Cyanidin-3-O-glucoside drives metabolic reprogramming and modulates PKA-dependent signaling in Leydig cells

Anthocyanins are widely regarded as beneficial antioxidants and are abundant in commonly consumed foods and nutraceutical formulations. However, their impact on Leydig cells metabolic and signaling responses under non-stressed conditions remain poorly defined. This study investigated how cyanidin-3-O-glucoside (C3G), a major dietary anthocyanin, modulates redox balance, mitochondrial function, endocrine signalling, and cellular metabolism in a model of progenitor Leydig cells. Cells were exposed to increasing C3G concentrations, and cell viability, redox and mitochondrial parameters, gene expression, protein phosphorylation and exometabolomic profiles were assessed using colorimetric-fluorescence assays, ELISA, immunoblotting, RT-qPCR, and ¹H-NMR. Low C3G concentrations reduced intracellular ROS and preserved mitochondrial membrane potential and mtDNA content, while 50.00 μM induced mitochondrial depolarization, mtDNA expansion, Sirt1 downregulation, and cytotoxicity. At 0.50 and 5.00 μM, C3G was associated with decreased androstenedione production and global PKA-dependent protein phosphorylation, together with changes in genes linked to endocrine and mitochondrial function. However, this mechanistic data supports a putative association between C3G exposure and impaired PKA-linked signaling events, rather than direct evidence of PKA II-specific interference. Concurrently, 0.50 and 5.00 μM C3G were associated with a trend toward a proliferative metabolic phenotype, with changes in glycolytic activity, amino acid turnover, and changes in tricarboxylic acid cycle intermediates, together with increased cell proliferation at 0.50 μM. Overall, C3G exposure was associated with concentration-dependent, exploratory changes across the redox, metabolic and signalling landscape of progenitor Leydig cells, highlighting that diet-derived anthocyanins may exert differential, concentration-specific effects on cellular function.