Top

Published in:

01-04-2011

Detoxification of Ammonia in Mouse Cortical GABAergic Cell Cultures Increases Neuronal Oxidative Metabolism and Reveals an Emerging Role for Release of Glucose-Derived Alanine

Authors: Renata Leke, Lasse K. Bak, Malene Anker, Torun M. Melø, Michael Sørensen, Susanne Keiding, Hendrik Vilstrup, Peter Ott, Luis V. Portela, Ursula Sonnewald, Arne Schousboe, Helle S. Waagepetersen

Published in: Neurotoxicity Research | Issue 3/2011

Abstract

Cerebral hyperammonemia is believed to play a pivotal role in the development of hepatic encephalopathy (HE), a debilitating condition arising due to acute or chronic liver disease. In the brain, ammonia is thought to be detoxified via the activity of glutamine synthetase, an astrocytic enzyme. Moreover, it has been suggested that cerebral tricarboxylic acid (TCA) cycle metabolism is inhibited and glycolysis enhanced during hyperammonemia. The aim of this study was to characterize the ammonia-detoxifying mechanisms as well as the effects of ammonia on energy-generating metabolic pathways in a mouse neuronal–astrocytic co-culture model of the GABAergic system. We found that 5 mM ammonium chloride affected energy metabolism by increasing the neuronal TCA cycle activity and switching the astrocytic TCA cycle toward synthesis of substrate for glutamine synthesis. Furthermore, ammonia exposure enhanced the synthesis and release of alanine. Collectively, our results demonstrate that (1) formation of glutamine is seminal for detoxification of ammonia; (2) neuronal oxidative metabolism is increased in the presence of ammonia; and (3) synthesis and release of alanine is likely to be important for ammonia detoxification as a supplement to formation of glutamine.

Ahboucha S, Butterworth RF (2004) Pathophysiology of hepatic encephalopathy: a new look at GABA from the molecular standpoint. Metab Brain Dis 19(3–4):331–343CrossRefPubMed

Ahboucha S, Butterworth RF (2008) The neurosteroid system: implication in the pathophysiology of hepatic encephalopathy. Neurochem Int 52(4):575–587CrossRefPubMed

Albrecht J, Jones EA (1999) Hepatic encephalopathy: molecular mechanisms underlying the clinical syndrome. J Neurol Sci 170(2):138–146CrossRefPubMed

Bak LK, Sickmann HM, Schousboe A, Waagepetersen HS (2005) Activity of the lactate-alanine shuttle is independent of glutamate–glutamine cycle activity in cerebellar neuronal-astrocytic cultures. J Neurosci Res 79(1–2):88–96CrossRefPubMed

Bak LK, Schousboe A, Sonnewald U, Waagepetersen HS (2006) Glucose is necessary to maintain neurotransmitter homeostasis during synaptic activity in cultured glutamatergic neurons. J Cereb Blood Flow Metab 26(10):1285–1297CrossRefPubMed

Bak LK, Iversen P, Sørensen M, Keiding S, Vilstrup H, Ott P, Waagepetersen HS, Schousboe A (2009) Metabolic fate of isoleucine in a rat model of hepatic encephalopathy and in cultured neural cells exposed to ammonia. Metab Brain Dis 24(1):135–145CrossRefPubMed

Basile AS (2002) Direct and indirect enhancement of GABAergic neurotransmission by ammonia: implications for the pathogenesis of hyperammonemic syndromes. Neurochem Int 41(2–3):115–122

Berl S, Takagari G, Clarke DD, Waelsch H (1962) Metabolic compartments in vivo ammonia and glutamic acid metabolism in brain and liver. J Biol Chem 237:2562–2599PubMed

Bessman SP, Bessman AN (1955) The cerebral and peripheral uptake of ammonia in liver disease with an hypothesis for the mechanism of hepatic coma. J Clin Invest 34(4):622–628CrossRefPubMed

Biemann K (1962) Mass spectrometry. In: Organic chemistry applications. McGraw, New York, pp 223–227

Blei AT, Córdoba J (2001) Encephalopathy. Practice Parameters Committee of the American College of Gastroenterology. Am J Gastroenterol 96(7):1968–1976CrossRefPubMed

Bosman DK, Deutz NE, De Graaf AA, vd Hulst RW, Van Eijk HM, Bovée WM, Maas MA, Jörning GG, Chamuleau RA (1990) Changes in brain metabolism during hyperammonemia and acute liver failure: results of a comparative 1H-NMR spectroscopy and biochemical investigation. Hepatology 12(2):281–290CrossRefPubMed

