Special Article

Vitamin D and adipogenesis: new molecular insights

Richard J Wood

The focus of the current review is to highlight some new insights into the molecular mechanism by which vitamin D, a potentially nutritionally modulated factor, influences adipo genesis. Recent studies, predominantly using the mouse 3T3-L 1 pre-adipocyte cell culture model, have shown that the role of vitamin D in inhibiting adipo genesis is mediated at the molecular level through a vitamin D (VDR)-dependent inhibition of CCAAT enhancer binding -alpha (C/EBPcx) and peroxisome proliferator-activated receptor-gamma (PPARy,l expression and a decrease in PPARy transactivating activity in the pre-adipocyte. The latter action may reflect a vitamin D-induced decrease in endogenous PPARyligand availability and a competition between VDR and PPAR1 for a limiting amount of (RXR), a common heterodimeric binding partner of both nuclear receptors. © 2008 International Life Sciences Institute

INTRODUCTION PPARy is necessary early in the initiation of differentia- tion, but it is not required for maintaining PPARy- The process of cell type-specific differentiation from mul- dependent expression in the mature adipocyte.12 tipotent mesenchymal stem cells is complex. The relative In addition to a series of complex intracellular balance of different molecular factors determines factors that regulate adipogenesis, a large number of whether a mesenchymal stem cell will undergo adipogen- extracellular factors have also been identified that can esis, osteogenesis, or myogenesis. This initial determina- influence adipogenesis. 1 In light of the global obesity epi- tion step, of which we know few details, seals the fate demic, there is increasing interest in the role of poten- of the cell along a given cell lineage. In the case of adipo- tially modifiable factors in adipogenesis. The focus of the genesis, a mesenchymal stem cell develops into a pre- current review is to describe some recent insights into the adipocyte, which can undergo terminal differentiation molecular mechanism by which vitamin D, a potentially into a mature adipocyte, but has lost the ability to become nutritionally modulated factor, influences adipogenesis. any of the other cell types. During the process of terminal differentiation, the pre-adipocyte undergoes changes in morphology, biochemical expression, and cellular func- OBESITY AND VITAMIN D STATUS tion as it is transformed into a mature adipocyte, which is able to transport and synthesize lipids, respond metaboli- Accumulating evidence suggests there is a potential link cally to insulin, and secrete adipocyte -specific between obesity and vitamin D insufficiency among (Figure 1). many populations worldwide? Although there is a sub- The process of adipogenesis requires a sequential stantial body of circumstantial evidence that excess body series of events. However, specific fat is associated with an increased risk of suboptimal molecular factors that may be critical during one phase of vitamin D status,` it is unclear to what extent poor differentiation may not be as important at another phase. vitamin D status is a consequence of obesity or is in some For example, cyclic AMP-dependent ligand activation of way involved in its development.

Affiliation: Ri Wood is with the Mineral Bioavailability Laboratory at Jean Mayer USDA Human Nutrition Research Center on Aging at Tufts University, Boston, Massachusetts, USA. Correspondence: RI Wood, Mineral Bioavailability Laboratory, Jean Mayer USDA Human Nutrition Research Center on Aging at Tufts University, 711 Washington Street, Boston, MA 02111, USA. E-mail: [email protected], Phone: +1-617-556-3192, Fax: +1-617-556-3344 Key words: adipo genesis, calcitriol, preadipocyte, vitamin 0 receptor

