Hyperammonemia in gene-targeted mice lacking functional hepatic glutamine synthetase

Abstract

Urea cycle defects and acute or chronic liver failure are linked to systemic hyperammonemia and often result in cerebral dysfunction and encephalopathy. Although an important role of the liver in ammonia metabolism is widely accepted, the role of ammonia metabolizing pathways in the liver for maintenance of whole-body ammonia homeostasis in vivo remains ill-defined. Here, we show by generation of liver-specific Gln synthetase (GS)-deficient mice that GS in the liver is critically involved in systemic ammonia homeostasis in vivo. Hepatic deletion of GS triggered systemic hyperammonemia, which was associated with cerebral oxidative stress as indicated by increased levels of oxidized RNA and enhanced protein Tyr nitration. Liver-specific GS-deficient mice showed increased locomotion, impaired fear memory, and a slightly reduced life span. In conclusion, the present observations highlight the importance of hepatic GS for maintenance of ammonia homeostasis and establish the liver-specific GS KO mouse as a model with which to study effects of chronic hyperammonemia.

Keywords: RNA oxidation; glutamine; hepatic encephalopathy; metabolic zonation; oxidative stress.

Fig. 1.

Organ-specific deletion of GS in the Glul^fl/fl × Alb-Cre⁺ mice. (A) Gene-targeted mice lacking functional hepatic GS were generated as described in Materials and Methods. (B) GS and GAPDH expression in liver tissue from Glul^fl/fl × Alb-Cre⁺, Alb-Cre⁺, and Glul^fl/fl mice is shown using Western blot analysis. One representative of n = 3 is shown. (C) Snap-frozen sections from liver tissue harvested from Alb-Cre⁺, Glul^fl/fl, and Glul^fl/fl × Alb-Cre⁺ mice were stained with anti-GS (green) and F-actin (red) antibodies. One representative of n = 3 is shown. (Scale bar: 200 μm.) (D, Left) Western blot analysis for GS and GAPDH on protein samples obtained from brain tissue from Glul^fl/fl × Alb-Cre⁺, Alb-Cre⁺, and Glul^fl/fl mice was performed. (D, Right) GS expression and GAPDH expression were assessed in muscle tissue of Glul^fl/fl × Alb-Cre⁺ and Glul^fl/fl mice by Western blot analysis. One representative of n = 3 is shown. (E) Immunostaining of snap-frozen sections from brain tissue harvested from Alb-Cre⁺, Glul^fl/fl, and Glul^fl/fl × Alb-Cre⁺ mice with an anti-GS antibody (green) and Hoechst 34580 (blue) was performed. One representative of n = 3 is shown. (Scale bar: 50 μm.) (F) GS activity was assessed in liver tissue harvested from Glul^fl/fl and Glul^fl/fl × Alb-Cre⁺ mice (n = 3–7, respectively).

Fig. 2.

Intact liver architecture/zonation and elevated systemic ammonia levels by liver-specific deletion of GS. (A) Representative H&E-stained sections of snap-frozen liver tissue obtained from Glul^fl/fl × Alb-Cre⁺ and Glul^fl/fl mice of n = 3 is shown. (Scale bar: 100 μm.) (B) Asp aminotransferase (AST) and Ala aminotransferase (ALT) activity was determined in the serum of Glul^fl/fl (n = 10) and Glul^fl/fl × Alb-Cre⁺ mice (n = 12). n.s., not statistically significantly different. (C) Immunofluorescence analyses of snap-frozen liver tissue from Glul^fl/fl × Alb-Cre⁺ (Lower) and Glul^fl/fl (Upper) mice were performed for GS (red), ornithine aminotransferase (OAT; green), and Hoechst 34580 (blue). One representative set of images of n = 3 is shown. (Scale bar: 20 μm.) (D) Immunofluorescence analyses of snap-frozen liver tissue from Glul^fl/fl × Alb-Cre⁺ (Right) and Glul^fl/fl (Left) mice were performed for ammonia transporter Rh family B glycoprotein (RhBG; red) and Hoechst 34580 (gray). One representative set of images of n = 3 (Glul^fl/fl) and n = 4 (Glul^fl/fl × Alb-Cre⁺) is shown. (Scale bar: 200 μm.) (E) Ammonia levels were assessed in blood samples collected by cardiac puncture from 8- to 9-wk-old Glul^fl/fl × Alb-Cre⁺ mice and Glul^fl/fl mice (Left, n = 7–8, respectively) and from 12- to 14-month-old animals (Right, n = 3).

