{"id":{"repo_id":"edinburgh","oai_identifier":"oai:era.ed.ac.uk:1842/43881"},"canonical_url":"https://search.dev.ndltd.org/etd/edinburgh/oai:era.ed.ac.uk:1842/43881","repository":{"repo_id":"edinburgh","name":"University of Edinburgh","base_url":"https://era.ed.ac.uk/server/oai/request"},"display":{"title":"Impaired ketogenesis is implicated in intestinal stem cell function and epithelial regeneration in ulcerative colitis","abstract":"Epithelial repair and regeneration is key to mucosal healing in Ulcerative Colitis (UC). Intestinal stem cells (ISCs) drive this response, proliferating and differentiating from the base of the crypt to regenerate the entire epithelium. ISCs therefore exist in a highly active state and resultantly are vulnerable to changes in energy dynamics in the intestine. Mitochondria, which amongst other functions are critical for energy production are therefore crucial for maintenance of the stem cell niche are significantly implicated in UC pathogenesis. Up to now, the mechanisms of how mitochondrial dysfunction influence ISC function in UC is not known. To investigate this, we used single cell RNA sequencing (scRNA-seq), single cell quantitative immunofluorescence and single cell energetic metabolism by profiling translation inhibition (SCENITH) on treatment-naïve UC patient epithelial samples and on UC patient derived organoids. Single-cell quantitative immunofluorescence showed a loss of mitochondrial oxidative phosphorylation proteins Complex I and IV, necessary for ATP production in the UC epithelium. Moreover, in UC-derived organoids from ISCs, we show persistence in mitochondrial damage as evidenced by immunofluorescence and scRNA-seq data that showed also defective mitophagy (PINK1) and mitochondrial biogenesis (PPARGC1α) in ex vivo culture conditions. This data suggests that mitochondrial dysfunction is a stable feature of ISCs and potentially can be propagated during division and differentiation into multiple progeny epithelial lineages to occupy the entire epithelial compartment. In parallel, scRNA-seq of the UC epithelial cells showed significant downregulation of metabolic pathways in ISCs, with de-regulation of fatty acid metabolism pathways, particularly ketogenesis, with HMGCS2 as the top downregulated gene in UC ISCs. HMGCS2, the rate limiting mitochondrial matrix enzyme in the ketogenesis pathway, producing βHB, which alongside providing metabolites for the mitochondrial tricarboxylic acid TCA cycle, has important roles in stem cell fate-signalling. We show that HGMCS2 expression was reduced throughout the intestinal crypt in active UC, and is increased in patients responding to therapy with mucosal healing. We find via colorimetric assay that colonic organoids secrete βHB, and production is reduced in active UC. We identify PPARα, a known key inducer of HGMCS2 transcription as being significantly downregulated in UC. We find HGMCS2 expression and βHB production can be restored in UC organoids via administration of PPARα agonist, fenofibrate. We performed scRNA-seq of βHB treated UC organoids finding that βHB specifically enhance the transcriptome of pathways that regulate mitochondrial function and reduces cellular stress, particularly in ISCs, by downregulating genes associated with cellular stress and protein misfolding (SOD2, HSPD1), as well as mitochondrial fission (FIS1, DNM1L). Specifically, βHB induces expression of key mitophagy related genes, aiding clearance of faulty mitochondria (PINK1). Functionally, we showed that βHB restored OXPHOS capacity in UC organoids as measured by SCENITH and reduced the production of cellular and mitochondrial reactive oxygen species, as measured by CellRox. Finally, HMGCS2 protein expression significantly increases in responders to medical therapy with mucosal healing. In newly diagnosed UC patients we provide evidence to show a crucial role in HMGCS2-mediated ketogenesis in ISC function. This phenotype can be rescued with βHB supplementation or pharmacologic induction of ketogenesis, thereby implicating the ketogenic pathway in UC pathogenesis and the potential for a novel metabolic therapy.","abstract_html":"Epithelial repair and regeneration is key to mucosal healing in Ulcerative Colitis (UC). Intestinal stem cells (ISCs) drive this response, proliferating and differentiating from the base of the crypt to regenerate the entire epithelium. ISCs therefore exist in a highly active state and resultantly are vulnerable to changes in energy dynamics in the intestine. Mitochondria, which amongst other functions are critical for energy production are therefore crucial for maintenance of the stem cell niche are significantly implicated in UC pathogenesis. Up to now, the mechanisms