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Figure 1 Vitamin D and adipogenesis. Current evidence suggests that as pre-adipocytes differentiate from spindle-like fibroblast-like cells into mature lipid storing functional adipocytes (left boxed panel) 1,25-dihydroxyvitamin D and the (VDR) may play an important role in adipogenesis. An early response to the adipogenic stimulus in pre-adipocytes is a rapid increase, then decrease, in the availability of VDR, which creates a short window of opportunity for 1,25-dihydroxyvitamin D to influence differentiation of pre-adipocytes into adipocytes. These early changes in VDR expression are accompanied by an increase in the transcription factors C/EBPö and C/EBPf3. 1,25-dihydroxyvitamin D may influence C/EBP-dependent gene expression via effects on CIEBPf3 or ETO, an inhibitor of C/EBPI3 activity, but these effects are currently controversial and require further study. Nevertheless, 1 ,25-dihydroxyvitamin D and the VDR can clearly inhibit the expression of C/EBPx and PPARy, which is the master regulator of adipogenesis, disrupt PPARy-dependent gene expression, and inhibit the subsequent maturation of adipocytes. The molecular mechanism by which 1,25-dihydroxyvitamin D/VDR inhibit PPARy and PPARy- dependent gene expression remains to be fully resolved, but may work on several fronts including inhibition of endogenous PPAR7 ligand production and sequestration of limiting amounts of RXR.

The metabolism and cellular action of vitamin D is prohormone 25-hydroxyvitamin D,which is a reflection of quite complex! Vitamin D is produced in the skin in overall vitamin D status. Locally, the prohormone can be response to ultraviolet B radiation and then converted converted to the bioactive hormone by the relative rates of in the liver to 25-hydroxyvitamin D, which acts as a cellular 1,25-dihydroxyvitamin D synthesis via CYP27B1 prohormone for the renal production of the 1,25- (1-hydroxylase) and hormone breakdown via CYP24 dihydroxyvitamin D hormone. This bioactive vitamin D (24-hydroxylase).38 metabolite acts as a steroid hormone and a high-affinity Despite the ability to synthesize vitamin D in the skin ligand for the cellular vitamin D receptor (VDR). The and the availability of vitamin D in some foods, vitamin D ligand- activatedvitamin D receptor forms a heterodimer deficiency is increasingly recognized as a widespread with the retinoid X receptor (RXR), which can bind to global nutritional problem.-" Vitamin D insufficiency vitamin D response elements in various and has ramifications not only for bone health, but also cause the transactivation or repression of vitamin D- in other non-skeletal areas of vitamin D function, responsive genes in a variety of tissues. Intracellular such as immune cells, 12,13 muscle cells 14 and, perhaps, 1,25-dihydroxyvitamin D activates the VDR in a adipocytes.5"6 dose-dependent manner. The concentration of 1,25- dihydroxyvitamin D in the cell is controlled by a number PPARy: A MASTER REGULATOR OF ADIPOGENESIS of factors, including the following: circulating plasma 1,25-dihydroxyvitamin D concentrations, which depends Peroxisome proliferator-activated receptor-gamma primarily on renal 1,25-dihydroxyvitamin D production; (PPARy) is both sufficient and necessary for conversion of and, in some cell types, the intracellular availability of the pre-adipocytes into adipocytes, and is the likely "master

Nutrition Reviews ® VoL 66(1):40-46 41 regulator" of adipogenesis. Peroxisome proliferator- (CCAAT-enhancer binding protein) family (C/EBP(X, activated receptors are members of the nuclear-receptor C/EBPI3, CIEBPy, CIEBP6 and CHOP) and Kruppel-like superfamily of proteins and act as nuclear family (KLF2, KLF5, KLF15), which bind to PPAR factors where they form a heterodimer with the retinoid promoters, as do GATA transcription factors. Depending X receptor (RXR). The PPAR-RXR heterodimer binds on the balance of these factors bound to gene promoters, to PPAR response elements (PPRE) in the promoter of the effect on the pre-adipocyte may be either pro- or anti- PPAR-responsive genes. There are three different types adipogenic. 7 In addition, certain transcription factors, of PPARs (PPAR(x, PPARy, and PPAR6), which are such as sterol response element-binding protein-ic expressed from three different genes. In addition, PPARy (SREBP1c)8 and C/EBP3, 9 can influence the production has three different isoforms (PPARy1, PPARy2, and of an endogenous PPARy ligand early in adipogenesis, PPARy3) that result from alternative processing of PPARy which can influence PPARy transactivation activity. This mRNA. The protein product for PPARy1 and PPARy3 are panoply of adipogenic regulators indicates there are likely identical, but PPARy2 is 30 amino acids longer. PPARy2 many layers of transcriptional control woven into a is predominantly expressed in adipose tissue, while the complex temporal network of gene expression control other PPARy isoforms are expressed in adipocytes and points that regulate adipogenesis. Relatively little is other tissues.7 known about the role of the VDR and its activating ligand 1,25-dihydroxyvitamin D in adipogenesis. GENETIC PROGRAMMING IN ADIPOGENESIS