Brusilow SW, Traystman R (1986) Hepatic encephalopathy. N Engl J Med 314(12):786–787PubMed

Butterworth RF (2000) Hepatic encephalopathy: a neuropsychiatric disorder involving multiple neurotransmitter systems. Curr Opin Neurol 13(6):721–727CrossRefPubMed

Butterworth RF (2002) Pathophysiology of hepatic encephalopathy: a new look at ammonia. Metab Brain Dis 17(4):221–227CrossRefPubMed

Cauli O, Mansouri MT, Agusti A, Felipo V (2009) Hyperammonemia increases GABAergic tone in the cerebellum but decreases it in the rat cortex. Gastroenterology 136(4):1359–1367CrossRefPubMed

Cesar M, Hamprecht B (1995) Immunocytochemical examination of neural rat and mouse primary cultures using monoclonal antibodies raised against pyruvate carboxylase. J Neurochem 64(5):2312–2318CrossRefPubMed

Chatauret N, Rose C, Therrien G, Butterworth RF (2001) Mild hypothermia prevents cerebral edema and CSF lactate accumulation in acute liver failure. Metab Brain Dis 16(1–2):95–102CrossRefPubMed

Chatauret N, Zwingmann C, Rose C, Leibfritz D, Butterworth RF (2003) Effects of hypothermia on brain glucose metabolism in acute liver failure: a H/C-nuclear magnetic resonance study. Gastroenterology 125(3):815–824CrossRefPubMed

Cooper AJ, Plum F (1987) Biochemistry and physiology of brain ammonia. Physiol Rev 67(2):440–519PubMed

Cooper AJ (2001) Role of glutamine in cerebral nitrogen metabolism and ammonia neurotoxicity. Ment Retard Dev Disabil Res Rev 7(4):280–286CrossRefPubMed

Cooper AJ, McDonald JM, Gelbard AS, Gledhill RF, Duffy TE (1979) The metabolic fate of 13N-labeled ammonia in rat brain. J Biol Chem 254(12):4982–4992PubMed

Cooper AJ, Mora SN, Cruz NF, Gelbard AS (1985) Cerebral ammonia metabolism in hyperammonemic rats. J Neurochem 44(6):1716–1723CrossRefPubMed

Cruz NF, Lasater A, Zielke HR, Dienel GR (2005) Activation of astrocytes in brain of conscious rats during acoustic stimulation: acetate utilization in working brain. J Neurochem 92(4):934–947CrossRefPubMed

Gegelashvili G, Danbolt NC, Schousboe A (1997) Neuronal soluble factors differentially regulate the expression of the GLT1 and GLAST glutamate transporters in cultured astroglia. J Neurochem 69(6):2612–2615CrossRefPubMed

Haghighat N, McCandless DW (1997) Effect of ammonium chloride on energy metabolism of astrocytes and C6-glioma cells in vitro. Metab Brain Dis 12(4):287–298PubMed

Hassel B, Sonnewald U, Fonnum F (1995) Glial-neuronal interactions as studied by cerebral metabolism of [2–13C]acetate and [1–13C]glucose: an ex vivo 13C NMR spectroscopic study. J Neurochem 64(6):2773–2782CrossRefPubMed

Hawkins RA, O’Kane RL, Simpson IA, Viña JR (2006) Structure of the blood–brain barrier and its role in the transport of amino acids. J Nutr 136(Suppl 1):218S–226SPubMed

Hazell AS, Butterworth RF (1999) Hepatic encephalopathy: an update of pathophysiologic mechanisms. Proc Soc Exp Biol Med 222(2):99–112CrossRefPubMed

Hertz L, Kala G (2007) Energy metabolism in brain cells: effects of elevated ammonia concentrations. Metab Brain Dis 22(3–4):199–218CrossRefPubMed

Hertz L, Juurlink BHJ, Fosmark H, Schousboe A (1982) Astrocytes in primary cultures. In: Pfeiffer SE (ed) Neuroscience approached through cell culture, vol 1. CRC Press, Boca Raton, FL, pp 175–186

Hertz E, Yu ACH, Hertz L, Juurlink BHJ, Schousboe A (1989) Preparation of primary cultures of mouse cortical neurons. In: Shahar A, De Vellis J, Vernadakis A, Haber B (eds) A dissection and tissue culture manual for the nervous system. Alan R. Liss, New York, pp 183–186