of how mitochondrial dysfunction influence ISC function in UC is not known. To investigate this, we used single cell RNA sequencing (scRNA-seq), single cell quantitative immunofluorescence and single cell energetic metabolism by profiling translation inhibition (SCENITH) on treatment-naïve UC patient epithelial samples and on UC patient derived organoids. Single-cell quantitative immunofluorescence showed a loss of mitochondrial oxidative phosphorylation proteins Complex I and IV, necessary for ATP production in the UC epithelium. Moreover, in UC-derived organoids from ISCs, we show persistence in mitochondrial damage as evidenced by immunofluorescence and scRNA-seq data that showed also defective mitophagy (PINK1) and mitochondrial biogenesis (PPARGC1α) in ex vivo culture conditions. This data suggests that mitochondrial dysfunction is a stable feature of ISCs and potentially can be propagated during division and differentiation into multiple progeny epithelial lineages to occupy the entire epithelial compartment. In parallel, scRNA-seq of the UC epithelial cells showed significant downregulation of metabolic pathways in ISCs, with de-regulation of fatty acid metabolism pathways, particularly ketogenesis, with HMGCS2 as the top downregulated gene in UC ISCs. HMGCS2, the rate limiting mitochondrial matrix enzyme in the ketogenesis pathway, producing βHB, which alongside providing metabolites for the mitochondrial tricarboxylic acid TCA cycle, has important roles in stem cell fate-signalling. We show that HGMCS2 expression was reduced throughout the intestinal crypt in active UC, and is increased in patients responding to therapy with mucosal healing. We find via colorimetric assay that colonic organoids secrete βHB, and production is reduced in active UC. We identify PPARα, a known key inducer of HGMCS2 transcription as being significantly downregulated in UC. We find HGMCS2 expression and βHB production can be restored in UC organoids via administration of PPARα agonist, fenofibrate. We performed scRNA-seq of βHB treated UC organoids finding that βHB specifically enhance the transcriptome of pathways that regulate mitochondrial function and reduces cellular stress, particularly in ISCs, by downregulating genes associated with cellular stress and protein misfolding (SOD2, HSPD1), as well as mitochondrial fission (FIS1, DNM1L). Specifically, βHB induces expression of key mitophagy related genes, aiding clearance of faulty mitochondria (PINK1). Functionally, we showed that βHB restored OXPHOS capacity in UC organoids as measured by SCENITH and reduced the production of cellular and mitochondrial reactive oxygen species, as measured by CellRox. Finally, HMGCS2 protein expression significantly increases in responders to medical therapy with mucosal healing. In newly diagnosed UC patients we provide evidence to show a crucial role in HMGCS2-mediated ketogenesis in ISC function. This phenotype can be rescued with βHB supplementation or pharmacologic induction of ketogenesis, thereby implicating the ketogenic pathway in UC pathogenesis and the potential for a novel metabolic therapy.","abstract_has_math":false,"creators":["Rutherford, Duncan Glendinning"],"institution":"The University of Edinburgh","degree_name":null,"degree_level":null,"degree_discipline":null,"degree_department":null,"school":null,"contributors":[],"advisors":["Ho, Gwo-Tzer","Jones, Gareth"],"committee_chairs":[],"committee_members":[],"year":2025,"date_issued":"2025-08-28","date_published":"2025-08-28","updated_at":"2026-07-24T02:13:53Z","subjects":["Ulcerative Colitis (UC)","Intestinal Stem Cells (ISCs)","Ketogenesis","Mitochondrial Dysfunction","Epithelial Regeneration"],"languages":["en"],"rights":[],"rights_urls":[],"identifier_entries":[{"key":"dc:identifier.uri","label":"Identifier URI","values":["http://dx.doi.org/10.7488/era/6412"],"render_values":[{"text":"http://dx.doi.org/10.7488/era/6412","href":"http://dx.doi.org/10.7488/era/6412","code":true}]}]},"links":{"outbound_url":"https://hdl.handle.net/1842/43881","outbound_label":"Handle","outbound_source":"dc:identifier.uri"},"metadata_groups":[{"id":"people","label":"People","entries":[{"key":"dc:contributor.advisor","label":"Advisor","values":["Ho, Gwo-Tzer","Jones, Gareth"]},{"key":"dc:creator","label":"Author","values":["Rutherford, Duncan Glendinning"]}]},{"id":"academic_context","label":"Academic Context","entries":[{"key":"dc:date.accessioned","label":"Dc Date Accessioned","values":["2025-08-28T14:45:41Z"]},{"key":"dc:date.available","label":"Dc Date Available","values":["2025-08-28T14:45:41Z"]},{"key":"dc:date.issued","label":"Date","values":["2025-08-28"]},{"key":"dc:publisher","label":"Institution","values":["The University