Background: Age induced decline in stemness of Intestinal Stem Cells (ISCs) was reported to be associated with a metabolic shift from mitochondrial oxidation to anaerobic utilization of pyruvate. Hypothesis: We hypothesized that increasing mitochondrial oxidation of pyruvate by inhibiting pyruvate dehydrogenase 4 (Pdk4) will enhance stemness of older and geriatric ISCs. Methods and Results: Enteroids were generated from duodenal and jejunal enteroid lines from young (20-35y) and aged (>55y) healthy individuals, and proximal small intestinal crypts from young (2-4 months), older (8-10 months) and geriatric mice (18-22 months). Mitochondrial basal and maximal oxygen consumption rates (OCRs), mitochondrial ATP (mitoATP), and glycolytic ATP (glycoATP) production rates were measured using Seahorse XF Analyzer with access to full metabolic substrates or single substrates for isolated mitochondria. Transmission electron microscopy performed on ISC mitochondria revealed that geriatric mitochondria were swollen and stunted (38% lower ferret distance, 55% lower branch length). Basal and maximal OCRs in enteroids from older individuals were 43% and 52% lower than younger individuals, respectively. Similarly, in mice, geriatric basal OCRs were 55% and 44% lower than younger and older enteroids, respectively. Concomitantly, mitoATP production declined (-67% and -115%, respectively). In contrast, glycoATP was elevated at baseline and in response to mitochondrial inhibition by oligomycin. To discern differences in mitochondrial handling of metabolites, we isolated mitochondria from enteroids and measured responses to single metabolic substrates. In the presence of palmitic, linoleic, and linolenic acid, mitochondrial OCR decreased progressively with age (-35%, -43%, and -38% for older; -55%, -67%, -70% for geriatric, respectively). However, in the presence of pyruvate, OCR of older and geriatric ISC mitochondria was comparable to that of the young, demonstrating a shift in preference for pyruvate over fatty acids in the ISC mitochondria. Additionally, geriatric ISC mitochondria had 60% higher pyruvate, 66% lower acetyl CoA, and 76% lower pyruvate dehydrogenase (PDH) activity than younger enteroids. Suppression of Pdk4 (that phosphorylates and inactivates PDH) by dichloroacetate (DCA, 4mM) and transient silencing of Pdk4 gene (5-10nM siRNA) robustly enhanced growth and budding capacities of both human (enteroid area: 75%, bud numbers: 67%) and mice enteroids (enteroid area: 53-69%, bud numbers: 55-68%) and increased expression of Lgr5 (35%) and Olfm4 (42%). Mechanistically, suppression of Pdk4 increased PDH activity (35-62%) and mitochondrial acetyl CoA, consequently increasing pyruvate oxidation, mitochondrial OCR (DCA: 34%, Pdk4 siRNA: 43%), and mitoATP (35-48%). Conclusion: Induction of mitochondrial oxidation robustly enhances stemness of human and mice ISCs.

Aasem Awwad

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