In vitro and in vivo induction of brown adipocyte uncoupling protein (thermogenin) by retinoic acid.
ABSTRACT: The effects of retinoic acid (RA) isomers (all-trans-RA and 9-cis-RA) on the appearance of uncoupling protein (UCP; thermogenin), the only unequivocal molecular marker of the brown adipocyte differentiated phenotype, have been investigated in primary cultures of brown adipocytes, in the brown adipocyte cell line HIB 1B and directly in intact mice. The results obtained with cultured cells indicate that retinoids function as inducers of the appearance of UCP and, at the same time, partially inhibit brown adipocyte cell proliferation. The two RA isomers displayed similar effectiveness as UCP inducers, their effect being comparable with that triggered by noradrenaline, so far considered to be the main modulator of UCP gene expression. The effectiveness of retinoids as UCP inducers was dependent on the stage of brown adipocyte differentiation, being maximal in confluent primary cells and in the medium-late differentiation stage of HIB 1B cells. Corroborating the results obtained in vitro, we show that administration of all-trans-RA or 9-cis-RA to mice leads to an increase in their brown adipose tissue specific UCP content. 9-cis-RA treatment also prevented the loss of UCP on cold deacclimation. To our knowledge, this is the first report of a stimulatory effect of retinoid compounds on UCP induction in vivo.
Project description:The intracellular pathways and receptors mediating the effects of retinoic acid (RA) on the brown-fat-uncoupling-protein-1 gene (ucp-1) have been analysed. RA activates transcription of ucp-1 and the RA receptor (RAR) is known to be involved in this effect. However, co-transfection of an expression vector for retinoid-X receptor (RXR) increases the action of 9-cis RA but not the effects of all-trans RA on the ucp-1 promoter in brown adipocytes. Either RAR-specific ¿p-[(E)-2-(5,6,7,8,-tetrahydro-5,5,8, 8-tetramethyl-2-naphthalenyl)-1-propenyl]benzoic acid¿ or RXR-specific [isopropyl-(E,E)-(R,S)-11-methoxy-3,7, 11-trimethyldodeca-2,4-dienoate, or methoprene] synthetic compounds increase the expression of UCP-1 mRNA and the activity of chloramphenicol acetyltransferase expression vectors driven by the ucp-1 promoter. The RXR-mediated action of 9-cis RA requires the upstream enhancer region at -2469/-2318 in ucp-1. During brown-adipocyte differentiation RXRalpha and RXRgamma mRNA expression is induced in parallel with UCP-1 mRNA, whereas the mRNA for the three RAR subtypes, alpha, beta and gamma, decreases. Co-transfection of murine expression vectors for the different RAR and RXR subtypes indicates that RARalpha and RARbeta as well as RXRalpha are the major retinoid-receptor subtypes capable of mediating the responsiveness of ucp-1 to retinoids. It is concluded that the effects of retinoids on ucp-1 transcription involve both RAR- and RXR-dependent signalling pathways. The responsiveness of brown adipose tissue to retinoids in vivo relies on a complex combination of the capacity of RAR and RXR subtypes to mediate ucp-1 induction and their distinct expression in the differentiated brown adipocyte.
Project description:The regulation of transcription of the gene for the beta subunit of the FoF1 ATP synthase (ATPsynbeta) in brown adipose tissue has been studied as a model to determine the molecular mechanisms for mitochondrial biogenesis associated with brown adipocyte differentiation. The expression of the ATPsynbeta mRNA is induced during the brown adipocyte differentiation that occurs during murine prenatal development or when brown adipocytes differentiate in culture. This induction occurs in parallel with enhanced gene expression for other nuclear and mitochondrially-encoded components of the respiratory chain/oxidative phosphorylation system (OXPHOS). Transient transfection assays indicated that the expression of the ATPsynbeta gene promoter is higher in differentiated HIB-1B brown adipocytes than in non-differentiated HIB-1B cells. A major transcriptional regulatory site was identified between nt -306 and -266 in the ATPsynbeta promoter. This element has a higher enhancer capacity in differentiated brown adipocyte HIB-1B cells than in non-differentiated cells. Electrophoretic shift analysis indicated that Sp1and nuclear respiratory factor-2/GA-binding protein (NRF2/GABP) were the main nuclear proteins present in brown adipose tissue that bind this site. Double-point mutant analysis indicated a major role for the NRF2/GABP site in the enhancer capacity of this element in brown fat cells. It is proposed that NRF2/GABP plays a pivotal role in the co-ordinated enhancement of OXPHOS gene expression associated with mitochondrial biogenesis in brown adipocyte differentiation.