of Edinburgh"]},{"key":"dc:type","label":"Dc Type","values":["Thesis or Dissertation"]},{"key":"dc:type.qualificationlevel","label":"Dc Type Qualificationlevel","values":["Doctoral"]},{"key":"dc:type.qualificationname","label":"Dc Type Qualificationname","values":["PhD Doctor of Philosophy"]}]},{"id":"subjects_keywords","label":"Subjects and Keywords","entries":[{"key":"dc:subject","label":"Dc Subject","values":["Ulcerative Colitis (UC)","Intestinal Stem Cells (ISCs)","Ketogenesis","Mitochondrial Dysfunction","Epithelial Regeneration"]}]},{"id":"language_rights","label":"Language and Rights","entries":[{"key":"dc:language.iso","label":"Language (ISO)","values":["en"]}]},{"id":"identifiers","label":"Identifiers","entries":[{"key":"dc:identifier.uri","label":"Identifier URI","values":["https://hdl.handle.net/1842/43881","http://dx.doi.org/10.7488/era/6412"]}]},{"id":"additional","label":"Additional Metadata","entries":[{"key":"dc:description.abstract","label":"Abstract","values":["Epithelial repair and regeneration is key to mucosal healing in Ulcerative Colitis (UC). Intestinal stem cells (ISCs) drive this response, proliferating and differentiating from the base of the crypt to regenerate the entire epithelium. ISCs therefore exist in a highly active state and resultantly are vulnerable to changes in energy dynamics in the intestine. Mitochondria, which amongst other functions are critical for energy production are therefore crucial for maintenance of the stem cell niche are significantly implicated in UC pathogenesis. Up to now, the mechanisms of how mitochondrial dysfunction influence ISC function in UC is not known. To investigate this, we used single cell RNA sequencing (scRNA-seq), single cell quantitative immunofluorescence and single cell energetic metabolism by profiling translation inhibition (SCENITH) on treatment-naïve UC patient epithelial samples and on UC patient derived organoids. Single-cell quantitative immunofluorescence showed a loss of mitochondrial oxidative phosphorylation proteins Complex I and IV, necessary for ATP production in the UC epithelium. Moreover, in UC-derived organoids from ISCs, we show persistence in mitochondrial damage as evidenced by immunofluorescence and scRNA-seq data that showed also defective mitophagy (PINK1) and mitochondrial biogenesis (PPARGC1α) in ex vivo culture conditions. This data suggests that mitochondrial dysfunction is a stable feature of ISCs and potentially can be propagated during division and differentiation into multiple progeny epithelial lineages to occupy the entire epithelial compartment. In parallel, scRNA-seq of the UC epithelial cells showed significant downregulation of metabolic pathways in ISCs, with de-regulation of fatty acid metabolism pathways, particularly ketogenesis, with HMGCS2 as the top downregulated gene in UC ISCs. HMGCS2, the rate limiting mitochondrial matrix enzyme in the ketogenesis pathway, producing βHB, which alongside providing metabolites for the mitochondrial tricarboxylic acid TCA cycle, has important roles in stem cell fate-signalling. We show that HGMCS2 expression was reduced throughout the intestinal crypt in active UC, and is increased in patients responding to therapy with mucosal healing. We find via colorimetric assay that colonic organoids secrete βHB, and production is reduced in active UC. We identify PPARα, a known key inducer of HGMCS2 transcription as being significantly downregulated in UC. We find HGMCS2 expression and βHB production can be restored in UC organoids via administration of PPARα agonist, fenofibrate. We performed scRNA-seq of βHB treated UC organoids finding that βHB specifically enhance the transcriptome of pathways that regulate mitochondrial function and reduces cellular stress, particularly in ISCs, by downregulating genes associated with cellular stress and protein misfolding (SOD2, HSPD1), as well as mitochondrial fission (FIS1, DNM1L). Specifically, βHB induces expression of key mitophagy related genes, aiding clearance of faulty mitochondria (PINK1). Functionally, we showed that βHB restored OXPHOS capacity in UC organoids as measured by SCENITH and reduced the production of cellular and mitochondrial reactive oxygen species, as measured by CellRox. Finally, HMGCS2 protein expression significantly increases in responders to medical therapy with mucosal healing. In newly diagnosed UC patients we provide evidence to show a crucial role in HMGCS2-mediated ketogenesis in ISC function. This phenotype can be rescued with βHB supplementation or pharmacologic induction of ketogenesis, thereby implicating the ketogenic pathway in UC pathogenesis and the potential for a novel metabolic therapy."]