Dietary Supplementation of Glutamate Alleviates LPS-Induced Intestinal Injury Associated with m⁶A Modification of...

Acute inflammatory challenge can impair intestinal barrier function and bile acid homeostasis in weaned piglets. Glutamate serves as an important energy substrate and functional amino acid for intestinal epithelial cells, yet the epitranscriptomic mechanisms underlying its potential protective effects remain poorly defined. This study investigated whether dietary glutamate supplementation is associated with m⁶A-dependent regulation of SLC10A2 and bile acid signaling in weaned piglets subjected to an acute LPS challenge, with supporting validation in IPEC-J2 cells. LPS challenge (100 μg/kg body weight) impaired ileal morphological structure, activated the TLR4/NF-κB signaling pathway, and aggravated intestinal inflammatory responses in piglets. In contrast, glutamate supplementation was associated with restored ileal mucosal morphology, up-regulated expression of intestinal tight junction proteins, and suppressed TLR4/NF-κB pathway activation. Further metabolomic analysis indicated that LPS stimulation and glutamate intervention altered ileal bile acid metabolism: LPS decreased total bile acid (TBA) content in ileal tissue while increasing TBA accumulation in ileal chyme, whereas glutamate treatment partially reversed this pattern, by elevating ileal tissue TBA and reducing chyme TBA, suggesting a facilitatory effect of glutamate on intestinal bile acid reabsorption. Glutamate supplementation was also associated with increased ileal ASBT (SLC10A2) and FXR (NR1H4) expression. The SLC10A2 transcript exhibited detectable m⁶A modification; glutamate supplementation rescued the LPS-induced downregulation of m⁶A demethylases FTO and ALKBH5, which was associated with reduced global and SLC10A2-specific m⁶A modification. In vitro experiments in IPEC-J2 cells suggested that inhibition of FTO or glutamate dehydrogenase attenuated the effects of glutamate on m⁶A modification and SLC10A2 expression, and that YTHDF2 may contribute to the degradation of hyper-m⁶A-modified SLC10A2 mRNA. Glutamate-derived α-KG may serve as a cofactor supporting demethylase expression and activity after LPS stimulation. Collectively, these findings suggest that glutamate may alleviate LPS-induced intestinal inflammation in weaned piglets through a pathway involving α-KG-dependent maintenance of m⁶A demethylase expression, reduced m⁶A modification of SLC10A2, and preserved ASBT/FXR signaling. This study provides evidence for a nutritional-epitranscriptomic axis that may contribute to intestinal protection under acute inflammatory stress.