Late-Life Short-Term Dietary Restriction Improves Intestinal Stem Cell Function and Alters Intestinal Stem Cell Deoxyribonucleic Acid Methylation in Mice

BACKGROUND: Intestinal regenerative capacity declines with age due to the declining intestinal stem cell (ISC) function. Dietary restriction (DR), the most robust anti-aging intervention to date, has been shown to improve ISC function in young mice. OBJECTIVES: The goal of this study is to evaluate the effect of short-term DR (stDR) on ISC function when implemented late in life. METHODS: Two- and twenty-one-month-old male C57BL/6 mice, fed on National Institutes of Health-31 (NIH-31), an open-source, unpurified, natural ingredient diet, were subjected to ad libitum feeding or stDR (40% DR for 4-mo) with 6 mice/age/treatment. Effects of age and stDR on ISC (leucine-rich repeat-containing G-protein coupled receptor 5) function was evaluated using in vitro enteroid-forming assay followed by ribonucleic acid sequencing (RNA-seq) analysis (using strand next-generation sequencing analysis software) and oxidative whole-genome bisulfite sequencing for deoxyribonucleic acid (DNA) methylation (using the R package methyl kit). Statistical analysis was conducted, where appropriate, using a 1-way analysis of variance with multiple testing corrections or Student's t-test. RESULTS: Our data shows that stDR increases enteroid formation when implemented late in life by ≥10-fold (P ≤ 0.05). RNA-Seq analysis identified 38 genes not commonly linked to ISC function but showed a 2-fold change (P ≤ 0.05) with age, which was prevented by stDR. Finally, evaluation of the methylation status of selected genes from RNA-seq (Slc28a3, Ly9, Cd2, Platr4) showed changes in methylation at Cytosine-Guanine (CG) sites in the cis-acting regions of the genes with age which were also prevented by stDR (P ≤ 0.05). CONCLUSIONS: Our study shows for the first time that late-life stDR can improve the function of leucine-rich repeat-containing G-protein coupled receptor 5 ISCs from old mice. These data suggest that late-life nutritional strategies might be effective in improving gut function that declines with age and reduce the risk of age-related intestinal disorders. Our study also provides the first data showing that DNA methylation may play a role in the ability of DR to improve ISC function.