Project description:Adipocyte precursor cells give raise to two major cell populations with different physiological roles: white and brown adipocytes. Here we demonstrate that the retinoblastoma protein (pRB) regulates white vs. brown adipocyte differentiation. Functional inactivation of pRB in wild-type mouse embryo fibroblasts (MEFs) and white preadipocytes by expression of simian virus 40 large T antigen results in the expression of the brown fat-specific uncoupling protein 1 (UCP-1) in the adipose state. Retinoblastoma gene-deficient (Rb-/-) MEFs and stem cells, but not the corresponding wild-type cells, differentiate into adipocytes with a gene expression pattern and mitochondria content resembling brown adipose tissue. pRB-deficient MEFs exhibit an increased expression of the Forkhead transcription factor Foxc2 and its target gene cAMP-dependent protein kinase regulatory subunit RIalpha, resulting in increased cAMP sensitivity. Suppression of cAMP-dependent protein kinase activity in Rb(-/-)MEFs blocked the brown adipocyte-like gene expression pattern without affecting differentiation per se. Immunohistochemical studies revealed that pRB is present in the nuclei of white but not brown adipocyte precursor cells at a developmental stage where both cell types begin to accumulate lipid and brown adipocytes express UCP-1. Furthermore, pRB rapidly undergoes phosphorylation upon cold-induced neodifferentiation and up-regulation of UCP-1 expression in brown adipose tissue. Finally, down-regulation of pRB expression accompanies transdifferentiation of white into brown adipocytes in response to beta3-adrenergic receptor agonist treatment. We propose that pRB acts as a molecular switch determining white vs. brown adipogenesis, suggesting a previously uncharacterized function of this key cell cycle regulator in adipocyte lineage commitment and differentiation.
Project description:The retinoid X receptor (RXR) is activated by its often elusive cognate ligand, 9-cis-retinoic acid (9-cis-RA). In flies and moths, molting is mediated by a heterodimer ecdysone receptor consisting of the ecdysone monomer (EcR) and an RXR homolog, ultraspiracle (USP); the latter is believed to have diverged from its RXR origin. In the more primitive insect, Locusta migratoria (Lm), RXR is more similar to human RXRs than to USPs. LmRXR was detected in early embryos when EcR transcripts were absent, suggesting another role apart from ecdysone signaling. Recombinant LmRXRs bound 9-cis-RA and all-trans-RA with high affinity (IC(50) = 61.2-107.7 nM; K(d) = 3 nM), similar to human RXR. To determine whether specific binding had functional significance, the presence of endogenous retinoids was assessed. Embryos were extracted by using modified Bligh and Dyer and solid-phase protocols to avoid the oily precipitate that makes this material unsuitable for assay. These extracts contained retinoids (5.4 nM) as assessed by RA-inducible Cyp26A1-promoter luciferase reporter cell lines. Furthermore, the use of HPLC and MS confirmed the presence of retinoids and identified in any embryo, 9-cis-RA, in addition to all-trans-RA. We estimate that whole embryos contain 3 nM RA, including 9-cis-RA at a concentration of 1.6 nM. These findings strongly argue for a functional role for retinoids in primitive insects and favor a model where signaling through the binding of 9-cis-RA to its RXR is established relatively early in evolution and embryonic development.
