Glutamine is the most abundant circulating amino acid and plays a versatile role in cell metabolism. Glutamine is primarily metabolized to glutamate and ammonia by the mitochondrial enzyme glutaminase-1 (GLS1). Glutamate is then converted to α-ketoglutarate (αKG) by glutamate dehydrogenase 1 (GLUD1) or aminotransferases (AT). Mitochondrial αKG enters the tricarboxylic acid cycle yielding adenosine triphosphate, lipids, and proteins that fulfill cellular energetic and biosynthetic demands. While glutamine regulates the function of vascular smooth muscle cells (VSMCs), the transcriptional and molecular pathways underlying this response remain incompletely understood. This study investigated how glutamine influences VSMC gene expression and determined the enzymatic pathways and metabolites mediating glutamine’s cellular effects. Bulk RNA sequencing of glutamine-starved rat aortic VSMCs revealed a distinct transcriptomic response, with 196 genes significantly upregulated and 218 genes significantly downregulated. Gene set enrichment analysis using Gene Ontology identified 282 differentially enriched categories. Upregulated genes were enriched in stress response and intrinsic apoptotic pathways, while downregulated genes were enriched in regulation of cell population proliferation, positive regulation of cell migration, and collagen-containing extracellular matrix. Consistent with the gene expression data, functional studies demonstrated that removal of glutamine from the culture media markedly attenuated VSMC proliferation, migration, and collagen synthesis, while modestly decreasing cell viability. Similarly, pharmacological inhibition of glutaminolysis with either the GLS1 inhibitor CB-839 or the pan AT inhibitor aminooxyacetic acid suppressed growth, motility, collagen production, and viability of VSMCs cultured in glutamine-replete media. In contrast, the GLUD1 inhibitor R162 had no effect on cellular responses to glutamine. Metabolite analysis revealed that glutamine deprivation or aminooxyacetic acid treatment, but not R162, reduced intracellular αKG concentrations, establishing AT as the primary enzyme mediating αKG production from glutamine. To determine which glutamine metabolite drives VSMC responses, glutamine-starved cells were supplemented with cell-permeable dimethyl-αKG or the ammonia donor ammonium chloride. Dimethyl-αKG significantly restored proliferation, migration, collagen synthesis, and viability whereas ammonium chloride only enhanced survival, demonstrating that αKG, rather than ammonia, mediates most glutamine-dependent VSMC functions. In conclusion, this study reveals that glutamine shapes the transcriptional landscape to favor VSMC activation and survival. Mechanistically, glutamine metabolism via the GLS1-AT pathway promotes VSMC proliferation, migration, collagen synthesis, and survival by generating αKG. These results identify the glutamine-αKG metabolic axis as a promising therapeutic target for occlusive vascular disease This work was supported by the National Institutes of Health, National Heart, Lung, and Blood Institute, Award Number R01 HL149727. This abstract was presented at the American Physiology Summit 2026 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.
Durante et al. (Fri,) studied this question.