Although it is increasingly recognized that the interaction of the diet and host specific genetic factors in the gut play an important role in intestinal and systemic disease, our understanding of in this emerging field is still limited and this represents an important knowledge gap. Here we propose to bridge this gap by advancing our knowledge how telomere shortening in the gut impacts the maturation of enterocytes, the terminally differentiated cells that are essential for barrier maintenance and absorption. Telomeres are important for the regeneration of stem cell-dependent tissues such as the intestine. Telomere shortening occurs during normal aging and is accelerated in patients with mutations in telomerase or in high cell turnover conditions such as ulcerative colitis and Crohn’s disease. Telomere shortening causes several pathologies in the intestine including atrophy, inflammation, and progression of colitis to cancer in patients and all these pathologies are faithfully recapitulated in telomerase knockout mice (TKO). Mechanistically, it is believed that short telomeres drive these pathologies through continuous apoptosis-mediated depletion of intestinal stem cells. Beyond stem cell- depletion, other pathogenic mechanisms are not known. In particular, it is not known whether telomere shortening compromises differentiated cell lineages in the gut, of which enterocytes represent the vast majority. Here we have found that telomere shortening impairs the maturation towards the enterocyte lineage leading to the generation of immature and functionally compromised enterocytes. This is supported by a multi- level analysis including RNAseq, proteomics, transmission and scanning electron microscopy demonstrating that the expression of digestive enzymes, transporters and structural components of microvilli are repressed in the proximal intestine in TKO mice. Importantly, preliminary studies indicate that this enterocyte compromise is preserved in human enterocytes with short telomeres. Mechanistically, deletion of p53 in TKO epithelium rescues enterocyte defects. Furthermore, these enterocytes are characterized by mitochondrial dysfunction and have low ATP levels. When exposed to a fructose-rich diet, they show pronounced propensity to steep decline in ATP levels and subsequent apoptosis, which exacerbates the barrier defect and malabsorption. Here we propose to establish the mechanisms through which p53 causes differentiation defects (Aim 1), establish whether the low ATP levels are the driving source for fructose toxicity by increasing ATP levels either through inactivation of the ATP depleting enzyme fructokinase or improving mitochondrial function through NAD supplementation. In Aim 3 we establish the relevance of short telomeres in causing enterocyte defects in humans using cell lines, organoids and enteroids with various telomere length.