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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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Presence of detergent-insoluble and nuclear GAPDH in C57BL hepatocytes. (A) Hepatocytes from C57BL and C3H mice were cultured in presence of the indicated concentrations of DDC or 0.1% DMSO vehicle (0) or in the absence of DDC/DMSO (Control). Detergent-insoluble equal fractions were prepared and then blotted with antibodies to actin (loading control) and GAPDH. (B) Immunofluorescence-based localization of GAPDH in vehicle (DMSO; panels a–d) or DDC-treated hepatocytes (panels e–h) that were also double stained with DAPI (nuclear staining). Note that after DDC treatment, GAPDH translocates to the nucleus in C3H hepatocytes (arrows in panels e and f) and appears to aggregate in the perinuclear region of C57BL hepatocytes (arrows in panels g and h). Bar, 20 µm.
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fig6: Presence of detergent-insoluble and nuclear GAPDH in C57BL hepatocytes. (A) Hepatocytes from C57BL and C3H mice were cultured in presence of the indicated concentrations of DDC or 0.1% DMSO vehicle (0) or in the absence of DDC/DMSO (Control). Detergent-insoluble equal fractions were prepared and then blotted with antibodies to actin (loading control) and GAPDH. (B) Immunofluorescence-based localization of GAPDH in vehicle (DMSO; panels a–d) or DDC-treated hepatocytes (panels e–h) that were also double stained with DAPI (nuclear staining). Note that after DDC treatment, GAPDH translocates to the nucleus in C3H hepatocytes (arrows in panels e and f) and appears to aggregate in the perinuclear region of C57BL hepatocytes (arrows in panels g and h). Bar, 20 µm.

Mentions: We also investigated the presence of GAPDH in the detergent-insoluble fraction of isolated hepatocytes and compared it with actin in the same fractions. C57BL, but not C3H, hepatocytes contained significant amounts of insoluble GAPDH (Fig. 6 A). Furthermore, immunofluorescence staining showed that DDC induced the nuclear translocation of GAPDH in C3H hepatocytes (Fig. 6 B, panels a, b, e, and f). In stark contrast, GAPDH is already present in the nuclei of C57BL hepatocytes under basal conditions (Fig. 6 B, panels c and d) but undergoes aggregation upon exposure to DDC (Fig. 6 B, panels g and h).


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)

Presence of detergent-insoluble and nuclear GAPDH in C57BL hepatocytes. (A) Hepatocytes from C57BL and C3H mice were cultured in presence of the indicated concentrations of DDC or 0.1% DMSO vehicle (0) or in the absence of DDC/DMSO (Control). Detergent-insoluble equal fractions were prepared and then blotted with antibodies to actin (loading control) and GAPDH. (B) Immunofluorescence-based localization of GAPDH in vehicle (DMSO; panels a–d) or DDC-treated hepatocytes (panels e–h) that were also double stained with DAPI (nuclear staining). Note that after DDC treatment, GAPDH translocates to the nucleus in C3H hepatocytes (arrows in panels e and f) and appears to aggregate in the perinuclear region of C57BL hepatocytes (arrows in panels g and h). Bar, 20 µm.
© Copyright Policy - openaccess
Related In: Results  -  Collection

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

fig6: Presence of detergent-insoluble and nuclear GAPDH in C57BL hepatocytes. (A) Hepatocytes from C57BL and C3H mice were cultured in presence of the indicated concentrations of DDC or 0.1% DMSO vehicle (0) or in the absence of DDC/DMSO (Control). Detergent-insoluble equal fractions were prepared and then blotted with antibodies to actin (loading control) and GAPDH. (B) Immunofluorescence-based localization of GAPDH in vehicle (DMSO; panels a–d) or DDC-treated hepatocytes (panels e–h) that were also double stained with DAPI (nuclear staining). Note that after DDC treatment, GAPDH translocates to the nucleus in C3H hepatocytes (arrows in panels e and f) and appears to aggregate in the perinuclear region of C57BL hepatocytes (arrows in panels g and h). Bar, 20 µm.
Mentions: We also investigated the presence of GAPDH in the detergent-insoluble fraction of isolated hepatocytes and compared it with actin in the same fractions. C57BL, but not C3H, hepatocytes contained significant amounts of insoluble GAPDH (Fig. 6 A). Furthermore, immunofluorescence staining showed that DDC induced the nuclear translocation of GAPDH in C3H hepatocytes (Fig. 6 B, panels a, b, e, and f). In stark contrast, GAPDH is already present in the nuclei of C57BL hepatocytes under basal conditions (Fig. 6 B, panels c and d) but undergoes aggregation upon exposure to DDC (Fig. 6 B, panels g and h).

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