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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 in C3H and C57BL hepatocytes is augmented by GAPDH and NDPK knockdown. (A) Biochemical analysis of protein expression of GAPDH, NDPK-B, and actin (loading control) 24 h after transfection of the isolated primary hepatocytes with the indicated siRNA. (B) Primary hepatocytes were transfected with the indicated siRNA for 24 h followed by the addition of 100 µM DDC for an additional 24 h. Representative differential interference contrast fluorescence images of ROS levels (green) in C3H and C57BL hepatocytes with DAPI counterstain (blue) are shown. Bar, 20 µm. (C) Quantification of hepatocyte ROS levels upon GAPDH and NDPK knockdown and DDC treatment. **, P < 0.01; ***, P < 0.001 using a one-way analysis of variance and relative to the respective control group. ROS levels in control-transfected DDC-treated C57BL hepatocytes were significantly higher compared with control-transfected DDC-treated C3H hepatocytes (P = 0.0009 using an unpaired t test). Results are represented as the mean and the SD (n = 15 cells/group), and the data for each tested group are representative of three independent hepatocyte isolations.
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fig8: DDC-induced ROS formation in C3H and C57BL hepatocytes is augmented by GAPDH and NDPK knockdown. (A) Biochemical analysis of protein expression of GAPDH, NDPK-B, and actin (loading control) 24 h after transfection of the isolated primary hepatocytes with the indicated siRNA. (B) Primary hepatocytes were transfected with the indicated siRNA for 24 h followed by the addition of 100 µM DDC for an additional 24 h. Representative differential interference contrast fluorescence images of ROS levels (green) in C3H and C57BL hepatocytes with DAPI counterstain (blue) are shown. Bar, 20 µm. (C) Quantification of hepatocyte ROS levels upon GAPDH and NDPK knockdown and DDC treatment. **, P < 0.01; ***, P < 0.001 using a one-way analysis of variance and relative to the respective control group. ROS levels in control-transfected DDC-treated C57BL hepatocytes were significantly higher compared with control-transfected DDC-treated C3H hepatocytes (P = 0.0009 using an unpaired t test). Results are represented as the mean and the SD (n = 15 cells/group), and the data for each tested group are representative of three independent hepatocyte isolations.

Mentions: We compared ROS formation in DDC-treated C3H and C57BL hepatocytes upon NDPK and GAPDH knockdown. The remaining protein levels were 1.5–5% for GAPDH and 23–55% for NDPK after the treatment with the specific siRNA (Fig. 8 A). NDPK knockdown also caused a decrease in GAPDH levels to 68% in C3H and 41% in C57BL hepatocytes, indicating a coregulation between GAPDH and NDPK, which may be a result of changes in the cellular availability of nucleoside triphosphates. For example, a decrease in NDPK, which uses ATP to generate non–ATP nucleoside triphosphates (Boissan et al., 2009), could result in an increase in cytoplasmic ATP, which is an allosteric inhibitor of rate-limiting enzymes in glycolysis and thus may affect GAPDH levels (Berg et al., 2002). The representative images in Fig. 8 B show elevated ROS levels after GAPDH or NDPK knockdown in DDC-treated C3H and C57BL hepatocytes. Quantification of the data from three independent images of each treatment group revealed statistically significant increases in ROS levels in C3H and C57BL hepatocytes upon GAPDH and NDPK knockdown (Fig. 8 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)

DDC-induced ROS formation in C3H and C57BL hepatocytes is augmented by GAPDH and NDPK knockdown. (A) Biochemical analysis of protein expression of GAPDH, NDPK-B, and actin (loading control) 24 h after transfection of the isolated primary hepatocytes with the indicated siRNA. (B) Primary hepatocytes were transfected with the indicated siRNA for 24 h followed by the addition of 100 µM DDC for an additional 24 h. Representative differential interference contrast fluorescence images of ROS levels (green) in C3H and C57BL hepatocytes with DAPI counterstain (blue) are shown. Bar, 20 µm. (C) Quantification of hepatocyte ROS levels upon GAPDH and NDPK knockdown and DDC treatment. **, P < 0.01; ***, P < 0.001 using a one-way analysis of variance and relative to the respective control group. ROS levels in control-transfected DDC-treated C57BL hepatocytes were significantly higher compared with control-transfected DDC-treated C3H hepatocytes (P = 0.0009 using an unpaired t test). Results are represented as the mean and the SD (n = 15 cells/group), and the data for each tested group are representative of three independent hepatocyte isolations.
© Copyright Policy - openaccess
Related In: Results  -  Collection

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fig8: DDC-induced ROS formation in C3H and C57BL hepatocytes is augmented by GAPDH and NDPK knockdown. (A) Biochemical analysis of protein expression of GAPDH, NDPK-B, and actin (loading control) 24 h after transfection of the isolated primary hepatocytes with the indicated siRNA. (B) Primary hepatocytes were transfected with the indicated siRNA for 24 h followed by the addition of 100 µM DDC for an additional 24 h. Representative differential interference contrast fluorescence images of ROS levels (green) in C3H and C57BL hepatocytes with DAPI counterstain (blue) are shown. Bar, 20 µm. (C) Quantification of hepatocyte ROS levels upon GAPDH and NDPK knockdown and DDC treatment. **, P < 0.01; ***, P < 0.001 using a one-way analysis of variance and relative to the respective control group. ROS levels in control-transfected DDC-treated C57BL hepatocytes were significantly higher compared with control-transfected DDC-treated C3H hepatocytes (P = 0.0009 using an unpaired t test). Results are represented as the mean and the SD (n = 15 cells/group), and the data for each tested group are representative of three independent hepatocyte isolations.
Mentions: We compared ROS formation in DDC-treated C3H and C57BL hepatocytes upon NDPK and GAPDH knockdown. The remaining protein levels were 1.5–5% for GAPDH and 23–55% for NDPK after the treatment with the specific siRNA (Fig. 8 A). NDPK knockdown also caused a decrease in GAPDH levels to 68% in C3H and 41% in C57BL hepatocytes, indicating a coregulation between GAPDH and NDPK, which may be a result of changes in the cellular availability of nucleoside triphosphates. For example, a decrease in NDPK, which uses ATP to generate non–ATP nucleoside triphosphates (Boissan et al., 2009), could result in an increase in cytoplasmic ATP, which is an allosteric inhibitor of rate-limiting enzymes in glycolysis and thus may affect GAPDH levels (Berg et al., 2002). The representative images in Fig. 8 B show elevated ROS levels after GAPDH or NDPK knockdown in DDC-treated C3H and C57BL hepatocytes. Quantification of the data from three independent images of each treatment group revealed statistically significant increases in ROS levels in C3H and C57BL hepatocytes upon GAPDH and NDPK knockdown (Fig. 8 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