Glutamine in the pathogenesis of hepatic encephalopathy: the trojan horse hypothesis revisited

Abstract

Hepatic encephalopathy (HE) is major neuropsychiatric disorder occurring in patients with severe liver disease and ammonia is generally considered to represent the major toxin responsible for this condition. Ammonia in brain is chiefly metabolized ("detoxified") to glutamine in astrocytes due to predominant localization of glutamine synthetase in these cells. While glutamine has long been considered innocuous, a deleterious role more recently has been attributed to this amino acid. This article reviews the mechanisms by which glutamine contributes to the pathogenesis of HE, how glutamine is transported into mitochondria and subsequently hydrolyzed leading to high levels of ammonia, the latter triggering oxidative and nitrative stress, the mitochondrial permeability transition and mitochondrial injury, a sequence of events we have collectively termed as the Trojan horse hypothesis of hepatic encephalopathy.

Figure 1

Immunohistochemistry (IHC) of L-type glutaminase (LGA) (green fluorescence) in normal mouse brain cortical sections. Frozen brain sections were performed as described previously [45]. Briefly, sections were fixed in ice-cold methanol and incubated overnight at 4°C with antibodies to LGA (goat polyclonal, 1:100), cytochrome oxidase subunit IV (red fluorescence, CO-IV, mouse monoclonal, 1:100) and GFAP (blue fluorescence, rabbit polyclonal, 1:400); washed 3-times with phosphate-buffered saline containing 0.1% Triton X 100; incubated with fluorescent secondary antibodies with different excitation wavelengths; and fluorescence was visualized with a confocal microscope. Note the merged image showing marked co-localization of LGA with CO-IV consistent with the mictochondrial localization of LGA.

Figure 2

Immunohistochemistry (IHC) of the glutamine transporter SNAT5 (green fluorescence) in brain cortical sections from control and mice treated with hepatotoxin thioacetamide (TAA) to induce ALF . Immunohistochemistry of frozen brain sections were performed as described in Figure 1. The antibodies used included SNAT5 (goat polyclonal, 1:100) and GFAP (red fluorescence, rabbit polyclonal, 1:400). Note that SNAT5 expression did not change in mice with TAA-induced ALF as compared to control animals (SNAT5-C).

Figure 3

Effect of ammonia (5 mM NH₄Cl) treatment to cultured astrocytes on SNAT5 protein expression. A. Representative immunoblot of SNAT5 protein density. B. Immunoblot of tubulin protein density (loading control) corresponding to the immunoblot of SNAT5. C. Quantification of SNAT5 protein densities. Note that ammonia treatment of astrocytes had no significant effect on SNAT5 expression at any time point. Values in each group are mean ± S.E.M of 2 individual culture plates taken from 2 separate seeding batches (n=4).