Retinoic acid has different effects on UCP1 expression in mouse and human adipocytes
- Maria Murholm†1, 2,
- Marie S Isidor†1, 2,
- Astrid L Basse1, 2,
- Sally Winther1,
- Cathrine Sørensen1,
- Jonas Skovgaard-Petersen1,
- Maja M Nielsen1,
- Aina S Hansen1,
- Bjørn Quistorff2 and
- Jacob B Hansen1Email author
© Murholm et al.; licensee BioMed Central Ltd. 2013
Received: 1 May 2013
Accepted: 18 September 2013
Published: 23 September 2013
Increased adipose thermogenesis is being considered as a strategy aimed at preventing or reversing obesity. Thus, regulation of the uncoupling protein 1 (UCP1) gene in human adipocytes is of significant interest. Retinoic acid (RA), the carboxylic acid form of vitamin A, displays agonist activity toward several nuclear hormone receptors, including RA receptors (RARs) and peroxisome proliferator-activated receptor δ (PPARδ). Moreover, RA is a potent positive regulator of UCP1 expression in mouse adipocytes.
The effects of all-trans RA (ATRA) on UCP1 gene expression in models of mouse and human adipocyte differentiation were investigated. ATRA induced UCP1 expression in all mouse white and brown adipocytes, but inhibited or had no effect on UCP1 expression in human adipocyte cell lines and primary human white adipocytes. Experiments with various RAR agonists and a RAR antagonist in mouse cells demonstrated that the stimulatory effect of ATRA on UCP1 gene expression was indeed mediated by RARs. Consistently, a PPARδ agonist was without effect. Moreover, the ATRA-mediated induction of UCP1 expression in mouse adipocytes was independent of PPARγ coactivator-1α.
UCP1 expression is differently affected by ATRA in mouse and human adipocytes. ATRA induces UCP1 expression in mouse adipocytes through activation of RARs, whereas expression of UCP1 in human adipocytes is not increased by exposure to ATRA.
KeywordsAdipogenesis ATRA Brown adipocyte UCP1 White adipocyte
Mammals have two types of fat, white and brown adipose tissue (WAT and BAT, respectively), that carry out essentially opposite functions in whole body energy metabolism [1, 2]. White adipocytes are specialized in energy storage and their content of triglyceride constitutes the largest energy reserve of the body. Contrary, brown adipocytes have a high capacity for energy dissipation through adaptive thermogenesis due to the presence of the brown adipocyte-specific uncoupling protein 1 (UCP1) in the inner membrane of the abundant mitochondria. BAT has been shown to counteract obesity and is important for rodents to defend their body temperature in response to prolonged cold exposure . Brown-like adipocytes expressing UCP1 appear in some rodent WAT depots after cold exposure or treatment with β-adrenergic agonists [3, 4]. Recent studies have suggested a negative correlation between body mass index and the amount of active BAT in humans . Strategies aiming at increasing levels of UCP1 in WAT have become of interest as reduced expression of brown adipocyte-enriched genes in WAT is associated with obesity and type 2 diabetes in humans [6–8].
Retinoic acid (RA) is a derivative of vitamin A that affects cellular growth, differentiation and apoptosis in various embryonic and adult tissues [9, 10]. All-trans RA (ATRA) has been reported being an agonist for multiple nuclear receptors, including RA receptors (RARs) [11, 12], peroxisome proliferator-activated receptor δ (PPARδ, also designated PPARβ) , testicular orphan receptor 4 (TR4)  and chicken ovalbumin upstream promoter transcription factor II (COUP-TFII) . It has been proposed that PPARδ mediates part of the metabolic effects of ATRA . Additionally, ATRA has been shown to regulate gene expression in a nongenomic manner . However, it is believed that most effects of ATRA are mediated by RARs that upon heterodimerization with retinoid X receptors control gene expression through binding to RA response elements in regulatory regions of target genes [9, 18].
