubMed Central - PubMed

Affiliation: 1. Department of Toxicology, the Ministry of Education Key Lab of Hazard Assessment and Control in Special Operational Environment, Shaanxi Provincial Key Lab of Free radical biology and medicine, School of Public Health, The Fourth Military Medical University, Xi'an, 710032, P. R. China;

ABSTRACT
Alcoholic liver disease (ALD) has become an important liver disease hazard to public and personal health. Oxidative stress is believed to be responsible for the pathological changes in ALD. Previous studies have showed that insulin, a classic regulator of glucose metabolism, has significant anti-oxidative function and plays an important role in maintaining the redox balance. For addressing the effects and mechanisms of insulin pre-administration on ethanol-induced liver oxidative injury, we investigated histopathology, inflammatory factors, apoptosis, mitochondrial dysfunction, oxidative stress, antioxidant defense system, ethanol metabolic enzymes and lipid disorder in liver of ethanol-exposed mice pretreatment with insulin or not. There are several novel findings in our study. First, we found insulin pre-administration alleviated acute ethanol exposure-induced liver injury and inflammation reflected by the decrease of serum AST and ALT activities, the improvement of pathological alteration and the inhibition of TNF-α and IL-6 expressions. Second, insulin pre-administration could significantly reduce apoptosis and ameliorate mitochondrial dysfunction in liver of mice exposed to ethanol, supporting by decreasing caspases-3 activities and the ratio of Bax/Bcl-2, increasing mitochondrial viability and mitochondrial oxygen consumption, inhibition of the decline of ATP levels and mitochondrial ROS accumulation. Third, insulin pre-administration prevented ethanol-mediated oxidative stress and enhance antioxidant defense system, which is evaluated by the decline of MDA levels and the rise of GSH/GSSG, the up-regulations of antioxidant enzymes CAT, SOD, GR through Nrf-2 dependent pathway. Forth, the modification of ethanol metabolism pathway such as the inhibition of CYP2E1, the activation of ALDH might be involved in the anti-oxidative and protective effects exerted by insulin pre-administration against acute ethanol exposure in mice. Finally, insulin pre-administration deteriorated hepatic steatosis in mice exposed to ethanol might be through SRBEP-1c activation. In summary, these results indicated that insulin pre-administration effectively alleviated liver oxidative injury through anti-inflammatory, anti-oxidative and anti-apoptotic activities but also deteriorated hepatic steatosis through SRBEP-1c activation in mice exposed to ethanol. Our study provided novel insight about the effects and mechanisms of insulin on ethanol-induced liver injury.

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Insulin pre-administration enhanced antioxidant defense system in liver of mice exposed to ethanol. (A) Hepatic GSH level; (B) The value of GSH/GSSG in liver; (C) CAT, SOD-2, GR and total Nrf-2 protein expressions in liver determinate by Western blotting; (D) Value of the CAT, SOD-2, GR and total Nrf-2 protein expressions expressed as the folds of control; (E) Protein expression of Nrf-2 in cytoplasm and nuclear fractions in liver determinate by Western blotting; (F) Value of the Nrf-2 protein expression in cytoplasm and nuclear fractions expressions expressed as the folds of control; (G) Hepatic CAT activity; (H) Hepatic SOD activity; (I) Hepatic GR activity. Normalizations of Western blots were ensured by Tubulin in total protein, β-actin in cytoplasm fraction and Lamin B in nuclear fraction. Data of A-B and G-I were expressed as mean ± SD (n = 8). Data of D and F were expressed as the mean ± SD of three independent experiments (n = 3). *P< 0.05, compared to control group; #P < 0.05, compared to EtOH group.
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Figure 5: Insulin pre-administration enhanced antioxidant defense system in liver of mice exposed to ethanol. (A) Hepatic GSH level; (B) The value of GSH/GSSG in liver; (C) CAT, SOD-2, GR and total Nrf-2 protein expressions in liver determinate by Western blotting; (D) Value of the CAT, SOD-2, GR and total Nrf-2 protein expressions expressed as the folds of control; (E) Protein expression of Nrf-2 in cytoplasm and nuclear fractions in liver determinate by Western blotting; (F) Value of the Nrf-2 protein expression in cytoplasm and nuclear fractions expressions expressed as the folds of control; (G) Hepatic CAT activity; (H) Hepatic SOD activity; (I) Hepatic GR activity. Normalizations of Western blots were ensured by Tubulin in total protein, β-actin in cytoplasm fraction and Lamin B in nuclear fraction. Data of A-B and G-I were expressed as mean ± SD (n = 8). Data of D and F were expressed as the mean ± SD of three independent experiments (n = 3). *P< 0.05, compared to control group; #P < 0.05, compared to EtOH group.

