Regulation And Molecular Mechanism Of Serotonin On Lipid Synthesis In Goat Mammary Epithelial Cells

Lipid is one of the important nutrients in goat milk.Its content and composition determine the special flavor and nutritional value of goat milk.Mammary epithelial cells are the main site of milk fat synthesis.Serotonin(5-HT),also known as serotonin,is a monoamine.Its receptors(HTRs)mediate various regulatory functions of 5-HT in central nervous system and peripheral system.5-HT has been reported to be involved in lipid metabolism in liver and adipose tissue.In the mammary gland,5-HT has the functions of promoting the development of acinus,regulating the synthesis of milk protein,and maintaining the calcium homeostasis,etc.GMEC can sense and respond to the organism’s functional regulation of the mammary gland through the HTRs on the cell membrane.Through the construction of competitive endogenous RNA(Ce RNA)regulatory network,the team found that 5-HT was involved in the regulation of calcium metabolism,lipid metabolism,immune response and other biological processes in dairy goats during the early lactation period.However,the regulation of 5-HT on lipid metabolism of goat mammary gland has not been reported.The elucidation of the effect and molecular mechanism of 5-HT on lipid metabolism of goat mammary epithelial cells is of great significance and value in regulating the lipid content and composition of goat milk,promoting the physiological health of goat mammary gland and improving the quality of goat milk.In this study,the 5-HT treatment was used to construct a model of cultured mammary epithelial cells in vitro by analyzing the correlation between 5-HT levels and milk fat content and milk fatty acid composition in dairy goats.By detecting the lipid content in GMEC,the expression of genes related to lipid synthesis and protein expression,and verifying the signal transduction pathway and protein-protein interaction effects shown by bioinformatics analysis,the regulation of 5-HT on cellular lipid synthesis was clarified,and the key receptors for regulating goat mammary lipid synthesis by 5-HT and their molecular mechanisms were screened and elucidated.The specific research results are as follows:1.Analysis of correlation between 5-HT concentration and milk composition,milk fatty acid composition in dairy goats at early lactation periodBlood and milk samples were collected from 30 early lactating dairy goats.The correlation among 5-HT concentration,calcium concentration,PTHr P concentration,milk component composition and milk fatty acid composition in the samples was analyzed.There was a positive correlation between serum 5-HT concentration in dairy goats and milk5-HT concentration(R= 0.431,P < 0.05),milk calcium concentration(R= 0.442,P < 0.05),milk PTHr P concentration(R= 0.421,P < 0.05).There was a positive correlation between milk 5-HT concentration and milk calcium concentration(R= 0.442,P < 0.05).There was a negative correlation between serum 5-HT and fatty acid contents of C17: 0(r =-0.438,P <0.05),C17: 1(R =-0.497,P < 0.05)and C18: 2(R =-0.516,P <0.05)in milk.There was a negative correlation between milk 5-HT concentration and the content of C10: 0(R =-0.479,P < 0.05),C17: 0(R =-0.433,P < 0.05)and C18: 0(R =-0.441,P <0.05)fatty acids in milk.2.Regulation of 5-HT on lipid synthesis in goat mammary gland epithelial cellsIn order to explore the regulatory effect and molecular pathway of 5-HT on lipid synthesis in milk goat mammary gland,a model of cultured 5-HT treated goat mammary epithelial cell was constructed.The results of real-time analysis of cell activity showed that GMEC cell activity increased with the increase of 5-HT treatment concentrations(0,6.25,12.5,25,50,100 and 150 μM).The results of CCK-8 test showed that the cell proliferative activity was significantly increased at the 5-HT treatment concentrations of 150 μM and200 μM(P < 0.05).Treatment of 100 μM 5-HT increased the level of calcium and reduced the lipid droplet accumulation,triglyceride(TAG)content,cholesterol(TC)content,and C12:0 and C14:0 fatty acid contents in cells(P < 0.05).In addition,treatment of 100 μM5-HT also reduced the m RNA expressions of lipid synthesis related genes such as ACC,FASN,SREBP1 and SCD1,and inhibited the expression of SREBP1 c,phosphorylated m TOR,and phosphorylated S6 K proteins in cells.The activities of CAMKKβ-AMPK signaling pathway,which negatively regulates cellular lipid synthesis,were detected.It was found that 5-HT treatment significantly promoted the activation of CAMKKβ and AMPK.Further experiments confirmed that Compound C(specific AMPK inhibitor)and STO-609(specific CAMKKβ inhibitor)could eliminate the negative regulatory effects of 5-HT on GMEC lipid synthesis-related indicators.These results indicated that 5-HT played a negative regulatory role in the lipid synthesis of GMEC by inducing and activating the CAMKKβ-AMPK signaling pathway.3.Molecular mechanism of negative regulation of GMEC lipid synthesis by 5-HT through HTR2AIn order to further explore the key receptors of 5-HT negatively regulating the lipid synthesis