Effects of 1,25-Dihydroxyvitamin D₃ on Adipocyte Differentiation

Several recent reports from this laboratory demonstrate a regulatory role for intracellular Ca²⁺([Ca²⁺]_i) in modulating lipid metabolism in both human and murine adipocytes, with increased [Ca²⁺]_i coordinately stimulating lipogenesis and inhibiting lipolysis, thereby expanding lipid mass. Further, we have recently demonstrated that

1,25-dihydroxyvitamin-D3 [1,25-(0H)2-D₃] stimulates adipocyte membrane vitamin D receptor (m VDR)-mediated rapid Ca²⁺ influx into adipocytes, resulting in the stimulation of lipogenesis and inhibition of lipolysis. However, increasing [Ca²⁺]_i in the early stages of differentiation inhibits human adipocyte differentiation, whereas increasing [Ca²⁺]_i in the late stage promotes human adipocyte differentiation.

Accordingly, we have investigated the role of 1,25-(0H)2-D₃ in the differentiation of 3T3-L1 preadipocytes, using triglyceride (TG) accumulation and glycerol-3-phosphate dehydrogenase (GPDH) as markers. 3T3-Ll preadipocytes were placed in differentiation media upon confluence, and exposed to varying amounts (0, 1nM, and 10nM) of 1,25-(0H)₂-D3 for either one hour pulses or for sustained (24 hrs or 48 hrs) amounts of time throughout the differentiation process. Exposure to one-hour pulses of 1nM 1,25-(0H)₂-D3 throughout differentiation caused modest decreases (31%-38%) in TG accumulation (p<0.0001), with one-hour pulse exposure to 1 0nM 1,25-(0H)2-D₃ having little to no effect on TG accumulation. One-hour pulse exposure to both 1nM and 10nM 1,25-(0H)2-D3 suppressed GPDH activity early, but not late in differentiation. Sustained (24-hour) exposure to 1,25-(0H)₂-D₃ (1nM and 10nM) inhibited differentiation at 0-24 hrs, with decreases in both TG and GPDH of 41-81 % (p<0.0001).

Similarly, sustained exposure of 1,25-(0H)₂-D3 resulted in marked inhibition of GPDH activity and TG accumulation early in differentiation. In contrast, sustained exposure late in differentiation exerted no significant effects on either marker of differentiation. PPAR-y and pref-1 expression were also used as markers of differentiation. One-hour pulses of 10nM 1,25-(0H)₂-D3 did not cause any changes in PPAR-γ expression compared to control. Sustained exposure to 1,25-(OH)₂-D₃ throughout differentiation decreased PPAR- γ expression, with a 92% decrease from 0-48h (p<0.0001). One-hour pulses of 1,25-(OH)₂-D₃ had no effect on Pref-1 expression, with the exception of an increase in expression at 47-48 hr (p<0.0001). Sustained exposure to 10nM 1,25-(OH)₂-D₃ at 0-48 hrs, 24-48 hrs 47-48 hrs. all caused significant increases (125%-146%) in the expression of Pref-1 (p<0.001). Thus, although 1,25-(OH)₂-D₃ stimulates lipogenesis, inhibits lipolysis and increases TG accumulation in mature human and murine adipocytes, it also modestly inhibits the differentiation of preadipocytes into mature adipocytes.