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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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DDC-induced ROS formation is associated with a depletion of energy metabolism and antioxidant enzymes and is regulated by GAPDH. (A) Hepatocytes from C3H and C57BL mice were left untreated (−) or treated with either 0.1% DMSO vehicle (0) or the indicated concentrations of DDC for 48 h. Equal protein amounts (NP-40 lysates) were analyzed for expression of the indicated proteins. Samples from each strain were analyzed on separate gels. PDIA4 levels were unaltered (loading control). (B) C57BL hepatocytes were transfected with Control or GAPDH siRNA for 24 h and were then cultured for an additional 24 h in the presence of 0.025% DMSO vehicle (0) or 100 µM DDC or were left untreated (−). The NP-40 cell lysates were analyzed for the expression of the various proteins indicated. (C) Overexpression of Flag-tagged mouse GAPDH in isolated hepatocytes. NP-40 lysates were analyzed for expression of Flag-tagged GAPDH and total GAPDH. The Coomassie stain is included as a loading control. (D) Flag-GAPDH expression in C57BL hepatocytes, as determined by immunostaining with a mouse anti-Flag antibody (representing overexpressed GAPDH; green) and a rabbit anti-GAPDH antibody (representing total GAPDH; red). (E) C57BL hepatocytes were mock transfected (Control) or transfected with GAPDH siRNA or Flag-GAPDH mouse cDNA for 24 h and were then treated with 100 µM DDC for an additional 24 h. Representative images of ROS signal (green) with DAPI nuclear counterstain (blue) are shown. ROS levels (quantified as described in Materials and methods) exhibited a 2.2-fold increase after GAPDH knockdown and a 10-fold decrease after GAPDH overexpression relative to control. Bars, 20 µm.
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fig7: DDC-induced ROS formation is associated with a depletion of energy metabolism and antioxidant enzymes and is regulated by GAPDH. (A) Hepatocytes from C3H and C57BL mice were left untreated (−) or treated with either 0.1% DMSO vehicle (0) or the indicated concentrations of DDC for 48 h. Equal protein amounts (NP-40 lysates) were analyzed for expression of the indicated proteins. Samples from each strain were analyzed on separate gels. PDIA4 levels were unaltered (loading control). (B) C57BL hepatocytes were transfected with Control or GAPDH siRNA for 24 h and were then cultured for an additional 24 h in the presence of 0.025% DMSO vehicle (0) or 100 µM DDC or were left untreated (−). The NP-40 cell lysates were analyzed for the expression of the various proteins indicated. (C) Overexpression of Flag-tagged mouse GAPDH in isolated hepatocytes. NP-40 lysates were analyzed for expression of Flag-tagged GAPDH and total GAPDH. The Coomassie stain is included as a loading control. (D) Flag-GAPDH expression in C57BL hepatocytes, as determined by immunostaining with a mouse anti-Flag antibody (representing overexpressed GAPDH; green) and a rabbit anti-GAPDH antibody (representing total GAPDH; red). (E) C57BL hepatocytes were mock transfected (Control) or transfected with GAPDH siRNA or Flag-GAPDH mouse cDNA for 24 h and were then treated with 100 µM DDC for an additional 24 h. Representative images of ROS signal (green) with DAPI nuclear counterstain (blue) are shown. ROS levels (quantified as described in Materials and methods) exhibited a 2.2-fold increase after GAPDH knockdown and a 10-fold decrease after GAPDH overexpression relative to control. Bars, 20 µm.

Mentions: We performed additional ex vivo experiments of isolated hepatocytes from C3H and C57BL mice to further explore the effects that were seen after DDC treatment in vivo. Similar to the in vivo findings (Fig. 2), ex vivo analysis showed that NDPK-B, CA3, and PRDX6 expression decreased after DDC treatment, whereas PDIA4 levels remained unchanged (Fig. 7 A; quantification is shown in Fig. S3 A). There was also a significant dose-dependent decrease in GAPDH and the tyrosine catabolism enzyme fumarylacetoacetate hydrolase (FAH) expression upon DDC treatment (Fig. 7 A), indicating a significant impairment in metabolic activity. These changes were more striking in C57BL hepatocytes, particularly in the case of NDPK-B. DMSO vehicle alone strongly decreased GAPDH, NDPK-B, and CA3 levels in C57BL hepatocytes (Fig. 7 A). The mechanism behind the DMSO effect is unclear; however, DMSO is known to exert control over the expression of several nuclear transcription factors (Su and Waxman, 2004), which may have contributed to its effects observed herein. DDC treatment augmented the expression of Cyp2e1 in both C3H and C57BL hepatocytes (Fig. 7 A).


