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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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Hepatic differences in the expression, activity, and distribution of the cytoplasmic enzyme NDPK. (A) Relative expression level of NDPK-B in extrahepatic organs from C3H and C57BL mice. The actin blot serves as a loading control. (B) Total NDPK activity in C3H and C57BL mouse livers under basal conditions and after DDC treatment (top) and in untreated isolated hepatocytes (bottom). **, P < 0.01 using an unpaired t test; ***, P < 0.001 using a two-way analysis of variance. Each tested group included three to four mice, and samples were analyzed in triplicates. Results are represented as the mean and the SD. (C) Primary cultured hepatocytes from C3H (panels a–c) and C57BL (panels d–f) mice were triple immunostained for NDPK-B and K8 and were then mounted in the presence of DAPI as described in Materials and methods. Bar, 10 µm.
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fig3: Hepatic differences in the expression, activity, and distribution of the cytoplasmic enzyme NDPK. (A) Relative expression level of NDPK-B in extrahepatic organs from C3H and C57BL mice. The actin blot serves as a loading control. (B) Total NDPK activity in C3H and C57BL mouse livers under basal conditions and after DDC treatment (top) and in untreated isolated hepatocytes (bottom). **, P < 0.01 using an unpaired t test; ***, P < 0.001 using a two-way analysis of variance. Each tested group included three to four mice, and samples were analyzed in triplicates. Results are represented as the mean and the SD. (C) Primary cultured hepatocytes from C3H (panels a–c) and C57BL (panels d–f) mice were triple immunostained for NDPK-B and K8 and were then mounted in the presence of DAPI as described in Materials and methods. Bar, 10 µm.

Mentions: Major differences in the expression of NDPK-A and -B isoforms were observed between the two strains, with a significantly lower expression in C57BL livers under both control and DDC-treatment conditions (Fig. 2 B). The NDPK protein differences were not reflected at the mRNA level (Table S1), implying a posttranslational control of NDPK expression. NDPK activity is important for the synthesis of non–ATP nucleoside triphosphates (i.e., CTP, GTP, and UTP) via the transfer of a phosphate group from ATP (Boissan et al., 2009). NDPK also plays a role in the protection from oxidative stress–induced damage (Arnaud-Dabernat et al., 2004; Lee et al., 2009). The striking strain differences in liver NDPK expression (Fig. 2 B) were liver specific, as kidney, lung, heart, and spleen NDPK-B levels were similar between the two strains (Fig. 3 A). Further, total NDPK activity was significantly higher in C3H livers after DDC treatment in comparison with control C3H and DDC-treated C57BL livers (Fig. 3 B, top). The differences were more dramatic when assessing total NDPK activity in isolated C57BL hepatocytes, which was threefold lower compared with C3H hepatocytes (Fig. 3 B, bottom). NDPK exhibited a cytoplasmic filamentous organization and partial colocalization with keratin filaments in the hepatocytes (Fig. 3 C), although coimmunoprecipitation experiments did not show a direct interaction between the two (not depicted).


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)

Hepatic differences in the expression, activity, and distribution of the cytoplasmic enzyme NDPK. (A) Relative expression level of NDPK-B in extrahepatic organs from C3H and C57BL mice. The actin blot serves as a loading control. (B) Total NDPK activity in C3H and C57BL mouse livers under basal conditions and after DDC treatment (top) and in untreated isolated hepatocytes (bottom). **, P < 0.01 using an unpaired t test; ***, P < 0.001 using a two-way analysis of variance. Each tested group included three to four mice, and samples were analyzed in triplicates. Results are represented as the mean and the SD. (C) Primary cultured hepatocytes from C3H (panels a–c) and C57BL (panels d–f) mice were triple immunostained for NDPK-B and K8 and were then mounted in the presence of DAPI as described in Materials and methods. Bar, 10 µm.
© Copyright Policy - openaccess
Related In: Results  -  Collection

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

fig3: Hepatic differences in the expression, activity, and distribution of the cytoplasmic enzyme NDPK. (A) Relative expression level of NDPK-B in extrahepatic organs from C3H and C57BL mice. The actin blot serves as a loading control. (B) Total NDPK activity in C3H and C57BL mouse livers under basal conditions and after DDC treatment (top) and in untreated isolated hepatocytes (bottom). **, P < 0.01 using an unpaired t test; ***, P < 0.001 using a two-way analysis of variance. Each tested group included three to four mice, and samples were analyzed in triplicates. Results are represented as the mean and the SD. (C) Primary cultured hepatocytes from C3H (panels a–c) and C57BL (panels d–f) mice were triple immunostained for NDPK-B and K8 and were then mounted in the presence of DAPI as described in Materials and methods. Bar, 10 µm.
Mentions: Major differences in the expression of NDPK-A and -B isoforms were observed between the two strains, with a significantly lower expression in C57BL livers under both control and DDC-treatment conditions (Fig. 2 B). The NDPK protein differences were not reflected at the mRNA level (Table S1), implying a posttranslational control of NDPK expression. NDPK activity is important for the synthesis of non–ATP nucleoside triphosphates (i.e., CTP, GTP, and UTP) via the transfer of a phosphate group from ATP (Boissan et al., 2009). NDPK also plays a role in the protection from oxidative stress–induced damage (Arnaud-Dabernat et al., 2004; Lee et al., 2009). The striking strain differences in liver NDPK expression (Fig. 2 B) were liver specific, as kidney, lung, heart, and spleen NDPK-B levels were similar between the two strains (Fig. 3 A). Further, total NDPK activity was significantly higher in C3H livers after DDC treatment in comparison with control C3H and DDC-treated C57BL livers (Fig. 3 B, top). The differences were more dramatic when assessing total NDPK activity in isolated C57BL hepatocytes, which was threefold lower compared with C3H hepatocytes (Fig. 3 B, bottom). NDPK exhibited a cytoplasmic filamentous organization and partial colocalization with keratin filaments in the hepatocytes (Fig. 3 C), although coimmunoprecipitation experiments did not show a direct interaction between the two (not depicted).

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