[Figure: see text].

Disrupted endothelial metabolism is linked to endothelial dysfunction and cardiovascular disease. Targeted metabolic inhibitors are potential therapeutics; however, their systemic impact on endothelial metabolism remains unknown. In this study, we combined stable isotope labeling with 13C metabolic flux analysis (13C MFA) to determine how targeted inhibition of the polyol (fidarestat), pentose phosphate (DHEA), and hexosamine biosynthetic (azaserine) pathways alters endothelial metabolism. Glucose, glutamine, and a four-carbon input to the malate shuttle were important carbon sources in the baseline human umbilical vein endothelial cell (HUVEC) 13C MFA model. We observed two to three times higher glutamine uptake in fidarestat and azaserine-treated cells. Fidarestat and DHEA-treated HUVEC showed decreased 13C enrichment of glycolytic and TCA metabolites and amino acids. Azaserine-treated HUVEC primarily showed 13C enrichment differences in UDP-GlcNAc. 13C MFA estimated decreased pentose phosphate pathway flux and increased TCA activity with reversed malate shuttle direction in fidarestat and DHEA-treated HUVEC. In contrast, 13C MFA estimated increases in both pentose phosphate pathway and TCA activity in azaserine-treated cells. These data show the potential importance of endothelial malate shuttle activity and suggest that inhibiting glycolytic side branch pathways can change the metabolic network, highlighting the need to study systemic metabolic therapeutic effects.