Hypothesis In aging colonocytes, sustained low luminal butyrate produces a chronic energy deficit that activates AMPK‑dependent autophagy as a short‑term rationing mechanism. However, the same deficit limits ATP‑dependent lysosomal acidification and cathepsin activity, causing autophagosome accumulation without successful degradation (stalled flux). The resulting buildup of damaged mitochondria releases mitochondrial DNA into the cytosol, triggering cGAS‑STING and NLRP3 inflammasome activation, which drives low‑grade inflammation, apoptosis of G0/G1 cells, and gradual epithelial hypoplasia. Thus, what appears as protective autophagy in acute butyrate loss becomes maladaptive when the siege is prolonged, linking microbiota‑derived butyrate decline to age‑related gut barrier erosion.

Mechanistic rationale

1. Butyrate fuels colonocyte β‑oxidation, maintaining high ATP/AMPK low state. When butyrate falls, AMPK phosphorylates Thr198, stabilizing p27Kip1 and promoting LC3‑II lipidation [1]. This initiates autophagosome formation to recycle intracellular components and sustain basal ATP.
2. Lysosomal acidification requires V‑ATPase activity, which is ATP‑intensive. Chronic ATP shortage reduces lysosomal proton pumping, impairing cathepsin maturation and autophagosome‑lysosome fusion. Consequently, LC3‑II accumulates (as observed in starvation models) without proportional increase in lysosomal degradation markers (e.g., LAMP1, cathepsin B activity).
3. Damaged mitochondria that escape mitophagy release mtDNA into the cytosol. Cytosolic mtDNA engages cGAS, producing cGAMP that activates STING, leading to type I IFN signaling and NF‑κB–mediated NLRP3 inflammasome priming. Simultaneously, mitochondrial ROS provides the second signal for NLRP3 assembly, culminating in caspase‑1 activation, IL‑1β/IL‑18 secretion, and pyroptotic or apoptotic epithelial loss [2].
4. The resulting low‑grade inflammation further suppresses stem‑cell proliferation and promotes G0/G1 arrest, contributing to the hypoplasia seen after prolonged butyrate withdrawal [2]. Barrier function remains intact until cumulative cell loss exceeds regenerative capacity, manifesting as increased permeability in aged individuals.

Testable predictions

• In colonocytes from aged mice or germ‑free mice, LC3‑II will be elevated while lysosomal cathepsin activity and LAMP1 expression are reduced relative to young, conventionally raised controls.
• mtDNA will be detectable in the cytosolic fraction of aged colonocytes, correlating with increased phospho‑STING and cleaved caspase‑1 levels.
• Pharmacological lysosomal enhancement (e.g., trehalose or TFEB overexpression) will restore autophagic flux (decreased LC3‑II/p62 ratio) and attenuate inflammasome markers, rescuing epithelial cell density without altering luminal butyrate concentration.
• Conversely, acute lysosomal inhibition (bafilomycin A1) in young butyrate‑sufficient colonocytes will mimic the aged phenotype: LC3‑II accumulation, mtDNA release, and inflammasome activation.

Experimental approach

1. Isolate colonic crypts from young (3 mo) and aged (24 mo) mice, and from germ‑free versus conventionally raised controls. Measure LC3‑II, p62, cathepsin B/L activity, LAMP1 by western blot and immunofluorescence.
2. Subcellular fractionation to quantify cytosolic mtDNA via qPCR; assess phospho‑STING, TBK1, and NLRP3 inflammasome components.
3. Treat aged crypts with trehalose (50 mM) or adenoviral TFEB for 48 h; repeat flux and inflammasome readouts.
4. Perform complementary in vitro experiments using Caco‑2 cells cultured with/without butyrate (5 mM) and lysosomal modulators, confirming causality.

If lysosomal rescue normalizes autophagic flux and reduces inflammasome activation despite persistent butyrate scarcity, the hypothesis is supported. Failure to observe flux restoration or inflammasome suppression would falsify the claim that stalled autophagy, rather than autophagy per se, drives age‑related epithelial attrition.