William H. Okamura

RESEARCH directed at defining the molecular mode of action of vitamin D is currently at its apex. There is now evidence implicating the essential involvement of vitamin D metabolites in a host of cellular processes, including calcium homeostasis, immunology, cell differentiation, and regulation of gene transcription. Further, there is evidence that the hormonally active form of vitamin D, 1α,25-dihydroxyvitamin D3 [1α,25(OH)2D3], may generate biological responses via both regulation of gene transcription as well as via nongenomic pathways, some of which involve opening of voltage-gated Ca2+ channels. In addition, there are many examples of pathological disruption of the normal state in which a drug form of a vitamin D metabolite is proposed to be a (potentially) useful form of treatment, e.g. renal osteodystrophy, psoriasis, leukemia, breast cancer, and osteoporosis. The importance of the molecule vitamin D in the biological systems of higher animals has been recognized since its discovery by Mellanby in 1920 (1). It was in the interval of 1920–1930 that vitamin D officially became classified as a “vitamin” that was essential for the normal development of the skeleton and maintenance of Ca2+ homeostasis. The chemical structure of vitamin D was not determined until 1932 (2), and it was only then that it was apparent that this important nutritional substance was in reality a steroid, more specifically, a secosteroid, indicating that one of the rings of the cyclopentanoperhydrophenanthrene ring structure (the 9–10 carbon-carbon bond of ring B) was broken (see Section II).

ADVERTISEMENT RETURN TO ISSUEPREVArticleNEXTSynthesis of Vitamin D (Calciferol)Gui-Dong Zhu and William H. OkamuraCite this: Chem. Rev. 1995, 95, 6, 1877–1952Publication Date (Print):September 1, 1995Publication History Published online1 May 2002Published inissue 1 September 1995https://pubs.acs.org/doi/10.1021/cr00038a007https://doi.org/10.1021/cr00038a007research-articleACS PublicationsRequest reuse permissionsArticle Views5319Altmetric-Citations301LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InRedditEmail Other access optionsGet e-Alertsclose Get e-Alerts

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.

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.

Recent studies have shown that 1alpha,25-dihydroxyvitamin D3 [1alpha,25-(OH)2D3] actions in cell growth and differentiation are mediated by both its nuclear receptor (VDRnuc) and its rapid membrane-related effects. In the present study, we investigated the effect of 1alpha,25-(OH)2D3 on p42mapk phosphorylation using human acute promyelocytic leukemia cells (NB4). 1Alpha,25-(OH)2D3 (10[-8] M) significantly increased p42mapk phosphorylation in a time- and dose-dependent manner, with the earliest response detectable at 30 sec. Because 1alpha,25-(OH)2D3 is a conformationally flexible molecule, we have used a series of conformationally locked (6-s-cis vs. 6-s-trans) analogs to evaluate which shape is optimal for activation. Four 6-s-cis-locked analogs (HF, JM, JN, and JP) and two 6-s-trans-locked analog (JB and JD) were studied. HF, JM, JN, and JP all increased p42mapk phosphorylation at 1 and 5 min (10[-8] M), but JB and JD had little effect. Analog HL [1beta,25-(OH)2D3], a specific antagonist for only the rapid effects of 1alpha,25-(OH)2D3, attenuated 1alpha,25-(OH)2D3-induced p42mapk phosphorylation 65-90%. To assess the potential involvement of the VDRnuc in mediating the analog's action, the relative abilities of the analogs to compete with [3H]1alpha,25-(OH)2D3 for binding in vitro to the VDRnuc of NB4 cells was measured. All 6-s-cis analogs bound poorly to VDRnuc (relative competitive index, 0.5-2%) compared with 1alpha,25-(OH)2D3 (relative competitive index, 100%). The present studies demonstrate for the first time that in NB4 cells 1alpha,25-(OH)2D3 rapidly activates the p42mapk pathway, and that this effect can be selectively mediated by analogs that can assume a 6-s-cis conformation.