Glutamine Regulates Cell Growth and Casein Synthesis through the CYTHs/ARFGAP1-Arf1-mTORC1 Pathway in Bovine Mammary Epithelial Cells

Glutamine (Gln) is a critical nutrient required by neonatal mammals for intestinal growth, especially for newborn piglets. However, the mechanisms underlying the role of Gln in porcine intestinal epithelium development are not fully understood. The objective of the current study was to explore the possible signaling pathway involved in the promotion of porcine intestinal epithelial cell (IPEC-J2) proliferation by Gln. The results showed that 1 mM Gln promoted IPEC-J2 cell proliferation, and tandem mass tag proteomics revealed 973 differentially expressed proteins in Gln-treated IPEC-J2 cells, 824 of which were upregulated and 149 of which were downregulated. Moreover, gene set enrichment analysis indicated that the Wnt signaling pathway is activated by Gln treatment. Western blotting analysis further confirmed that Gln activated the Wnt/β-catenin signaling pathway. In addition, Gln increased not only cytosolic β-catenin but also nuclear β-catenin protein expression. LF3 (a β-catenin/TCF4 interaction inhibitor) assay and β-catenin knockdown demonstrated that Gln-mediated promotion of Wnt/β-catenin signaling and cell proliferation were blocked. Furthermore, the inhibition of TCF4 expression suppressed Gln-induced cell proliferation. These findings further confirmed that Wnt/β-catenin signaling is involved in the promotion of IPEC-J2 cell proliferation by Gln. Collectively, these findings demonstrated that Gln positively regulated IPEC-J2 cell proliferation through the Wnt/β-catenin pathway. These data greatly enhance the current understanding of the mechanism by which Gln regulates intestinal development.

Section: ■ Discussionsupporting

confidence: 55%

Glutamine Promotes Porcine Intestinal Epithelial Cell Proliferation through the Wnt/β-Catenin Pathway

Fang,

Lu,

Cheng

et al. 2024

“…This finding was consistent with those of studies on functional cells, such as bovine mammary epithelial cells (BMECs). 40 Therefore, Arf1 was identified as an important positive regulator involved in Gln sensing by mTORC1 and a promoter of IPEC-J2 cell proliferation. The present study elucidates the role of Arf1 in mediating Gln's effects on mTORC1 activation and IPEC-J2 cell proliferation.…”

Section: ■ Resultsmentioning

confidence: 99%

“…This result suggested that Arf1 was required for Gln-induced mTORC1 activation, which promoted cell proliferation in IPEC-J2 cells. This finding was consistent with those of studies on functional cells, such as bovine mammary epithelial cells (BMECs) . Therefore, Arf1 was identified as an important positive regulator involved in Gln sensing by mTORC1 and a promoter of IPEC-J2 cell proliferation.…”

Section: Discussionmentioning

confidence: 99%

Extracellular Glutamine Promotes Intestinal Porcine Epithelial Cell Proliferation via Arf1-mTORC1 Pathway Independently of Rag GTPases

Yan,

Fang,

et al. 2023

Glutamine (Gln) is the major energy source of intestinal porcine epithelial cells (IPEC-J2 cells) and plays a critical role in the nutritional physiological function of the intestine. However, the underlying mechanism requires further investigation. Here, the Gln-sensing pathway in IPEC-J2 cells was investigated. The results showed that Gln increased the cell proliferation. Subsequently, an analysis of the phosphorylated proteome revealed that Gln markedly upregulated ribosomal protein S6 (RPS6) phosphorylation at serine 235/236, suggesting that Gln activated the mTORC1 pathway. mTOR inhibition revealed that Gln promotes cell proliferation through the mTORC1 pathway. Similarly, blocking ADP-ribosylation factor 1 (Arf1) activity indicated that Gln-induced mTORC1 activation promoted cell proliferation in an Arf1-dependent manner. Additionally, the RagA/B pathway did not participate in Gln-induced mTORC1 activation. Collectively, these findings suggest that Gln-induced mTORC1 activation promotes IPEC-J2 cell proliferation via Arf1, not Rag GTPases. These results broaden our understanding of functional-cell-sensing amino acids, particularly Gln, that are regulated by mTORC1.

“…20 In addition, a recent report showed that Arf1, a small GTPase transmitting glutamine signal to mTORC1, can regulate β-casein synthesis through mTORC1 signaling. 21 A previous report revealed that heat stress stimulation promoted the expression of CASTOR1 in the lactating mammary gland of dairy cows. 22 As a crucial upstream regulator of mTORC1, the specific roles of CASTOR1 in heatstress-induced apoptosis are still obscure.…”

Section: ■ Introductionmentioning

confidence: 97%

“…The mTORC1 signaling has a close connection to cellular apoptotic events, and many mTORC1 signaling components, like Akt, AMPK, and GS3Kβ, also exert important roles in regulating apoptosis . In addition, a recent report showed that Arf1, a small GTPase transmitting glutamine signal to mTORC1, can regulate β-casein synthesis through mTORC1 signaling . A previous report revealed that heat stress stimulation promoted the expression of CASTOR1 in the lactating mammary gland of dairy cows .…”

Section: Introductionmentioning

confidence: 99%

Downregulation of CASTOR1 Inhibits Heat-Stress-Induced Apoptosis and Promotes Casein and Lipid Synthesis in Mammary Epithelial Cells

Gai

Wang

et al. 2022

Heat stress is one of the most important factors limiting the milk yields of dairy animals. This decline can be attributed to the heat-stress-induced apoptosis of mammary epithelial cells (MECs). The cytosolic arginine sensor for mTORC1 subunit 1 (CASTOR1) is a crucial upstream regulator of the mechanistic target of rapamycin complex 1 (mTORC1) signaling, which has close connections with apoptosis. However, the specific roles of CASTOR1 in regulating the apoptosis and lactation of MECs are still obscure. In the present study, we found that heat stress promotes apoptosis and CASTOR1’s expression in HC11 cells. Downregulation of CASTOR1 inhibits heat-stress-induced apoptosis through a ROS-independent pathway. In addition, silencing of CASTOR1 promotes cell proliferation, cell cycle progression, and milk component synthesis, and overexpressing of CASTOR1 reverses these observations. Furthermore, we found that silencing of CASTOR1 contributes to the nuclear transport of SREBP1 and promotes lipid synthesis. This study demonstrates the pivotal roles of CASTOR1 in heat-stress-induced apoptosis and milk component synthesis in MECs.