High concentrations of ATRA inhibit differentiation of 3T3-L1 white preadipocytes and C3H10T½ mesenchymal stem cells [19–21], whereas low concentrations have been shown to stimulate white adipogenesis of Ob1771 cells . The inhibition of adipogenesis by ATRA is mediated by RARs and is linked to suppression of CCAAT/enhancer-binding protein β activity and induction of anti-adipogenic genes [19, 21, 23].
The UCP1 gene of mice, rats and humans contains RAR-responsive elements in its enhancer region and ATRA has been shown to promote UCP1 expression and oxidative metabolism in cultured rodent adipocytes [24–31]. Moreover, treatment of mice with ATRA causes increased expression of UCP1 in WAT and BAT [17, 28, 31, 32].
In the present study we compared the response of mouse and human preadipocytes and mature adipocytes to ATRA, with emphasis on the effects on differentiation and UCP1 expression. In addition, we have studied the importance of RARs, PPARδ and PGC-1α for the regulation of UCP1 expression by ATRA. We find that ATRA increases UCP1 expression in all mouse adipocyte models studied, including 3T3-L1 white adipocytes, and that this induction is mediated by RARs and is independent of PPARδ and PGC-1α. Finally, ATRA does not increase UCP1 expression in any of the human adipocytes examined in this study.
Exposure of differentiating mouse adipocytes to ATRA increases UCP1 expression
In order to examine the effects of ATRA on differentiating mouse adipocytes, we exposed four cell models of adipogenesis to a range of ATRA concentrations (10 nM to 10 μM) throughout the course of the differentiation process, i.e. between days −2 (the time of confluence) and 8 (designated chronic exposure). Gene expression was analyzed at day 8. We estimated the degree of differentiation by measuring mRNA levels of the adipocyte marker gene fatty acid-binding protein 4 (FABP4, also designated aP2). Expression of the brown fat-specific UCP1 gene was determined at both the mRNA and protein levels, and the expression of RARβ was used to estimate the degree of activation of RARs, as the RARβ gene is responsive to retinoids [33, 34]. The cells used were 3T3-L1 preadipocytes, wild-type (WT) mouse embryo fibroblasts (MEFs) and the mesenchymal stem cell line C3H10T½ as models of white adipocyte differentiation [35, 36], and MEFs lacking a functional retinoblastoma gene (Rb−/−) as a model of brown adipocyte differentiation .
Exposing mature mouse adipocytes to ATRA enhances expression of UCP1
RARs mediate the effects of ATRA
When the three agonists were acutely supplemented to mature WT MEF-derived adipocytes, UCP1 mRNA levels were significantly induced after 24 h, with AM580 and tazarotene displaying the most potent effect (Figure 4B).
PPARδ activation does not increase UCP1 expression in MEF-derived white adipocytes
The effects of ATRA on UCP1 expression is not dependent on PGC-1α
Exposing mature PGC-1α+/+ and PGC-1α−/− adipocytes to 1 μM ATRA elicited significant induction of UCP1 after 24 h (Figure 6B). Thus, enhanced expression of UCP1 caused by ATRA does not require PGC-1α.
ATRA inhibits human adipocyte differentiation in a dose-dependent manner and does not increase UCP1 expression
In this study, we report the effects of ATRA on differentiation and UCP1 expression in various mouse and human adipocytes. We find that high concentrations of ATRA inhibit mouse and human adipogenesis, whereas lower concentrations enhance UCP1 expression in mouse, but not in human, adipocytes. In addition, we show that the effects of ATRA are mediated by RARs and not by PPARδ or other ATRA-activated nuclear receptors. Moreover, the enhanced expression of UCP1 in response to ATRA is independent of PGC-1α.
