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Mechanisms of action of compounds that enhance storage lipid accumulation in Daphnia magna Rita Gomes Jordão, Bruno Campos, Benjamin Piña, Romà Tauler, Amadeu M.V.M. Soares, and Carlos Barata Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.6b04768 • Publication Date (Web): 10 Nov 2016 Downloaded from http://pubs.acs.org on November 21, 2016
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TITLE PAGE:
Mechanisms of action of compounds that enhance storage lipid accumulation in Daphnia magna.
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Rita Jordão1,2, Bruno Campos1, Benjamín Piña 1, Romà Tauler1, Amadeu M.V.M. Soares 2 and Carlos Barata1* 1
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Department of Environmental Chemistry, Institute of Environmental Assessment and Water Research (IDAEA), Spanish Research Council (IDAEA, CSIC), Jordi Girona 18, 08034 Barcelona, Spain 2
Centre for Environmental and Marine studies (CESAM), Department of Biology, University of Aveiro, Campus Universitário de Santiago, 3810-193 Aveiro , Portugal.
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*Address correspondence to Carlos Barata, Institute of Environmental Assessment
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and Water Research (IDAEA-CSIC), Jordi Girona 18, 08034 Barcelona, Spain.
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Telephone: ± 34-93-4006100. Fax: ± 34-93-2045904. E-mail:
[email protected] 18 19
Funding: This work was funded by the Spanish Ministry of Science and Innovation
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project (CTM2014-51985-R) and by the Advance grant of the European Research
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Foundation ERC-2012-AdG-320737. Funders had no role in study design, data collection
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and analysis, decision to publish, or preparation of the manuscript.
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Competing interests: The authors have declared that no competing interests exist.
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ABSTRACT
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Accumulation of storage lipids in the crustacean Daphnia magna can be altered by a
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number of exogenous and endogenous compounds, like 20- hydroxyecdysone (natural
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ligand of the ecdysone receptor, EcR), methyl farnesoate, pyrirproxyfen (agonists of the
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methyl farnesoate receptor, MfR) and tributyltin (agonist of the retinoid X acid receptor,
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RXR). This effect, analogous to the obesogenic disruption in mammals, alters Daphnia's
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growth and reproductive investment. Here we propose that storage lipid accumulation in
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droplets is regulated in Daphnia by the interaction between the nuclear receptor
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heterodimer EcR:RXR and MfR. The model was tested by determining changes in
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storage lipid accumulation and on gene transcription in animals exposed to different
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effectors of RXR, EcR and MfR signaling pathways, either individually or in
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combination. RXR, EcR and MfR agonists increased storage lipid accumulation, whereas
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fenarimol and testosterone (reported inhibitors of ecdysteroid synthesis and an EcR
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antagonist, respectively) decreased it. Joint effects of mixtures with fenarimol,
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testosterone and ecdysone were antagonistic, mixtures of juvenoids showed additive
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effects following a concentration addition model, and combinations of tributyltin with
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juvenoids resulted in greater than additive effects. Co-exposures of ecdysone with
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juvenoids resulted in de-regulation of ecdysone- and farnesoid-regulated genes,
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accordingly with the observed changes in lipid accumulation These results indicate the
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requirement of ecdysone binding to the EcR:RXR: MfR complex to regulate lipid
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storage, and that an excess of ecdysone disrupts the whole process, probably by
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triggering negative feedback mechanisms.
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Keywords: obesogen, metabolic disruption, nuclear receptor, arthropod, reproduction,
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juvenile receptor 2
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INTRODUCTION
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Recent studies have suggested the involvement of endocrine disrupting chemicals in the
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obesity epidemia occurring in many modern human societies 1. Obesity increases the risk
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of coronary artery diseases, diabetes and related health detrimental effects, such as
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hypertension and lipidemia 1, 2. Many widely used chemicals are known or suspected
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promoters of weight gain at low doses, in an extensive list that includes organotin
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antifouling agents, among others 3. It has been proposed that exposure to these so called
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obesogens in the uterus may lead to obesity later in life 4.