function of GMEC,the expression profiles of 14 serotonin receptors(HTRs)in the mammary gland of dairy goats at different lactation periods(early lactation,peak lactation,mid lactation,and late lactation)were detected.It was found that 13 receptors except HTR5 B were expressed in the mammary gland tissues of dairy goats,among which7 receptors of type HTR1 A,1D,2A,3A,4,and 6 were expressed in four lactation periods;The expression level of HTR2 A in mammary gland tissue in early lactation was significantly lower than that in the other three lactation periods.In GMEC,5-HT treatment significantly increased the m RNA expression of HTR2A(P<0.05),while PCPA(5-HT synthetase inhibitor)treatment significantly decreased the m RNA expression of HTR2A(P< 0.05).The DNA fragment of milk goat HTR2 A gene CDS region was cloned and analyzed.The protein interaction network prediction results show that there is protein interaction effect between goat HTR2 A,Calmodulin(CaM),CAMKKβ and AMPK protein.Using si RNA to interfere HTR2 A significantly up-regulate the expression of lipid synthesis related genes ACC,FASN and SREBP1 and the expression of calcium metabolism related genes PMCA1 and SPCA1,and inhibitted the expression of HTR2 A and the activity of CAMKKβ and AMPK in cells(P < 0.05);SAR(a specific antagonist of HTR2A)treatment of GMEC significantly increased cell lipid droplet accumulation,TAG content and TC content,and rescused the inhibitory effect of 5-HT on cell TAG and TC content(P < 0.05).The over-expression plasmid vector of goat HTR2 A gene was constructed and transfected into GMEC.It was found that the over-expression of HTR2 A down-regulated the m RNA expression of lipid synthesis related genes FASN and ELOVL6 and up-regulated the activity of CAMKKβ and AMPK(P < 0.05).Overexpression of HTR2 A increased the level of calcium ions in cells and decreased the accumulation of lipid droplets and the contents of TAG and TC in cells.Compound C and STO-609 blocked the activation of CAMKKβ-AMPK by overexpression of HTR2A(P <0.05).The results indicate that 5-HT exerts a negative regulatory effect on GMEC lipid synthesis through the activation of CAMKKβ-AMPK signaling pathway via HTR2 A,and si RNA or specific antagonist targeting HTR2 A can promote lipid synthesis in GMEC.4.Study on molecular mechanism of interaction between HTR2 A and CaM protein in GMECIn order to further explore the regulatory effect protein of HTR2 A and its mediating5-HT negative regulatory function on GMEC lipid synthesis,a DNA fragment in CDS region of CaM gene of dairy goat was cloned.The results of calmodulin target database software analysis showed that there were two CaM protein binding domains on goat HTR2 A protein,located at amino acids 185–200 and 377–393 respectively,and the phosphorylation activities at S188 and S391 sites in the binding domain were significant.Plasmid vectors p-Bi FC-VN155-HTR2 A and p-Bi FC-VC155-CaM were constructed for bimolecular fluorescence complementation assay.Fluorescence expression was detected after co-transfection with GMEC,indicating that there was an interaction between goat HTR2 A protein and CaM protein.The tag protein fusion expression vectors p EF-Neo-Flag-HTR2 A and p EF-Neo-Myc-CaM for Co-IP were constructed and co-transfected with GMEC.The results showed that the HTR2 A protein fused with Flag tag and the CaM protein fused with Myc tag were successfully expressed,and there was an interaction between the two.A tag protein fusion expression vector p EF-Neo-Flag-HTR2A-S188 E and p EF-Neo-Flag-HTR2A-S391 E which mutate serine(S)at positions S188 and S391(simulating HTR2 A continuous phosphorylation activation)into glutamic acid(E)are constructed by using segmental cloning and fusion PCR.After co-transfecting GMEC with p EF-Neo-Myc-CaM vector respectively,Co-IP found that the interaction between HTR2 A and CaM protein was lower after the mutation at S188 E and S391 E sites.After the calcium in cells was chelated with EGTA,5-HT was unable to exert the negative regulatory effect on GMEC lipid synthesis.These results indicated that there was an interaction between goat HTR2 A and CaM protein,and the S188 and S391 sites in the CaM binding domain on HTR2 A were the key receptor activation sites for regulating the interaction.Calcium in the cells was involved in the negative regulation effect of 5-HT on GMEC lipid synthesis.In summary,this study firstly revealed the negative correlation between circulation5-HT concentration and milk fatty acid content in dairy goats at the early lactation period.Taking goat mammary epithelial cells as the research object,it was found that 5-HT negatively regulated the lipid synthesis of goat mammary epithelial cells through the activation of the CaMKKβ-AMPK signal pathway via HTR2 A receptor,and determined that there was an interaction between HTR2 A and CaM protein.The S188 and S391 sites of HTR2 A receptor were the key receptor activation sites.The results of this study have provided information for elucidating the molecular regulatory mechanism of 5-HT on lipid metabolism in goat mammary epithelial cells.