To better understand the signaling pathways that VITAMIN D AND ADIPOGENESIS promote terminal differentiation, and factors that can modulate adipocyte function, many studies have utilized Studies conducted in Japan almost 20 years ago indicated cell culture models that have already been committed to that treatment of 3T3-Ll pre-adipocytes in culture with an adipocyte lineage, such as mouse 3T3-Ll, 3T3-F442A, 1,25-dihydroxyvitamin D, the bioactive form of vitamin and C3H1OT1/2 cells and human pre-adipocytes. 7 For D, affected differentiation and adipocyte metabolism.20 example, the mouse 3T3-Ll pre-adipocyte experimental These early studies indicated that nanomolar concentra- system has proven to be a useful and popular cell culture tions of 1,25-dihydroxyvitamin D could inhibit adipo- model to investigate the molecular steps involved in regu- genesis and reduce the accumulation of triacylglycerol lating the progression of pre-adipocytes to mature lipid- by 50% compared to fully differentiated control cells. In accumulating adipocytes. The 3T3-Ll pre-adipocytes can addition, treatment of pre-adipocytes with other vitamin be induced to differentiate into adipocytes by treatment D metabolites, such as 24,25-dihydroxyvitamin D, could with DIM, a hormonal cocktail of dexamethasone, also inhibit pre-adipocyte differentiation, but at higher insulin, and 3-isobutyl-l-methylxanthine, which is a concentrations than 1,25-dihydroxyvitamin D that paral- phosphodiesterase inhibitor that causes an increase in leled their reduced affinity for the vitamin D receptor. intracellular cyclic AMP. The differentiation program in These early studies also noted that specific 1,25- the 3T3-L1 pre-adipocyte cell line proceeds in a predict- dihydroxyvitamin D binding was evident in pre- able temporal pattern consisting of one or two rounds of adipocyte 3T3-Li cells, but there was no evidence of clonal expansion within the first 24 h or so followed by specific binding for 1,25-dihydroxyvitamin D in mature cell cycle arrest. Continued stimulation with the DIM adipocytes.21 This finding is consistent with the idea that cocktail for 48 h promotes the development of evident any influence of vitamin D on adipogenesis would likely morphologic changes in most of the cells, from spindle- be exerted early in the pre-adipocyte to adipocyte transi- shaped fibroblast-like pre-adipocytes to a more rounded tion when more VDR was available. Theoretically, the adipocyte phenotype. Continued insulin exposure is sensitivity of the pre-adipocyte to 1,25-dihydroxyvitamin needed to complete the morphologic, biochemical and D would be increased by a greater amount of vitamin D functional terminal differentiation of all the cells into receptor or other limiting VDR-associated proteins. More mature lipid-synthesizing adipocytes over the next four than a decade ago, it was shown that one of the early to five days in culture (Figure 1). effects of 1,25-dihydroxyvitamin D treatment of 3T3-L1 Our current understanding of genetic programming pre-adipocytes was an increase in vitamin D receptor in adipocytes reveals a great deal of underlying complex- mRNA expression in pre-adipocytes, which occurs within ity. 1,17 For example, in addition to the master regulator of 4 h of treatment in cell culture. 22 Little was appreciated, adipogenesis PPARy, there are more than 100 other tran- however, about any direct importance of the unliganded scriptional factors in adipocytes, including all the nuclear VDR in adipogenesis, or the molecular details whereby receptor families. Other important adipogenic transcrip- 1,25-dihydroxyvitamin D treatment inhibited differentia- tional factors include several members of the CIEBP tion of pre-adipocytes.