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Obesogenic effects in vertebrates have often been related to the disruption of the
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peroxisome proliferator-activated receptor (PPARγ) signaling pathway. This receptor is a
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master regulator of adipocyte differentiation and lipid metabolism in vertebrates, binding
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to the promoter of target genes and forming an heterodimer with the retinoid X receptor
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(RXR) 3. Although PPAR has not been found outside deuterostomes, a recent study
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showed that the suspected vertebrate obesogen tributyltin (TBT), also disrupts the
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dynamics of neutral lipids' storage in the crustacean Daphnia magna 5. As TBT is the
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only known ligand of RXR in arthropods 6, this increases the scope of the search for
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obesogenic effects to Arthropods and other Protostomata through the interaction with this
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nuclear receptor, which is present in virtually all Metazoans. In Daphnia, TBT impairs
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the transfer of triacylglycerols to eggs and hence promotes their accumulation in lipid
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droplets inside fat cells in post-spawning adult females 5, resulting in a lower fitness for
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offspring and adults. TBT increased mRNA levels of the RXR gene and of several genes
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regulated by the ecdysteroid (EcR) and the methyl farnesoate hormone (MfR) receptors 5.
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These results suggest a genetic interaction between the regulation of lipid storage by RXR
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and other endocrine signaling pathways in D. magna.
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More recently Jordão, et al. 7 found that in addition of TBT, agonists of EcR (20-
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hydroxyecdysone), of RXR (tributyltin), and of MfR (methyl farnesoate, pyriproxyfen),
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increased the accumulation of storage lipids in a concentration-related manner.
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Conversely fenarimol, which is known to deplete the levels of ecdysone in D. magna 8,
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decreased storage lipids. These previous results suggest that the accumulation of storage
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lipids in D. magna is promoted by agonists of the three transcription factors and that anti-
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ecdysteriods inhibited the whole process. There is therefore a need to understand how the
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different transcription factors interact regulating storage lipid dynamics in Daphnia.
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In D. magna, like in other crustacean and arthropods, storage lipid dynamics varied along
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the molt and reproduction cycle, which is regulated by the ecdysteroid and juvenile
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hormone receptor signaling pathways 9. Ecdysone exerts its effects through the interaction
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with the ecdysteroid receptor (EcR), known to heterodimerize with RXR and to bind to
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the promoters of ecdysone-regulated genes (i.e. HR3, Neverland) 10-12. TBT, which is an
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agonist of RXR together with methyl farnesoate and other juvenoids, enhanced the
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ecdysteroid-dependent activation of the EcR: RXR heterodimer 12. The previous study
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provided the first evidence for a ternary receptor complex in Daphnia (MfR, EcR, RXR)
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that would require ecdysteriods to elicit transcriptional responses to the other ligands13.
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Recent findings indicate that MfR in Daphnia is itself a complex of two nuclear proteins
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of the bHLH-PAS family of transcription factors: the methoprene-tolerant coactivator
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proteins (MET), which binds to methyl farnesoate and other juvenoid compounds, and the
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steroid receptor co-activator (SRC) 14, 15. Juvenoids promote expression of hemoglobin 4
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genes, such as Hb2 16, and of male sex determining genes in the latter stages of ovarian
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oocyte maturation 17. In this study we propose a conceptual model in which EcR, RXR,
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and MfR act as a molecular complex to regulate lipid accumulation and other key
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physiological functions through the modulation of the expression of key genes. This
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model is based in our current knowledge of the PPARγ mechanistic mode of action and
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incorporates physiological, life-history, and gene expression data from D. magna
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responses to effectors of the different receptors, both in single exposures and in
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combination mixtures.
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EXPERIMENTAL SECTION
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Studied compounds
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Studied compounds included the juvenile crustacean’s hormone methyl farnesoate (MF,
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CAS 10485-70-8) and the molting hormone 20- hydroxyecdysone (20E, CAS 5289-74-7)
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tributyltin (TBT, CAS 1461-22-9) 11 , the ecdysone synthesis inhibitor fenarimol (FEN,
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CAS 60168-88-9) 8 and the ecdysone receptor antagonist testosterone (T, 58-22-0) 8. All
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the compounds were obtained from Sigma Aldrich (U.S.A/Netherlands) except MF,
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which was supplied by Echelon Bioscience, Utah, U.S.A.
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Experimental animals
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All experiments were performed using the well-characterized single clone F of D. magna
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maintained indefinitely as pure parthenogenetic cultures 18. Individual cultures were
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maintained in 100 ml of ASTM hard synthetic water at high food ration levels (5x105
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cells/ml of Chlorella vulgaris), as described in Barata and Baird 18.
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Experimental procedures
; the juvenoid pesticide pyriproxyfen (PP, CAS 95737-68-1); the RXR agonist
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Experiments follow previous procedures
. Briefly experiments were initiated with
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newborn neonates
