13C metabolic flux analysis in neurons utilizing a model that accounts for hexose phosphate recycling within the pentose phosphate pathway

Abstract

Glycolysis, mitochondrial substrate oxidation, and the pentose phosphate pathway (PPP) are critical for neuronal bioenergetics and oxidation-reduction homeostasis, but quantitating their fluxes remains challenging, especially when processes such as hexose phosphate (i.e., glucose/fructose-6-phosphate) recycling in the PPP are considered. A hexose phosphate recycling model was developed which exploited the rates of glucose consumption, lactate production, and mitochondrial respiration to infer fluxes through the major glucose consuming pathways of adherent cerebellar granule neurons by replicating [13C]lactate labeling from metabolism of [1,2-13C2]glucose. Flux calculations were predicated on a steady-state system with reactions having known stoichiometries and carbon atom transitions. Non-oxidative PPP activity and consequent hexose phosphate recycling, as well as pyruvate production by cytoplasmic malic enzyme, were optimized by the model and found to account for 28±2 % and 7.7±0.2 % of hexose phosphate and pyruvate labeling, respectively. From the resulting fluxes, 52±6 % of glucose was metabolized by glycolysis, compared to 19±2 % by the combined oxidative/non-oxidative pentose cycle that allows for hexose phosphate recycling, and 29±8 % by the combined oxidative PPP/de novo nucleotide synthesis reactions. By extension, 62±6 % of glucose was converted to pyruvate, the metabolism of which resulted in 16±1 % of glucose oxidized by mitochondria and 46±6 % exported as lactate. The results indicate a surprisingly high proportion of glucose utilized by the pentose cycle and the reactions synthesizing nucleotides, and exported as lactate. While the in vitro conditions to which the neurons were exposed (high glucose, no lactate or other exogenous substrates) limit extrapolating these results to the in vivo state, the approach provides a means of assessing a number of metabolic fluxes within the context of hexose phosphate recycling in the PPP from a minimal set of measurements.

Glucose promotes resistance in lymphocytes against oxidative stress-induced apoptosis through signaling and metabolic pathways. Implications for Parkinson’s disease

“…Recent data suggest that dysregulation of G metabolism might represent an early metabolic biomarker of sporadic PD (6). Because lymphocytes express GLU-1 and GLU-3 transporter proteins (49), display molecular death machinery (50), and G metabolism (25) similar to neurons (8,47,51), we consider that lymphocyte cells represent a remarkable non-neural cell model to understanding the signaling and metabolic regulation of apoptosis in response to stressful stimuli. Therefore, the metabolism-OS-cell death axis is a fundamental issue in the therapeutic design to prevent, delay, or ameliorate the treatment of individuals at risk of PD as encountered in Antioquia, Colombia (52).…”

Updates to a 13 C metabolic flux analysis model for evaluating energy metabolism in cultured cerebellar granule neurons from neonatal rats

“…These constraints include (a) the number, connectedness, and compartmentation of the reactions (i.e., the system structure), (b) the carbon atom rearrangements for each substrate/product pair, (c) the stoichiometries of the reactions, and (d) any assumptions about pathway behavior (e.g., reversibility). We recently developed a model for quantifying fluxes through the major glucose consuming reaction blocks in primary cerebellar granule neurons metabolizing [1,2- 13 C 2 ]glucose (Gebril et al, 2016). The model quantitatively tracks 13 C distribution in glucose-6-phosphate/fructose-6-phosphate (hexose phosphates-HxP) from the recycling of glucose carbons via the non-oxidative PPP back to the oxidative PPP, and 13 C distribution in malate by carbon rearrangements within the tricarboxylic acid (TCA) cycle.…”

“…The latter distribution was considered since 13 C in pyruvate entering the TCA cycle can be recycled back to pyruvate via cytoplasmic and mitochondrial malic enzyme. The quantitative analysis is based on measurements of glucose consumption, lactate production, and mitochondrial respiration, and theoretical predictions on the extent of HxP recycling within the PPP (Gebril et al, 2016; WOOD and KATZ, 1958). The measured 13 C distribution in exported lactate (i.e., the fraction having zero, one, two, or three 13 C atoms- denoted as M, M1, M2, and M3, respectively) is reproduced from calculations (predictions) of 13 C distributions in HxP, malate, triose phosphates (TrP; glyceraldehyde-3-phosphate and dihydroxyacetone phosphate), and pyruvate, which in turn are determined from optimizing the relative contributions of the non-oxidative PPP, glycolysis, the TCA cycle, and malic enzyme to producing these metabolites.…”

“…The measured 13 C distribution in exported lactate (i.e., the fraction having zero, one, two, or three 13 C atoms- denoted as M, M1, M2, and M3, respectively) is reproduced from calculations (predictions) of 13 C distributions in HxP, malate, triose phosphates (TrP; glyceraldehyde-3-phosphate and dihydroxyacetone phosphate), and pyruvate, which in turn are determined from optimizing the relative contributions of the non-oxidative PPP, glycolysis, the TCA cycle, and malic enzyme to producing these metabolites. One notable assumption made about pathway behavior was unidirectional flux through the non-oxidative PPP (Gebril et al, 2016), despite the known reversible nature of this pathway; this served to minimize the number of calculations required for assessing 13 C distribution in HxP and TrP. This article describes revisions to the original model that improve its accuracy and applicability to different cell types by accounting for 13 C randomization in HxP, pentose phosphates, and TrP resulting from reverse non-oxidative PPP flux, and in malate by symmetrical metabolism of succinate in the TCA cycle.…”

“…This article describes revisions to the original model that improve its accuracy and applicability to different cell types by accounting for 13 C randomization in HxP, pentose phosphates, and TrP resulting from reverse non-oxidative PPP flux, and in malate by symmetrical metabolism of succinate in the TCA cycle. The updates prompted re-analysis of 13 C distribution in lactate from cerebellar granule neurons metabolizing [1,2- 13 C 2 ]glucose previously reported in (Gebril et al, 2016). The revised model and measurements indicate negligible non-oxidative PPP flux, contrary to our original conclusion of extensive pentose cycle activity.…”