Targeted Metabolomics Reveals a Metabolic Signature Derived From the Stimulation of Cells With the Specific AT2R Agonist C21

Abstract

Aim

Despite increasing evidence for the cardiometabolic protective effects of angiotensin II Type 2 receptor (AT₂R) activation, the underlying molecular mechanisms remain incompletely understood. This study aimed to determine how AT₂R activation modulates cellular metabolism and to identify pathways relevant to its tissue-protective actions.

Methods

3T3-L1 adipocytes, endothelial cells, and renal proximal tubular epithelial cells were treated with the specific AT₂R agonist Compound 21 (C21). Targeted liquid chromatography–tandem mass spectrometry was used to quantify amino acids and metabolites involved in energy metabolism and redox homeostasis. Functional analyses included nitrate/nitrite determination and cystathionine β-synthase (CBS) activity.

Results

Compound 21 induced shared and cell type-specific metabolic changes. Arginine, citrulline, glucose, and glyceraldehyde 3-phosphate increased consistently across all cell types, whereas citrate decreased. Sulfur-containing metabolites and glutathione-related intermediates increased predominantly in adipocytes and renal cells. Pathway analysis highlighted three metabolic axes affected by C21: arginine biosynthesis, central carbon metabolism, and amino acid-linked redox pathways. Functionally, C21 increased nitrate/nitrite accumulation in BAECs and CBS activity in RPTECs.

Conclusion

Treatment with C21 was associated with a conserved metabolic signature across adipocytes, renal epithelial cells, and endothelial cells involving arginine biosynthesis, central carbon metabolism, and amino acid-linked redox homeostasis. While the response differed among cell types, coordinated changes in arginine, citrulline, glucose, glyceraldehyde 3-phosphate, citrate, and GSH-related metabolites suggest that AT₂R signaling is associated with cellular pathways involved in nitric oxide metabolism, energy metabolism, and redox homeostasis. Increased NOx production in BAECs and CBS activity in RPTECs support the cell type-specific metabolic effects of C21.