Summary Hexokinases (HK) catalyze the first step of glycolysis and thereby limit its pace. HK2 is highly expressed in the gut epithelium, plays a role in immune responses and is upregulated in inflammation and ulcerative colitis 1–3 . Here, we examined the microbial regulation of HK2 and its impact on intestinal inflammation by generating mice lacking HK2 specifically in intestinal epithelial cells ( Hk2 Δ IEC ). Hk2 Δ IEC mice were less susceptible to acute intestinal inflammation upon challenge with dextran sodium sulfate (DSS). Analyzing the epithelial transcriptome from Hk2 Δ IEC mice during acute colitis revealed downregulation of cell death signaling and mitochondrial dysfunction dependent on loss of HK2. Using intestinal organoids derived from Hk2 Δ IEC mice and Caco-2 cells lacking HK2, we identified peptidyl-prolyl cis-trans isomerase (PPIF) as a key target of HK2-mediated regulation of mitochondrial permeability and repression of cell-death during intestinal inflammation. The microbiota strongly regulated HK2 expression and activity. The microbially-derived short-chain fatty acid (SCFA) butyrate repressed HK2 expression and oral supplementation protected wildtype but not Hk2 Δ IEC mice from DSS colitis. Our findings define a novel mechanism how butyrate may act as a protective factor for intestinal barrier homeostasis and suggest targeted HK2 inhibition as a promising therapeutic avenue in intestinal inflammation.

Microbial regulation of hexokinase 2 links mitochondrial metabolism and cell death in colitis