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Energy determinants GAPDH and NDPK act as genetic modifiers for hepatocyte inclusion formation.

Snider NT, Weerasinghe SV, Singla A, Leonard JM, Hanada S, Andrews PC, Lok AS, Omary MB - J. Cell Biol. (2011)

Bottom Line: Prominent histological features of some chronic human liver diseases are hepatocyte ballooning and Mallory-Denk bodies.GAPDH knockdown depleted bioenergetic and antioxidant enzymes and elevated hepatocyte ROS, whereas GAPDH overexpression decreased hepatocyte ROS.We propose that GAPDH and NDPK are genetic modifiers of murine DDC-induced liver injury and potentially human liver disease.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Molecular and Integrative Physiology, University of Michigan, Ann Arbor, MI 48109, USA. nsnider@umich.edu

ABSTRACT
Genetic factors impact liver injury susceptibility and disease progression. Prominent histological features of some chronic human liver diseases are hepatocyte ballooning and Mallory-Denk bodies. In mice, these features are induced by 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) in a strain-dependent manner, with the C57BL and C3H strains showing high and low susceptibility, respectively. To identify modifiers of DDC-induced liver injury, we compared C57BL and C3H mice using proteomic, biochemical, and cell biological tools. DDC elevated reactive oxygen species (ROS) and oxidative stress enzymes preferentially in C57BL livers and isolated hepatocytes. C57BL livers and hepatocytes also manifested significant down-regulation, aggregation, and nuclear translocation of glyceraldehyde 3-phosphate dehydrogenase (GAPDH). GAPDH knockdown depleted bioenergetic and antioxidant enzymes and elevated hepatocyte ROS, whereas GAPDH overexpression decreased hepatocyte ROS. On the other hand, C3H livers had higher expression and activity of the energy-generating nucleoside-diphosphate kinase (NDPK), and knockdown of hepatocyte NDPK augmented DDC-induced ROS formation. Consistent with these findings, cirrhotic, but not normal, human livers contained GAPDH aggregates and NDPK complexes. We propose that GAPDH and NDPK are genetic modifiers of murine DDC-induced liver injury and potentially human liver disease.

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Strain-specific and DDC-induced changes in the expression of regulators of protein processing, energy metabolism, and oxidative stress. (A–C, top panels) Equal amounts of total liver protein from three untreated and four DDC-fed C3H or C57BL mice were resolved on SDS-PAGE gels and immunoblotted with antibodies to the indicated proteins. Blots separated by solid lines indicate nonconsecutive lanes of the same equally exposed membrane. The expression of some proteins (PDIA4, FAH, and ME1) was relatively unaffected across groups, and they serve as loading controls. (bottom panels) Relative band intensity values were plotted for each protein in the three different groups. ME1, malic enzyme 1; mono, monomer; oligo, oligomer. *, P < 0.05; **, P < 0.01; ***, P < 0.001 using a two-way analysis of variance. Each tested group included three to four mice. Results are represented as the mean and the SD.
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fig2: Strain-specific and DDC-induced changes in the expression of regulators of protein processing, energy metabolism, and oxidative stress. (A–C, top panels) Equal amounts of total liver protein from three untreated and four DDC-fed C3H or C57BL mice were resolved on SDS-PAGE gels and immunoblotted with antibodies to the indicated proteins. Blots separated by solid lines indicate nonconsecutive lanes of the same equally exposed membrane. The expression of some proteins (PDIA4, FAH, and ME1) was relatively unaffected across groups, and they serve as loading controls. (bottom panels) Relative band intensity values were plotted for each protein in the three different groups. ME1, malic enzyme 1; mono, monomer; oligo, oligomer. *, P < 0.05; **, P < 0.01; ***, P < 0.001 using a two-way analysis of variance. Each tested group included three to four mice. Results are represented as the mean and the SD.

Mentions: The proteomic data were subsequently subjected to further biochemical validation (Fig. 2). The expression of proteasome 26S ATPase subunit 6 (PSMC6), which catalyzes the ATP-dependent 26S proteasomal degradation of ubiquitinated proteins (Smith et al., 2006), was significantly different between control and DDC-treated livers with respect to the existence of oligomeric PSMC6 complexes (Fig. 2 A, brackets), which is consistent with proteasomal impairments in this model (Harada et al., 2008), although there were no significant strain differences. At the mRNA level, PSMC6 was higher in the C3H strain under control conditions and elevated upon DDC treatment, whereas it was diminished upon DDC treatment in the C57BL livers (Table S1). The expression levels of monomeric protein disulfide isomerase A4 (PDIA4), which catalyzes oxidative protein folding in the ER (Gruber et al., 2006), were identical between the two strains. In C57BL livers, there were slightly higher levels of selenium-binding protein 1 (SBP1; Fig. 2 A), which has been implicated in intra-Golgi transport and in selenium-dependent ubiquitination- and deubiquitination-mediated protein degradation pathways (Porat et al., 2000; Jeong et al., 2009). This was in contrast to the mRNA levels, which showed the opposite patterns (i.e., higher SBP1 mRNA in C3H livers) both before and after DDC treatment. The discordance between the protein and mRNA levels may reflect extensive posttranslational regulation of SBP1 and/or antibody reactivity with other SBP isoforms such as SBP2 (Lanfear et al., 1993). Although there were no major strain differences with regards to the total expression levels of PSMC6, PDIA4, and SBP1, the requirement of energy and reducing equivalents for their proper function suggests that differences may exist at the functional level. Therefore, we next examined the expression of energy metabolism regulators.


