Fig. 2

E4 increases lactate production in mouse brain and E4 astrocytes show increased glycolytic flux and lower oxidative respiration. a Experimental design (¹³C, blue filled circles; ¹²C, white circles; (m + n, where n is the number of ¹³C labeled carbons within a metabolite). [U-¹³C] glucose was administered in vivo to E3 (n = 6) and E4 (n = 8) mice via oral gavage, brain tissue was collected after 45 min, and metabolites analyzed for ¹³C enrichment in pyruvate and lactate. E3 and E4 expressing astrocytes were cultured in [U-¹³C] glucose media for 24 h, media collected, cells washed, and metabolites analyzed for ¹³C enrichment (n = 6). b While fully labeled pyruvate is present in similar amounts in E3 and E4 brains, lactate synthesized from ¹³C-glucose is higher in E4 mouse brains. c-e Primary astrocytes expressing E4 show increased ¹³C enrichment in lactate (c), higher LDH activity (d), and decreased ¹³C enrichment in the TCA cycle (average of all detected TCA intermediates) (e). f Increased lactate synthesis as measured by HSQCAD NMR spectroscopy (n = 3). Representative NMR spectra (f) showing E4 astrocytes have both increased intracellular ¹³C-lactate and export more lactate into extracellular media (bar graph insert). g Extracellular acidification rate (ECAR) of E3 and E4 primary astrocytes shown over time during the glycolysis stress test (n = 24 for both groups). h Contributions to ECAR at baseline, in response to glucose (glycolysis), in response to stress (glycolytic capacity), and un-tapped reserve were calculated. i Oxygen consumption rate (OCR) during the glycolysis stress test assay was graphed over time and j represented as average respiration before and after glucose. k Metabolic phenotypes of E3 and E4 astrocytes were characterized by plotting ECAR vs. OCR. l E3 and E4 astrocytes were incubated in glucose free media (−) or glucose rich media (+) and oxidation of 1.0 μCi/mL ¹⁴C-glucose was measured by trapping ¹⁴CO₂ and counting radio activity. (*P < 0.05 unpaired t-test, two-tailed, n = 4 per genotype). m Glucose oxidation capacity, dependency, and flexibility was assessed in E3 and E4 astrocytes via the Mito Fuel Flex Assay. n E3 and E4 astrocytes were incubated in 1.0 μCi/mL ¹⁴C-glucose with (+) or without (−) 12.5 mM lactate (n = 3). (b-l,n, *P < 0.05, ***P < 0.001, ****P < 0.0001, unpaired t-test, two tailed) (m, *P < 0.05 Two-way ANOVA, Sidak’s multiple comparisons test)