Epithelial cells of ulcerative colitis patients require normal levels of mitochondria and beta-catenin to utilize butyrate metabolism and promote ulcer healing

Abstract BACKGROUND Under normal conditions butyrate produced by obligate anaerobes (e.g. firmicutes) provides the dominant energy source for colonocytes via β-oxidation and the tricarboxylic acid cycle. Ulcerative Colitis (UC) is an inflammatory bowel disease (IBD) characterized by reduced butyrate metabolism and intestinal epithelial cell (IEC) mitochondria (needed for β-oxidation). Given that nonhealing ulcers in UC are associated with reduced crypt fissioning and branching, we asked whether there was a relationship between mitochondrial deficiency and butyrate-induced crypt branching using colonoid cultures grown from human biopsies. METHODS IECs from biopsies taken from active and uninvolved areas of the same UC patient were grown in colonoid cultures. After the second passage, the colonoids with crypt branching were quantified. In separate studies, we modeled mitochondrial deficiency by shRNA knockdown of transcription mitochondrial factor A (Tfam) in human colonoids, a gene responsible for mitochondrial DNA stability and transcription. Both shControl and shTfam colonoids were treated with 5mM of Na-butyrate overnight. NCM460 cells were also treated with 5mM of butyrate overnight, and both ATP levels and TCF/LEF (b-catenin transcriptional activity) were examined. RESULTS Colonoids from active UC segments demonstrated 27% less crypt branching than uninvolved areas. These areas also displayed reduced mitochondria complex levels by WB of IEC. To directly examine the role of mitochondria in these effects, crypt branching was examined in shTFAM-treated colonoids where crypt branching levels were 31% less than shCont-treated colonoids (Fig. 1). Butyrate treatment of both shCont and shTfam colonoids increased crypt formation by 11% and 8%, respectively compared to untreated groups. In addition, butyrate increased levels of ATP by 55% and b-catenin transcriptional activity (TCF-LEF) by >600%. CONCLUSION This data was consistent with the notion that the failure to heal ulceration in UC patients involves both 1) reduction in butyrate availability from the microbiome (ie reduced stimulation) and 2) reduced mitochondria needed to metabolize butyrate. Upon effective anti-inflammatory treatment, we believe butyrate levels and IEC mitochondrial metabolism increase which contributes to IEC b-catenin signaling and enhanced crypt fissioning/branching needed for effective ulcer healing. We believe this data provide a potential mechanism for how enhanced mitochondrial function contributes to ulcer healing in IBD.