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)

DDC-induced ROS formation is associated with a depletion of energy metabolism and antioxidant enzymes and is regulated by GAPDH. (A) Hepatocytes from C3H and C57BL mice were left untreated (−) or treated with either 0.1% DMSO vehicle (0) or the indicated concentrations of DDC for 48 h. Equal protein amounts (NP-40 lysates) were analyzed for expression of the indicated proteins. Samples from each strain were analyzed on separate gels. PDIA4 levels were unaltered (loading control). (B) C57BL hepatocytes were transfected with Control or GAPDH siRNA for 24 h and were then cultured for an additional 24 h in the presence of 0.025% DMSO vehicle (0) or 100 µM DDC or were left untreated (−). The NP-40 cell lysates were analyzed for the expression of the various proteins indicated. (C) Overexpression of Flag-tagged mouse GAPDH in isolated hepatocytes. NP-40 lysates were analyzed for expression of Flag-tagged GAPDH and total GAPDH. The Coomassie stain is included as a loading control. (D) Flag-GAPDH expression in C57BL hepatocytes, as determined by immunostaining with a mouse anti-Flag antibody (representing overexpressed GAPDH; green) and a rabbit anti-GAPDH antibody (representing total GAPDH; red). (E) C57BL hepatocytes were mock transfected (Control) or transfected with GAPDH siRNA or Flag-GAPDH mouse cDNA for 24 h and were then treated with 100 µM DDC for an additional 24 h. Representative images of ROS signal (green) with DAPI nuclear counterstain (blue) are shown. ROS levels (quantified as described in Materials and methods) exhibited a 2.2-fold increase after GAPDH knockdown and a 10-fold decrease after GAPDH overexpression relative to control. Bars, 20 µm.
© Copyright Policy - openaccess
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3198167&req=5

fig7: DDC-induced ROS formation is associated with a depletion of energy metabolism and antioxidant enzymes and is regulated by GAPDH. (A) Hepatocytes from C3H and C57BL mice were left untreated (−) or treated with either 0.1% DMSO vehicle (0) or the indicated concentrations of DDC for 48 h. Equal protein amounts (NP-40 lysates) were analyzed for expression of the indicated proteins. Samples from each strain were analyzed on separate gels. PDIA4 levels were unaltered (loading control). (B) C57BL hepatocytes were transfected with Control or GAPDH siRNA for 24 h and were then cultured for an additional 24 h in the presence of 0.025% DMSO vehicle (0) or 100 µM DDC or were left untreated (−). The NP-40 cell lysates were analyzed for the expression of the various proteins indicated. (C) Overexpression of Flag-tagged mouse GAPDH in isolated hepatocytes. NP-40 lysates were analyzed for expression of Flag-tagged GAPDH and total GAPDH. The Coomassie stain is included as a loading control. (D) Flag-GAPDH expression in C57BL hepatocytes, as determined by immunostaining with a mouse anti-Flag antibody (representing overexpressed GAPDH; green) and a rabbit anti-GAPDH antibody (representing total GAPDH; red). (E) C57BL hepatocytes were mock transfected (Control) or transfected with GAPDH siRNA or Flag-GAPDH mouse cDNA for 24 h and were then treated with 100 µM DDC for an additional 24 h. Representative images of ROS signal (green) with DAPI nuclear counterstain (blue) are shown. ROS levels (quantified as described in Materials and methods) exhibited a 2.2-fold increase after GAPDH knockdown and a 10-fold decrease after GAPDH overexpression relative to control. Bars, 20 µm.
Mentions: We performed additional ex vivo experiments of isolated hepatocytes from C3H and C57BL mice to further explore the effects that were seen after DDC treatment in vivo. Similar to the in vivo findings (Fig. 2), ex vivo analysis showed that NDPK-B, CA3, and PRDX6 expression decreased after DDC treatment, whereas PDIA4 levels remained unchanged (Fig. 7 A; quantification is shown in Fig. S3 A). There was also a significant dose-dependent decrease in GAPDH and the tyrosine catabolism enzyme fumarylacetoacetate hydrolase (FAH) expression upon DDC treatment (Fig. 7 A), indicating a significant impairment in metabolic activity. These changes were more striking in C57BL hepatocytes, particularly in the case of NDPK-B. DMSO vehicle alone strongly decreased GAPDH, NDPK-B, and CA3 levels in C57BL hepatocytes (Fig. 7 A). The mechanism behind the DMSO effect is unclear; however, DMSO is known to exert control over the expression of several nuclear transcription factors (Su and Waxman, 2004), which may have contributed to its effects observed herein. DDC treatment augmented the expression of Cyp2e1 in both C3H and C57BL hepatocytes (Fig. 7 A).

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