Hertz L, Dringen R, Schousboe A, Robinson SR (1999) Astrocytes: glutamate producers for neurons. J Neurosci Res 57(4):417–428CrossRefPubMed

Hindfelt B, Plum F, Duffy TE (1977) Effect of acute ammonia intoxication on cerebral metabolism in rats with portacaval shunts. J Clin Invest 59(3):386–396CrossRefPubMed

Iversen P, Sørensen M, Bak LK, Waagepetersen HS, Vafee MS, Borghammer P, Mouridsen K, Jensen SB, Vilstrup H, Schousboe A, Ott P, Gjedde A, Keiding S (2009) Low cerebral oxygen consumption and blood flow in patients with cirrhosis and an acute episode of hepatic encephalopathy. Gastroenterology 136:863–871CrossRefPubMed

Jayakumar AR, Rama Rao KV, Schousboe A, Norenberg MD (2004) Glutamine-induced free radical production in cultured astrocytes. Glia 46(3):296–301CrossRefPubMed

Jayakumar AR, Rao KV, ChR Murthy, Norenberg MD (2006) Glutamine in the mechanism of ammonia-induced astrocyte swelling. Neurochem Int 48(6–7):623–628PubMed

Johansen ML, Bak LK, Schousboe A, Iversen P, Sørensen M, Keiding S, Vilstrup H, Gjedde A, Ott P, Waagepetersen HS (2007) The metabolic role of isoleucine in detoxification of ammonia in cultured mouse neurons and astrocytes. Neurochem Int 50(7–8):1042–1051CrossRefPubMed

Jones EA (2003) Potential mechanisms of enhanced GABA-mediated inhibitory neurotransmission in liver failure. Neurochem Int 43(4–5):509–516CrossRefPubMed

Kala G, Hertz L (2005) Ammonia effects on pyruvate/lactate production in astrocytes-interaction with glutamate. Neurochem Int 47(1–2):4–12CrossRefPubMed

Kanamatsu T, Tsukada Y (1999) Effects of ammonia on the anaplerotic pathway and amino acid metabolism in the brain: an ex vivo 13C NMR spectroscopic study of rats after administering [2–13C] glucose with or without ammonium acetate. Brain Res 841(1–2):11–19CrossRefPubMed

Kanamori K, Ross BD, Chung JC, Kuo EL (1996) Severity of hyperammonemic encephalopathy correlates with brain ammonia level and saturation of glutamine synthetase in vivo. J Neurochem 67(4):1584–1594CrossRefPubMed

Keiding S, Sørensen M, Bender D, Munk OL, Ott P, Vilstrup H (2006) Brain metabolism of 13N-ammonia during acute hepatic encephalopathy in cirrhosis measured by PET. Hepatology 43:42–50 (Correction in Hepatology 2006;44:1056)

Kosenko E, Kaminsky Y, Kaminsky A, Valencia M, Lee L, Hermenegildo C, Felipo V (1997) Superoxide production and antioxidant enzymes in ammonia intoxication in rats. Free Radic Res 27(6):637–644CrossRefPubMed

Kramer L, Tribl B, Gendo A, Zauner C, Schneider B, Ferenci P, Madl C (2000) Partial pressure of ammonia versus ammonia in hepatic encephalopathy. Hepatology 31(1):30–34CrossRefPubMed

Kundra A, Jain A, Banga A, Bajaj G, Kar P (2005) Evaluation of plasma ammonia levels in patients with acute liver failure and chronic liver disease and its correlation with the severity of hepatic encephalopathy and clinical features of raised intracranial tension. Clin Biochem 38(8):696–699CrossRefPubMed

Lai JC, Cooper AJ (1986) Brain alpha-ketoglutarate dehydrogenase complex: kinetic properties, regional distribution, and effects of inhibitors. J Neurochem 47(5):1376–1386CrossRefPubMed

Lapidot A, Gopher A (1997) Quantitation of metabolic compartmentation in hyperammonemic brain by natural abundance 13C-NMR detection of 13C–15N coupling patterns and isotopic shifts. Eur J Biochem 243(3):597–604CrossRefPubMed

Leke R, Bak LK, Schousboe A, Waagepetersen HS (2008) Demonstration of neuron-glia transfer of precursors for GABA biosynthesis in a co-culture system of dissociated mouse cerebral cortex. Neurochem Res 33(12):2629–2635CrossRefPubMed

Lockwood AH (2004) Blood ammonia levels and hepatic encephalopathy. Metab Brain Dis 19(3–4):345–349CrossRefPubMed

