ECTS Abstracts (2015) 1 P217

Estradiol modulates and recovers osteocyte metabolic/lipid profiles after ovariectomy in acute and long-term in vivo hormone replacement therapeutics

Ana M Silva1,2, Ana C Moreira3, Maria S Santos1,2, Juliana R Dias4,5, Nuno Alves4,5, Romeu A Videira6, Rui A Carvalho1,2 & Vilma A Sardão1

1CNC - Center for Neuroscience and Cell Biology, Coimbra, Portugal; 2Department of Life Sciences, University of Coimbra, Coimbra, Portugal; 3IBMC.INEB, University of Porto, Porto, Portugal; 4CDRSP - Centre for Rapid and Sustainable Product Development, Marinha Grande, Portugal; 5IPL – Instituto Politécnico de Leiria, Leiria, Portugal; 6CQ-VR – Chemistry Centre – Vila Real, University of Trás-os-Montes e Alto Douro, Vila Real, Portugal.

For the first time, we assessed the metabolic and lipid profiles of osteocytes ex vivo. During menopause, the appearance of an osteoporotic condition can be associated with an overall metabolic decline in bone cells, and we hypothesised that it is mainly attributed to osteocyte metabolic and lipid changes, which are attenuated after increasing blood oestradiol (E2) levels. To test this, we considered control and ovariectomised (OVX) female rats in order to compare metabolic/lipid profiles of bone-embedded osteocytes, in the presence or absence of E2. Animal groups (used accordingly with FELASA approved procedures) were: a) Controls SHAM, CTL; b) ovariectomised animals, OVX; and c) OVX+E2 (single bolus injection 30 μg/Kg, 24 hours prior sacrifice for the acute study. For the sub-chronic study, rats were implanted with 0.5mg E2 slow release pellets for 21 days. 24 hours prior sacrifice, animals were I.P. injected with deuterated water for metabolic fluxes analyses. Left and right femurs and tibia were surgically removed and freeze-clamped or preserved for DXA and μCT analysis. Extracted metabolites from those cells as well as total lipids were analysed by 1H nuclear magnetic resonance (NMR) spectroscopy and 2H NMR for de novo lipogenesis analyses. Total lipids were extracted, quantified and analysed by HPLC-MS and fatty acids analysed by GC-MS. Ovariectomy clearly changed lipid profile inducing significant changes in both diacyl- and choline-plasmalogens content (comparatively with SHAM and OVX+E2 groups). Also, an increase of the relative proportion of long chain fatty acids was observed in the OVX group, being attenuated by 24h-treatment with E2. Total lipids analysis revealed that E2 was able to recover the CTL profile in OVX+E2, and decrease de novo synthesis of lipids after 21 days treatment. In terms of metabolites profile, the OVX group presented a slight decrease of lactate/alanine ratio, although osteocytes were forced to produce high levels of lactate after E2 treatment, increasing this ratio. Our results show a change in the process of lipid remodelling as a result of ovaries removal, with E2 partially compensating the alteration in long chain fatty acids. High levels of PC-Plasmalogens measured in OVX animals may be related to a signalling/protective action against the damaging effects of oxidative stress triggered by the E2 decline. Acute E2 administration in OVX animals induced osteocytes to increase aerobic glycolysis in an attempt to compensate for the metabolic deficit associated with ovaries removal. Long-term therapeutics supported the effects of the acute study, indicative of the high impact of this hormone in osteocytes metabolism.

Disclosure: The authors declared no competing interests. This Work was supported by PTDC/AGR-ALI/108326/2008 and PEst-C/SAU/LA0001/2013-2014 grants from Fundação para a Ciência e Tecnologia (FCT), co-funded by FEDER/Compete/National Funds. FCT PhD and Post-doc fellowships (SFRH/BD/33892/2009, SFRH/BD/76086/2011 and SFRH/BPD/31549/2006, respectively). The work was also supported by QREN project #4832 ”Stemcell-based platforms for Regenerative and Therapeutic Medicine”, reference CENTRO-07-ST24-FEDER-002008.

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