Severe Calorie Restriction Induces Gut Microbiota-Dependent Intestinal Stem Cell Dysfunction

Abstract Objectives Anorexia nervosa (AN) is a life-threatening illness, primarily characterized by extreme calorie restriction (CR). Given the substantial undernourishment in patients with AN, it is essential to understand the effects of severe CR on the small intestinal epithelium which is the primary tissue responsible for nutrient absorption and is therefore critical for the refeeding process. While we have previously reported that patients with AN harbor an altered intestinal microbiota, it remains unknown whether an AN-associated gut microbial community, induced by a nutrient-deprived environment, impacts intestinal epithelial physiology. Methods Conventionally-raised (CONVR) and germ-free (GF) mice were individually housed and placed on a 60% CR (60CR) diet for 9 days. CONVR mice were housed in standard specific-pathogen free conditions while GF mice were bred and housed in sterile isolators to occlude exposure to microorganisms. Mice were injected intraperitoneally with 4 mg/kg 5-ethynyl-2’deoxyuridine (EdU) 90 minutes before necropsy to mark proliferating cells. Intestinal tissue was collected for histology and intestinal crypts were isolated for ex vivo organoid culture. Results CONVR and GF mice on a 60CR diet lost significant body weight (28%±1 and 24%±1, respectively). 60CR CONVR and GF mice both displayed a significant increase in ISCs (+1.27 ± 0.02-fold and + 1.15 ± 0.05-fold, respectively) yet a significant decrease in proliferating cells (1.7 ± 0.2-fold and 1.7 ± 0.1-fold, respectively) compared to ad libitum controls. Interestingly, ex vivo organoids derived from crypts of CONVR 60CR mice were significantly smaller compared to organoids yielded from control crypts, but, strikingly, no differences in organoid size were observed between GF crypts from either diet group. Conclusions While ISC from 60CR CONVR and GF mice exhibited similar features, 60CR GF ISC function was maintained as indicated by the similar organoid growth relative to GF ad libitum controls. These data suggest that severe CR induces ISC dysfunction resulting in impaired de novo generation of digestive epithelial mass, and that these changes are microbiota-dependent. Funding Sources National Institutes of Health, North Carolina Translational and Clinical Sciences Institute, NCSU College of Veterinary Medicine, UNC Center for Gastrointestinal Biology and Disease.