| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
|
FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online September 5, 2002 as doi:10.1096/fj.02-0255fje. |
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
,25-dihydroxyvitamin D3 inhibits uncoupling protein 2 expression in human adipocytes 1
University of Tennessee, Knoxville, Tennessee, 37996, USA;
* University of California, Riverside, California, USA; and
Zen-Bio, Research Triangle Park, North Carolina, USA
2Correspondence: University of Tennessee, 1215 West Cumberland Avenue, #229, Knoxville, TN 37996-1900, USA. E-mail: mzemel{at}utk.edu
SPECIFIC AIMS
This study was conducted to determine the role of1
,25-dihydroxyvitamin D3 (1,25-(OH)2-D3) in modulating human adipocyte uncoupling protein 2 (UCP2) expression via genomic action mediated by nuclear vitamin D receptor.
PRINCIPAL FINDINGS
1. 1,25-(OH)2-D3 inhibits human adipocyte basal, isoproterenol, and fatty acid-stimulated UCP2 expression
Figure 1
, top panel, shows that 48 h treatment of human adipocytes with 1 nM 1,25-(OH)2-D3 caused a 40% decrease in UCP2 mRNA (P<0.003) whereas direct stimulation of Ca2+ influx with 10 mM KCl, a cell membrane depolarization agent, exerted no effect. Similar results were observed on UCP2 protein levels measured by Western blot (P<0.002, Fig. 1
, bottom panel). 10 nM isoproterenol caused a 
twofold increase in adipocyte UCP2 mRNA (P<0.002), which was completely blocked by 1,25-(OH)2-D3 but by only 20% by KCl. Although KCl inhibited isoproterenol-stimulated increases in UCP2 protein (P<0.006), 1,25-(OH)2-D3 exerted a more potent effect, reducing UCP2 protein below basal levels. Free fatty acids (oleic acid, linoleic acid, and stearic acid mixture) caused a twofold increase in UCP2 mRNA that was completely prevented by 1,25-(OH)2-D3, but not by KCl. These data indicate that 1,25-(OH)2-D3 inhibition of UCP2 expression is largely independent of its effects on Ca2+ influx or fatty acid flux.
|
2. Membrane vitamin D receptor does not mediate the inhibitory effect of 1,25-(OH)2-D3 on human adipocyte UCP2 expression
To study whether membrane vitamin D receptor (mVDR) mediates this inhibition of 1,25-(OH)2-D3 on adipocyte UCP2 expression, 1,25-dihydroxylumisterol3 (1,25-(OH)2-lumisterol3), a specific mVDR agonist, and 1ß,25-dihydroxyvitamin D (1ß,25-(OH)2-D3), a specific mVDR antagonist, were used to treat human adipocytes. 1,25-(OH)2-lumisterol3 failed to exert an inhibitory effect on UCP2 mRNA, whereas 1ß,25-(OH)2-D3 was unable to reverse 1,25-(OH)2-D3-induced inhibition on UCP2 mRNA. Similarly, the mVDR agonist and antagonist exerted no effect on isoproterenol- and fatty acid-stimulated UCP2 expression. These data indicate that mVDR does not mediate the inhibitory effect of 1,25-(OH)2-D3 on adipocyte UCP2 expression.
3. Nuclear vitamin D receptor mediates the inhibitory effect of 1,25-(OH)2-D3 on human adipocyte UCP2 expression
We next investigated the role of the nuclear vitamin D receptor (nVDR) in mediating the inhibitory effect of 1,25-(OH)2-D3 on adipocyte UCP2 expression. Using RT-PCR, we detected a 465 bp nVDR fragment in human adipocytes and preadipocytes. This was confirmed by Western blot analysis; using a nVDR monoclonal antibody, we detected a 50 kDa protein. We then performed a transient transfection of antisense ODN to knock out the nVDR. A time course study shows that treatment with nVDR antisense ODN inhibited nVDR protein from 48 h through 96 h, whereas the mutant antisense ODN was without effect (Fig. 2
, top panel). We then treated the nVDR knockout adipocytes with 1,25-(OH)2-D3. Figure 2
(bar graph) shows that 1,25-(OH)2-D3 inhibited UCP2 mRNA by 60% in either control adipocytes or adipocytes treated with mutant ODN. However, 1,25-(OH)2-D3 was unable to exert the inhibitory effect in nVDR knockout adipocytes. Similar results were observed on UCP2 protein levels measured by Western blot (Fig. 2
, bottom). These data indicate that this inhibitory effect of 1,25-(OH)2-D3 on UCP2 expression is mediated by the nVDR.
|
CONCLUSIONS
It is now well recognized that 1,25-(OH)2-D3 generates biological responses via both genomic and nongenomic pathways. 1,25-(OH)2-D3 generates genomic actions via binding to a specific nuclear hormone receptor, nVDR. Moreover, 1,25-(OH)2-D3 generates rapid, nongenomic signal transduction, including modulation of calcium channels, via a putative membrane mVDR in a wide variety of cells.
Our previous and present data extend these observations by demonstrating that 1,25-(OH)2-D3 elicits genomic and nongenomic action in adipocytes. We previously reported that 1,25-(OH)2-D3 stimulates adipocyte [Ca2+]i, promotes lipogenesis, and inhibits lipolysis via a rapid nongenomic action. Data presented here further demonstrate that 1,25-(OH)2-D3 exerts an inhibitory effect on adipocyte UCP2 expression via a genomic action. Therefore, 1,25-(OH)2-D3 appears to play an important role in modulating adipocyte lipid metabolism and energy homeostasis.