Project description:<h4>Objective</h4>Perivascular adipose tissue depots around the aorta are regionally distinct and have specific functional properties. Thoracic aorta perivascular adipose tissue (tPVAT) expresses higher levels of thermogenic genes and lower levels of inflammatory genes than abdominal aorta perivascular adipose tissue (aPVAT). It is not known whether this distinction is due to the in-vivo functional environment or to cell-autonomous traits that persist outside the in-vivo setting. In this study, we asked whether the progenitor cells in tPVAT and aPVAT have cell-autonomous traits that lead to formation of regionally distinct PVAT.<h4>Methods</h4>We performed microarray analysis of thoracic and abdominal peri-aortic adipose tissues of C57Bl/6J mice to define gene expression profile of each depot. To derive adipocyte progenitor cells, C57Bl/6J mice were sacrificed and thoracic and abdominal aorta fragments were embedded in Matrigel and cultured under pro-angiogenic conditions. Adipogenesis was induced using the Ppar-? agonist rosiglitazone, a thiazolidinedione (TZD). TZD-induced adipocyte populations were analyzed using immunofluorescence and qRT-PCR.<h4>Results</h4>Microarray analysis showed that tPVAT expressed higher levels of transcription factors related brown adipose tissue development compared to aPVAT. Classic brown adipose tissue (BAT) genes such as Ucp-1, Prdm16, Dio2, Slc27a displayed a concordant trend of higher level expression in tPVAT, while white adipose tissue (WAT) genes such as Hoxc8, Nnat, Sncg, and Mest were expressed at a higher level in aPVAT. The adipokines resistin and retinol binding protein 4 were also higher in aPVAT. Furthermore, adipocyte progenitors from abdominal and thoracic aortic rings responded to TZD with expression of canonical adipocyte genes Acrp30, Plin1, and Glut4. Adipocytes differentiated from thoracic aorta progenitors displayed markedly higher induction of Ucp-1 and Cidea.<h4>Conclusions</h4>Thoracic aorta PVAT expresses higher levels of brown adipocyte transcription factors than aPVAT. Precursor cells from the thoracic aorta give rise to adipocytes that express significantly higher levels of Ucp-1 and Cidea ex vivo, suggesting that progenitor cells in tPVAT and aPVAT have cell-autonomous properties that dictate adipocyte phenotype.
Project description:The synthesis and release of 11-cis-retinoids by primary cultures of human retinal pigment epithelium (RPE) and the transfer of these retinoids to co-incubated human rod outer segments (ROS) were studied. Monolayers of 2-3-week-old cultured RPE incorporate tritiated all-trans-retinol, esterify it to the corresponding retinyl palmitate, form 11-cis-retinol and 11-cis-retinaldehyde and release retinaldehyde into the culture medium. The ratio of 11-cis to all-trans isomers of retinol, retinyl palmitate and retinaldehyde formed in the cells along with retinaldehyde released and incorporated into the ROS progressively increases, indicating a progressive increase in the concentration of 11-cis isomer from the time it is formed in RPE cells until its transfer to ROS. Incorporation of 11-cis-retinaldehyde into the ROS is directly related to the amount of albumin present in the media, suggesting the transfer of retinoids from RPE to photoreceptor to be a protein-mediated process. Events leading to isomerization, esterification, oxidation and release of retinoids by human RPE and incorporation of retinoids into ROS can therefore be examined in vitro.
Project description:Retinoids are essential in the proper establishment and maintenance of immunity. Although retinoids are implicated in immune related processes, their role in immune cell adhesion has not been well established. In this study, the effect of 9-cis-retinoic acid (9-cis-RA) on human hematopoietic cell adhesion was investigated. 9-cis-RA treatment specifically induced cell adhesion of the human immune cell lines HuT-78, NB4, RPMI 8866 and U937. Due to the prominent role of integrin receptors in mediating immune cell adhesion, we sought to evaluate if cell adhesion was integrin-dependent. By employing a variety of integrin antagonist including function-blocking antibodies and EDTA, we establish that 9-cis-RA prompts immune cell adhesion through established integrin receptors in addition to a novel integrin-independent process. The novel integrin-independent adhesion required the presence of retinoid and was attenuated by treatment with synthetic corticosteroids. Finally, we demonstrate that 9-cis-RA treatment of primary murine B-cells induces ex vivo adhesion that persists in the absence of integrin function. Our study is the first to demonstrate that 9-cis-RA influences immune cell adhesion through at least two functionally distinct mechanisms.