},{"key":"dc:title","label":"Title","values":["Impaired ketogenesis is implicated in intestinal stem cell function and epithelial regeneration in ulcerative colitis"]}]}],"canonical_facts":{"dc:contributor.advisor":["Ho, Gwo-Tzer","Jones, Gareth"],"dc:creator":["Rutherford, Duncan Glendinning"],"dc:date.accessioned":["2025-08-28T14:45:41Z"],"dc:date.available":["2025-08-28T14:45:41Z"],"dc:date.issued":["2025-08-28"],"dc:description.abstract":["Epithelial repair and regeneration is key to mucosal healing in Ulcerative Colitis (UC). Intestinal stem cells (ISCs) drive this response, proliferating and differentiating from the base of the crypt to regenerate the entire epithelium. ISCs therefore exist in a highly active state and resultantly are vulnerable to changes in energy dynamics in the intestine. Mitochondria, which amongst other functions are critical for energy production are therefore crucial for maintenance of the stem cell niche are significantly implicated in UC pathogenesis. Up to now, the mechanisms of how mitochondrial dysfunction influence ISC function in UC is not known. To investigate this, we used single cell RNA sequencing (scRNA-seq), single cell quantitative immunofluorescence and single cell energetic metabolism by profiling translation inhibition (SCENITH) on treatment-naïve UC patient epithelial samples and on UC patient derived organoids. Single-cell quantitative immunofluorescence showed a loss of mitochondrial oxidative phosphorylation proteins Complex I and IV, necessary for ATP production in the UC epithelium. Moreover, in UC-derived organoids from ISCs, we show persistence in mitochondrial damage as evidenced by immunofluorescence and scRNA-seq data that showed also defective mitophagy (PINK1) and mitochondrial biogenesis (PPARGC1α) in ex vivo culture conditions. This data suggests that mitochondrial dysfunction is a stable feature of ISCs and potentially can be propagated during division and differentiation into multiple progeny epithelial lineages to occupy the entire epithelial compartment. In parallel, scRNA-seq of the UC epithelial cells showed significant downregulation of metabolic pathways in ISCs, with de-regulation of fatty acid metabolism pathways, particularly ketogenesis, with HMGCS2 as the top downregulated gene in UC ISCs. HMGCS2, the rate limiting mitochondrial matrix enzyme in the ketogenesis pathway, producing βHB, which alongside providing metabolites for the mitochondrial tricarboxylic acid TCA cycle, has important roles in stem cell fate-signalling. We show that HGMCS2 expression was reduced throughout the intestinal crypt in active UC, and is increased in patients responding to therapy with mucosal healing. We find via colorimetric assay that colonic organoids secrete βHB, and production is reduced in active UC. We identify PPARα, a known key inducer of HGMCS2 transcription as being significantly downregulated in UC. We find HGMCS2 expression and βHB production can be restored in UC organoids via administration of PPARα agonist, fenofibrate. We performed scRNA-seq of βHB treated UC organoids finding that βHB specifically enhance the transcriptome of pathways that regulate mitochondrial function and reduces cellular stress, particularly in ISCs, by downregulating genes associated with cellular stress and protein misfolding (SOD2, HSPD1), as well as mitochondrial fission (FIS1, DNM1L). Specifically, βHB induces expression of key mitophagy related genes, aiding clearance of faulty mitochondria (PINK1). Functionally, we showed that βHB restored OXPHOS capacity in UC organoids as measured by SCENITH and reduced the production of cellular and mitochondrial reactive oxygen species, as measured by CellRox. Finally, HMGCS2 protein expression significantly increases in responders to medical therapy with mucosal healing. In newly diagnosed UC patients we provide evidence to show a crucial role in HMGCS2-mediated ketogenesis in ISC function. This phenotype can be rescued with βHB supplementation or pharmacologic induction of ketogenesis, thereby implicating the ketogenic pathway in UC pathogenesis and the potential for a novel metabolic therapy."],"dc:identifier.uri":["https://hdl.handle.net/1842/43881","http://dx.doi.org/10.7488/era/6412"],"dc:language.iso":["en"],"dc:publisher":["The University of Edinburgh"],"dc:subject":["Ulcerative Colitis (UC)","Intestinal Stem Cells (ISCs)","Ketogenesis","Mitochondrial Dysfunction","Epithelial Regeneration"],"dc:title":["Impaired ketogenesis is implicated in intestinal stem cell function and epithelial regeneration in ulcerative colitis"],"dc:type":["Thesis or Dissertation"],"dc:type.qualificationlevel":["Doctoral"],"dc:type.qualificationname":["PhD Doctor of Philosophy"]},"updated_at":"2026-07-24T02:13:53Z"}