The metabolic role of isoleucine in detoxification of ammonia in cultured mouse neurons and astrocytes

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Abstract

Cerebral hyperammonemia is a hallmark of hepatic encephalopathy, a debilitating condition arising secondary to liver disease. Pyruvate oxidation including tricarboxylic acid (TCA) cycle metabolism has been suggested to be inhibited by hyperammonemia at the pyruvate and α-ketoglutarate dehydrogenase steps. Catabolism of the branched-chain amino acid isoleucine provides both acetyl-CoA and succinyl-CoA, thus by-passing both the pyruvate dehydrogenase and the α-ketoglutarate dehydrogenase steps. Potentially, this will enable the TCA cycle to work in the face of ammonium-induced inhibition. In addition, this will provide the α-ketoglutarate carbon skeleton for glutamate and glutamine synthesis by glutamate dehydrogenase and glutamine synthetase (astrocytes only), respectively, both reactions fixing ammonium. Cultured cerebellar neurons (primarily glutamatergic) or astrocytes were incubated in the presence of either [U-13C]glucose (2.5 mM) and isoleucine (1 mM) or [U-13C]isoleucine and glucose. Cell cultures were treated with an acute ammonium chloride load of 2 (astrocytes) or 5 mM (neurons and astrocytes) and incorporation of 13C-label into glutamate, aspartate, glutamine and alanine was determined employing mass spectrometry. Labeling from [U-13C]glucose in glutamate and aspartate increased as a result of ammonium-treatment in both neurons and astrocytes, suggesting that the TCA cycle was not inhibited. Labeling in alanine increased in neurons but not in astrocytes, indicating elevated glycolysis in neurons. For both neurons and astrocytes, labeling from [U-13C]isoleucine entered glutamate and aspartate albeit to a lower extent than from [U-13C]glucose. Labeling in glutamate and aspartate from [U-13C]isoleucine was decreased by ammonium treatment in neurons but not in astrocytes, the former probably reflecting increased metabolism of unlabeled glucose. In astrocytes, ammonia treatment resulted in glutamine production and release to the medium, partially supported by catabolism of [U-13C]isoleucine. In conclusion, i) neuronal and astrocytic TCA cycle metabolism was not inhibited by ammonium and ii) isoleucine may provide the carbon skeleton for synthesis of glutamate/glutamine in the detoxification of ammonium.

Introduction

Cerebral hyperammonemia is thought to play a pivotal role in hepatic encephalopathy (HE), a debilitating neurological condition arising secondary to severe liver disease. The majority of patients suffering from liver cirrhosis develop at least one episode of HE during the cause of the disease, varying from minor disturbances in personality to frank coma. The pathogenesis of HE is largely unknown; however, cerebral intoxication caused by ammonium derived from the intestines (usually detoxified by the liver) is believed to play a role and hyperammonemia is a hallmark of liver cirrhosis (e.g. review by Albrecht and Dolinska, 2001). The glutamine synthetase (GS) reaction, forming glutamine from glutamate by amidation, is the quantitatively most important way for disposal of excess ammonium in the mammalian brain (review, Hawkins et al., 2002). Interestingly, GS is selectively localized in astrocytes (Norenberg and Martinez-Hernandez, 1979). In addition, the mitochondrial glutamate dehydrogenase (GDH; both neurons and astrocytes) reaction may also to some extent serve this purpose, as it fixes ammonium while forming glutamate from α-ketoglutarate by reductive amination, a mechanism shown to operate in cultured neurons (Yudkoff et al., 1990).

