From samples obtained from Sham-operated (SHAM), ovariectomized (OVX), and ovariectomized rats
From samples obtained from Sham-operated (SHAM), ovariectomized (OVX), and ovariectomized rats treated with NMP **P < 0.01 vs. SHAM; P < 0.01 vs. OVXwere treated with adipogenic medium (MDI) then treated with Tariquidar chemical information different doses of NMP (5 and 10 mM) or without NMP for 10 days. To determine the effect of NMP on adipogenic differentiation, the accumulated intracellular lipid was stained with Oil Red O dye and quantified. Oil Red O staining performed on day 10 of the treatment shown in Fig. 4b suggests that 3T3-L1 preadipocytes differentiate into mature adipocytes. However, cells co-treated with 5 mM NMP for 10 days showed inhibition of MDI-induced lipid droplet accumulation and 10 mM NMP nearly fully blocked lipid droplet accumulation. As shown in Fig. 4a, a dose-dependent reduction in lipid accumulation was observed in the cells treated with NMP, suggesting that NMP inhibits adipocyte transcription factors during adipocyte differentiation. To further evaluate the effect of NMP on adipocyte formation, the expression level of PPAR was investigated. The mRNA levels of PPAR were significantly lower in 10-mM NMP-treated 3T3-L1 pre-adipocytes compared to 5-mM NMP or MDI alone treated cells (Fig. 4c).Effect of NMP PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/27906190 on the binding ability of BET bromodomainsTo assess the possibility of NMP acting as a bromodomain inhibitor particularly for BRD2 and BRD4, both associated with adipogenesis, we used an AlphaScreen assay format to determine the effect of NMP on recombinant human BET bromodomains. Our results suggest that NMP inhibits the binding of acetyl-lysines to BRD2-BD1BD2, a member of the “BET” subfamily of bromodomain-containing proteins [29]. AlphaScreen dose response experiments performed against BRD2 and BRD4 as a whole and BRD2 BD1 and BRD2 BD2 component independently gave raise to millimolar half-maximum inhibitory concentration (IC50) values for NMP (Fig. 5). Affinity to BRD2 and BRD4 as a whole are 3.3 and 3.4 mM, respectively, and therefore very similar.Discussion In an ovariectomized rat model, mimicking menopause, we recently showed that N-methyl pyrrolidone (NMP) prevents bone loss and improves both mass and quality ofGjoksi et al. Clinical Epigenetics (2016) 8:Page 5 ofFig. 3 Influence of NMP on adipocyte differentiation from hMCS. a Human bone marrow-derived mesenchymal cells (MSCs) were induced into adipogenesis by differentiation medium (MDI) containing IBMX, dexamethasone, indomethacin, and insulin as described in the “Methods” section. After 14 days, the lipid droplets were stained with Oil Red O. For the quantitative analysis, Oil Red O staining was extracted with isopropyl alcohol and the absorbance at 490 nm was measured. Data are presented as mean ?SD. b Representative micrographs showing the cell monolayers stained with Oil Red O. As seen from the staining and the quantification in cells treated with differentiation medium alone, there is much more lipid accumulation than in cells treated with both differentiation medium and NMP (5 and 10 mM). However, no significant difference was observed between the two different doses of NMP. c Real-time PCR analysis for PPAR mRNA expression. The levels of PPAR were normalized to the levels of PPIA, suitable reference genes during adipogenic differentiation of MSCs. Result is presented as CT. *P < 0.01 MDI-treated cells vs. control; P < 0.05 NMP + MDI-treated cells vs. MDI-treated cellsFig. 4 Influence of NMP on T3-L1 adipogenic maturation. a Two-day post-confluent 3T3-L1 pre-adipocytes.
