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We reported recently that suppression of the renal 1alpha,25-dihyroxyvitamin D3 (1lpha,25-(OH)2-D3) production in aP2-agouti transgenic mice by increasing dietary calcium decreases adipocyte intracellular Ca2+ ([Ca2+]i), stimulates lipolysis, inhibits lipogenesis, and reduces adiposity. However, it was not clear whether this modulation of adipocyte metabolism by dietary calcium is a direct effect of inhibition of 1alpha,25-(OH)2-D3-induced [Ca2+]i. Accordingly, we have now evaluated the direct role of 1alpha,25-(OH)2-D3. Human adipocytes exhibited a 1alpha,25-(OH)2-D3 dose-responsive (1-50 nM) increase in [Ca2+]i (P<0.01). This action was mimicked by 1alpha,25-dihyroxylumisterol3 (1alpha,25-(OH)2-lumisterol3) (P<0.001), a specific agonist for a putative membrane vitamin D receptor (mVDR), and completely prevented by 1b,25-dihydroxyvitamin D3 (1beta,25-(OH)2-D3), a specific antagonist for the mVDR. Similarly, 1alpha,25-(OH)2-D3 (5 nM) caused 50%-100% increases in adipocyte fatty acid synthase (FAS) expression and activity (P<0.02), a 61% increase in glycerol-3-phosphate dehydrogenase (GPDH) activity (P<0.01), and an 80% inhibition of isoproterenol-stimulated lipolysis (P<0.001), whereas 1beta,25-(OH)2-D3 completely blocked all these effects. Notably, 1alpha,25-(OH)2-lumisterol3 exerted more potent effects in modulating adipocyte lipid metabolism, with 2.5- to 3.0-fold increases in FAS expression and activity (P<0.001) and a threefold increase in GPDH activity (P<0.001). Also 1alpha,25-(OH)2-lumisterol3 was approximately twice as potent in inhibiting basal lipolysis (P<0.025), whereas 1beta,25-(OH)2-D3 completely blocked all these effects. These data suggest that 1alpha,25-(OH)2-D3 modulates adipocyte Ca2+ signaling and, consequently, exerts a coordinated control over lipogenesis and lipolysis. Thus, a direct inhibition of 1alpha,25-(OH)2-D3-induced [Ca2+]i may contribute to an anti-obesity effect of dietary calcium, and the mVDR may represent an important target for obesity.

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We recently demonstrated that suppressing 1alpha,25-(OH)2-D3 by increasing dietary calcium decreases adipocyte intracellular Ca2+ ([Ca2+]i), stimulates lipolysis, and inhibits lipogenesis. High calcium diets also increase core temperature and white adipose tissue uncoupling protein 2 (UCP2) expression in aP2-agouti transgenic mice. Accordingly, we have evaluated the role of 1alpha,25-(OH)2-D3 in regulating human adipocyte UCP2 expression. Treatment of human adipocytes for 48 h with 1 nM 1alpha,25-(OH)2-D3 inhibited UCP2 mRNA and protein levels by 50% (P<0.002) and completely blocked isoproterenol- or fatty acid-stimulated two- to threefold increases in UCP2 expression. However, a specific agonist for the membrane vitamin D receptor (mVDR), 1alpha,25-dihydroxylumisterol3, was unable to inhibit basal, isoproterenol-stimulated, or fatty acid-stimulated UCP2 expression, whereas a specific mVDR antagonist,1beta,25-dihydroxyvitamin D3, was unable to prevent the 1alpha,25-(OH)2-D3 inhibition of UCP2 expression. In contrast, nuclear vitamin D receptor (nVDR) knockout via antisense oligodeoxynucleotide (ODN) prevented the inhibitory effect of 1alpha,25-(OH)2-D3 on adipocyte UCP2 expression and protein levels. These data indicate that 1a,25-(OH)2-D3 exerts an inhibitory effect on adipocyte UCP2 expression via the nVDR. Thus, suppression of 1alpha,25-(OH)2-D3 and consequent up-regulation of UCP2 may contribute to our previous observation of increased thermogenesis in mice fed with high calcium diets.

