Phospholipase D Mediates Glutamine-Induced mTORC1 Activation to Promote Porcine Intestinal Epithelial Cell Proliferation

Background: The intestinal epithelium is one of the fastest self-renewal tissues in the body, and glutamine plays a crucial role in providing carbon and nitrogen for biosynthesis. In intestinal homeostasis, phosphorylation-mediated signaling networks that cause altered cell proliferation, differentiation, and metabolic regulation have been observed. However, our understanding of how glutamine affects protein phosphorylation in the intestinal epithelium is limited, and identifying the essential signaling pathways involved in regulating intestinal epithelial cell growth is particularly challenging. Objectives: This study aimed to identify the essential proteins and signaling pathways involved in glutamine ' s promotion of porcine intestinal epithelial cell proliferation. Methods: Phosphoproteomics was applied to describe the protein phosphorylation landscape under glutamine treatment. Kinase-substrate enrichment analysis was subjected to predict kinase activity and validated by qRT-PCR and Western blotting. Cell Counting Kit -8, glutamine rescue experiment, chloroquine treatment, and 5- fl uoro-2-indolyl deschlorohalopemide inhibition assay revealed the possible underlying mechanism of glutamine promoting porcine intestinal epithelial cell proliferation. Results: In this study, glutamine starvation was found to signi fi cantly suppress the proliferation of intestinal epithelial cells and change phosphoproteomic pro fi les with 575 downregulated sites and 321 upregulated sites. Interestingly, phosphorylation of eukaryotic initiation factor 4E-binding protein 1 at position Threonine70 was decreased, which is a crucial downstream of the mechanistic target of rapamycin complex 1 (mTORC1) pathway. Further studies showed that glutamine supplementation rescued cell proliferation and mTORC1 activity, dependent on lysosomal function and phospholipase D activation. Conclusion: In conclusion, glutamine activates mTORC1 signaling dependent on phospholipase D and a functional lysosome to promote intestinal epithelial cell proliferation. This discovery provides new insight into regulating the homeostasis of the intestinal epithelium, particularly in pig production.