Adipocyte and adipose tissue function are impacted by ATRA [47, 48]. Expression of UCP1 is reduced in BAT of mice fed vitamin A-depleted feed [17, 49] and exogenous ATRA enhances expression of UCP1 in both WAT and BAT of mice and rats [16, 17, 28, 32, 49, 50]. Expression of UCP1 is induced by ATRA in primary brown adipocytes from mice and rats as well as in mouse brown adipocyte cell lines [24, 26, 28, 29, 51–53]. Moreover, UCP1 expression is strongly induced in MEF-derived white adipocytes . Contrary, ATRA has been reported not to induce expression of UCP1 in mature 3T3-L1 adipocytes and mouse primary white adipocytes [16, 30, 54]. Exposure to ATRA leads to activation of p38 mitogen-activated protein kinase (MAPK), an activation that is required for full induction of UCP1 expression by ATRA [29, 31].
ATRA has been reported to activate three nuclear receptors besides RARs, namely PPARδ, COUP-TFII and TR4. The induction of UCP1 observed in the mouse cells applied in this study is unlikely to be mediated by COUP-TFII and TR4, as the app. EC50 of ATRA are 20 μM  and 24 μM , respectively, which is 20 to 240 times higher than the concentrations inducing UCP1. Although the EC50 of ATRA for PPARδ is much lower than for COUP-TFII and TR4 (app. 200 nM) , PPARδ is not mediating the effects of ATRA either. Firstly, the RAR agonist TTNPB mimics the effects of ATRA (see Figure 3), but does not bind to PPARδ . Secondly, a potent PPARδ agonist does not enhance expression of UCP1 (see Figure 5). Thirdly, a RAR antagonist attenuates the effects of ATRA (see Figure 3). In this study, we have not addressed the potential involvement of nongenomic effects of ATRA, e.g. activation of p38 MAPK and the cell surface receptor responsible for retinol uptake called stimulated by retinoic acid gene 6 .
At intermediate concentrations of ATRA, we consistently observe an induction of UCP1 expression in mouse adipocytes. This does not only occur in the mouse cells shown here, but was also observed with WT-1 brown adipocytes [56, 57] and 3T3-F442A white adipocytes (data not shown). Thus, our results demonstrate that ATRA can cause an induction of UCP1 expression in white adipocyte cell models of mouse origin. It is tempting to speculate that exposure to ATRA will cause white preadipocytes and mature adipocytes to transdifferentiate into brown-like adipocytes in vitro. However, in order to confirm if a transdifferentiation event has taken place in our study, a more detailed gene expression analysis is required combined with a characterization of mitochondrial function.
Using three cell models of human origin, SGBS and hMADS cells as well as primary subcutaneous adipocytes from two different donors, we failed to detect an induction of UCP1 expression by ATRA (see Figures 7 and 8). hMADS cells have been proposed to represent brown or brown-like adipocytes, the latter due to the induction of UCP1 expression in response to prolonged culture in the presence of rosiglitazone [58, 59] or upon treatment with atrial natriuretic peptide . Despite being considered white fat cells, SGBS and primary subcutaneous human adipocytes have the ability to induce expression of UCP1 in response to genetic manipulation [61, 62]. Thus, the lack of effect of ATRA in the human adipocyte models studied here cannot be explained by an inherent inability to induce expression of UCP1. Consistently, to our knowledge, an induction of the endogenous human UCP1 gene by ATRA has never been reported. Nevertheless, we cannot rule out that the lack of response in our study is due to the experimental setup or the human cell models used. In particular, it remains to be shown if primary human brown adipocytes respond to ATRA by increasing UCP1 expression. However, as we consistently observe enhanced expression of UCP1 by intermediate concentrations of ATRA in mouse fat cells, we find this difference between mouse and human adipocytes noteworthy.
In conclusion, we demonstrate that ATRA is a powerful inducer of UCP1 expression in mouse white and brown adipocytes, supporting that ATRA has the capacity to increase the potential for uncoupled respiration in those cells. The increased expression of UCP1 in response to ATRA is mediated by RARs, not PPARδ, and is independent of PGC-1α. We do not find induction of UCP1 gene expression by ATRA in the human adipocytes studied here, but whether this applies to the human UCP1 gene in general remains to be determined. Nevertheless, differences between rodents and humans in terms of regulation of UCP1 expression are highly relevant, as modulation of BAT activity and browning of WAT are being considered as potential anti-obesity targets. More studies comparing rodent and human adipocytes are needed to understand their similarities and differences with respect to regulation of UCP1 expression.
