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

Abstract

A hexose phosphate recycling model previously developed to infer fluxes through the major glucose consuming pathways in cultured cerebellar granule neurons (CGNs) from neonatal rats metabolizing [1,2-13C2]glucose was revised by considering reverse flux through the non-oxidative pentose phosphate pathway (PPP) and symmetrical succinate oxidation within the tricarboxylic acid (TCA) cycle. The model adjusts three flux ratios to effect 13C distribution in the hexose, pentose, and triose phosphate pools, and in TCA cycle malate to minimize the error between predicted and measured 13C labeling in exported lactate (i.e., unlabeled, single-, double-, and triple-labeled; M, M1, M2, and M3, respectively). Inclusion of reverse non-oxidative PPP flux substantially increased the number of calculations but ultimately had relatively minor effects on the labeling of glycolytic metabolites. From the error-minimized solution in which the predicted M-M3 lactate differed by 0.49% from that measured by liquid chromatography-triple quadrupole mass spectrometry, the neurons exhibited negligible forward non-oxidative PPP flux. Thus, no glucose was used by the pentose cycle despite explicit consideration of hexose phosphate recycling. Mitochondria consumed only 16% of glucose while 45% was exported as lactate by aerobic glycolysis. The remaining 39% of glucose was shunted to pentose phosphates presumably for de novo nucleotide synthesis, but the proportion metabolized through the oxidative PPP vs. the reverse non-oxidative PPP could not be determined. The lactate exported as M1 (2.5%) and M3 (1.2%) was attributed to malic enzyme, which was responsible for 7.8% of pyruvate production (vs. 92.2% by glycolysis). The updated model is more broadly applicable to different cell types by considering bi-directional flux through the non-oxidative PPP. Its application to cultured neurons utilizing glucose as the sole exogenous substrate has demonstrated substantial oxygen-independent glucose utilization by aerobic glycolysis as well as the oxidative PPP and/or reverse non-oxidative PPP, but negligible glucose consumption by the pentose cycle.

Brain Glucose Metabolism: Integration of Energetics with Function

“…Because most postsynaptic spines, many presynaptic varicosities, and some synaptosomes do not have mitochondria, specific neuronal subcellular compartments and functions depend on glycolysis and they would produce and release lactate. Under resting and depolarizing conditions, cultured cerebellar glutamatergic neurons release nearly half of the glucose metabolized to the medium as lactate (216,325,668), as do cultured cortical neurons (378). However, lactate production by cultured cells is quite unlikely to be predictive of cellular sources of lactate in living brain because the large extracellular medium volume dilutes the released lactate and pulls lactate from all cells via the equilibrative LDH and MCT reactions to inflate glycolytic rates (162,274).…”

“…Parallel assays showed that lactate oxidation did not increase under these conditions. Cultured cerebellar neurons do oxidize lactate but are highly glycolytic under resting (668) and depolarizing conditions (216,325), with~45% of the glucose consumed being released to the medium as lactate in both cases. The anomaly that astrocyte-neuron shuttling of glycogen-derived lactate should be needed for neurotransmission in cerebellar cocultures when lactate is not used by and is massively released by neuronal monocultures requires explanation.…”

Breast cancer cells that preferentially metastasize to lung or bone are more glycolytic, synthesize serine at greater rates, and consume less ATP and NADPH than parent MDA-MB-231 cells

“…1 and predicts the fractional abundance of 13 C lactate isotopologues (designated M, M1, M2, and M3 lactate, for zero, one, two, or three, respectively, 13 C atoms) for cells metabolizing [1,2- 13 C] glucose as the sole exogenous substrate. Details of this model have been previously described [ 15 , 16 ].

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13C metabolic flux analysis: Classification and characterization from the perspective of mathematical modeling and application in physiological research of neural cell

“…Meanwhile, it determined that 16% of glucose used by mitochondria comparing to 46% by lactate dehydrogenase ( Gebril et al, 2016 ). By revising bi-directional reaction of the non-oxidative PPP pathway and TCA cycle, this method became more broadly applicable to different cell types ( Jekabsons et al, 2017 ). Patel et al (2021) investigated TCA cycle and neurotransmitter cycle fluxes ratio by FR method from a steady-state [2- 13 C] acetate experiment and the 13 C turnover rates of neurotransmitter by a KFP-like fitting on labeling kinetics of amino acids in 13 C glucose infusion experiment.…”