Comparative Study
doi: 10.1002/hep.25543.
Argininosuccinate synthase conditions the response to acute and chronic ethanol-induced liver injury in mice
Affiliations
- PMID: 22213272
- PMCID: PMC4632528
- DOI: 10.1002/hep.25543
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Comparative Study
Argininosuccinate synthase conditions the response to acute and chronic ethanol-induced liver injury in mice
Hepatology.
2012 May.
Abstract
Argininosuccinate synthase (ASS) is the rate-limiting enzyme in both the urea and the L-citrulline/nitric oxide (NO·) cycles regulating protein catabolism, ammonia levels, and NO· generation. Because a proteomics analysis identified ASS and nitric oxide synthase-2 (NOS2) as coinduced in rat hepatocytes by chronic ethanol consumption, which also occurred in alcoholic liver disease (ALD) and in cirrhosis patients, we hypothesized that ASS could play a role in ethanol binge and chronic ethanol-induced liver damage. To investigate the contribution of ASS to the pathophysiology of ALD, wildtype (WT) and Ass(+/-) mice (Ass(-/-) are lethal due to hyperammonemia) were exposed to an ethanol binge or to chronic ethanol drinking. Compared with WT, Ass(+/-) mice given an ethanol binge exhibited decreased steatosis, lower NOS2 induction, and less 3-nitrotyrosine (3-NT) protein residues, indicating that reducing nitrosative stress by way of the L-citrulline/NO· pathway plays a significant role in preventing liver damage. However, chronic ethanol-treated Ass(+/-) mice displayed enhanced liver injury compared with WT mice. This was due to hyperammonemia, lower phosphorylated AMP-activated protein kinase alpha (pAMPKα) to total AMPKα ratio, decreased sirtuin-1 (Sirt-1) and peroxisomal proliferator-activated receptor coactivator-1α (Pgc1α) messenger RNAs (mRNAs), lower fatty acid β-oxidation due to down-regulation of carnitine palmitoyl transferase-II (CPT-II), decreased antioxidant defense, and elevated lipid peroxidation end-products in spite of comparable nitrosative stress but likely reduced NOS3.
Conclusion: Partial Ass ablation protects only in acute ethanol-induced liver injury by decreasing nitrosative stress but not in a more chronic scenario where oxidative stress and impaired fatty acid β-oxidation are key events.
Copyright © 2011 American Association for the Study of Liver Diseases.
Figures
Liver H&E staining from rats fed the control diet showed minimal steatosis (A, left), while rats fed the ethanol diet showed periportal and pericentral micro- (
) and macrovesicular (
) steatosis (A, right. PV: portal vein). Serum ammonia and urea. Results are average values ± SEM. n=10/group; *p<0.05 and ***p<0.001 for chronic ethanol vs control (B). Transmission electron microscopy showing ultrastructural features (micro- (
) and macrovesicular steatosis (
), 
ER: endoplasmic reticulum, L: lysosome, M: mitochondria, N: nucleus). Livers from control rats show normal mitochondria, minimal microvesicular steatosis and stacked ER (left), while livers from ethanol-fed rats show greater micro- and macrovesicular steatosis, disrupted ER and dilated mitochondria (right) (C). Western blot analysis for CYP2E1, NOS2, DYNAMIN, HSP70 and ACTIN in HControl and HEthanol. The quantification of the intensity of the signals corrected by that of ACTIN is indicated under the blots. Results are average values ± SEM. n=3/group; **p<0.01 and ***p<0.001 for HEthanol
vs HControl
(D). Tryptic peptides of ASS were identified by mass spectrometry as highlighted in orange in the sequence. The calculation of their abundance ratios is shown in the chromatograms reconstructed from LC/MS-MS data. Red lines are raw spectra, blue lines are smoothed spectra, green lines are the area under the curve used in the ratio calculation and horizontal cyan lines are the background in the spectra. n=5/group (E).
) and macrovesicular () steatosis (A, right. PV: portal vein). Serum ammonia and urea. Results are average values ± SEM. n=10/group; *p<0.05 and ***p<0.001 for chronic ethanol vs control (B). Transmission electron microscopy showing ultrastructural features (micro- () and macrovesicular steatosis (), ER: endoplasmic reticulum, L: lysosome, M: mitochondria, N: nucleus). Livers from control rats show normal mitochondria, minimal microvesicular steatosis and stacked ER (left), while livers from ethanol-fed rats show greater micro- and macrovesicular steatosis, disrupted ER and dilated mitochondria (right) (C). Western blot analysis for CYP2E1, NOS2, DYNAMIN, HSP70 and ACTIN in HControl and HEthanol. The quantification of the intensity of the signals corrected by that of ACTIN is indicated under the blots. Results are average values ± SEM. n=3/group; **p<0.01 and ***p<0.001 for HEthanol
vs HControl
(D). Tryptic peptides of ASS were identified by mass spectrometry as highlighted in orange in the sequence. The calculation of their abundance ratios is shown in the chromatograms reconstructed from LC/MS-MS data. Red lines are raw spectra, blue lines are smoothed spectra, green lines are the area under the curve used in the ratio calculation and horizontal cyan lines are the background in the spectra. n=5/group (E).
