Study On The Mechanism Underlying The Potentiation Of 17-? Estradiol On The Response To Sweet Tastants In NCI-H716 Cells

In order to provide more biological basis for the resarch on E2 affecting sweetness perception capacity,feeding behavior and obesity and so on,the human L enteroendocrine cell line NCI-H716 was used as a research tool,which could sense sweet tastants.The present study probed to reveal the mechanism underlying the potentiation of 17-? estradiol on the sweetness signal transduction in NCI-H716 cells via the cell functional level and the molecular level.The research outline was below:NCI-H716 cells were stimulated with different concentrations of E2(1,10,50 and 100 nM)and(or)sweeteners(sucrose,AK),and then the effects of extracellular ATP and intracellular Ca2+ release were detected by ATP test kit and calcium flow detection technology respectively.Besides,the gene expression of the key sweet signaling molecules(T1R2/T1R3,Ga,PLCP2 and TRPM5)were tested by semiquantitative analysis qRT-PCR.Moreover,the sweet signaling proteins(T1R3,PLC?2 and TRPM5)were detected by Western blotting analysis.The main findings were as follows:As for the cell functional level,the secretion of extracellular ATP and the release of intracellular Ca2+ were increased when the NCI-H716 cells were stimulated by sucrose alone.However,AK incubation alone only increased the secretion of extracellular ATP but not the release of intracellular Ca2+.Further,a long term(12 h)or short term(5 min)pretreatment with certain concentrations of E2 was able to promote not only the secretion of extracellular ATP induced by sucrose or AK,but also the sucrose-induced intracellular Ca2+ release.These data suggest that E2 can potentiate the response to sweet tastants in NCI-H716 cells,and that there might be differences in sweet signal transduction triggered by sucrose and AK.At the molecular level,NCI-H716 cells were treated with E2 at different concentrations(1,10,50 and 100 nM)for different time periods(3,12,18 and 24 h).The results showed that:the mRNA expressions of sweet signal molecules were upregulated significantly when cells were treated with 10 nM E2 for 12 h.Compared with the sweeteners group,the pretreatment with 10 nM E2 for 12 h promoted the sucrose-induced mRNA expression of T1R2/PLC?2 and the AK-induced mRNA expression of G?/PLC?2/TRPM5 significanty.Moreover,the pretreatment with 10 nM E2 for 12 h promoted the sucrose-induced protein expression of T1R3/PLCP2/TRPM5 significantly,but not the AK-induced protein expression of T1R3/PLC?2/TRPM5.To sum up,E2 could potentiate the sweet signal transduction to increase the capacity of sweet taste perception of NCI-H716 cells by the genomic and the non-genomic mechanisms;and there might be differences in sweet signal transduction triggered by sucrose and AK.