Hypothesis

Chronic elevation of hexosamine biosynthetic pathway (HBP) flux leads to sustained O-GlcNAcylation of the V-ATPase subunit ATP6V0D1, impairing lysosomal acidification and autophagic clearance, thereby promoting lysosomal aggregate accumulation and senescence-associated secretory phenotype (SASP) in non-dividing cells. This mechanism connects nutrient stress to lysosomal dysfunction, a core SENS damage category, independent of hyperglycemia.

Rationale

• HBP flux generates UDP-GlcNAc, the donor for O-GlcNAc transferase (OGT). In diabetes and aging, HBP flux is elevated even when glucose is normal [[https://pmc.ncbi.nlm.nih.gov/articles/PMC10209545/]].
• O-GlcNAcylation of lysosomal proteins has been shown to alter their activity; for example, O-GlcNAc modification of LAMP2 reduces lysosomal stability [[https://doi.org/10.1073/pnas.1205748109]].
• V-ATPase pumps protons into lysosomes; its activity is essential for autophagosome-lysosome fusion and degradation. Phosphorylation and acetylation regulate V-ATPase assembly, but O-GlcNAcylation remains unexplored.
• We propose that OGT-mediated addition of a single GlcNAc to a serine/threonine residue in the cytosolic tail of ATP6V0D1 sterically hinders V1-V0 assembly, lowering V-ATPase activity and lysosomal pH.
• Elevated lysosomal pH impairs cathepsin activity, leading to incomplete degradation of ubiquitinated proteins and accumulation of p62-positive aggregates, a hallmark of lysosomal storage and aging [[https://doi.org/10.1002/advs.202309211]].
• Lysosomal dysfunction triggers TFEB nuclear translocation failure, reducing lysosomal biogenesis and amplifying SASP secretion via NF-κB activation, which is itself sensitized by O-GlcNAcylation of p65 [[https://doi.org/10.1161/JAHA.119.014046]].

Testable Predictions

1. In vitro: Treating HEK293 or primary fibroblasts with glucosamine (to boost HBP) will increase O-GlcNAcylation of ATP6V0D1 (detected by immunoprecipitation + lectin blot) and decrease Lysotracker fluorescence, indicating higher lysosomal pH. Knockdown of OGT or overexpression of OGA will rescue both signals.
2. In vivo: Aged mice (≥24 mo) fed a low‑dose glucosamine supplement will show elevated hippocampal ATP6V0D1 O-GlcNAcylation, reduced lysosomal acidification, and increased p62 aggregates compared with controls. Genetic lysosomal targeting of OGT (AAV‑ATP6V0D1‑OGT) should exacerbate these phenotypes, while lysosomal-targeted OGA overexpression will mitigate them.
3. Human relevance: Post‑mortem brain tissue from individuals with Alzheimer’s disease (non‑diabetic) will display higher ATP6V0D1 O-GlcNAcylation correlating with lysosomal pH probes (e.g., Magic Red cathepsin activity) and p62 load, independent of diabetes status.
4. Falsifiability: If glucosamine treatment does not alter ATP6V0D1 O-GlcNAcylation or lysosomal pH, or if manipulating O-GlcNAc on ATP6V0D1 fails to affect autophagic flux (measured by LC3-II turnover with bafilomycin A1), the hypothesis is refuted.

Mechanistic Insight Beyond Current Data

The hypothesis extends the view of HBP as merely modifying transcription factors or cytoskeletal proteins. By targeting the V-ATPase, a central regulator of organelle homeostasis, HBP flux couples nutrient sensing directly to lysosomal integrity. This creates a feed‑forward loop: lysosomal failure impairs clearance of damaged mitochondria, increasing ROS, which further activates GFAT via oxidative stress‑responsive pathways, amplifying HBP flux. Such a loop could explain why transient hyperglycemia leaves a lasting “metabolic memory” of lysosomal dysfunction even after glucose normalization.

Final remarks

Targeting the O-GlcNAcylation site on ATP6V0D1—either with selective OGT inhibitors or lysosome‑directed OGA activators—offers a promising avenue to decouple nutrient stress from lysosomal decline, potentially ameliorating both diabetic complications and age‑related proteostasis collapse.