Effects of Ammonia and β‐Methylene‐dl‐Aspartate on the Oxidation of Glucose and Pyruvate by Neurons and Astrocytes in Primary Culture

Abstract

Both ammonia and beta-methylene-DL-aspartate (beta-MA), an irreversible inhibitor of aspartate aminotransferase activity and thus of the malate-aspartate shuttle, were found previously to decrease oxidative metabolism in cerebral cortex slices. In the present work, the possibility that ammonia and beta-MA affect energy metabolism by a common mechanism (i.e., via inhibition of the malate-aspartate shuttle) was investigated using primary cultures of neurons and astrocytes. Incubation of astrocytes for 30 min with 5 mM beta-MA resulted in a decreased production of 14CO2 from [U-14C]glucose, but did not affect 14CO2 production from [2-14C]pyruvate. Conversely, incubation of astrocytes with 3 mM ammonium chloride resulted in decreased 14CO2 production from [2-14C]pyruvate, but 14CO2 production from [U-14C]glucose was not significantly affected. Ammonium chloride had no significant effect on 14CO2 production from either [U-14C]glucose or [2-14]pyruvate by neurons. However, incubation of neurons with beta-MA or beta-MA plus ammonium chloride resulted in a approximately 45% decrease of 14CO2 production from both [U-14C]glucose and [2-14C]pyruvate. A 2-h incubation of astrocytes with beta-MA resulted in no change in ATP levels, but a 35% decrease in phosphocreatine. Similar treatment of neurons resulted in greater than 50% decrease in ATP, but had little effect on phosphocreatine. beta-MA also caused a decrease in glutamate and aspartate content of neurons, but not of astrocytes. The different metabolic responses of neurons and astrocytes towards beta-MA were probably not due to a differential inhibition of aspartate aminotransferase which was inhibited by approximately 45% in astrocytes and by approximately 55% in neurons.

Role of Pyruvate Carboxylase in Facilitation of Synthesis of Glutamate and Glutamine in Cultured Astrocytes

“…As malate, oxaloacetate, and aspartate are likely in isotopic equilibrium, this implies that the mass amount of aspartate is lower than that of malate. Low levels of aspartate were reported previously in cultured astrocytes (Fitzpatrick et a!., 1988). Low aspartate availability is likely responsible for the slow conversion of a-ketog!utarate to glutamate observed in Fig.…”

Increased brain lactate is central to the development of brain edema in rats with chronic liver disease

“…In addition, we observed an increase in glucose-derived lactate, and an increase in glycolysis flux, possibly a result of ammonia-stimulated phosphofructokinase activity [34]. However, in spite of these TCA cycle alterations (inhibition of α-ketoglutarate dehydrogenase), ATP levels remain maintained [35] and [36]. Therefore, ammoniainduced increase in brain lactate is a not a result of energy failure (activated anaerobic metabolism); rather, the increase in lactate synthesis may occur as a compensatory mechanism to maintain ATP levels.…”

Ammonium Induces Aberrant Blastocyst Differentiation, Metabolism, pH Regulation, Gene Expression and Subsequently Alters Fetal Development in the Mouse

“…Ammonium has been shown to reduce the oxidative capacity of tissues by interfering with the transport of reducing equivalents between the cytoplasm and the mitochondria by disrupting the malate-aspartate shuttle activity [56]. Disruption of this shuttle results in an inability of cells to maintain the balance of NAD ϩ /NADH within the cytoplasm and mitochondria and results in a decrease in tricarboxylic acid-cycle activity [57,58]. Ammonium also causes changes in the mitochondrial NAD ϩ :NADH ratios [59].…”