Lovatt D, Sonnewald U, Waagepetersen HS, Schousboe A, He W, Lin JH, Han X, Takano T, Wang S, Sim FJ, Goldman SA, Nedergaard M (2007) The transcriptome and metabolic gene signature of protoplasmic astrocytes in the adult murine cortex. J Neurosci 27(45):12255–12266CrossRefPubMed

Lowry OH, Rosebrough NJ, Farr AL, Randall RJ (1951) Protein measurement with the folin phenol reagent. J Biol Chem 193:265–275PubMed

Merle M, Martin M, Villégier A, Canioni P (1996) [1–13C]Glucose metabolism in brain cells: isotopomer analysis of glutamine from cerebellar astrocytes and glutamate from granule cells. Dev Neurosci 18(5–6):460–468CrossRefPubMed

Murthy CR, Rama Rao KV, Bai G, Norenberg MD (2001) Ammonia-induced production of free radicals in primary cultures of rat astrocytes. J Neurosci Res 66(2):282–288CrossRefPubMed

Norenberg MD (1998) Astroglial dysfunction in hepatic encephalopathy. Metab Brain Dis 13(4):319–335CrossRefPubMed

Norenberg MD, Martinez-Hernandez A (1979) Fine structural localization of glutamine synthetase in astrocytes of rat brain. Brain Res 161(2):303–310CrossRefPubMed

Norenberg MD, Rao KV, Jayakumar AR (2005) Mechanisms of ammonia-induced astrocyte swelling. Metab Brain Dis 20(4):303–318CrossRefPubMed

Ott P, Clemmesen O, Larsen FS (2005) Cerebral metabolic disturbances in the brain during acute liver failure: from hyperammonemia to energy failure and proteolysis. Neurochem Int 47(1–2):13–18CrossRefPubMed

Patel MS (1974) The relative significance of CO₂-fixing enzymes in the metabolism of rat brain. J Neurochem 22(5):717–724CrossRefPubMed

Rama Rao KV, Jayakumar AR, Norenberg MD (2003) Induction of the mitochondrial permeability transition in cultured astrocytes by glutamine. Neurochem Int 43(4–5):517–523CrossRefPubMed

Rama Rao KV, Jayakumar AR, Norenberg MD (2005) Role of oxidative stress in the ammonia-induced mitochondrial permeability transition in cultured astrocytes. Neurochem Int 47(1–2):31–38CrossRefPubMed

Rao KV, Norenberg MD (2001) Cerebral energy metabolism in hepatic encephalopathy and hyperammonemia. Metab Brain Dis 16(1–2):67–78CrossRefPubMed

Ratnakumari L, Murthy CR (1992) In vitro and in vivo effects of ammonia on glucose metabolism in the astrocytes of rat cerebral cortex. Neurosci Lett 148(1–2):85–88CrossRefPubMed

Ratnakumari L, Murthy CR (1993) Response of rat cerebral glycolytic enzymes to hyperammonemic states. Neurosci Lett 161(1):37–40CrossRefPubMed

Schlag BD, Vondrasek JR, Munir M, Kalandadze A, Zelenaia OA, Rothstein JD, Robinson MB (1998) Regulation of the glial Na⁺-dependent glutamate transporters by cyclic AMP analogs and neurons. Mol Pharmacol 53(3):355–369PubMed

Schousboe A, Waagepetersen HS (2007) GABA: homeostatic and pharmacological aspects. Prog Brain Res 160:9–19CrossRefPubMed

Shank RP, Bennett GS, Freytag SO, Campbell GL (1985) Pyruvate carboxylase: an astrocyte specific enzyme implicated in the replenishment of amino acid neurotransmitter pools. Brain Res 329(1–2):364–367CrossRefPubMed

Swain M, Butterworth RF, Blei AT (1992) Ammonia and related amino acids in the pathogenesis of brain edema in acute ischemic liver failure in rats. Hepatology 15(3):449–453CrossRefPubMed

Swanson RA, Liu J, Miller JW, Rothstein JD, Farrell K, Stein BA, Longuemare MC (1997) Neuronal regulation of glutamate transporter subtype expression in astrocytes. J Neurosci 17(3):932–940PubMed

Tofteng F, Larsen FS (2002) Monitoring extracellular concentrations of lactate, glutamate, and glycerol by in vivo microdialysis in the brain during liver transplantation in acute liver failure. Liver Transpl 8(3):302–305CrossRefPubMed

