Background: Environmental factors, including changes in diet and microbiota, have been identified as key contributors to the development of inflammatory bowel diseases (IBD). The prevalence of IBD is on the rise, particularly in industrialized nations with populations consuming western-style diets rich in fat and protein. While elevated total and animal protein intake have been associated with IBD, their specific impact on IBD remains poorly understood. Branch-chain amino acids found in different protein sources possess bioactive properties and can interact with the mechanistic target of rapamycin (mTOR), a cellular growth regulator that controls autophagy and inflammation. Given the observed mTOR hyperactivation in IBD patients, coupled with its correlation to exacerbated colitis severity in mice, the study of the relationship between dietary protein intake, mTOR activation, autophagy, and intestinal inflammation is warranted.

Aim: To study the influence of an enriched protein diet on mTOR activation, autophagy, and intestinal inflammation during experimental colitis.

Methods: Specific pathogen free C57BL/6 mice were fed an enriched protein diet (EPD; 40% casein) or control diet (CD; 14% casein) for three weeks prior to tissue collection (N=6 per diet) or colitis induction (N=8 per diet). Colitis was induced using dextran sulfate sodium (DSS; 2% w/v) in drinking water. DSS was provided for five days, followed by two days of water recovery before tissue collection. Rapamycin was provided intraperitoneally to a subset of mice as an mTOR inhibitor. mTOR activation (p-mTOR, p-S6) and autophagy proteins (LC3, Beclin-1, Atg7, Atg12-5) were assessed by western blot of colon tissue extracts pre- and post-colitis. Colitis severity was assessed by colonic histologic analysis, intestinal permeability (Ussing chambers), weight loss, and clinical scores. Inflammatory gene transcripts were quantified in colon tissue by Nanostring. 16s rRNA microbiome sequencing by Illumina and metabolomic analysis by liquid chromatography-mass spectrometry were performed.

Results: EPD consumption increased mTOR activation as evidenced by elevated p-mTOR and p-S6 compared to the CD. Autophagy proteins were downregulated in colon tissue of EPD-fed mice before colitis. After DSS, mice fed the EPD experienced greater weight loss, tissue damage, inflammatory gene signaling, intestinal permeability, diarrhea, and stool blood. Inhibition of mTOR with rapamycin restored autophagy proteins and reduced colitis severity. Elevated branch-chain fatty acids were observed in intestinal content of EPD-fed mice.

Conclusions: These findings highlight the potential contribution of protein-enriched diets to mTOR hyperactivation in IBD. Further research should be conducted to understand the impact of protein concentration and sources on inflammation through mTOR to help guide clinical dietary advice.

Pranshu Muppidi

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