Glutamate regulating autophagy-related gene expression in Leydig cells of Xiang pig through T1R1/T1R3-ERK1/2 pathway

【Objective】This paper explored the effect of L-glutamate stimulation on autophagy in Xiang pig Leydig cells，providing a theoretical basis for further research on the effects of taste receptor T1R1/T1R3 on male reproduction under the regulation of rapamycin target protein（mTOR）through autophagy.【Method】Selecting Leydig cells isolated and cultured from healthy 30 days old Congjiang Xiang pigs，different concentrations of L-glutamate were used to stimulate Leydig cells T1R1/T1R3，to investigate the effect of activating T1R1/T1R3 on expression of autophagy-related genes of Leydig cell（TAS1R1，TAS1R3，ERK1，ERK2，mTOR，Beclin 1 and MAP1LC3B）.【Result】 The proliferation of Leydig cells from Congjiang Xiang pig was the fastest during 72-96 h of cultivation，and reached a logarithmic growth phase at 96 h of cultivation. 10 mmol/L was the effective concentration of L-glutamate to stimulate the taste receptor T1R1/T1R3 in Leydig cells. After stimulation with 10 mmol/L L-glutamate，the relative expression levels of mTOR，ERK1，and ERK2 genes in Leydig cells were extremely significantly higher than those in the control group（P＜0.01，the same below）. The relative expression levels of Beclin 1 gene were extremely significantly lower than those in the control group. And the relative expression levels of MAP1LC3B gene were significantly lower than those in the control group（P＜0.05， the same below）. The relative expression levels of ERK1/2 and p-S6K1 proteins were significantly higher than those in the control group， while the relative expression levels of Beclin 1 protein were significantly lower than those in the control group. The staining results of dansylcadaverine（MDC）showed that after stimulation with 10 mmol/L L-glutamate，the fluorescence intensity of acidic autophagic vesicles in Leydig cells was significantly lower than that of the control group，indicating that intracellular autophagy was inhibited.【Conclusion】10 mmol/L of L-glutamate can activate the Leydig cells T1R1/ T1R3 of Congjiang Xiang pig，and the expression of autophagy related genes TAS1R1，TAS1R3，ERK1，ERK2 and mTOR，as well as related proteins T1R1，T1R3，ERK1/2，p-S6K1，and p-mTOR increase. However，the expression of Beclin 1 and MAP1LC3B genes and Beclin 1 protein decrease. Therefore，it is speculated that glutamate can participate in the negative regulation of male reproductive autophagy by activating taste receptor T1R1/T1R3.