42 Nutrition Reviews ® Vol. 66(1):40-46 The anti-diabetic drug thiazolidinedione is a specific Clonal expansion in 3T3-L1 cells does not mediate the ligand for PPARy, the master regulator of adipogenesis, effects of vitamin D on adipogenesis and acts as a strong inducer of terminal differentiation in pre-adipocytes. An important molecular insight was In 3T3-Ll cells, there is an obligatory clonal cell gained several years ago by Hida et al. 23 who found that population expansion step, involving one to two cell treatment of 3T3-L1 cells with 1,25-dihydroxyvitamin D divisions, that occurs immediately following exposure inhibited thiazolidinedione- induced pre-adipocyte dif- of the cells to the differentiating cocktail. Given that ferentiation. Moreover, this effect of the bioactive vitamin 1,25-dihydroxyvitamin D is well known to have anti- D metabolite was associated with inhibition of the proliferative properties, the Chicago investigators upregulation of PPARy2 protein expression, which nor- expected that the early vitamin D effect would be evident mally occurs during the first 48 h after initiation of at the clonal expansion step. However, they observed that pre-adipocyte differentiation. 1,25-dihydroxyvitamin D had no effect on mitotic clonal expansion. A similar finding was also made by the Boston group" in 3T3-L1 cells. NEW MOLECULAR INSIGHTS INTO THE MECHANISM In addition, Kong and Li" provided further support- OF 1,25-DIHYDROXYVITAMIN D ACTION IN ing evidence that a clonal expansion step was not critical PRE-ADIPOCYTES to the vitamin D effect on adipogenesis by studying the effects of 1,25-dihydroxyvitamin D in primary cultures of Although it has been known for many years that 1,25- mouse epididymal fat pads, an experimental system that dihydroxyvitamin D can affect PPARy expression and does not undergo an initial clonal expansion step. Ex vivo block pre-adipocyte differentiation, the molecular treatment of primary cultures of these fat cells with 1,25- mechanism responsible for this action had not been well dihydroxyvitamin D also caused a decrease in the expres- clarified. Recently, independent work from the research sion of C/EBPa and PPARy mRNA seen in 3T3-L1 cells, laboratories of Kong and Li in Chicago" and Blumberg confirming that a clonal expansion step is not necessary et al. in Boston" have elucidated some of the layers of to observe an effect of vitamin D on adipogenesis. complexity of the vitamin D effect on adipogenesis.

A short window of opportunity for vitamin D 1 ,25-dihydroxyvitamin D inhibits endogenous action in adipogenesis PPARyligand activity

