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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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Differences in GAPDH aggregation and Siah1 levels in C57BL and C3H mouse livers and effect of pioglitazone on DDC-modulated GAPDH nuclear localization in C57BL hepatocytes. (A) Nuclear fractions were prepared from C3H and C57BL mouse livers (livers from control diet [n = 3] or DDC-fed mice [n = 4]; groups are separated by dotted lines) and analyzed on the same gel by SDS-PAGE followed by immunoblotting for GAPDH under reducing or nonreducing conditions. Lamin B1 was used as a loading control. Significant levels of high molecular weight nuclear GAPDH aggregates were detected only in C57BL livers after DDC exposure. (B) C57BL livers (three independent control livers/strain and four separate livers from DDC-fed mice) express significantly higher levels of Siah1 protein after DDC treatment. Coomassie stain serves as loading control. (C) Biochemical analysis on total, cytoplasmic, and nuclei-enriched fractions from C57BL hepatocytes. The hepatocytes were cultured in the presence of vehicle (DMSO), 100 µM DDC, or 3 µM pioglitazone (Pio) plus DDC for 48 h. Detergent lysates were then prepared and blotted with antibodies to GAPDH, lamin B1 (nuclear marker), β-tubulin (cytoplasmic marker), and pan-actin (loading control).
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fig5: Differences in GAPDH aggregation and Siah1 levels in C57BL and C3H mouse livers and effect of pioglitazone on DDC-modulated GAPDH nuclear localization in C57BL hepatocytes. (A) Nuclear fractions were prepared from C3H and C57BL mouse livers (livers from control diet [n = 3] or DDC-fed mice [n = 4]; groups are separated by dotted lines) and analyzed on the same gel by SDS-PAGE followed by immunoblotting for GAPDH under reducing or nonreducing conditions. Lamin B1 was used as a loading control. Significant levels of high molecular weight nuclear GAPDH aggregates were detected only in C57BL livers after DDC exposure. (B) C57BL livers (three independent control livers/strain and four separate livers from DDC-fed mice) express significantly higher levels of Siah1 protein after DDC treatment. Coomassie stain serves as loading control. (C) Biochemical analysis on total, cytoplasmic, and nuclei-enriched fractions from C57BL hepatocytes. The hepatocytes were cultured in the presence of vehicle (DMSO), 100 µM DDC, or 3 µM pioglitazone (Pio) plus DDC for 48 h. Detergent lysates were then prepared and blotted with antibodies to GAPDH, lamin B1 (nuclear marker), β-tubulin (cytoplasmic marker), and pan-actin (loading control).

Mentions: Given the major oxidative and metabolic differences between the MDB-resistant and MDB-susceptible mice, we focused our attention on the status of GAPDH as a potential common regulator of these pathways. Prior studies showed that oxidative stress induces extensive GAPDH modification and aggregation, which leads to its inactivation and compromised glycolysis (Nakajima et al., 2007, 2009; Tristan et al., 2011). Furthermore, upon nitrosylation (Hara et al., 2005) and association with the E3 ubiquitin ligase Siah1, GAPDH translocates to the nucleus and regulates autophagy induction (Colell et al., 2007), nuclear protein nitrosylation (Kornberg et al., 2010), and apoptotic cell death (Hara et al., 2005). To determine whether GAPDH aggregation and nuclear translocation occur in vivo after DDC treatment, we analyzed the nuclear fractions of livers from both mouse strains and found that total and aggregated nuclear GAPDH were significantly more abundant in C57BL livers (Fig. 5 A). Of note, the GAPDH aggregates were observed only when analyzed under nonreducing conditions, which is consistent with their intermolecular disulfide-bonded nature. The increased C57BL nuclear GAPDH after DDC exposure (Fig. 5 A) correlated with significantly higher levels of Siah1 protein (which is involved in GAPDH nuclear translocation) in C57BL livers after DDC treatment (Fig. 5 B). Biochemical analysis of C57BL hepatocyte total, cytoplasmic, and nuclei-enriched fractions showed that DDC caused a significant reduction in the total and cytoplasmic levels of GAPDH to 12 and 14%, respectively, as compared with vehicle levels (Fig. 5 C). In contrast, there was an increase of 215% in the nuclear levels of GAPDH after exposure to DDC. Of note, cotreatment with the insulin sensitizer pioglitazone almost completely reversed the total and cytoplasmic DDC-induced GAPDH depletion to 80% of vehicle and decreased the nuclear accumulation to 23% of vehicle (Fig. 5 C). Collectively, these data demonstrate a pharmacologically amenable role for GAPDH in hepatocellular injury.


