The increasing prevalence of obesity has been paralleled by a rise in the consumption of caloric sweeteners over the past five decades. All of the most common caloric sweeteners contain a significant proportion of fructose, including table sugar (sucrose), honey, and high-fructose corn syrup (HFCS). Although fructose has likely been part of the diet since the beginning of mankind, humans in the western world now consume more than ever recorded. It is imperative that we understand how fructose impacts our bodies and develop novel treatment strategies to prevent its complications. We and others have shown that moderate daily exposure to dietary fructose exacerbates the obesogenic effects of a high fat diet, however the mechanisms by which this occurs remain uncertain. Our preliminary data identify villous hypertrophy and enhanced lipid uptake in mice fed diets containing sucrose or HFCS. A metabolomic and biochemical analysis of the intestinal epithelium show that fructose 1-phosphate (F1P), the primary fructose metabolite, is increased after dietary fructose and reduces pyruvate kinase (PK) activity. Genetic and pharmacologic activation of PK reduces villous length and protects against diet-induced weight gain. Therefore, we hypothesize that fructose promotes villous hypertrophy and lipid uptake by acting as an inhibitor of the M2 isozyme of PK (PKM2) in the enterocyte, which can be prevented and reversed with PK activators. We will test this hypothesis using dietary and pharmacologic interventions in mice and intestinal organoids. In Aim 1, we will genetically and pharmacologically manipulate ketohexokinase, the enzyme that produces F1P from fructose, and PKM2 in the intestines of mice to interrogate their role as mediators of diet-induced obesity. In Aim 2, we will define the effects of PK activity on enterocyte lipid metabolism to elucidate the mechanistic link between dietary fructose and obesity. Together, these aims will change our fundamental understanding of how fructose alters intestinal metabolism, define the fructose/F1P/PKM2 axis as a metabolic mediator of lipid uptake, and provide pre-clinical evidence for PKM2 activators as a novel therapeutic modality to combat obesity.