In Chicago, Kong and Li" confirmed in 3T3-L1 mouse PPARy is a ligand- activatednuclear receptor. There is pre-adipocytes that 1,25-dihydroxyvitamin D treatment evidence that 3T3-L1 cells produce increased amounts of a inhibits adipocyte differentiation, and they observed that novel endogenous PPARy ligand on day 1 and day 2 fol- the normal induction of a number of genes involved with lowing exposure to the adipogenic cocktail. The Boston the early and later stages of adipocyte development, such group" focused their attention on determining to what as CCAAT/enhancer binding protein alpha (C/EBP(x), extent 1 ,25-dihydroxyvitamin D influenced the formation PPARy, lipoprotein lipase (LPL) and adipocyte protein of this unidentified endogenous PPARy ligand. Using a 2/adipocyte-specific fatty acid-binding protein (02), unique stable 3T3-L1 5132 cell line expressing an ingenious as well as sterol-regulatory element-binding protein reporter gene that is sensitive to changes in the availability (SREBP)- 1 and fatty acid synthase (FAS), which are genes of endogenous ligand for PPARy, Blumberg et al. 2 demon- characteristic of the mature adipocyte, were blunted in a strated that 1,25-dihydroxyvitamin D treatment partially dose-dependent manner by 1,25-dihydroxyvitamin D. inhibited endogenous PPAR7 ligand formation, suggest- Interestingly, however, they also observed that 1,25- ing that 1,25-dihydroxyvitamin D-mediated inhibition of dihydroxyvitamin D treatment was only efficacious in PPARy ligand production could account, at least in part, blocking fat cell differentiation and gene expression when for the reduced PPARy transactivation activity in 1,25- administered within the first 48 h of initiating differen- dihydroxyvitamin D-treated cells." Although the identity tiation with the hormonal cocktail. In addition, they of the endogenous PPAR7 ligand has not yet been deter- observed that removal of 1,25-dihydroxyvitamin D after mined, the observations of the Boston group using three days of treatment allowed the differentiation the functional PPARy-ligand assay indicate that at least process to be reinitiated. This important observation sug- one of the 1,25-dihydroxyvitamin D-mediated effects on gests that the main locus of the vitamin D effect on pre-adipocyte differentiation in 3T3-L1 cells may be adipogenesis must reside in the suppression of a key upstream of the endogenous PPARy ligand production reversible molecular event very early in the pre-adipocyte step, which is evident within the first 24 h following DIM differentiation process. administration.