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)

Differences in GAPDH aggregation and Siah1 levels in C57BL and C3H mouse livers and effect of pioglitazone on DDC-modulated GAPDH nuclear localization in C57BL hepatocytes. (A) Nuclear fractions were prepared from C3H and C57BL mouse livers (livers from control diet [n = 3] or DDC-fed mice [n = 4]; groups are separated by dotted lines) and analyzed on the same gel by SDS-PAGE followed by immunoblotting for GAPDH under reducing or nonreducing conditions. Lamin B1 was used as a loading control. Significant levels of high molecular weight nuclear GAPDH aggregates were detected only in C57BL livers after DDC exposure. (B) C57BL livers (three independent control livers/strain and four separate livers from DDC-fed mice) express significantly higher levels of Siah1 protein after DDC treatment. Coomassie stain serves as loading control. (C) Biochemical analysis on total, cytoplasmic, and nuclei-enriched fractions from C57BL hepatocytes. The hepatocytes were cultured in the presence of vehicle (DMSO), 100 µM DDC, or 3 µM pioglitazone (Pio) plus DDC for 48 h. Detergent lysates were then prepared and blotted with antibodies to GAPDH, lamin B1 (nuclear marker), β-tubulin (cytoplasmic marker), and pan-actin (loading control).
© Copyright Policy - openaccess
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC3198167&req=5

fig5: Differences in GAPDH aggregation and Siah1 levels in C57BL and C3H mouse livers and effect of pioglitazone on DDC-modulated GAPDH nuclear localization in C57BL hepatocytes. (A) Nuclear fractions were prepared from C3H and C57BL mouse livers (livers from control diet [n = 3] or DDC-fed mice [n = 4]; groups are separated by dotted lines) and analyzed on the same gel by SDS-PAGE followed by immunoblotting for GAPDH under reducing or nonreducing conditions. Lamin B1 was used as a loading control. Significant levels of high molecular weight nuclear GAPDH aggregates were detected only in C57BL livers after DDC exposure. (B) C57BL livers (three independent control livers/strain and four separate livers from DDC-fed mice) express significantly higher levels of Siah1 protein after DDC treatment. Coomassie stain serves as loading control. (C) Biochemical analysis on total, cytoplasmic, and nuclei-enriched fractions from C57BL hepatocytes. The hepatocytes were cultured in the presence of vehicle (DMSO), 100 µM DDC, or 3 µM pioglitazone (Pio) plus DDC for 48 h. Detergent lysates were then prepared and blotted with antibodies to GAPDH, lamin B1 (nuclear marker), β-tubulin (cytoplasmic marker), and pan-actin (loading control).
Mentions: Given the major oxidative and metabolic differences between the MDB-resistant and MDB-susceptible mice, we focused our attention on the status of GAPDH as a potential common regulator of these pathways. Prior studies showed that oxidative stress induces extensive GAPDH modification and aggregation, which leads to its inactivation and compromised glycolysis (Nakajima et al., 2007, 2009; Tristan et al., 2011). Furthermore, upon nitrosylation (Hara et al., 2005) and association with the E3 ubiquitin ligase Siah1, GAPDH translocates to the nucleus and regulates autophagy induction (Colell et al., 2007), nuclear protein nitrosylation (Kornberg et al., 2010), and apoptotic cell death (Hara et al., 2005). To determine whether GAPDH aggregation and nuclear translocation occur in vivo after DDC treatment, we analyzed the nuclear fractions of livers from both mouse strains and found that total and aggregated nuclear GAPDH were significantly more abundant in C57BL livers (Fig. 5 A). Of note, the GAPDH aggregates were observed only when analyzed under nonreducing conditions, which is consistent with their intermolecular disulfide-bonded nature. The increased C57BL nuclear GAPDH after DDC exposure (Fig. 5 A) correlated with significantly higher levels of Siah1 protein (which is involved in GAPDH nuclear translocation) in C57BL livers after DDC treatment (Fig. 5 B). Biochemical analysis of C57BL hepatocyte total, cytoplasmic, and nuclei-enriched fractions showed that DDC caused a significant reduction in the total and cytoplasmic levels of GAPDH to 12 and 14%, respectively, as compared with vehicle levels (Fig. 5 C). In contrast, there was an increase of 215% in the nuclear levels of GAPDH after exposure to DDC. Of note, cotreatment with the insulin sensitizer pioglitazone almost completely reversed the total and cytoplasmic DDC-induced GAPDH depletion to 80% of vehicle and decreased the nuclear accumulation to 23% of vehicle (Fig. 5 C). Collectively, these data demonstrate a pharmacologically amenable role for GAPDH in hepatocellular injury.

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