UCP2, a homologue of UCP1, is ubiquitously expressed, with the highest level in white adipose tissue. UCP2 has been shown to stimulate mitochondrial proton leak and therefore to exhibit a potential role in thermogenesis, energy metabolism, and obesity. Functional characterization of UCP2 promoter region has demonstrated several potent cis-acting regulatory elements, including multiple PPAR
and thyroid hormone responsive elements. However, little is known regarding negative regulatory factors of UCP2 expression. Here we report that 1,25-(OH)2-D3 exerts an inhibitory effect on UCP2 expression. The mechanism of this nVDR-mediated inhibition of UCP2 is not known. However, human and mouse UCP2 promoters do contain several cis-acting negative regulatory elements that strongly repress promoter activity, although it is not clear whether nVDR acts on one of these silencers. Using a promoter analysis program (http://www.lsi.upc.es/cgi-bin/user/alggen/promo/promo/dynmat.cgi), we analyzed the human (Genbank accession no. AF208500) and mouse (Genbank accession no. AF115319) UCP2 promoters, which showed that multiple putative nVDR binding sites may exist on UCP2 promoter regions; at least one is located on the silencer regions. Alternatively, nVDR may also compete with other positive transcriptional factors containing similar DNA binding domains on the responsive element binding or a similar protein–protein interaction domain (such as PPAR or TR) on heterodimerization with the same transcriptional factor (RXR). This has been evidenced by studies demonstrating that up-regulation or activation of nVDR by 1,25-(OH)2-D3 antagonizes the effects of PPAR or TR on adipocyte differentiation. However, further studies are required to address the mechanism whereby 1,25-(OH)2-D3 inhibits UCP2 expression.
Regulation of adipocyte metabolism via 1,25-(OH)2-D3 signaling pathways may play an important role in the development of obesity in vivo. Several lines of evidence demonstrate that the circulating 1,25-(OH)2-D3 level is elevated in obese humans. Since increasing dietary calcium suppresses 1,25-(OH)2-D3 levels, we used this strategy to demonstrate that suppression of 1,25-(OH)2-D3 by increasing dietary calcium decreases adipocyte intracellular Ca2+, stimulates lipolysis, inhibits lipogenesis, and increases adipocyte UCP2 expression and core temperature in aP2-agouti transgenic mice, thereby reducing body weight and fat mass in these animals. Recent data demonstrate comparable effects in humans.
In summary, these data indicate that 1,25-(OH)2-D3 exerts an inhibitory effect on white adipocyte basal, isoproterenol, and fatty acid-stimulated UCP2 expression and that this effect is mediated via a genomic action. Thus, suppression of 1,25-(OH)2-D3 and consequent up-regulation of UCP2 may contribute to our earlier observation of increased thermogenesis in mice fed a high-calcium diet. This effect, coupled with decreased lipogenesis and increased lipolysis secondary to decreased [Ca2+]i mediated by nongenomic action, may contribute to an anti-obesity effect of dietary calcium.
|
FOOTNOTES
1 To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.02-0255fje; to cite this article, use FASEB J. (September 5, 2002) 10.1096/fj.02-0255fje 
This article has been cited by other articles:
|
|
 |
|
  M. B. Zemel and X. Sun Dietary Calcium and Dairy Products Modulate Oxidative and Inflammatory Stress in Mice and Humans J. Nutr., June 1, 2008; 138(6): 1047 - 1052. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 B. W. Bailey, D. K. Sullivan, E. P. Kirk, S. Hall, and J. E. Donnelly The Influence of Calcium Consumption on Weight and Fat Following 9 Months of Exercise in Men and Women J. Am. Coll. Nutr., August 1, 2007; 26(4): 350 - 355. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 H. Shi, B. Cave, K. Inouye, C. Bjorbaek, and J. S. Flier Overexpression of Suppressor of Cytokine Signaling 3 in Adipose Tissue Causes Local but Not Systemic Insulin Resistance Diabetes, March 1, 2006; 55(3): 699 - 707. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 N. Boon, G. B. Hul, N. Viguerie, A. Sicard, D. Langin, and W. H. Saris Effects of 3 diets with various calcium contents on 24-h energy expenditure, fat oxidation, and adipose tissue message RNA expression of lipid metabolism-related proteins Am. J. Clinical Nutrition, December 1, 2005; 82(6): 1244 - 1252. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. B. Zemel The Role of Dairy Foods in Weight Management J. Am. Coll. Nutr., December 1, 2005; 24(suppl_6): 537S - 546S. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 N. Boon, L. L. J. Koppes, W. H. M. Saris, and W. Van Mechelen The Relation between Calcium Intake and Body Composition in a Dutch Population: The Amsterdam Growth and Health Longitudinal Study Am. J. Epidemiol., July 1, 2005; 162(1): 27 - 32. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Schrager Dietary Calcium Intake and Obesity J Am Board Fam Med, May 1, 2005; 18(3): 205 - 210. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 G. M. Zinser and J. Welsh Vitamin D receptor status alters mammary gland morphology and tumorigenesis in MMTV-neu mice Carcinogenesis, December 1, 2004; 25(12): 2361 - 2372. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 H. Shi, I. Tzameli, C. Bjorbaek, and J. S. Flier Suppressor of Cytokine Signaling 3 Is a Physiological Regulator of Adipocyte Insulin Signaling J. Biol. Chem., August 13, 2004; 279(33): 34733 - 34740. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. B Zemel Role of calcium and dairy products in energy partitioning and weight management Am. J. Clinical Nutrition, May 1, 2004; 79(5): 907S - 912S. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