Nutrition Reviews ® Vol. 66(1):40-46 43 Vitamin D receptor is necessary for adipocytes is likely governed by the temporary increased 1,25-di hydroxyvita min D-mediated effects availability of VDR, or some VDR transactivation factor, in pre-adipocytes within the first day or two following the triggering of the differentiation program. The importance ofVDR on adipogenesis was investigated by the Chicago group" in VDR knockout mice by using Controversial role of C/EBPP in 1,25-dihydroxyvitamin mouse embryonic fibroblasts (MEFs). The MEFs, which D-induced inhibition of CIEBPa and PPARy expression could be stimulated to differentiate into adipocytes, were isolated from wild-type and vitamin D receptor knockout Kong and Li" and Blumberg et al." both observed that mice. Differentiation was monitored by measuring the vitamin D-induced block of pre-adipocyte differen- PPARy expression. 1,25-dihydroxyvitamin D was able to tiation in 3T3-Ll cells was associated with an inhibition block the expected increase in PPARy expression in the of PPARy and C/EBPcL expression. C/EBP(x and PPARy wild-type MEFs, but not in the MEFs from the VDR are critical molecular components of adipogenesis and knockout, supporting the absolute importance of the can be regulated by C/EBPf3.24 Blumberg et al. 5 found VDR in mediating the effects of 1,25-dihydroxyvitamin D that C/EBPI3 mRNA and protein rose equally in control on PPARy expression and pre-adipocyte differentiation. and 1,25-dihydroxyvitamin D-treated cells, but declined Kong and Li" also found that VDR protein expres- more precipitously in the vitamin D-treated group. A sion was quite low in mouse 3T3-Ll pre-adipocyte. sharper decline in C/EBPI3 in the vitamin D-treated However, VDR increased dramatically by 4 h following 3T3-L1 cells could indicate that reduced C/EBPI3 is a treatment with the differentiation cocktail, and then possible proximate cause for the inhibition of C/EBPo declined back to baseline levels by day two. Treatment and PPARy expression, or the production of endogenous with 1,25-dihydroxyvitamin D was able to stabilize PPARy ligand 9 and subsequent derailment of the termi- the expression of VDR for at least one additional nal differentiation program. In support of this idea, day. The importance of VDR stabilization by 1,25- Blumberg et al. 15 investigated the effects of 1,25- dihydroxyvitamin D treatment in mediating the effects of dihydroxyvitamin D on the expression of ETO/MTG8, 1,25-dihydroxyvitamin D on adipogenesis is currently which has been recently identified as a potent inhibitor of unknown, but overall, these observations are consistent adipogenesis because it binds to C/EBPI3 and prevents its with the idea of an early VDR-dependent window of interaction with the C/EBPa gene promoter . 25 Normally, opportunity for 1,25-dihydroxyvitamin D to inhibit adi- ETOIMTG8 mRNA expression declines quite quickly pogenesis, at least in the 3T3-L1 cell culture model. over the first 12 h following administration of differenti- The role of the VDR in pre-adipocyte differentiation ating medium to 3T3-L1 cells. However, in the presence in 3T3-L1 cells was also addressed in the studies con- of 1,25-dihydroxyvitamin D, the Boston group" found ducted in Boston by Blumberg et al. 15 In searching for the there was a clear rebound of ETOIMTG8 mRNA expres- early induction of cAMP-dependent genes using cDNA sion back to baseline levels by 24 h and these levels microarray analyses in 3T3-L1 cells, the researchers iden- persisted throughout differentiation. The continued pres- tified the VDR gene as a potential early cAMP target in ence of ETO/MTG8 in 1,25-dihydroxyvitamin D-treated the adipogenesis pathway. Similar to the Chicago group, pre-adipocytes would presumably bind to any C/EBPI3 they found that VDR mRNA increased to a maximum by still present and inhibit C/EBPa expression, which could 6 h following initiation of differentiation, and VDR in turn inhibit adipogenesis. 26 A key unanswered ques- protein accumulated in the nucleus as early as 4 h post tion, however, was whether C/EBP activity was reduced in initiation reaching a maximum by 12 h and then declin- pre-adipocytes triggered to differentiate and treated with ing over the next 2 days. Consistent with their observa- 1 ,25-dihydroxyvitamin D. 15 tion that the early induction of VDR gene expression In contrast to the perspective of the Boston group in 3T3-L1 cells was cAMP-dependent, they identified on the role of C/EBPI3 in vitamin D-induced inhibition of 3-isobutyl- 1 -methyixanthine, the phosphodiesterase pre-adipocyte differentiation, the Chicago group" found inhibitor in the DIM hormonal cocktail, as the critical that the early increase (4 h to 39 h) in C/EBPf3 mRNA cocktail component responsible for the induction of expression in 3T3-L1 cells after the initiation of differen- VDR. tiation was not affected by 1,25-dihydroxyvitamin D Overall, then, the observations of both the Chicago" treatment. The reason for this apparent discrepancy and Boston" groups are consistent with earlier findings" between the two groups is unknown, but could relate to that the availability of the VDR is not equal at all points the Chicago groups use of a Northern blot method and during differentiation of pre-adipocytes into adipocytes. the Boston groups use of a more sensitive quantitative This early temporal window of opportunity for vitamin real-time PCR method. Nevertheless, when the Chicago D-mediated effects on the differentiation program in pre- group transfected 3T3-L1 cells with a reporter gene

