1,25-Dihydroxyvitamin D Regulates Lipid Metabolism in 3T3-L1 Adipocytes

It is well established that vitamin D is sequestered in adipose tissue. Many studies have demonstrated vitamin D regulation of adipocyte differentiation, inflammation, and energy metabolism, highlighting the multifaceted role that vitamin D plays in adipose tissue physiology. However, whether vitamin D impacts lipid metabolism in terminally differentiated adipocytes is not yet known. Our studies aim to address this knowledge gap. To study this, 3T3-L1 adipocytes were differentiated for 9 days, followed by stimulation with 1,25-dihydroxyvitamin D (1,25(OH)₂D; 10 nM) or vehicle for the times indicated. Triacylglycerol (TAG) accumulation and glycerol release were determined using commercially available kits. Fatty acid uptake was assessed using BODIPY FL C16. Fatty acid oxidation was assessed by quantifying the production of ¹⁴CO₂ from [1-¹⁴C] palmitic acid. De novo lipogenesis was assessed by quantifying D-[U-¹³C]glucose and [¹³C₂]acetate incorporation into fatty acids using liquid chromatography-mass spectrometry. All data are normalized to protein content, determined using the bicinchoninic acid (BCA) assay. We have previously demonstrated that 1,25(OH)₂D, the bioactive vitamin D metabolite, stimulates a 21% reduction in TAG accumulation in differentiated 3T3-L1 adipocytes after 4 days (p=0.01). This occurs despite a significant increase in fatty acid uptake following 1-4 days of treatment (p<0.01), and with concomitant stimulation of PKA-dependent lipolysis (p<0.01). In our current studies, we demonstrate that 1,25(OH)₂D stimulates a 2.5-fold increase in complete fatty acid oxidation (p<0.01), assessed by quantifying the production of ¹⁴CO₂ from [1-¹⁴C] palmitic acid. Further, 1,25(OH)₂D stimulates slight inhibition of fatty acid synthesis, evidenced by a 10% reduction in [¹³C₂]acetate incorporation into palmitic, palmitoleic, stearic, and oleic acids (p=0.03), determined by LC-MS. Interestingly, D-[U-¹³C]glucose incorporation into these fatty acids was reduced by 30% in response to 1,25(OH)₂D (p<0.01), indicating a reduced contribution of glucose as a substrate for de novo lipogenesis. In summary, 1,25(OH)₂D stimulates fatty acid turnover in 3T3-L1 adipocytes, characterized by stimulation of fatty acid uptake and PKA-dependent lipolysis. Further, 1,25(OH)₂D stimulates fatty acid oxidation, while reducing glucose utilization as a substrate for fatty acid synthesis. Collectively, these changes in lipid metabolism may contribute to the reduction in TAG accumulation that is stimulated by 1,25(OH)₂D, and may be protective against excessive fat mass accumulation and associated metabolic disorders.