Project description:Browning white adipocytes may be a new target in anti-obesity therapy. Pentamethylquercetin (PMQ) has been shown to have anti-obesity effects in monosodium glutamate-induced obese mice. Here, we aimed to study the anti-obesity effects of PMQ in vitro and in vivo and to determine if adipose browning is involved in the mechanism underlying the anti-obesity effects of PMQ. We evaluated the effects of PMQ on cell proliferation, cell differentiation, glucose consumption, cellular lipid metabolism, and related brown gene expression in 3T3-L1 adipocytes. We also investigated the effects of PMQ in a mouse model of high-fat diet (HFD)-induced obesity. Our results demonstrated that PMQ increased the consumption of glucose, inhibited the accumulation of cellular triglycerides (TGs), and induced the expression of brown adipocyte-specific genes, such as uncoupling protein 1 (UCP-1), during the early stage of differentiation in 3T3-L1 adipocytes. In HFD mice, PMQ treatment reduced waist circumference, LEE index, white adipose tissue (WAT) weight and white adipocyte size and increased brown adipose tissue (BAT) weight. Moreover, PMQ treatment induced mitochondrial biogenesis and upregulated UCP-1 expression in WAT. These findings suggest that PMQ may induce browning of adipose tissue, a phenomenon that is at least partly related to its anti-obesity effects.
Project description:Carotenoids and retinoids are known to alter the allergic response with important physiological roles in the skin and the immune system. In the human organism various carotenoids are present, some of which are retinoid precursors. The bioactive derivatives of these retinoids are the retinoic acids, which can potently activate nuclear hormone receptors such as the retinoic acid receptor and the retinoid X receptor. In this study, we aimed to assess how plasma carotenoid and retinoid concentrations along with the ratio of their isomers are altered in atopic dermatitis (AD) patients (n = 20) compared to healthy volunteers (HV, n = 20). The study indicated that plasma levels of the carotenoids lutein (HV 198 ± 14 ng/mL, AD 158 ± 12 ng/mL, p = 0.02; all values in mean ± SEM), zeaxanthin (HV 349 ± 30 ng/mL, AD 236 ± 18 ng/mL, p ? 0.01), as well as the retinoids retinol (HV 216 ± 20 ng/mL, AD 167 ± 17 ng/mL, p = 0.04) and all-trans-retinoic acid (HV 1.1 ± 0.1 ng/mL, AD 0.7 ± 0.1 ng/mL, p = 0.04) were significantly lower in the AD-patients, while lycopene isomers, ?-carotene, and ?-carotene levels were comparable to that determined in the healthy volunteers. In addition, the ratios of 13-cis- vs. all-trans-lycopene (HV 0.31 ± 0.01, AD 0.45 ± 0.07, p = 0.03) as well as 13-cis- vs. all-trans-retinoic acid (HV 1.4 ± 0.2, AD 2.6 ± 0.6, p = 0.03) were increased in the plasma of AD-patients indicating an AD-specific 13-cis-isomerisation. A positive correlation with SCORAD was calculated with 13-cis- vs. all-trans-lycopene ratio (r = 0.40, p = 0.01), while a negative correlation was observed with zeaxanthin plasma levels (r = -0.42, p = 0.01). Based on our results, we conclude that in the plasma of AD-patients various carotenoids and retinoids are present at lower concentrations, while the ratio of selected lycopene isomers also differed in the AD-patient group. An increase in plasma isomers of both lycopene and retinoic acid may cause an altered activation of nuclear hormone receptor signaling pathways and thus may be partly responsible for the AD-phenotype.
Project description:The pleiotropic activities of retinoids are mediated by two types of nuclear receptors, the retinoic acid receptors (RARs) and the retinoid X receptors (RXRs). All-trans-retinoic acid (RA) transcriptionally activates RARs, but not RXRs, whereas its natural stereoisomer, 9-cis-RA, is the ligand for RXRs. Here, we demonstrate that 9-cis-RA did not transcriptionally activate RARs, whereas in the presence of all-trans-RA the transactivation of RARs was inhibited in a dose-dependent manner by 9-cis-RA. RAR homodimer complexes were destabilized in vitro in the presence of 9-cis-RA. This suggests that 9-cis-RA may be a natural antagonist of all-trans-RA for binding to RAR complexes. The levels of 9-cis-RA may determine by which pathway the transcription of retinoid-responsive genes is modulated.