Extended Data Fig. 4: Aspa^−/− adipocytes shunt glucose-derived carbon into pyrimidines.

a, Schematic of metabolite profiling of SVF-derived differentiated adipocytes generated from Aspa^WT and Aspa^KO mice. Glucose incorporation into metabolite pools was measured by incubating cells with U-¹³C glucose. Isotopolog distribution for indicated metabolites was measured after 24 h using IC-MS targeted profiling. b, Relative abundance of intracellular NAA (n = 3 biological replicates/group). Data represent mean ± s.e.m. ****P < 0.0001 by unpaired two-tailed Student’s t-test. c, Schematic showing contribution of ¹³C₆-labeled glucose tracing into early pyrimidines via aspartate (m + 2 and m + 3 isotopologs), and R5P and downstream pyrimidines (m + 5 isotopologs). G6P, glucose-6-phosphate; 6-PG, 6-phosphogluconate; R5P, ribose-5-phosphate; F6P, fructose-6-phosphate; 3PG, 3-phosphoglycerate. d, Fractional labeling of aspartate, early and later pyrimidines, and the pentose phosphate pathway metabolite, R5P. DHOA, dihydroorotate; OMP, orotidine 5’-monophosphate; UMP, uridine monophosphate. Isotopolog data are corrected for ¹³C natural abundance (n = 3 replicates/group). Data are mean ± s.e.m. *P < 0.05, **P < 0.01, ***P < 0.001 by unpaired two-tailed Student’s t-test. e, Immunoblot of Aspa^WT and Aspa^KO primary SVF cells ± differentiation, probed for ASPA, phospho-CAD (S1859), total CAD, phospho-S6K (T389), total S6K, and ADIPOQ. HSP90 served as the loading control. Representative of 3 independent experiments. f, Representative Oil-Red O (ORO) staining of lipid accumulation in differentiated Aspa^WT and Aspa^KO SVF adipocytes. Scale bar is 0.783 cm. Panels a and c created with BioRender.com.

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