Analysis of the liver mitochondrial proteome in response to ethanol and S-adenosylmethionine treatments: novel molecular targets of disease and hepatoprotection

Abstract

S-adenosylmethionine (SAM) minimizes alcohol hepatotoxicity; however, the molecular mechanisms responsible for SAM hepatoprotection remain unknown. Herein, we use proteomics to determine whether the hepatoprotective action of SAM against early-stage alcoholic liver disease is linked to alterations in the mitochondrial proteome. For this, male rats were fed control or ethanol-containing liquid diets +/- SAM and liver mitochondria were prepared for proteomic analysis. Two-dimensional isoelectric focusing (2D IEF/SDS-PAGE) and blue native gel electrophoresis (BN-PAGE) were used to determine changes in matrix and oxidative phosphorylation (OxPhos) proteins, respectively. SAM coadministration minimized alcohol-dependent inflammation and preserved mitochondrial respiration. SAM supplementation preserved liver SAM levels in ethanol-fed rats; however, mitochondrial SAM levels were increased by ethanol and SAM treatments. With use of 2D IEF/SDS-PAGE, 30 proteins showed significant changes in abundance in response to ethanol, SAM, or both. Classes of proteins affected by ethanol and SAM treatments were chaperones, beta oxidation proteins, sulfur metabolism proteins, and dehydrogenase enzymes involved in methionine, glycine, and choline metabolism. BN-PAGE revealed novel changes in the levels of 19 OxPhos proteins in response to ethanol, SAM, or both. Ethanol- and SAM-dependent alterations in the proteome were not linked to corresponding changes in gene expression. In conclusion, ethanol and SAM treatment led to multiple changes in the liver mitochondrial proteome. The protective effects of SAM against alcohol toxicity are mediated, in part, through maintenance of proteins involved in key mitochondrial energy conserving and biosynthetic pathways. This study demonstrates that SAM may be a promising candidate for treatment of alcoholic liver disease.

Fig. 1.

S-adenosylmethionine (SAM) reduces ethanol-induced hepatic steatosis. Representative photomicrographs of rats maintained on control (A), ethanol (B), control + SAM (C), and ethanol + SAM (D) diets. Note that the inclusion of SAM in the ethanol diet (D) reduced the extent of steatosis compared with the ethanol-alone group (B); however, this difference was not statistically significant (P = 0.21). Representative photomicrographs are shown for n = 6 pairs of animals per group.

Fig. 2.

Representative 2D gel images of liver mitochondrial proteins. A: mitochondrial protein from livers of control, ethanol, control + SAM-, and ethanol + SAM-treated groups were separated by use of pH 3–10 isoelectric focusing (IEF) gel strips and 10% SDS-PAGE gels. B demonstrates that there were no significant differences in total protein density on gels prepared from the 4 treatment groups. Data represent the means ± SE, n = 6 animals per each group. 2-factor ANOVA; ethanol P = 0.44, SAM P = 0.96, and interaction (ethanol × SAM) P = 0.75.

Fig. 3.

Master map of mitochondrial proteins differentially altered by ethanol and SAM. Analysis of gels shown in Fig. 2 revealed 30 proteins differentially altered in abundance in response to ethanol and SAM treatment alone or in combination (circled proteins). The mass spectrometry identification and statistical analyses for these proteins is presented in Table 3. Please note that a separate and different numbering system is used for the global proteome map and table provided in the supplemental data section.

Fig. 4.

Quantification of mitochondrial proteins differentially altered by ethanol and SAM. This series of bar graphs illustrates the change in abundance (increase or decrease) of mitochondrial proteins found to be altered by ethanol, SAM, or both. The numbers below each set of bars correspond to the protein spot number given in Table 3 and the master map shown in Fig. 3. For ease in graphical presentation, proteins were separated into 4 broad functional classifications: proteins associated with chaperone functions (A), fatty acid metabolism proteins (B), oxidoreductase (dehydrogenase) proteins of key metabolism pathways (C), and miscellaneous ligases, transferases, and other proteins (D). Statistical analysis was performed as stated in materials and methods, and P values are listed in Table 3. Data represent means ± SE, n = 6 animals per group.

Fig. 5.

Representative 2D blue native gel electrophoresis (BN-PAGE) gels of liver mitochondrial proteins. A: mitochondrial protein from livers of control, ethanol, control + SAM-, and ethanol + SAM-treated groups were subjected to BN-PAGE as described in materials and methods. B: comparison of the relative quantities of complexes I, V, III, and IV in liver mitochondria from the 4 treatment groups. Data represent means ± SE, n = 4 animals per each group; *P < 0.05 compared with control; **P < 0.05 compared with EtOH. Results from 2-factor ANOVA for each complex are reported in B.

Fig. 6.

BN-PAGE master map of oxidative phosphorylation system proteins. A: BN-PAGE master protein map of the oxidative phosphorylation (OxPhos) proteins that were found in common in all gels across the 4 treatment groups. The circled proteins and adjacent number labels correspond to those proteins listed in Table 4. B: relative abundances of those proteins (–9) present in the first lane (I), which corresponds to complex I and associated proteins. C: relative abundances of those proteins (–15) present in the second lane (V), which corresponds to complex V and associated proteins. D: relative abundances of those proteins (–24) present in the third lane (III), which corresponds to complex III and associated proteins. E shows the relative abundances of those proteins (–33) present in the fourth lane (IV), which corresponds to complex IV and associated proteins. Numbers listed under each correspond to circled proteins located on the master map (A) and in Table 4. Data represent the means ± SE, n = 4 animals per group. The mass spectrometry identification and statistical analyses for these proteins are presented in Table 4. Note that not all OxPhos proteins were identified by this “miniaturized” high-throughput version of BN-PAGE gel electrophoresis.