Energy determinants GAPDH and NDPK act as genetic modifiers for hepatocyte inclusion formation.

Snider NT, Weerasinghe SV, Singla A, Leonard JM, Hanada S, Andrews PC, Lok AS, Omary MB - J. Cell Biol. (2011)

Strain-specific and DDC-induced changes in the expression of regulators of protein processing, energy metabolism, and oxidative stress. (A–C, top panels) Equal amounts of total liver protein from three untreated and four DDC-fed C3H or C57BL mice were resolved on SDS-PAGE gels and immunoblotted with antibodies to the indicated proteins. Blots separated by solid lines indicate nonconsecutive lanes of the same equally exposed membrane. The expression of some proteins (PDIA4, FAH, and ME1) was relatively unaffected across groups, and they serve as loading controls. (bottom panels) Relative band intensity values were plotted for each protein in the three different groups. ME1, malic enzyme 1; mono, monomer; oligo, oligomer. *, P < 0.05; **, P < 0.01; ***, P < 0.001 using a two-way analysis of variance. Each tested group included three to four mice. Results are represented as the mean and the SD.
© Copyright Policy - openaccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC3198167&req=5

fig2: Strain-specific and DDC-induced changes in the expression of regulators of protein processing, energy metabolism, and oxidative stress. (A–C, top panels) Equal amounts of total liver protein from three untreated and four DDC-fed C3H or C57BL mice were resolved on SDS-PAGE gels and immunoblotted with antibodies to the indicated proteins. Blots separated by solid lines indicate nonconsecutive lanes of the same equally exposed membrane. The expression of some proteins (PDIA4, FAH, and ME1) was relatively unaffected across groups, and they serve as loading controls. (bottom panels) Relative band intensity values were plotted for each protein in the three different groups. ME1, malic enzyme 1; mono, monomer; oligo, oligomer. *, P < 0.05; **, P < 0.01; ***, P < 0.001 using a two-way analysis of variance. Each tested group included three to four mice. Results are represented as the mean and the SD.
Mentions: The proteomic data were subsequently subjected to further biochemical validation (Fig. 2). The expression of proteasome 26S ATPase subunit 6 (PSMC6), which catalyzes the ATP-dependent 26S proteasomal degradation of ubiquitinated proteins (Smith et al., 2006), was significantly different between control and DDC-treated livers with respect to the existence of oligomeric PSMC6 complexes (Fig. 2 A, brackets), which is consistent with proteasomal impairments in this model (Harada et al., 2008), although there were no significant strain differences. At the mRNA level, PSMC6 was higher in the C3H strain under control conditions and elevated upon DDC treatment, whereas it was diminished upon DDC treatment in the C57BL livers (Table S1). The expression levels of monomeric protein disulfide isomerase A4 (PDIA4), which catalyzes oxidative protein folding in the ER (Gruber et al., 2006), were identical between the two strains. In C57BL livers, there were slightly higher levels of selenium-binding protein 1 (SBP1; Fig. 2 A), which has been implicated in intra-Golgi transport and in selenium-dependent ubiquitination- and deubiquitination-mediated protein degradation pathways (Porat et al., 2000; Jeong et al., 2009). This was in contrast to the mRNA levels, which showed the opposite patterns (i.e., higher SBP1 mRNA in C3H livers) both before and after DDC treatment. The discordance between the protein and mRNA levels may reflect extensive posttranslational regulation of SBP1 and/or antibody reactivity with other SBP isoforms such as SBP2 (Lanfear et al., 1993). Although there were no major strain differences with regards to the total expression levels of PSMC6, PDIA4, and SBP1, the requirement of energy and reducing equivalents for their proper function suggests that differences may exist at the functional level. Therefore, we next examined the expression of energy metabolism regulators.

Bottom Line: Prominent histological features of some chronic human liver diseases are hepatocyte ballooning and Mallory-Denk bodies.GAPDH knockdown depleted bioenergetic and antioxidant enzymes and elevated hepatocyte ROS, whereas GAPDH overexpression decreased hepatocyte ROS.We propose that GAPDH and NDPK are genetic modifiers of murine DDC-induced liver injury and potentially human liver disease.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Molecular and Integrative Physiology, University of Michigan, Ann Arbor, MI 48109, USA. nsnider@umich.edu

ABSTRACT
Genetic factors impact liver injury susceptibility and disease progression. Prominent histological features of some chronic human liver diseases are hepatocyte ballooning and Mallory-Denk bodies. In mice, these features are induced by 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) in a strain-dependent manner, with the C57BL and C3H strains showing high and low susceptibility, respectively. To identify modifiers of DDC-induced liver injury, we compared C57BL and C3H mice using proteomic, biochemical, and cell biological tools. DDC elevated reactive oxygen species (ROS) and oxidative stress enzymes preferentially in C57BL livers and isolated hepatocytes. C57BL livers and hepatocytes also manifested significant down-regulation, aggregation, and nuclear translocation of glyceraldehyde 3-phosphate dehydrogenase (GAPDH). GAPDH knockdown depleted bioenergetic and antioxidant enzymes and elevated hepatocyte ROS, whereas GAPDH overexpression decreased hepatocyte ROS. On the other hand, C3H livers had higher expression and activity of the energy-generating nucleoside-diphosphate kinase (NDPK), and knockdown of hepatocyte NDPK augmented DDC-induced ROS formation. Consistent with these findings, cirrhotic, but not normal, human livers contained GAPDH aggregates and NDPK complexes. We propose that GAPDH and NDPK are genetic modifiers of murine DDC-induced liver injury and potentially human liver disease.

Show MeSH
Related in: MedlinePlus