Fig. 3.

Hepatic GS KO triggers PTN in mouse brain. Protein samples harvested from brain slices of the cerebellum, hippocampus, somatosensory cortex, and piriform cortex of Glul^fl/fl and Glul^fl/fl × Alb-Cre⁺ mice were tested for PTN using Western blot analysis. (A–D, Upper) Representative blots showing anti–3′-nitrotyrosine immunoreactivity. (A–D, Lower) GAPDH served as a loading control. (E) Mean ± SEM of the densitometry of the cerebellum (C), hippocampus (H), somatosensory cortex (S.C.), and cortex piriform (C.P.) is presented (n = 6 for Glul^fl/fl and n = 9 for Glul^fl/fl × Alb-Cre⁺ mice for the cerebellum and hippocampus, n = 6 for Glul^fl/fl and n = 8 for Glul^fl/fl × Alb-Cre⁺ mice for the somatosensory cortex, and n = 3 for Glul^fl/fl and n = 6 for Glul^fl/fl × Alb-Cre⁺ mice for the cortex piriform).

Fig. 4.

Liver-specific deletion of GS induces RNA oxidation in mouse brain. (A–D) Survey of 8-OH(d)G immunoreactivity in brain tissue from Glul^fl/fl (Left) and Glul^fl/fl × Alb-Cre⁺ (Right) mice is presented in the absence (Lower) or presence (Upper) of costaining with GFAP (green) and Hoechst 34580 (blue). One representative of the cerebellum (A), hippocampus (B), somatosensory cortex (C), and piriform cortex (D) of n = 5 is demonstrated. (Scale bars: A, 100 μm; B–D, 200 μm.) (E) Samples harvested from brain sections (from left to right: cerebellum, hippocampus, somatosensory cortex, and piriform cortex) of Glul^fl/fl and Glul^fl/fl × Alb-Cre⁺ mice were tested for RNA oxidation expression using Northwestern blotting [n = 6 for Glul^fl/fl mice, n = 9 (cerebellum, hippocampus, and piriform cortex) and n = 10 (somatosensory cortex) for Glul^fl/fl × Alb-Cre⁺ mice].

Fig. 5.

Microglia activation marker and proinflammatory cytokine mRNA expression in the cerebral cortex of WT and Glul^fl/fl × Alb-Cre⁺ mice. (A) Detection of Iba1 in mouse cerebral cortex by wide-field fluorescence microscopy. One representative immunofluorescence analysis of three is shown. (Scale bars: 200 μm; Inset, 20 μm.) mRNA expression levels in mouse cerebral cortex by RT-PCR of Iba1 and CD14 (B, n = 3) or IL-1β, I-L6, or TNF-α (C, n = 8 for Glul^fl/fl mice and n = 6 of for Glul^fl/fl × Alb-Cre⁺ mice) are shown. mRNA expression levels of Iba1, CD14, IL-1β, IL-6, or TNF-α were normalized to succinate dehydrogenase complex subunit A (SDHA) mRNA levels.

Fig. 6.

Glul^fl/fl × Alb-Cre⁺ mice show behavioral abnormalities compared with WT mice. (A and B) Glul^fl/fl and Glul^fl/fl × Alb-Cre⁺ mice were transferred into a light barrier-equipped cage and allowed to adapt to the new environment for 24 h prior to starting the measurement. Total activity time (Left, n = 22 for Glul^fl/fl mice and n = 23 for Glul^fl/fl × Alb-Cre⁺ mice) and counts (Right, n = 23 for Glul^fl/fl mice and n = 24 for Glul^fl/fl × Alb-Cre⁺ mice) (A) and distance traveled (n = 21 for Glul^fl/fl mice and n = 22 for Glul^fl/fl × Alb-Cre⁺ mice) (B) were determined after 24 h. Animals were monitored using the O-Maze test. Time (Left) and visits (Right) in the open arms (C) and time (Left) and visits (Right) in the closed arms (D) are presented (n = 10 for Glul^fl/fl mice and n = 11 for Glul^fl/fl × Alb-Cre⁺ mice).