Mentions: The mechanisms underlying the insulin-induced antioxidative protective effects against ethanol is not entirely clear until now. To further investigate the mechanisms of the antioxidative actions exerted by insulin pre-administration, we measured the antioxidant defense system such as antioxidant GSH, the value of GSH/GSSG, the protein expressions of Nrf-2 and related antioxidant enzymes, and the activities of SOD, CAT and GR. As shown in Fig.5 A and B, in ethanol-treated mice there was significant decrease both in hepatic GSH level and GSH/GSSG ratio compared with those in control group. However, insulin pre-administration notability suppressed the decrease of the level of GSH and the value of GSH/GSSG induced by ethanol exposure. Results in Fig.5 C and D showed that an increase of the protein expressions of GR, CAT and total Nrf-2 in insulin-treated mice and a decrease of the protein expressions of GR, SOD-2, CAT and total Nrf-2 in ethanol-treated mice were observed. As expect, in the insulin pre-administration mice the protein expressions of GR, SOD-2, CAT and total Nrf-2 were significantly up-regulated compared with these of mice treated with ethanol. For further and precisely investigate the role of Nrf-2 in the antioxidative actions against ethanol, we next examined the expressions of Nrf-2 in cytoplasm and nuclear extract. As demonstrated in Fig.5 E and F, insulin administration alone markedly increased and ethanol exposure slightly increased the nuclear Nrf-2 protein expressions, indicating both of insulin and ethanol treatment could promote the nuclear translocation of Nrf-2. Insulin pre-administration showed a notably increase of nuclear Nrf-2 expression compared with ethanol group, suggesting insulin significantly promoted the nuclear translocation of Nrf-2 induced by ethanol. We then determined the activities of antioxidant enzymes including SOD, CAT and GR to evaluate the antioxidative effects of insulin pre-administration. As illustrated in Fig.5 G-I, insulin administration alone increased and ethanol treatment decrease the activities of SOD, CAT and GR, and insulin pre-administration significantly suppressed the decline of these antioxidant enzymes activities mediated by ethanol exposure. Together with the oxidative data, these results indicated that insulin pre-administration reduced oxidative stress and enhanced antioxidant system in mice in mice subjected to ethanol exposure might through the activation of Nrf-2.


The effects of insulin pre-administration in mice exposed to ethanol: alleviating hepatic oxidative injury through anti-oxidative, anti-apoptotic activities and deteriorating hepatic steatosis through SRBEP-1c activation.

Liu J, Wang X, Peng Z, Zhang T, Wu H, Yu W, Kong D, Liu Y, Bai H, Liu R, Zhang X, Hai C - Int. J. Biol. Sci. (2015)

Insulin pre-administration enhanced antioxidant defense system in liver of mice exposed to ethanol. (A) Hepatic GSH level; (B) The value of GSH/GSSG in liver; (C) CAT, SOD-2, GR and total Nrf-2 protein expressions in liver determinate by Western blotting; (D) Value of the CAT, SOD-2, GR and total Nrf-2 protein expressions expressed as the folds of control; (E) Protein expression of Nrf-2 in cytoplasm and nuclear fractions in liver determinate by Western blotting; (F) Value of the Nrf-2 protein expression in cytoplasm and nuclear fractions expressions expressed as the folds of control; (G) Hepatic CAT activity; (H) Hepatic SOD activity; (I) Hepatic GR activity. Normalizations of Western blots were ensured by Tubulin in total protein, β-actin in cytoplasm fraction and Lamin B in nuclear fraction. Data of A-B and G-I were expressed as mean ± SD (n = 8). Data of D and F were expressed as the mean ± SD of three independent experiments (n = 3). *P< 0.05, compared to control group; #P < 0.05, compared to EtOH group.
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Related In: Results  -  Collection

