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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 differences in oxidative stress–related enzymes and ROS. (A, left) Equal amounts of total liver protein from three untreated and four DDC-fed C3H and C57BL mice were resolved by SDS-PAGE and immunoblotted with antibodies to Cyp2e1 or Cyp3a. The PRDX6 blot serves as a loading control. (right) Relative band intensity values of the blots were plotted for each strain/treatment group. ***, 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. (B) Isolated primary hepatocytes from C3H (panels a–d) and C57BL (panels e–h) mice were cultured in the presence of DDC or vehicle (0.1% DMSO), loaded with the ROS indicator CM-H2DCFDA (panels a, b, e, and f) or immunostained for CBR3 (panels c, d, g, and h), and then mounted in the presence of DAPI as described in Materials and methods. Qualitative assessment of ROS levels (arrows in b and f) is based on CM-H2DCFDA fluorescence (green). CBR3 appears as perinuclear particulates (arrows in panel h). Bars, 20 µm.
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fig4: Strain-specific differences in oxidative stress–related enzymes and ROS. (A, left) Equal amounts of total liver protein from three untreated and four DDC-fed C3H and C57BL mice were resolved by SDS-PAGE and immunoblotted with antibodies to Cyp2e1 or Cyp3a. The PRDX6 blot serves as a loading control. (right) Relative band intensity values of the blots were plotted for each strain/treatment group. ***, 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. (B) Isolated primary hepatocytes from C3H (panels a–d) and C57BL (panels e–h) mice were cultured in the presence of DDC or vehicle (0.1% DMSO), loaded with the ROS indicator CM-H2DCFDA (panels a, b, e, and f) or immunostained for CBR3 (panels c, d, g, and h), and then mounted in the presence of DAPI as described in Materials and methods. Qualitative assessment of ROS levels (arrows in b and f) is based on CM-H2DCFDA fluorescence (green). CBR3 appears as perinuclear particulates (arrows in panel h). Bars, 20 µm.

Mentions: Next, we examined the expression of prooxidant liver enzymes and ROS generation. The expression of Cyp2e1, a known ROS-generating enzyme implicated in the pathogenesis of ALD (Cederbaum et al., 2009), and Cyp3a, the enzyme responsible for mouse liver DDC metabolism (Hanada et al., 2010), was significantly different between the two strains after DDC treatment (Fig. 4 A). Specifically, Cyp2e1 expression was down-regulated by 90% in the C3H and by only 30% in the C57BL livers, resulting in a fivefold higher relative Cyp2e1 expression in C57BL compared with C3H livers after DDC treatment. The apparent decrease in Cyp2e1 protein was not a result of the contribution of bile ductular epithelium to total liver protein, as induction of K19 (ductal marker) was similar between the two strains (Fig. S2 A), which is in agreement with previous findings (Hanada et al., 2008). Cyp2e1 mRNA levels also decreased in both strains (Fig. S2 B). In C57BL but not C3H livers, there was a marked Cyp3a induction (Fig. 4 A), which we have previously shown to correlate with the propensity of male mice to form more MDBs compared with female mice (Hanada et al., 2010). The differences in ROS-generating cytochromes P450 (CYPs) and GST induction suggested possible glutathione depletion in the C57BL livers. However, measurement of the total and oxidized glutathione levels between the two strains did not reveal major differences (Fig. S2 C).


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 differences in oxidative stress–related enzymes and ROS. (A, left) Equal amounts of total liver protein from three untreated and four DDC-fed C3H and C57BL mice were resolved by SDS-PAGE and immunoblotted with antibodies to Cyp2e1 or Cyp3a. The PRDX6 blot serves as a loading control. (right) Relative band intensity values of the blots were plotted for each strain/treatment group. ***, 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. (B) Isolated primary hepatocytes from C3H (panels a–d) and C57BL (panels e–h) mice were cultured in the presence of DDC or vehicle (0.1% DMSO), loaded with the ROS indicator CM-H2DCFDA (panels a, b, e, and f) or immunostained for CBR3 (panels c, d, g, and h), and then mounted in the presence of DAPI as described in Materials and methods. Qualitative assessment of ROS levels (arrows in b and f) is based on CM-H2DCFDA fluorescence (green). CBR3 appears as perinuclear particulates (arrows in panel h). Bars, 20 µm.
© Copyright Policy - openaccess
Related In: Results  -  Collection

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

fig4: Strain-specific differences in oxidative stress–related enzymes and ROS. (A, left) Equal amounts of total liver protein from three untreated and four DDC-fed C3H and C57BL mice were resolved by SDS-PAGE and immunoblotted with antibodies to Cyp2e1 or Cyp3a. The PRDX6 blot serves as a loading control. (right) Relative band intensity values of the blots were plotted for each strain/treatment group. ***, 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. (B) Isolated primary hepatocytes from C3H (panels a–d) and C57BL (panels e–h) mice were cultured in the presence of DDC or vehicle (0.1% DMSO), loaded with the ROS indicator CM-H2DCFDA (panels a, b, e, and f) or immunostained for CBR3 (panels c, d, g, and h), and then mounted in the presence of DAPI as described in Materials and methods. Qualitative assessment of ROS levels (arrows in b and f) is based on CM-H2DCFDA fluorescence (green). CBR3 appears as perinuclear particulates (arrows in panel h). Bars, 20 µm.
Mentions: Next, we examined the expression of prooxidant liver enzymes and ROS generation. The expression of Cyp2e1, a known ROS-generating enzyme implicated in the pathogenesis of ALD (Cederbaum et al., 2009), and Cyp3a, the enzyme responsible for mouse liver DDC metabolism (Hanada et al., 2010), was significantly different between the two strains after DDC treatment (Fig. 4 A). Specifically, Cyp2e1 expression was down-regulated by 90% in the C3H and by only 30% in the C57BL livers, resulting in a fivefold higher relative Cyp2e1 expression in C57BL compared with C3H livers after DDC treatment. The apparent decrease in Cyp2e1 protein was not a result of the contribution of bile ductular epithelium to total liver protein, as induction of K19 (ductal marker) was similar between the two strains (Fig. S2 A), which is in agreement with previous findings (Hanada et al., 2008). Cyp2e1 mRNA levels also decreased in both strains (Fig. S2 B). In C57BL but not C3H livers, there was a marked Cyp3a induction (Fig. 4 A), which we have previously shown to correlate with the propensity of male mice to form more MDBs compared with female mice (Hanada et al., 2010). The differences in ROS-generating cytochromes P450 (CYPs) and GST induction suggested possible glutathione depletion in the C57BL livers. However, measurement of the total and oxidized glutathione levels between the two strains did not reveal major differences (Fig. S2 C).

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