Fig. 1.

LC-MS-based isotope tracking demonstrates gluconeogenesis in zebrafish embryo. A) Glucose level increases from 12 to 36 hpf in the zebrafish. B) ¹³C-labeled isotopologues (filled circle, ¹³C; open circle, ¹²C) resulted from the substrates-¹³C (glycerol, alanine, lactate, and glutamate). For most metabolites in upper glycolysis, enrichment of mass + 3 (M + 3) isotopologues, which scarcely exist in nature, best represents the ¹³C inherited from the substrates-¹³C. Entry of substrate-¹³C into Krebs cycle gives rise to M + 2 to M + 6 isotopologues of the intermediates (filled and filled hatched circles). C) 6- or 12-hpf embryos took the injections of substrates-¹³C and given 6 or 12 h of incubation, followed by metabolite extraction. D) A rescaled heatmap showing relative M + 3 isotopologue enrichment (M + 3/M + 0) of the metabolites in upper glycolysis, hexosamine synthesis, glycogen synthesis, and pentose phosphate pathways in natural (N) or ¹³C-labeled (¹³C) substrate-injected embryos. The duration (Dur.) and substrates (Subs.) of the experiments are shown below the heatmap. Glucose M + 3 was most highly enriched in glutamate-¹³C5-injected 24-hpf embryos. Because M + 3 level of lactate was incomparably high in lactate-¹³C3-injected embryos (asterisks), these were given the same value of third-highest ones (alanine-¹³C3-injected samples) to show relative levels of other samples. E) A rescaled heatmap for relative enrichment of M + 2 to M + 5 isotopologues (M + n/M + 0) in Krebs cycle intermediates. Additional mass (n) of each isotopologue was shown on the left. αKG, α-ketoglutarate; CIT, citrate; DHAP, dihydroxyacetone phosphate; FUM, fumarate; GAP, glyceraldehyde phosphate; G1P, glucose-1-phosphate; G3P, glycerol-3-phosphate; G6P, glucose-6-phosphate; MAL, malate; PEP, phosphoenolpyruvate; SUCC, succinate; S7P, sedoheptulose-7-phosphate; UDP-Glc, UDP-glucose; UDP-GlcNAc, UDP-N-acetylglucosamine.