WT and Rb−/− MEFs were propagated and differentiated as previously described . Immortalized PGC-1α+/+ and PGC-1α−/− brown preadipocyte cell lines were obtained from Dr. Bruce M. Spiegelman , and C3H10T½ mesenchymal stem cells  and 3T3-L1 white preadipocytes  were obtained from Dr. Karsten Kristiansen. Brown preadipocyte cell lines and C3H10T½ cells were propagated in Dulbecco’s Modified Eagle’s Medium (DMEM) (Life Technologies) supplemented with 10% foetal bovine serum (FBS) (Life Technologies) and differentiated as WT and Rb−/− MEFs. 3T3-L1 cells were propagated in DMEM supplemented with 10% bovine serum and differentiated as WT and Rb−/− MEFs. Thus, all mouse cells were cultured in the presence of rosiglitazone from day 0 until the time of harvesting. The SGBS white preadipocyte cell line was obtained from Dr. Martin Wabitch  and propagated in Advanced DMEM/F12 (Life Technologies) with 10% FBS and 2 mM L-glutamine (Life Technologies). Two days postconfluent cells (designated day 0) were induced to differentiate in Advanced DMEM/F12 with 2% FBS supplemented with 0.86 μM insulin (Roche), 1 μM dexamethasone (Sigma-Aldrich), 0.5 mM 3-isobutyl-1-methylxanthine (IBMX) (Sigma-Aldrich), 1 μM rosiglitazone (Cayman Chemical), 1 μM cortisol (Sigma-Aldrich) and 1 nM 3,3′,5-triiodo-L-thyronine (T3) (Sigma-Aldrich). On day 3 the cells were fed the same medium as on day 0. On days 6, 9 and 12 medium contained 2% FBS, 0.86 μM insulin, 1 μM rosiglitazone and 1 nM T3. hMADS cells were obtained by Dr. Christian Dani and their propagation and differentiation were carried out as described [45, 46] with minor modifications. Briefly, hMADS cells were cultured in low glucose DMEM (Lonza) supplemented with 10% FBS, 2 mM L-glutamine, 10 mM HEPES (Lonza) and 2.5 ng/ml human fibroblast growth factor 2 (Life Technologies). Two days postconfluent cells (designated day 0) were induced to differentiate in low glucose DMEM/Ham’s F12 medium (Lonza) with 10 mM HEPES, 2 mM L-glutamine supplemented with 10 μg/ml transferrin, 0.86 μM insulin, 0.1 μM rosiglitazone, 0.2 nM T3, 1 μM dexamethasone and 0.5 mM IBMX. At days 2, 4, 6, 8, 10 and 12 medium was supplemented with 10 mM HEPES, 2 mM L-glutamine, 10 μg/ml transferrin, 0.86 μM insulin, 0.1 μM rosiglitazone and 0.2 nM T3. Primary human white subcutaneous preadipocytes (Lonza) were cultured in PBM-2 medium (Lonza). Two days postconfluent preadipocytes (designated day 0) were induced to differentiate with PBM-2 medium supplemented with insulin, dexamethasone, IBMX and indomethacin (all supplied by Lonza) according to the instructions of the manufacturer. On day 3 the cells were refed the same medium as on day 0. On days 6, 9 and 12 cells were refreshed with PBM-2 medium containing insulin and indomethacin. All media described above were supplemented with 50 U/ml penicillin and 50 μg/ml streptomycin, and all cells were cultured at 37°C in humidified atmospheric air with 5% CO2, except for hMADS cells that were cultured with 10% CO2.