44 Nutrition Reviews Vol. 66(1):40-46 construct containing a C/EBP response element, which additional insights into how gene-diet interactions can would allow them to directly monitor C/EBP-mediated serve as risk factors for the development of obesity. transactivation activity in the presence or absence of 1,25- The Chicago group" pursued the question of the dihydroxyvitamin D treatment in pre-adipocytes, they unliganded VDR even further by investigating whether found there was no effect of 1,25-dihydroxyvitamin D overexpression of VDR alone, in the absence of added treatment on reporter gene activity after 16 h of treat- 1,25-dihydroxyvitamin D ligand, could affect PPARy ment. This latter observation would appear to argue transactivation activity in either the basal or induced strongly against an important role of increased ETO and state. They found that increased expression of VDR apparent decreased C/EBPI3, as found by the Boston reduced both the basal activity of a PPAR7-responsive group, as an apparent cause for the vitamin D-mediated reporter gene and the ability to induce reporter gene inhibition of C/EBPa and PPARy expression and adipo- activity by increasing PPARy protein expression. This genesis. However, it should be noted that CIEBP transac- intriguing observation directed their attention to the spe- tivation measurements were made at a time point (16 h) cific details by which the VDR might block PPARy activity that may precede the rebound in ETO expression (24 h). and adipogenesis (Figure 1). If CIEBP response element binding activity in 1,25- 3T3-Ll pre-adipocytes express relatively low levels dihydroxyvitamin D-treated pre-adipocyte cells is of RXR, the heterodimeric receptor partner of VDR. normal, then it suggests that the vitamin D-associated Several years ago, Hida et al .21 speculated that 1,25- decrease in C/EBPa and PPARyis likely to be a proximate dihydroxyvitamin D might inhibit pre-adipocyte differ- cause, rather than a consequence, of the vitamin entiation by a mechanism wherein VDR competes with D-dependent interruption of the terminal differentiation PPARy for their common heterodimeric partner RXR. program. The Chicago group" tested the hypothesis that additional VDR may influence PPARy-mediated gene expression Does the unhiganded vitamin D receptor control activity by sequestering low amounts of endogenous RXR adipogenesis by monopolizing RXR and blocking and thereby squelching PPARy transactivation activity in PPARy activity? 3T3-L1 cells. To test this squelching hypothesis, 3T3-L1 cells were co-transfected with the PPARy-responsive In cell culture studies, when VDR was overexpressed in PPRE-luciferase reporter gene cDNA and VDR cDNA, mouse 3T3-Ll cells, even in the absence of 1,25- either with or without RXR cDNA. Consistent with the dihydroxyvitamin D treatment, pre-adipocyte differen- squelching hypothesis, they observed that an increase in tiation was inhibited completely." This finding is VDR expression reduced basal PPRE-driven luciferase noteworthy because it points to a possible role of the reporter activity, which was also inhibited by 1,25- unliganded VDR in controlling adipogenesis. This obser- dihydroxyvitamin D treatment alone. However, increased vation is interesting because it could have ramifications expression of RXR protein was able to increase PPRE- for adipogenesis, irrespective of vitamin D status, in luciferase activity in the presence of increased VDR and people with different VDR polymorphisms, or suggest a prevent inhibition by 1,25-dihydroxyvitamin D. Thus, possible interaction between vitamin D status and VDR these observations are consistent with the hypothesis that polymorphisms on adipogenesis; thus, it warrants further the vitamin D receptor inhibits adipogenesis by blocking study in the future. Moreover, the availability of increased PPARy activity, at least in 3T3-L1 cells, by a mechanism amounts of the unliganded VDR early in the differentia- apparently due to VDR monopolizing the limiting tion process, identified by both the Chicago and Boston amounts of shared endogenous RXR. The importance research groups, is of biological and nutritional interest of this potential mechanism of 1,25-dihydroxyvitamin because these changes in unliganded VDR availability D-induced inhibition of adipogenesis will need addi- may have variable effects on adipogenesis and the risk of tional testing under in vivo conditions. developing obesity, depending upon the intracellular 1,25-dihydroxyvitamin D concentration. Intracellular CONCLUSION 1,25-dihydroxyvitamin D concentration can be con- trolled by a number of factors, including overall vitamin Overall, the recent observations made by the Chicago and D nutritional status and the relative activity of intracellu- Boston groups make a strong case for the role of vitamin lar enzymes involved in the cellular synthesis of 1,25- D in adipogenesis being mediated at the molecular dihydroxyvitamin D from the 25-hydroxyvitamin D level through VDR-dependent inhibition of C/EBPz and prohormone by CYP27B1 (1-hydroxylase) and the intra- PPARy expression and a decrease in PPARy transactiva- cellular catabolism of vitamin D metabolites by CYP24 tion activity. However, additional research is needed to (24-hydroxylase). Future investigations of polymor- fully clarify the role of CIEBPf3, to determine why phisms in these vitamin D metabolism genes may offer 1,25-dihydroxyvitamin D causes a decrease in PPARy

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