KETOGENIC PATHWAY MEDIATED BY HMGCS2 IS IMPLICATED IN STEM CELL FUNCTION AND INTESTINAL REGENERATION IN ULCERATIVE COLITIS

Background: Several lines of evidence show mitochondrial damage and bioenergetic failure in ulcerative colitis (UC). However, little is known about the specific pathogenic metabolic processes that influence epithelial function and repair in UC¹.
Aim/Methods: To investigate the role of mitochondrial dysfunction and consequent effect on the ketogenic pathway in UC in intestinal stem cell (iSC) and epithelial regeneration. We characterised mitochondrial oxidative phosphorylation (OXPHOS) proteins; performed single-cell RNA sequencing (scRNAseq) on colonic epithelial cells and performed growth assay experiments on organoids derived from newly diagnosed drug naïve UC patients.
Results: Using single-cell quantitative immunofluorescence, we showed a loss of mitochondrial Complex I and IV in both inflamed and non-inflamed UC (vs. non-IBD controls; mean 300 000 cells/group [n=10/group]; both p<0.0001). We also confirmed this loss of OXPHOS in newly diagnosed UC-derived organoids to show that mitochondrial dysfunction is potentially propagated from the iSC level. ScRNAseq analyses of epithelial cells isolated from active UC showed the most downregulated pathway is related to OXPHOS (KEGG for OXPHOS NES: -2.95; p<0.0001), within this pathway the contributing genes are mitochondrial encoded (min fold change -1.84; p<0.0001). Within iSCs the most significantly downregulated gene from scRNAseq was 3-hydroxy-3-methylgluraryl CoA synthetase 2 (Hmgcs2) (fold change -3.34; p<0.0001, Figure 1). HMGCS2 is the rate limiting mitochondrial matrix enzyme in the ketogenesis pathway, producing beta hydroxybutyrate (BHB), which provides metabolites for TCA cycle, and has important role in cell-fate-signalling. We show that whilst there is strong HMGCS2 expression in non-IBD controls and non-active UC, HGMCS2 expression is significantly reduced through the crypt-villus axis in active UC ([n=5/group]; p<0.001, Figure 2). We cultured organoids isolated from active and non-inflamed UC colon, we observed a significantly lower growth rate over 5 days (as measured by organoid area using, automated non-biased organoid tracking software: OrganoID; p<0.0001). The impaired iSC function detected in UC organoid cultures can be rescued with BHB supplementation (2.5-5 mM; p<0.0001).
Conclusions: In newly-diagnosed UC iSCs and organoids, we provide specific evidence to implicate OXPHOS failure and loss of HMGCS2 gene and protein expression, with resultant impairment of iSC function. This impairment can be rescued with supplementation of BHB, thereby implicating the ketogenic pathway in the pathogenesis of UC.

1. Ho, G. and Theiss, A.L. (2022) ‘Mitochondria and Inflammatory Bowel Diseases: Toward a Stratified Therapeutic Intervention’, Annual Review of Physiology, https://doi.org/10.1146/annurev-physiol-060821-083306.