Liver H&E staining from rats fed the control diet showed minimal steatosis (A, left), while rats fed the ethanol diet showed periportal and pericentral micro- () and macrovesicular () steatosis (A, right. PV: portal vein). Serum ammonia and urea. Results are average values ± SEM. n=10/group; *p<0.05 and ***p<0.001 for chronic ethanol vs control (B). Transmission electron microscopy showing ultrastructural features (micro- () and macrovesicular steatosis (), ER: endoplasmic reticulum, L: lysosome, M: mitochondria, N: nucleus). Livers from control rats show normal mitochondria, minimal microvesicular steatosis and stacked ER (left), while livers from ethanol-fed rats show greater micro- and macrovesicular steatosis, disrupted ER and dilated mitochondria (right) (C). Western blot analysis for CYP2E1, NOS2, DYNAMIN, HSP70 and ACTIN in HControl and HEthanol. The quantification of the intensity of the signals corrected by that of ACTIN is indicated under the blots. Results are average values ± SEM. n=3/group; **p<0.01 and ***p<0.001 for HEthanol
vs HControl
(D). Tryptic peptides of ASS were identified by mass spectrometry as highlighted in orange in the sequence. The calculation of their abundance ratios is shown in the chromatograms reconstructed from LC/MS-MS data. Red lines are raw spectra, blue lines are smoothed spectra, green lines are the area under the curve used in the ratio calculation and horizontal cyan lines are the background in the spectra. n=5/group (E).
) and macrovesicular () steatosis (A, right. PV: portal vein). Serum ammonia and urea. Results are average values ± SEM. n=10/group; *p<0.05 and ***p<0.001 for chronic ethanol vs control (B). Transmission electron microscopy showing ultrastructural features (micro- () and macrovesicular steatosis (), ER: endoplasmic reticulum, L: lysosome, M: mitochondria, N: nucleus). Livers from control rats show normal mitochondria, minimal microvesicular steatosis and stacked ER (left), while livers from ethanol-fed rats show greater micro- and macrovesicular steatosis, disrupted ER and dilated mitochondria (right) (C). Western blot analysis for CYP2E1, NOS2, DYNAMIN, HSP70 and ACTIN in HControl and HEthanol. The quantification of the intensity of the signals corrected by that of ACTIN is indicated under the blots. Results are average values ± SEM. n=3/group; **p<0.01 and ***p<0.001 for HEthanol
vs HControl
(D). Tryptic peptides of ASS were identified by mass spectrometry as highlighted in orange in the sequence. The calculation of their abundance ratios is shown in the chromatograms reconstructed from LC/MS-MS data. Red lines are raw spectra, blue lines are smoothed spectra, green lines are the area under the curve used in the ratio calculation and horizontal cyan lines are the background in the spectra. n=5/group (E).
The expression of ASS, ASL, ARG1, arginine residues, NOS2, 3-NT residues and ACTIN in HControl and HEthanol was analyzed by Western blot. n=4 (A). Human liver explants from healthy (n=5), stage-3 HCV-cirrhotic (n=4) and ALD (n=2) patients were evaluated for the expression of ASS, NOS2, 3-NT residues, COLLAGEN-I and ACTIN by Western blot (B). Rat hepatocytes were either treated with 5 μM citrulline, an inducer of ASS, transfected with control siRNA or Ass siRNA or incubated with inhibitors of ASS such as 15 μM fumonisin B1, 10 μM mithramycin A and 50 μM α-MDLA. Experiments were repeated three times and a representative blot for NOS2 and ASS is shown (C-E). The quantification of the intensity of the signal corrected by that of ACTIN is indicated under all blots. ***p<0.001 and **p<0.01 for treated or diseased vs control.
Western blot analysis for the urea cycle enzymes. The quantification of each signal corrected by that of CALNEXIN is shown for the ethanol binge model (A) and for the chronic ethanol feeding model (B). Liver and serum ammonia and urea (C-E). Results are average values ± SEM. n=8/group; *p<0.05 and **p<0.01 for binge or chronic ethanol vs control. •p<0.05 and ••p<0.01 for Ass+/− vs WT.
H&E staining showed increased micro- (
) and macrovesicular (
) steatosis in binged WT compared with Ass+/− mice (A). Steatosis grade (B), liver triglycerides (C) and ALT activity (D). The ethanol Lieber-DeCarli diet caused more inflammation (
), micro- (
) and macrovesicular (
) steatosis in Ass+/− than in WT mice as shown by the H&E staining (E). The scores for inflammation, necrosis, ductular reaction and steatosis are shown in (F). PV: portal vein. Results are average values ± SEM. n=8/group; *p<0.05 and ***p<0.001 for binge or chronic ethanol vs control; •p<0.05 and ••p<0.01 for Ass+/− vs WT.
