Mechanism Of Low-energy Sweeteners On The Sweetness Pathway Of STC-1 Cells

ObjectiveThe effect of low-energy sweetener(LCS)on the mechanism of cell sweetness pathway was studied by using mouse intestinal endocrine STC-1 cells.By detecting the effects of LCS on gastrointestinal hormones(CCK,GIP,GLP-1),glucose transporters(SGLT-1,GLUT-2),functional factors of sweet signal(Ca2+?ATP)and key factors of sweet pathway(T1R2,T1R3,G?,PLC?2,TRPM5)in STC-1 cells,this study revealed the mechanism of LCS affecting sweet pathway and the role of LCS in regulating energy metabolism.This study provides more scientific basis for the mechanism of LCS affecting sweet signal pathway and the relationship between LCS and sweet perception and metabolic diseases.MethodsAccording to the sweetness ratio,sucralose group,acesulfame group,aspartame group,blank control group and sucrose group were set up respectively.MTT assay was used to detect the effect of sweeteners on the survival rate of STC-1 cells.Enzyme linked immunosorbent assay(ELISA)kit was used to determine the contents of GIP,GLP-1 and CCK secreted by STC-1 cells.ATP detection kit and Ca2+detection kit were used to detect extracellular ATP and intracellular Ca2+ respectively.Real time quantitative RT-PCR was used to determine the mRNA expression of glucose transporters SGLT-1,GLUT-2 and sweet pathway factors T1R2,T1R3,G?,PLCP2 and TRPM5.Results(1)Effects of LCS on gastrointestinal hormones and glucose transporters in STC-1 cells:compared with the blank control group,LCS had no significant difference in the secretion of CCK and GIP in all dose groups of sucralose,acesulfame and aspartame(P>0.05).Low,medium and high dose of acesulfame and high dose of aspartame could significantly increase the secretion of GLP-1(P<0.05).There was no significant difference in GLP-1 secretion between each dose of LCS group and sucrose group(P>0.05).High doses of sucralose,acesulfame and aspartame significantly up-regulated the expression of SGLT1 and GLUT-2(P<0.05).Compared with the corresponding inhibitor group,there was no significant difference in the levels of CCK and GIP between the LCS group and the inhibitor group(P>0.05).The level of GLP-1 in medium and high dose of acesulfame group and high dose of aspartame group was significantly higher than that in inhibitor group(P<0.05).The expression level of SGLT-1 in sucrose,sucralose,acesulfame and aspartame groups was significantly higher than that in the corresponding inhibitor group(P<0.05).The expression of GLUT-2 in high-dose sucrose group and equal sweetness LCS group was significantly higher than that in inhibitor group(P<0.05).(2)Effects of LCS on extracellular ATP and intracellular Ca2+in STC-1 cells:compared with the blank control group,the secretion of ATP and Ca2+ in high-dose Acesulfame and aspartame group was significantly higher than that in the control group(P<0.05).Compared with the sucrose group with equal sweetness,the ATP secretion of medium and high dose sucralose group and medium dose aspartame group was significantly lower than that of sucrose group(P<0.05).There was no significant difference in ATP secretion level between the three LCS groups with low dose and the sucrose group with equal sweetness(P>0.05).There was no significant difference in Ca2+ level between LCS group and sucrose group with equal sweetness(P>0.05).Compared with the corresponding inhibitor group,the ATP secretion of high-dose Acesulfame and aspartame group was significantly higher than that of the corresponding inhibitor group(P<0.05).The Ca2+ level of medium and high dose of acesulfame group and high dose of aspartame group was significantly higher than that of the corresponding inhibitor group(P<0.05).There was no significant difference in ATP and Ca2+ secretion between each dose of sUcralose and the inhibitor group(P>0.05).(3)Effect of LCS on sweet signal transduction molecules in STC-1 cells:compared with the blank control group,high-dose sucrose,acesulfame and aspartame could significantly up regulate the mRNA expression of T1R2,T1R3,G?,PLCP2 and TRPM5(P<0.05).There was no statistical difference between PLC?2 and Ga expression of sucralose group and control group(P>0.05).Compared with the sucrose group with equal sweetness,the expression of T1R2,T1R3 and Ga in sucralose,acesulfame and aspartame group was significantly lower than 200 mM sucrose(P<0.05),and the expression of PLC?2 in sucralose and aspartame group was significantly lower than 200 mM sucrose(P<0.05).There was no significant difference in the expression of TRPM5 mRNA between the three LCS groups and 200 mM sucrose group(P>0.05).Compared with the corresponding inhibitor group,the gene expression of T1R2,T1R3,G?,PLC?2 and TRPM5 in the high-dose sucrose,acesulfame and aspartame group was significantly higher than that in the corresponding inhibitor group(P<0.05)and the PLC?2 and Ga expression of sucralose was not significantly different from that in the corresponding inhibitor group(P<0.05).ConclusionThis study proved that a certain dose of LCS promoted the secretion of GLP-1 in STC-1 cells,and found that it could up regulate the expression of glucose transporters SGLT-1 and GLUT-2.200 mM sucrose and equal sweetness acesulfame and aspartame can induce the release of intracellular Ca2+,so as to release ATP outside the cell.LCS can increase the expression of cell sweet receptors T1R2,T1R3 and sweet signal molecules G?,PLC?2,TRPM5,the expression of T1R2,T1R3,PLC?2 and Ga is significantly different from that of sucrose,which reveals that sucrose and LCS may have different sweet signal transduction pathways on STC-1 cells.In conclusion,LCS can stimulate the gene expression of sweetness receptor in the intestine,releases gastrointestinal hormones into the intestine through a series of cascade reactions,and regulates the transport efficiency of glucose transporters,so as to adjust the energy transport and metabolic balance of the body.