The Carbohydrate Environment During Differentiation Shapes the Functional Characteristics and Response to Acute Sugar Exposure of Human Intestinal Caco-2 Cells

Background: The intestinal epithelium continuously adapts to changes in nutrient availability, yet whether the carbohydrate environment experienced during epithelial differentiation contributes to shaping the mature enterocyte functional characteristics remains largely unexplored. Objectives: This study investigated whether replacing glucose with fructose or sucrose during Caco-2 cell differentiation influences epithelial maturation, the expression of genes involved in intestinal sugar digestion and transport, and the cellular response to a subsequent acute sugar challenge. Methods: Caco-2 cells were differentiated for 23 days in medium containing glucose, fructose, or sucrose. Barrier function was monitored throughout differentiation by measuring transepithelial electrical resistance and paracellular permeability. At the end of differentiation, dome formation, expression of genes involved in sugar digestion and transport, and extracellular sugar distribution were evaluated. Differentiated cells were then exposed for 24 h to glucose, fructose, or sucrose to assess their adaptive response. Results: All carbohydrate conditions supported epithelial differentiation, although with distinct maturation kinetics. Sucrose promoted earlier barrier maturation, reduced dome formation, and generated a transcriptional profile that differed from those induced by glucose or fructose. The transcriptional response to acute sugar exposure depended on both the carbohydrate present during differentiation and that used for the subsequent challenge, whereas extracellular carbohydrate distribution remained largely unchanged among experimental groups. Conclusions: These findings demonstrate that the carbohydrate environment experienced during differentiation contributes to shaping the functional characteristics of intestinal epithelial cells. Beyond emphasizing the importance of carefully defining differentiation conditions in Caco-2-based studies, this work supports the concept that nutrient availability during epithelial maturation may represent an underappreciated determinant of intestinal epithelial adaptation.