To validate the lineage-specific effects of TCA-cycle use in vivo, we examined the effect of Ogdh suppression on intestinal homeostasis in TRE-shOgdhCag-rtTA3 mice (Extended Data Fig. 7a). We disentangled the direct TCA-cycle effects from those mediated by increased αKG by comparing Ogdh knockdown with DM-αKG administration3. In contrast to the TRE-shRenCag-rtTA3 controls, TRE–shOgdhCag-rtTA3 mice exhibited severe weight loss and, developed bowel obstructions and required euthanasia 7–11 days after doxycycline treatment (Extended Data Fig. 7b–e). Although a high dose of DM-αKG (600 mg kg−1) was toxic, a lower dose (300 mg kg−1) was tolerated without significant weight loss or intestinal structure disruption (Extended Data Fig. 7f–h).
Histological and immunofluorescence analysis revealed that αKG supplementation and Ogdh knockdown had distinct effects on the physiology of the intestine. Ogdh knockdown reduced proliferation within three days of doxycycline treatment (Fig. 4a–c and Extended Data Fig. 7i). Apoptosis occurred later, by day 6, concentrated in the upper region of the crypts and associated with the emergence of crypt hypoplasia and a reduction in HNF4α-positive cells (Fig. 4a–c and Extended Data Fig. 7j–l). Consistent with the high metabolic demand of enterocytes for canonical TCA-cycle functions, supplementation with DM-succinate after OGDH depletion reduced apoptosis (Fig. 4d–f). By marked contrast, treatment with DM-αKG did not induce profound cell-cycle arrest or detectable apoptosis in the crypts (Extended Data Fig. 7m).
Fig. 4: Role of OGDH in gut homeostasis.
a,b, ABP and immunofluorescence for lysozyme, GFP and cleaved caspase 3 (CC3) in intestinal tissue from TRE–shRenCag-rtTA3 and TRE–shOgdhCag-rtTA3 mice (a) and vehicle-treated and DM-αKG-treated mice (b) at the indicated time points. c, Heat map depicting time-course quantification (D indicates day) of OGDH (fluorescence intensity), BrdU (positive cells), Spdef (mRNA levels), lysozyme (Lyz) (positive cells) and CC3 (positive cells) in intestinal crypts from doxycycline-treated TRE–shRenCag-rtTA3 and TRE–shOgdhCag-rtTA3 mice or DM-αKG-treated C57Bl/6 mice (n = 5 per group). d,e, ABP and immunofluorescence for OGDH, lysozyme (Paneth cells), HNF4α (enterocytes) and ACE2 (mature enterocytes) (d), and CC3 (cell death) (e) in intestinal sections from TRE–shOgdhCag-rtTA3 mice concomitantly treated with or without DM-succinate and doxycycline for 6–7 days. Data are representative of shRen n = 3, shRen + succinate n = 3, shOgdh n = 8 and shOgdh + succinate n = 4 mice. f, Quantification from e. Each dot represents one crypt or villus for lysozyme and CC3 and one mouse for HNF4α. a.u., arbitrary units. g, Immunofluorescence for 5hmC and HNF4α in crypts from TRE–shRenCag-rtTA3, TRE–shOgdhCag-rtTA3 and DM-αKG-treated mice. Dashed lines outline crypts (top) and 5hmC+HNF4α− cells (bottom). h, Top, enzyme-linked immunosorbent assay (ELISA) of crypt lysates to measure intestinal 5hmC abundance in TRE–shRenCag-rtTA3, TRE–shOgdhCag-rtTA3, DM-αKG-treated and vehicle-treated mice. Each dot represents one mouse (shRen n = 4, shOgdh n = 9, vehicle n = 4, αKG n = 6). Bottom, relative 5hmC levels in the Spdef promoter within isolated crypts from shRenCag-rtTA3, TRE–shOgdhCag-rtTA3 and DM-αKG-treated mice, measured by qPCR at the indicated time points. Each dot represents one mouse (n ≥ 3 mice per group). i, Venn diagrams of upregulated (red) and downregulated (blue) genes in TRE–shOgdhCag-rtTA3 and αKG-treated mice versus controls (TRE–shRenCag-rtTA3 and vehicle-treated), with gene ontology (GO) analysis of the overlapping genes. Abs(score), absolute value of enrichment score. This figure is adapted from our published patent (WO2024229094A1)50. Data are mean ± s.e.m. Statistical significance was determined using one-way ANOVA followed by Tukey’s HSD test in f and two-tailed t-test in h.
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Despite these differences in the absorptive lineage, both αKG supplementation and Ogdh suppression triggered an accumulation of 5hmC-high secretory cells and a depletion of ISCs in both the small intestine and the colon (Fig. 4g and Extended Data Fig. 7n–s). Loci-targeted bisulfite sequencing detected increased levels of 5hmC at the Spdef promoter, a Paneth cell and goblet cell transcription factor, correlating with early upregulation of Spdef (Fig. 4h,i) and a subsequent expansion of secretory cells (Fig. 4a–c and Extended Data Fig. 7n–s). Although the TRE-shOgdhCags-rtTA3 model induces systemic Ogdh suppression, similar effects were observed with intestine-specific Ogdh knockdown using TRE-shOgdhVillin-rtTA mice, which exhibited an increase in lysozyme-expressing secretory cells, along with high levels of ABP and 5hmC (Extended Data Fig. 8a,b).
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To further characterize the molecular changes induced by αKG supplementation and Ogdh suppression, we performed bulk RNA-seq analysis on isolated intestinal crypts (Extended Data Fig. 8c). Ogdh suppression, but not DM-αKG supplementation, significantly reduced transcriptional programs associated with cell proliferation and specification of the absorptive lineage (Extended Data Fig. 8d–i). By contrast, transcriptional signatures of the secretory lineage were enriched in both DM-αKG-treated and TRE-shOgdhCag-rtTA3 mice (Fig. 4i and Extended Data Fig. 8d–i). These findings indicate that OGDH is essential for enterocyte expansion and survival, and that its suppression contributes to secretory-lineage differentiation. Therefore, regulation of OGDH expression is crucial for lineage specification and balance during intestinal regeneration (Extended Data Fig. 8j).
Chaves-Perez, A., Millman, S.E., Janaki-Raman, S. et al. Metabolic adaptations direct cell fate during tissue regeneration.
Nature (2025).
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Chaves-Perez, A., Millman, S.E., Janaki-Raman, S. et al. Metabolic adaptations direct cell fate during tissue regeneration.
Nature (2025). https://doi.org/10.1038/s41586-025-09097-6
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Tags: bowel obstructions and intestinal physiologycrypt hypoplasia and cell apoptosiseffects of DM-αKG on proliferationhistological analysis of intestinal changesimplications of metabolic pathways in regenerationintestinal structure disruption in micelineage-specific metabolic effects in vivometabolic adaptations in tissue regenerationmetabolic demand of enterocytesOgdh suppression and intestinal healthTCA cycle effects on cell fateαKG and intestinal homeostasis
