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Classical lipid transporters are suggested to modulate cellular vitamin D uptake. This study investigated the vitamin D levels in serum and tissues of mice deficient in SR-B1 (Srb1^-/-), CD36 (Cd36^-/-) and ABC-G5/G8 (Abcg5/g8^-/-) and compared them with corresponding wild-type (WT) mice. All mice received triple-deuterated vitamin D₃ (vitamin D₃-d₃) for six weeks. All knockout mice vs. WT mice showed specific alterations in their vitamin D concentrations. Srb1^-/- mice had higher levels of vitamin D₃-d₃ in the serum, adipose tissue, kidney and heart, whereas liver levels of vitamin D₃-d₃ remained unaffected. Additionally, Srb1^-/- mice had lower levels of deuterated 25-hydroxyvitamin D₃ (25(OH)D₃-d₃) in the serum, liver and kidney compared to WT mice. In contrast, Cd36^-/- and WT mice did not differ in the serum and tissue levels of vitamin D₃-d₃, but Cd36^-/- vs. WT mice were characterized by lower levels of 25(OH)D₃-d₃ in the serum, liver and kidney. Finally, Abcg5/g8^-/- mice tended to have higher levels of vitamin D₃-d₃ in the serum and liver. Major alterations in Abcg5/g8^-/- mice were notably higher levels of 25(OH)D₃-d₃ in the serum and kidney, accompanied by a higher hepatic mRNA abundance of <i>Cyp27a1</i> hydroxylase. To conclude, the current data emphasize the significant role of lipid transporters in the uptake, tissue distribution and activation of vitamin D.