Abstract
Hepatic encephalopathy (HE) is a clinical manifestation of a low grade cerebral edema with a mutual interrelationship between osmotic- and oxidative stress. This leads to RNA oxidation and posttranslational protein modifications such as protein tyrosine nitration with pathophysiological relevance. Here, we report on O-GlcNAcylation as another ammonia-induced posttranslational protein modification in cultured rat astrocytes. NH4Cl induced O-GlcNAcylation of distinct proteins (25–250 kDa) in astrocytes in a dose- and time-dependent manner. Exposure of astrocytes to NH4Cl (5 mmol/l) for 48 h and 72 h significantly increased protein O-GlcNAcylation by about 2-fold and 4-fold, respectively. NH4Cl at a concentration of 1 mmol/l was sufficient to double protein O-GlcNAcylation in astrocytes after 72 h as compared to untreated controls. Ammonia-induced protein O-GlcNAcylation was sensitive towards glutamine-synthetase inhibition by methionine sulfoximine (MSO), but was not induced by hypoosmolarity (205 mosmol/l) or CH3NH3Cl (5 mmol/l). Increased protein O-GlcNAcylation in NH4Cl (5 mmol/l, 48 h)-treated astrocytes was fully reversible within 24 h after withdrawal of NH4Cl from culture medium. Amongst the proteins which are O-GlcNAcylated in response to ammonia, GAPDH was identified. It is concluded that ammonia induces reversible protein O-GlcNAcylation in astrocytes that depends on glutamine synthesis but not on astrocyte swelling per se or ammonia-induced pH-changes. In view of the complex involvement of O-GlcNAcylation in cell regulation, such as energy metabolism, apoptosis and circadian rhythmicity and in pathologies, such as neurodegenerative diseases, O-GlcNAcylation might contribute to the pathophysiology of hepatic encephalopathy.
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Abbreviations
- CHO:
-
Chinese hamster ovary cells
- GAPDH:
-
Glyceraldehyde-3-phosphate dehydrogenase
- GFAT:
-
Glucose-fructose amidotransferase
- GlcN6P:
-
Glucosamine-6-phosphate
- GlcNAc:
-
N-acetyl-D-glucosamine
- GS:
-
Glutamine synthetase
- HE:
-
Hepatic encephalopathy
- iNOS:
-
Inducible nitric oxide synthase
- MSO:
-
L-methionine sulfoximine
- O-GlcNAc:
-
O-linked N-acetyl-D-glucosamine
- OGT:
-
O-linked N-acetylglucosaminyltransferase
- PUGNAc:
-
O-(2-acetamido-2-deoxy-D-glucopyranosyl-idene) amino N-phenyl carbamate
- TNFα:
-
Tumor necrosis factor α
- UDP-GlcNAc:
-
Uridine-diphosphate-N-acetyl-D-glucosamine
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Acknowledgments
This study was supported by Deutsche Forschungsgemeinschaft through Sonderforschungsbereiche SFB 575 “Experimental Hepatology” and SFB 974 “Communication and Systems Relevance in Liver Injury and Regeneration”(Düsseldorf). Expert technical assistance by Torsten Janssen and Brigida Ziegler is gratefully acknowledged.
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Supplementary Figure 1
Biosynthesis of activated glucosamine in the hexosamine pathway. Fructose and glutamine are utilized by glucose:fructose-amido transferase (GFAT) for the synthesis of glucosamine-6-phosphate (GlcN6P) in the rate controlling step of the hexosamine-pathway to produce phosphorylated N-acetylglucosamine (UDP-GlcNAc) which serves as a substrate for protein glycosylation by specific transferases. GS: glutamine-synthetase; Acetyl-CoA: Acetyl-Coenzyme A; GlcNAc-6P: N-acetylglucosamine-6-phosphate; GlcNAc-1P: N-acetylglucosamine-1-phosphate; UTP: uridine 5′-triphosphate; OGT: O-GlcNAc transferase. (JPEG 262 kb)
Supplementary Figure 2
Validation of anti-O-GlcNAc antibody specifity. Protein lysates of untreated controls or (A/B) NH4Cl (5 mmol/l, 72 h)- or (C/D) PUGNAc (80 μmol/l, 24 h)- exposed astrocytes were analysed for anti-O-GlcNAc immunoreactivitiy by dot- (A/C) and Western-blot (B/D) in the presence or absence of free N-acetyl-D-glucosamine (30 mmol/l, 24 h pre-treatment). GAPDH served as loading control. (JPEG 302 kb)
(JPEG 349 kb)
Supplementary Figure 3
Effect of NH4Cl on O-GlcNAc transferase protein expression in cultured rat astrocytes. Astrocytes were either treated with NH4Cl (5 mmol/l) or left untreated for 72 h and analysed for O-GlcNAc transferase (OGT) expression by Western-blot (A). Densitometric quantification of OGT expression (B). OGT expression is given relative to untreated controls. n.s.: not significantly different compared to untreated controls. WB: Western-Blot. (JPEG 220 kb)
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Karababa, A., Görg, B., Schliess, F. et al. O-GlcNAcylation as a novel ammonia-induced posttranslational protein modification in cultured rat astrocytes. Metab Brain Dis 29, 975–982 (2014). https://doi.org/10.1007/s11011-013-9454-7
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DOI: https://doi.org/10.1007/s11011-013-9454-7