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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.

The current study was done to assess if heterogeneity existed in the degree of adipogenesis in stromal cells (preadipocytes) from multiple donors. In addition to conventional lipid-based methods, we have employed a novel signal amplification technology, known as branched DNA, to monitor expression of an adipocyte specific gene product aP2. The fatty acid binding protein aP2 increases during adipocyte differentiation and is induced by thiazolidinediones and other peroxisome proliferator activated receptor gamma ligands. The current work examined the adipogenic induction of aP2 mRNA levels in human adipose tissue stromal cells derived from 12 patients (mean age +/- SEM, 38.9 +/- 3.1) with mild to moderate obesity (mean body mass index +/- SEM, 27.8 +/- 2.4). Based on branched DNA technology, a rapid and sensitive measure of specific RNAs, the relative aP2 level in adipocytes increased by 679 +/- 93-fold (mean +/- SEM, n=12) compared to preadipocytes. Normalization of the aP2 mRNA levels to the housekeeping gene, glyceraldehyde phosphate dehydrogenase, did not significantly alter the fold induction in a subset of 4 patients (803.6 +/- 197.5 vs 1118.5 +/- 308.1). Independent adipocyte differentiation markers were compared between adipocytes and preadipocytes in parallel studies. Leptin secretion increased by up to three-orders of magnitude while measurements of neutral lipid accumulation by Oil Red O and Nile Red staining increased by 8.5-fold and 8.3-fold, respectively. These results indicate that preadipocytes isolated from multiple donors displayed varying degrees of differentiation in response to an optimal adipogenic stimulus in vitro. This work also demonstrates that branched DNA measurement of aP2 is a rapid and sensitive measure of adipogenesis in human stromal cells. The linear range of this assay extends up to three-orders of magnitude and correlates directly with independent measures of cellular differentiation.

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