Cerebral hyperammonemia has been suggested to inhibit tricarboxylic acid (TCA) cycle metabolism at the level of both pyruvate dehydrogenase (PDH) and α-ketoglutarate dehydrogenase (α-KGDH) which are both rate-limiting steps in oxidative metabolism (Lai and Cooper, 1986, Zwingmann et al., 2003). The consequences of this might be an overall reduction of oxidative (TCA cycle) metabolism, which in turn may result in increased glycolysis. In a recently published hypothesis, degradation of the carbon skeletons of the branched-chain amino acids (BCAAs) particularly isoleucine and valine, was suggested to be able to compensate for the ammonium-induced inhibition of α-KGDH by introducing anaplerosis subsequent to the α-KGDH step plus acetyl-CoA (Fig. 1) (Ott et al., 2005). The anaplerotic function of isoleucine/valine was suggested to provide the carbon skeletons for glutamate and glutamine synthesis by GDH and GS, respectively, both reactions consuming ammonium and thus contributing to detoxification of excess ammonium. There are important differences in the degradation of the BCAAs carbon skeleton; leucine ultimately ends up as acetyl-CoA and may then be oxidized in the TCA cycle, whereas valine ends up as succinyl-CoA, and may act as an anaplerotic substrate. Isoleucine, on the other hand, is degraded into both acetyl-CoA and succinyl-CoA. This would make isoleucine an ideal substrate to counteract both the inhibition of α-KGDH and PDH by ammonium.

In the present study, the role of isoleucine as a TCA cycle substrate and metabolism of glucose during exposure to ammonium chloride was investigated in cultured mouse cerebellar neurons or astrocytes. The objective was: (i) to detect any significant inhibitory effect of ammonium exposure on TCA cycle metabolism; and (ii) to determine the ability of isoleucine to support TCA cycle metabolism and formation of glutamate/glutamine in the face of an ammonium challenge. Both types of cell cultures were incubated in the presence of different levels of ammonium chloride and either [U-13C]isoleucine or [U-13C]glucose. Cell extracts were analyzed by mass spectrometry for incorporation of label into alanine, glutamate, glutamine (in astrocytes) and aspartate. Release of glutamine into the medium (astrocytes only) was quantified by HPLC.

Section snippets

Materials

Seven-day-old NMRI mice were obtained from Taconic M&B (Ry, Denmark). Plastic tissue culture dishes were purchased from NUNC A/S (Roskilde, Denmark), fetal calf serum from SeraLab Ltd. (Sussex, U.K.). Culture medium and poly-D-lysine (MW > 300,000) were from Sigma Chemical Co. (St. Louis, MO, U.S.A.). Penicillin was from Leo (Ballerup, Denmark). Isotopically labeled compounds were either from Cambridge Isotopes Laboratories, Inc. (Massachusetts, U.S.A.) or Isotec (a subsidiary of Sigma Chemical

Results

In the present study, 13C-labeling from either [U-13C]isoleucine or [U-13C]glucose observed in alanine, glutamate, glutamine (astrocytes only) and aspartate is employed to study the effect of ammonium on TCA cycle metabolism of these two substrates. TCA cycling may in any given microenvironment be monitored using labeling of glutamate since aspartate aminotransferase (AAT) activity is several-fold higher than that of the TCA cycle (Drejer et al., 1985, Fitzpatrick et al., 1990, Mason et al.,

Discussion

Cerebral metabolic disturbances caused by hyperammonemia which may be related to the development of HE have so far not been fully elucidated. It was shown more than 4 decades ago, that ammonium inhibits oxygen consumption in brain slices and isolated mitochondria (McKhann and Tower, 1961) and, in addition, it has been suggested that the malate-aspartate shuttle may be inhibited due to a lowered amount of cytosolic glutamate (e.g. Hindfelt et al., 1977). Two hypotheses evolving around

Acknowledgements

Mr. Lars Evje, M.Sc. (Norwegian University of Science and Technology) and Ms. Lene Vigh (Danish University of Pharmaceutical Sciences) are cordially acknowledged for their expert technical assistance. The Lundbeck, Alfred Benzons and Hørslev Foundations as well as the Danish Medical Research Council (grants 22-04-0314 and 22-03-0250) are cordially acknowledged for generous funding.

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