Waagepetersen HS, Sonnewald U, Larsson OM, Schousboe A (2000) A possible role of alanine for ammonia transfer between astrocytes and glutamatergic neurons. J Neurochem 75(2):471–479CrossRefPubMed

Waagepetersen HS, Qu H, Hertz L, Sonnewald U, Schousboe A (2002) Demonstration of pyruvate recycling in primary cultures of neocortical astrocytes but not in neurons. Neurochem Res 27(11):1431–1437CrossRefPubMed

Yu AC, Drejer J, Hertz L, Schousboe A (1983) Pyruvate carboxylase activity in primary cultures of astrocytes and neurons. J Neurochem 41(5):1484–1487CrossRefPubMed

Yudkoff M, Nissim I, Hertz L (1990) Precursors of glutamic acid nitrogen in primary neuronal cultures: studies with ¹⁵N. Neurochem Res 15(12):1191–1196CrossRefPubMed

Zwingmann C (2007) The anaplerotic flux and ammonia detoxification in hepatic encephalopathy. Metab Brain Dis 22(3–4):235–249CrossRefPubMed

Zwingmann C, Butterworth R (2005) An update on the role of brain glutamine synthesis and its relation to cell-specific energy metabolism in the hyperammonemic brain: further studies using NMR spectroscopy. Neurochem Int 47(1–2):19–30CrossRefPubMed

Zwingmann C, Leibfritz D (2005) Ammonia toxicity under hyponatremic conditions in astrocytes: de novo synthesis of amino acids for the osmoregulatory response. Neurochem Int 47(1–2):39–50CrossRefPubMed

Zwingmann C, Brand A, Richter-Landsberg C, Leibfritz D (1998) Multinuclear NMR spectroscopy studies on NH₄Cl-induced metabolic alterations and detoxification processes in primary astrocytes and glioma cells. Dev Neurosci 20(4–5):417–426CrossRefPubMed

Zwingmann C, Richter-Landsberg C, Brand A, Leibfritz D (2000) NMR Spectroscopic study on the metabolic fate of [3–13C]alanine in astrocytes, neurons, and cocultures: implications for glia-neuron interactions in neurotransmitter metabolism. Glia 32(3):286–303CrossRefPubMed

Zwingmann C, Chatauret N, Leibfritz D, Butterworth RF (2003) Selective increase of brain lactate synthesis in experimental acute liver failure: results of a [H–C] nuclear magnetic resonance study. Hepatology 37(2):420–428CrossRefPubMed

Title: Detoxification of Ammonia in Mouse Cortical GABAergic Cell Cultures Increases Neuronal Oxidative Metabolism and Reveals an Emerging Role for Release of Glucose-Derived Alanine
Authors: Renata Leke
Lasse K. Bak
Malene Anker
Torun M. Melø
Michael Sørensen
Susanne Keiding
Hendrik Vilstrup
Peter Ott
Luis V. Portela
Ursula Sonnewald
Arne Schousboe
Helle S. Waagepetersen
Publication date: 01-04-2011
Publisher: Springer-Verlag
Published in: Neurotoxicity Research / Issue 3/2011
Print ISSN: 1029-8428
Electronic ISSN: 1476-3524
DOI: https://doi.org/10.1007/s12640-010-9198-7

Keynote webinar | Spotlight on medication adherence

Springer Medicine

Detoxification of Ammonia in Mouse Cortical GABAergic Cell Cultures Increases Neuronal Oxidative Metabolism and Reveals an Emerging Role for Release of Glucose-Derived Alanine

Abstract

Keynote webinar | Spotlight on medication adherence

Springer Medicine

Abstract

Please log in to get access to this content

Other articles of this Issue 3/2011

Peroxisome Proliferator-Activated Receptor-γ (PPAR-γ) Activation Confers Functional Neuroprotection in Global Ischemia

Prenatal Exposure to PFOS or PFOA Alters Motor Function in Mice in a Sex-Related Manner

Neuroprotection of Interleukin-6 Against NMDA-Induced Apoptosis and Its Signal-Transduction Mechanisms

Accumulation of Non-Transferrin-Bound Iron by Neurons, Astrocytes, and Microglia

Neuroprotective Effects of PACAP Against Ethanol-Induced Toxicity in the Developing Rat Cerebellum

Highly Toxic Microcystis aeruginosa Strain, Isolated from São Paulo—Brazil, Produce Hepatotoxins and Paralytic Shellfish Poison Neurotoxins