Cellular turnover is a fundamental biological process that counteracts aging by activating stem cells to replace aging cells. This process is especially critical for maintaining the intestinal epithelium, one of the most rapidly renewing tissues in the body. However, the replacement of aging enterocytes cannot be explained solely by the generation of new cells from intestinal stem cells (ISCs); it also requires the complementary process of physically removing aging enterocytes through shedding. Compared with the well-characterized regulation of ISCs, the mechanisms governing enterocyte shedding remain poorly understood. Here, we identify pileup, a novel age-associated antimicrobial peptide (AMP) in Drosophila, as both a genetic marker and a key regulator of enterocyte shedding. Reactive oxygen species (ROS) generated through age-related mitochondrial dysfunction, a process facilitated by Autophagy-related gene 1 (ATG1) kinase, activate hypoxia-inducible factor 1α, which induces pileup expression specifically in aging enterocytes. Pileup then compromises the membrane of these cells and triggers c-Jun N-terminal kinase (JNK) activation, which we propose promotes their shedding. Notably, pileup has three homologs, and loss of all four genes leads to tumorous cell accumulation, gut barrier dysfunction, microbiome dysbiosis, and premature death. Our findings reveal a previously unrecognized role for AMPs in the turnover of aging host cells, extending their classical function beyond microbial defense. This work provides new mechanistic insights into enterocyte shedding and underscores its essential role in preserving gut homeostasis during aging.
