Vitamin D₃, 25-Hydroxyvitamin D₃, and 1,25-Dihydroxyvitamin D₃ Uptake in Cultured Human Mature Adipocytes

Background/Objectives: Vitamin D₃ is predominantly sequestered in adipose tissue, where it is slowly mobilized under conditions of deficiency in vivo. However, the kinetics of its uptake, release, and interaction with its major metabolites, 25(OH)D₃ and 1,25(OH)₂D₃, remain poorly understood. Given the close relationship between obesity, low-grade chronic inflammation, and disrupted vitamin D metabolism, a clearer understanding of these dynamics in adipocytes is essential. Thus, we sought to characterize time-dependent uptake and metabolites in differentiated human adipocytes. Methods: Human pre-adipocytes were differentiated in vitro and exposed to either vitamin D₃ and 1,25(OH)₂D₃ or the combination of vitamin D₃, 25(OH)D₃ and 1,25(OH)₂D₃. Intracellular concentrations were quantified through HPLC at various time points. A separate efflux experiment assessed vitamin D₃ release under basal and isoproterenol-stimulated conditions using ³H-vitamin D₃ and scintillation counting. Results: Vitamin D₃ uptake showed a gradual and sustained increase over 96 h, suggesting ongoing accumulation within lipid-rich compartments. In contrast, 25(OH)D₃ and 1,25(OH)₂D₃ peaked rapidly within the first hour and declined sharply. Isoproterenol stimulation significantly enhanced vitamin D₃ release into the extracellular medium from the adipocytes, indicating increased efflux during lipolytic activation. Conclusions: Adipocytes selectively retain vitamin D₃ while rapidly clearing its hydroxylated forms. These findings highlight the distinct intracellular handling of vitamin D metabolites and suggest that tailored supplementation strategies-particularly in individuals with excess adiposity-may improve bioavailability and metabolic efficacy.