The hormone 1alpha,25(OH)(2)-vitamin D(3) (125D) binds to its nuclear receptor (VDR) to stimulate gene transcription activity. Inversion of configuration at C-20 of the side chain to generate 20-epi-1alpha,25(OH)(2)D(3) (20E-125D) increases transcription 200-5000-fold over 125D with its 20-normal (20N) side chain. This enhancement has been attributed to the VDR ligand-binding domain (LBD) having different contact sites for 20N and 20E side chains that generate different VDR conformations. We synthesized 1alpha, 25-dihydroxy-21-(3-hydroxy-3-methylbutyl)vitamin D(3) (Gemini) with two six-carbon side chains (both 20N and 20E orientations). Energy minimization calculations indicate the Gemini side chain possesses significantly more energy minima than either 125D or 20E-125D (2346, 207, and 127 minima, respectively). We compared activities of 125D, 20E-125D, and Gemini, respectively, in several assays: binding to wild-type (100%, 147%, and 38%) and C-terminal-truncated mutant VDR; transcriptional activity (of the transfected osteopontin promoter in ROS 17/2.8 cells: ED(50) 10, 0.005, and 1.0 nM); mediation of conformational changes in VDR assessed by protease clipping (major trypsin-resistant fragment of 34, 34, and 28 kDa). For inhibition of cellular clonal growth of human leukemia (HL-60) and breast cancer (MCF7) cell lines, the ED(50)(125D)/ED(50)(Gem) was respectively 380 and 316. We conclude that while Gemini readily binds to the VDR and generates unique conformational changes, none of them is able to permit a superior gene transcription activity despite the presence of a 20E side chain.

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1Alpha,25(OH)2D3 is an important negative regulator of parathyroid hormone (PTH) gene transcription. In parathyroid cells, as in other target tissues, 1alpha,25(OH)2D3 is degraded by side chain oxidation by the inducible 24-hydroxylase. We have previously shown that one metabolite of this pathway, 1alpha,23(S),25-(OH)3-24-oxo-D3, potently suppresses PTH synthesis and secretion in cultured bovine parathyroid cells (bPTC). Further examination of the metabolites of 1alpha,25(OH)2D3 in bPTC has revealed another compound that is less polar than 1alpha,25(OH)2D3. By HPLC analysis and mass spectrometry, this metabolite was identified as 1alpha,25(OH)2-3-epi-D3. The activity of this metabol ite on PTH gene transcription was assessed by the steady-state PTH secretion by bPTC after 72-h treatment with concentrations from 10(-11) M to 10(-7) M. 1Alpha,25(OH)2-3-epi-D3 was found to be only slightly, but not significantly, less active than the native 1alpha,25(OH)2D3 in suppressing PTH secretion despite having 30 times lower affinity for the bPTC VDR. Both 1alpha,25(OH)2D3 and 1alpha,25(OH)2-3-epi-D3 maximally suppressed PTH secretion by 50%. Along with 1alpha,25(OH)2-3-epi-D3, the activities of the other two A-ring diastereomers were assessed. 1beta,25(OH)2D3 suppressed PTH only at 10(-7) M with a decrease of only 30%, in good agreement with its low VDR affinity. Surprisingly, 1beta,25(OH)2-3-epi-D3 stimulated PTH secretion by 30-50% at concentrations from 10(-11) M to 10(-8)M and fell to control (untreated) rates at 10(-7) M. The mechanism for this increase in PTH secretion is under investigation. Metabolism studies performed in bPTC cells using high concentrations of 1alpha,25(OH)2D3 substrate showed that in some incubations, the concentration of 1alpha,25(OH)2-3-epi-D3 was even higher than that of the parent substrate 1alpha,25(OH)2D3. This finding indicates a slower rate of metabolism for this diastereomer. Thus, production and accumulation of 1alpha,25(OH)2-3-epi-D3, as a major stable metabolite of 1alpha,25(OH)2D3 in parathyroid glands, may contribute to the prolonged suppressive effect of 1alpha,25(OH)2D3 on PTH gene transcription.