Quantification of Metabolic Rearrangements During Neural Stem Cells Differentiation into Astrocytes by Metabolic Flux Analysis

Abstract

Proliferation and differentiation of neural stem cells (NSCs) have a crucial role to ensure neurogenesis and gliogenesis in the mammalian brain throughout life. As there is growing evidence for the significance of metabolism in regulating cell fate, knowledge on the metabolic programs in NSCs and how they evolve during differentiation into somatic cells may provide novel therapeutic approaches to address brain diseases. In this work, we applied a quantitative analysis to assess how the central carbon metabolism evolves upon differentiation of NSCs into astrocytes. Murine embryonic stem cell (mESC)-derived NSCs and astrocytes were incubated with labelled [1-13 C]glucose and the label incorporation into intracellular metabolites was followed by GC-MS. The obtained 13 C labelling patterns, together with uptake/secretion rates determined from supernatant analysis, were integrated into an isotopic non-stationary metabolic flux analysis ( 13 C-MFA) model to estimate intracellular flux maps. Significant metabolic differences between NSCs and astrocytes were identified, with a general downregulation of central carbon metabolism during astrocytic differentiation. While glucose uptake was 1.7-fold higher in NSCs (on a per cell basis), a high lactate-secreting phenotype was common to both cell types. Furthermore, NSCs consumed glutamine from the medium; the highly active reductive carboxylation of alpha-ketoglutarate indicates that this was converted to citrate and used for biosynthetic purposes. In astrocytes, pyruvate entered the TCA cycle mostly through pyruvate carboxylase (81%). This pathway supported glutamine and citrate secretion, recapitulating well described metabolic features of these cells in vivo. Overall, this fluxomics study allowed us to quantify the metabolic rewiring accompanying astrocytic lineage specification from NSCs.

Multiple Pathways Involved in Palmitic Acid-Induced Toxicity: A System Biology Approach

“…The distribution of reactions in metabolic pathways is shown in Figure 2. These results are similar to those previously reported astrocytic models (Fitch and Silver, 1997;Ciccarelli et al, 2001;Çakir et al, 2007;Giaume et al, 2010;Sertbaş et al, 2014;Martín-Jiménez et al, 2017;Sá et al, 2017). For example, Martín-Jiménez et al (2017) developed a genome-scale metabolic reconstruction of human astrocyte, comprising of 5.659 reactions (237 exchange reactions and 1.948 transport reactions), 3.765 genes, 862 enzymes, 5.007 metabolites.…”

Unveiling dynamic metabolic signatures in human induced pluripotent and neural stem cells

“…This type of behavior has been also observed for adipocytes [25]. Interestingly, our results closely match the data obtained by carbon-labelling experiments, usually more expensive and labor-intensive [9,22]. Thus, the use of perturbation experiments could be developed in the future to benefit metabolic flux studies.…”

“…Instead, the number of lipidic pools for which the steady-state increased was much higher in hNSC than in hiPSC, suggesting an increase in metabolic flux through the upstream section of the TCA cycle. These observations corroborated our previous findings in metabolic flux studies which predicted reductive carboxylation of α-ketoglutarate to fuel fatty acids biosynthesis in NSC [22]. In contrast to the overall response of TCA cycle intermediates and lipids, most amino acid pools decreased their absolute levels (Fig 3).…”

Towards an Understanding of Energy Impairment in Huntington’s Disease Brain

“…This suggests that given chronic high energy demands, cells may switch their reliance upon specific energetic pathways attempting to optimize ATP production. Comparing differentiated astrocytes and neural stem cells with 13 C flux analysis after differentiation from embryonic stem cells, only astrocytes downregulated their carbon utilization [ 175 ]. Given that metabolic rerouting occurs during developmentally appropriate growth, similar metabolic adaptations could be expected to occur upon introduction of abnormal genotypes.…”