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bioRxiv · 10.1101/2024.09.23.614374

Mitochondrial oxidation of the carbohydrate fuel driven by pyruvate dehydrogenase robustly enhances stemness of older and geriatric Intestinal Stem Cells

Ahmed, S.·Awwad, A.·Eddy, N.·Weber, G.·Shahid, Z.·Sethi, Z.·Labampa, J.·Murphy, R.·Roth, E. W.·Gustafson, K.·Shah, H.·Sundaresan, S.
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Abstract

Background and AimsAging impairs Intestinal Stem Cell (ISC) function and attenuates their regenerative capacity. Although the transcriptional landscape governing ISC fate during aging has been described, almost nothing is known about how metabolite handling regulates ISC renewal and maintains stemness. We investigated how mitochondrial metabolism of glucose and fatty acid-derived carbons, regulated by the gatekeeper, pyruvate dehydrogenase (PDH) rescues ISC stemness in older and geriatric mice and humans. MethodsProximal small intestinal organoids (enteroids) generated from pinch biopsy specimens obtained from young (21-25y) and older individuals (64-75y), and GFP-sorted single ISCs from Lgr5-EGFP mice (2-24 months) were used to examine hallmarks of ISC stemness. Mitochondrial morphology was evaluated using transmission electron microscopy. Mitochondrial oxygen consumption rate (OCR), ATP (mitoATP), and glycolytic ATP production were measured in the presence of full and single metabolic substrates (pyruvate, glutamate, and fatty acids) in whole cell and isolated mitochondria using the high throughput Seahorse XF technology. Carbon flux through TCA cycle was determined by 13C6-glucose tracing and measuring 13C enrichment in TCA cycle intermediates using liquid chromatography mass spectrometry. ResultsAge induced decline in ISC stemness is driven by a dramatic decrease in PDH activity that shuttles pyruvate away from the TCA cycle. Restoring PDH activity by inhibition of pyruvate dehydrogenase kinase 4 (PDK4) drives glucose-derived carbon entry into TCA cycle and subsequently increases mitochondrial OCR and mitoATP, collectively rescuing the decline in stemness in aging ISCs. The observed shift in fuel preference from fatty acids to glucose is unaltered by PDK4 inhibition. ConclusionPDH upregulation rescues age-induced decline in ISC stemness in humans and mice via directing glucose derived carbons to TCA cycle and increasing mitoATP production.

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Ahmed, S., Awwad, A., Eddy, N., Weber, G., Shahid, Z., Sethi, Z., Labampa, J., Murphy, R., Roth, E. W., Gustafson, K., Shah, H., Sundaresan, S.. 2024-09-25. Mitochondrial oxidation of the carbohydrate fuel driven by pyruvate dehydrogenase robustly enhances stemness of older and geriatric Intestinal Stem Cells. https://doi.org/10.1101/2024.09.23.614374

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