) and macrovesicular () steatosis in binged WT compared with Ass+/− mice (A). Steatosis grade (B), liver triglycerides (C) and ALT activity (D). The ethanol Lieber-DeCarli diet caused more inflammation (), micro- () and macrovesicular () steatosis in Ass+/− than in WT mice as shown by the H&E staining (E). The scores for inflammation, necrosis, ductular reaction and steatosis are shown in (F). PV: portal vein. Results are average values ± SEM. n=8/group; *p<0.05 and ***p<0.001 for binge or chronic ethanol vs control; •p<0.05 and ••p<0.01 for Ass+/− vs WT.
H&E staining showed increased micro- () and macrovesicular () steatosis in binged WT compared with Ass+/− mice (A). Steatosis grade (B), liver triglycerides (C) and ALT activity (D). The ethanol Lieber-DeCarli diet caused more inflammation (), micro- () and macrovesicular () steatosis in Ass+/− than in WT mice as shown by the H&E staining (E). The scores for inflammation, necrosis, ductular reaction and steatosis are shown in (F). PV: portal vein. Results are average values ± SEM. n=8/group; *p<0.05 and ***p<0.001 for binge or chronic ethanol vs control; •p<0.05 and ••p<0.01 for Ass+/− vs WT.
) and macrovesicular () steatosis in binged WT compared with Ass+/− mice (A). Steatosis grade (B), liver triglycerides (C) and ALT activity (D). The ethanol Lieber-DeCarli diet caused more inflammation (), micro- () and macrovesicular () steatosis in Ass+/− than in WT mice as shown by the H&E staining (E). The scores for inflammation, necrosis, ductular reaction and steatosis are shown in (F). PV: portal vein. Results are average values ± SEM. n=8/group; *p<0.05 and ***p<0.001 for binge or chronic ethanol vs control; •p<0.05 and ••p<0.01 for Ass+/− vs WT.
IHC for NOS2 protein (
) (A) and 3-NT residues (
) (B) in binged WT and Ass+/− mice. Morphometry analysis for NOS2 and 3-NT staining (C). IHC and morphometry analysis for NOS2 and 3-NT in chronic ethanol-fed WT and Ass+/− mice (D-F). CV: central vein. Results are average values ± SEM. n=8/group; *p<0.05 and **p<0.01 for binge or chronic ethanol vs control; •p<0.05 for Ass+/− vs WT.
) (A) and 3-NT residues () (B) in binged WT and Ass+/− mice. Morphometry analysis for NOS2 and 3-NT staining (C). IHC and morphometry analysis for NOS2 and 3-NT in chronic ethanol-fed WT and Ass+/− mice (D-F). CV: central vein. Results are average values ± SEM. n=8/group; *p<0.05 and **p<0.01 for binge or chronic ethanol vs control; •p<0.05 for Ass+/− vs WT.
IHC for NOS2 protein () (A) and 3-NT residues () (B) in binged WT and Ass+/− mice. Morphometry analysis for NOS2 and 3-NT staining (C). IHC and morphometry analysis for NOS2 and 3-NT in chronic ethanol-fed WT and Ass+/− mice (D-F). CV: central vein. Results are average values ± SEM. n=8/group; *p<0.05 and **p<0.01 for binge or chronic ethanol vs control; •p<0.05 for Ass+/− vs WT.
) (A) and 3-NT residues () (B) in binged WT and Ass+/− mice. Morphometry analysis for NOS2 and 3-NT staining (C). IHC and morphometry analysis for NOS2 and 3-NT in chronic ethanol-fed WT and Ass+/− mice (D-F). CV: central vein. Results are average values ± SEM. n=8/group; *p<0.05 and **p<0.01 for binge or chronic ethanol vs control; •p<0.05 for Ass+/− vs WT.
Total and mitochondrial GSH levels in WT and Ass+/− mice chronically fed ethanol (A). Western blot analysis for GCLC and GCLM in WT and Ass+/− mice chronically fed ethanol. The quantification of the intensity of the signals corrected by that of ACTIN is indicated under the blots (B). Catalase (C), GT (D) and GR (E) activities. Results are average values ± SEM. n=8/group; *p<0.05, **p<0.01 and ***p<0.001 for chronic ethanol vs control; •p<0.05 and ••p<0.05 for Ass+/− vs WT.
Western blot analysis for PPARγ, SREBP-1, PPARα, pAMPKα, AMPKα1, AMPKα2, ACTIN and CALNEXIN in both models in WT and Ass+/− mice (A). Western blot analysis for FAS, ACC2 and CALNEXIN in both models in WT and Ass+/− mice (B). Sirt-1
(C), Pgc1-α
(D) and Cpt-1
(E) mRNAs in chronic ethanol-fed WT and Ass+/− mice. Western blot analysis for CPT-II and CALNEXIN in chronically ethanol-fed WT than in Ass+/− mice (F). The quantification of the intensity of all signals corrected by that of ACTIN or CALNEXIN is indicated under the blots. Results are average values ± SEM. n=8/group; *p<0.05 and ***p<0.001 for binge or chronic ethanol vs control; •p<0.05 and ••p<0.01 for Ass+/− vs WT.
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