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Figure 5: Insulin pre-administration enhanced antioxidant defense system in liver of mice exposed to ethanol. (A) Hepatic GSH level; (B) The value of GSH/GSSG in liver; (C) CAT, SOD-2, GR and total Nrf-2 protein expressions in liver determinate by Western blotting; (D) Value of the CAT, SOD-2, GR and total Nrf-2 protein expressions expressed as the folds of control; (E) Protein expression of Nrf-2 in cytoplasm and nuclear fractions in liver determinate by Western blotting; (F) Value of the Nrf-2 protein expression in cytoplasm and nuclear fractions expressions expressed as the folds of control; (G) Hepatic CAT activity; (H) Hepatic SOD activity; (I) Hepatic GR activity. Normalizations of Western blots were ensured by Tubulin in total protein, β-actin in cytoplasm fraction and Lamin B in nuclear fraction. Data of A-B and G-I were expressed as mean ± SD (n = 8). Data of D and F were expressed as the mean ± SD of three independent experiments (n = 3). *P< 0.05, compared to control group; #P < 0.05, compared to EtOH group.
Mentions: The mechanisms underlying the insulin-induced antioxidative protective effects against ethanol is not entirely clear until now. To further investigate the mechanisms of the antioxidative actions exerted by insulin pre-administration, we measured the antioxidant defense system such as antioxidant GSH, the value of GSH/GSSG, the protein expressions of Nrf-2 and related antioxidant enzymes, and the activities of SOD, CAT and GR. As shown in Fig.5 A and B, in ethanol-treated mice there was significant decrease both in hepatic GSH level and GSH/GSSG ratio compared with those in control group. However, insulin pre-administration notability suppressed the decrease of the level of GSH and the value of GSH/GSSG induced by ethanol exposure. Results in Fig.5 C and D showed that an increase of the protein expressions of GR, CAT and total Nrf-2 in insulin-treated mice and a decrease of the protein expressions of GR, SOD-2, CAT and total Nrf-2 in ethanol-treated mice were observed. As expect, in the insulin pre-administration mice the protein expressions of GR, SOD-2, CAT and total Nrf-2 were significantly up-regulated compared with these of mice treated with ethanol. For further and precisely investigate the role of Nrf-2 in the antioxidative actions against ethanol, we next examined the expressions of Nrf-2 in cytoplasm and nuclear extract. As demonstrated in Fig.5 E and F, insulin administration alone markedly increased and ethanol exposure slightly increased the nuclear Nrf-2 protein expressions, indicating both of insulin and ethanol treatment could promote the nuclear translocation of Nrf-2. Insulin pre-administration showed a notably increase of nuclear Nrf-2 expression compared with ethanol group, suggesting insulin significantly promoted the nuclear translocation of Nrf-2 induced by ethanol. We then determined the activities of antioxidant enzymes including SOD, CAT and GR to evaluate the antioxidative effects of insulin pre-administration. As illustrated in Fig.5 G-I, insulin administration alone increased and ethanol treatment decrease the activities of SOD, CAT and GR, and insulin pre-administration significantly suppressed the decline of these antioxidant enzymes activities mediated by ethanol exposure. Together with the oxidative data, these results indicated that insulin pre-administration reduced oxidative stress and enhanced antioxidant system in mice in mice subjected to ethanol exposure might through the activation of Nrf-2.

Bottom Line: Previous studies have showed that insulin, a classic regulator of glucose metabolism, has significant anti-oxidative function and plays an important role in maintaining the redox balance.First, we found insulin pre-administration alleviated acute ethanol exposure-induced liver injury and inflammation reflected by the decrease of serum AST and ALT activities, the improvement of pathological alteration and the inhibition of TNF-α and IL-6 expressions.Our study provided novel insight about the effects and mechanisms of insulin on ethanol-induced liver injury.

View Article: PubMed Central - PubMed

Affiliation: 1. Department of Toxicology, the Ministry of Education Key Lab of Hazard Assessment and Control in Special Operational Environment, Shaanxi Provincial Key Lab of Free radical biology and medicine, School of Public Health, The Fourth Military Medical University, Xi'an, 710032, P. R. China;

ABSTRACT
Alcoholic liver disease (ALD) has become an important liver disease hazard to public and personal health. Oxidative stress is believed to be responsible for the pathological changes in ALD. Previous studies have showed that insulin, a classic regulator of glucose metabolism, has significant anti-oxidative function and plays an important role in maintaining the redox balance. For addressing the effects and mechanisms of insulin pre-administration on ethanol-induced liver oxidative injury, we investigated histopathology, inflammatory factors, apoptosis, mitochondrial dysfunction, oxidative stress, antioxidant defense system, ethanol metabolic enzymes and lipid disorder in liver of ethanol-exposed mice pretreatment with insulin or not. There are several novel findings in our study. First, we found insulin pre-administration alleviated acute ethanol exposure-induced liver injury and inflammation reflected by the decrease of serum AST and ALT activities, the improvement of pathological alteration and the inhibition of TNF-α and IL-6 expressions. Second, insulin pre-administration could significantly reduce apoptosis and ameliorate mitochondrial dysfunction in liver of mice exposed to ethanol, supporting by decreasing caspases-3 activities and the ratio of Bax/Bcl-2, increasing mitochondrial viability and mitochondrial oxygen consumption, inhibition of the decline of ATP levels and mitochondrial ROS accumulation. Third, insulin pre-administration prevented ethanol-mediated oxidative stress and enhance antioxidant defense system, which is evaluated by the decline of MDA levels and the rise of GSH/GSSG, the up-regulations of antioxidant enzymes CAT, SOD, GR through Nrf-2 dependent pathway. Forth, the modification of ethanol metabolism pathway such as the inhibition of CYP2E1, the activation of ALDH might be involved in the anti-oxidative and protective effects exerted by insulin pre-administration against acute ethanol exposure in mice. Finally, insulin pre-administration deteriorated hepatic steatosis in mice exposed to ethanol might be through SRBEP-1c activation. In summary, these results indicated that insulin pre-administration effectively alleviated liver oxidative injury through anti-inflammatory, anti-oxidative and anti-apoptotic activities but also deteriorated hepatic steatosis through SRBEP-1c activation in mice exposed to ethanol. Our study provided novel insight about the effects and mechanisms of insulin on ethanol-induced liver injury.

Show MeSH
Related in: MedlinePlus