Mass Isotopomer Study of Glutamine Oxidation and Synthesis in Primary Culture of Astrocytes

Abstract

The metabolism of [1,2–¹³C₂] acetate via the tricarboxylic acid (TCA) cycle leads to the formation of a number of key mass isotopomers of glutamate. The distribution of these isotopomers which is a function of pyruvate carboxylase, pyruvate dehydrogenase and pyruvate recycling was used to determine the relative anaplerotic flux and glutamine oxidation of astrocytes in culture under different substrate conditions. Combinatory analysis of mass isotopomers formed from the condensation of labeled oxaloacetate with labeled acetyl-CoA was used to determine precursor enrichment and fractional glutamine synthesis. When glucose or glutamine was supplied in the medium, the effective anaplerotic flux (Y') was about 1.5 times that of the TCA cycle flux. Under substrate-limiting conditions, Y' and glutamine synthesis was significantly reduced. A unique feature of the use of [1, 2–¹³C₂] acetate in this study is the formation of singly labeled isotopomer of glutamine in the C4 or C5 position when glutamine is irreversibly loss in net oxidation. We observed very little [4-¹³C] or [5-¹³C] glutamine either because of the lack of pyruvate recycling or the lack of pyruvate dehydrogenase activity. The lack of ^l3C recycling to the C4 and C5 position of glutamine suggests that less than 10% of the glutamine is oxidized in astrocytes for energy production. Therefore, glutamine is not a major energy substrate for astrocytes in culture.

Increased Pentose Phosphate Pathway Flux after Clinical Traumatic Brain Injury: A [1,2-13C2]glucose Labeling Study in Humans

“…Finally, with higher enrichment of labeled glucose and use of additional labeled compounds such as 13 C-labeled lactate, it would be possible to evaluate other pathways. For example, comparisons of glutamate and glutamine isotopomers can give information about fluxes through the TCA cycle, anaplerosis, and the relative activity of the pyruvate dehydrogenase complex versus the pyruvate carboxylase enzyme (Hyder et al, 1996;Lee et al, 1996;Rothman et al, 2003). Detection of 13 C-label in proteins could show production of amino acids from glucose by way of TCA cycle intermediates.…”

GLP-1 stimulates glucose-derived de novo fatty acid synthesis and chain elongation during cell differentiation and insulin release

“…Proteins were removed by centrifugation, and the supernatant was neutralized with potassium hydroxide. The neutralized supernatant was passed through a set of Dowex-50 (H ϩ ) and Dowex-1 (acetate) columns for the isolation of glutamate and conversion to its trifluoroacetyl butyl ester (TAB) as described elsewhere (17,18). Under EI conditions, ionization of TAB-glutamate gives rise to two fragments, m/z 198 and m/z 152, corresponding to C 2 -C 5 and C 2 -C 4 of glutamate, respectively, which are routinely analyzed during the determination of TCA cycle carbon flow.…”

Regulation of glial metabolism studied by 13C‐NMR

“…145 Oxidative metabolism of glutamine, however, is much less efficient compared to glutamate, consistent with a limitation given by restricted PAG activity in astrocytes. 180,182 Besides glutamine degradation by glutaminase, reversible GS may be involved in glutamine consumption. 183 In addition, glutamine aminotranferase K may play a role for glial glutamine metabolism.…”