Additional ligands were used in concentrations stated in figures and figure legends and were added from either day −2 and onwards in chronic treatment experiments or from day 8 (mouse cells) or day 12 (human cells) in experiments with acute exposure of mature adipocytes. ATRA, TTNPB and AM580 were purchased from Sigma-Aldrich. Tazarotene, CD1530 and BMS493 were purchased from Tocris Bioscience, and GW501516 was kindly provided by Novo Nordisk A/S. All nuclear receptor ligands were dissolved in dimethyl sulfoxide (DMSO) (Sigma-Aldrich), and dishes not supplemented with ligands were treated with an equal volume of DMSO.
Reverse transcription-quantitative polymerase chain reaction
Total RNA was purified using TRI Reagent (Sigma-Aldrich). Reverse transcription (RT) and RT-quantitative polymerase chain reaction (RT-qPCR) were performed as previously described . Primers used were: ADRP (mouse), fw-GAATTTCTGGTTGGCACTGT, rev-GACCATTTCTCAGCTCCACTC (80 bp); FABP4 (mouse), fw-TGGAAGCTTGTCTCCAGTGA, rev-AATCCCCATTTACGCTGATG (111 bp); RARβ (mouse), fw-ACAGATCTCCGCAGCATCAG, rev-GCATTGATCCAGGAATTTCCA (76 bp); TBP (mouse), fw-ACCCTTCACCAATGACTCCTATG, rev-ATGATGACTGCAGCAAATCGC (190 bp); UCP1 (mouse), fw-GGCATTCAGAGGCAAATCAGCT, rev-CAATGAACACTGCCACACCTC (151 bp); FABP4 (human), fw-AGCACCATAACCTTAGATGGGG, rev-CGTGGAAGTGACGCCTTTCA (132 bp); RARβ (human), fw-AAGTGCTTTGAAGTGGGAATG, rev-GCTTTTCGGATCTTCTCTGTG (143 bp); TBP (human), fw-CCCGAAACGCCGAATATAA, rev-GAAAATCAGTGCCGTGGTTC (83 bp); UCP1 (human), fw-CCAACTGTGCAATGAAAGTGT, rev-CAAGTCGCAAGAAGGAAGGTA (81 bp).
Whole cell extracts and immunoblotting
Preparation of whole-cell extracts and immunoblotting were done as described . Antibodies used were against glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (Ab8245, Abcam) and UCP1 (Ab10983, Abcam).
All experiments were repeated at least three times and three dishes were harvested at each time point or treatment for each independent experiment. Data from a representative experiment are presented as mean of the three dishes (+SEM). Statistical significance was determined by Student’s t-test. Bonferroni correction was applied when multiple comparisons were carried out.
Adipose differentiation-related protein
Brown adipose tissue
Chicken ovalbumin upstream promoter transcription factor II
Dulbecco’s Modified Eagle’s Medium
Fatty acid-binding protein 4
Foetal bovine serum
Glyceraldehyde 3-phosphate dehydrogenase
Primary human white preadipocytes/adipocytes
Mitogen activated protein kinase
Mouse embryo fibroblast
Peroxisome proliferator-activated receptor
Reverse transcription-quantitative polymerase chain reaction
Testicular orphan receptor 4
Uncoupling protein 1
White adipose tissue
We appreciate the generous gift of reagents from Drs. Bruce M. Spiegelman (Harvard Medical School, Dana Farber Cancer Institute, Boston, USA), Martin Wabitch (University of Ulm, Germany), Christian Dani (University of Nice Sophia Antipolis, France), Karsten Kristiansen (University of Copenhagen, Denmark) and Novo Nordisk A/S (Måløv, Denmark). Sally Winther is the recipient of a Novo Scholarship. This work was supported by grants to Jacob B. Hansen from the EU FP7 project DIABAT (HEALTH-F2-2011-278373), The Danish Medical Research Council, The Novo Nordisk Foundation, The Carlsberg Foundation, The Aase and Ejnar Danielsen Foundation, The Augustinus Foundation, The Hartmann Brothers’ Foundation, The Beckett Foundation and Fonden til Laegevidenskabens Fremme.
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