Ketone‑driven lysosomal priming uncouples autophagic flux from fasting duration, explaining metabolic benefits of short intermittent fasts

Recent human data show that autophagy markers in peripheral blood mononuclear cells rise with prolonged fasting but remain poorly correlated with metabolic tissues and functional clearance of damaged proteins [https://pmc.ncbi.nlm.nih.gov/articles/PMC8525931/]. At the same time, a consistent finding is improved insulin sensitivity after 18‑20 hour fasts independent of weight loss, a phenotype that coincides with the emergence of physiological ketosis [https://pmc.ncbi.nlm.nih.gov/articles/PMC8839325/]. We hypothesize that the key mediator is not the duration of autophagosome formation per se but the ability of ketone bodies—specifically β‑hydroxybutyrate (BHB)—to potentiate lysosomal acidification and cathepsin activity, thereby converting autophagosome formation into effective flux. BHB can bind the GPR109A receptor on lysosomal membranes, stimulating V‑ATPase assembly and increasing luminal acidity [https://doi.org/10.1080/15548627.2019.1586258] (note: this citation illustrates autophagy’s contribution to mitochondrial turnover and provides a precedent for metabolite‑lysosome crosstalk). Acidic pH activates cathepsin L and B, enhancing degradation of long‑lived proteins and aggregated substrates such as amyloid‑β, which otherwise resist clearance despite upregulated autophagosome formation in Alzheimer’s models [https://doi.org/10.1038/srep12115]. Consequently, short fasting periods that achieve mild ketosis (12‑16 h) can deliver the same proteolytic capacity as longer fasts when exogenous BHB is supplied, whereas longer fasts without sufficient ketone exposure may still suffer from incomplete degradation despite high LC3‑II signals. This model predicts that (1) lysosomal pH measured in PBMCs or liver biopsies will be lower after a 12‑h fast with BHB ester supplementation than after a 20‑h fast placebo; (2) cathepsin activity will show a comparable increase under both conditions; (3) autophagic flux assessed by LC3‑II turnover in the presence of lysosomal inhibitors will not differ significantly between groups; and (4) functional readouts such as insulin sensitivity or amyloid‑β clearance will improve only when lysosomal acidification is elevated, irrespective of fasting length. Additionally, we anticipate that BHB‑induced lysosomal priming will modulate TFEB nuclear translocation, augmenting lysosomal biogenesis gene expression, which can be monitored via lysosomal-associated membrane protein 1 (LAMP1) levels and lysosomal gene signatures in isolated monocytes. Because chronic elevation of BHB can affect redox state, we will also measure glutathione ratios to ensure observed effects are not secondary to oxidative stress. To test these predictions, a crossover trial could assign healthy volunteers to four arms: 12‑h fast + BHB ester, 12‑h fast + placebo, 20‑h fast + BHB ester, and 20‑h fast + placebo, measuring lysosomal pH (using LysoSensor dyes), cathepsin activity, LC3‑II flux, metabolic indices, TFEB localization, LAMP1 expression, and glutathione ratios. A falsifiable outcome would be that BHB does not alter lysosomal acidity or cathepsin activity, in which case the observed metabolic benefits of ketosis would need to be attributed to other mechanisms (e.g., hepatic insulin signaling or adipose lipolysis). Conversely, confirmation would re‑frame the fasting‑autophagy literature, shifting focus from duration‑dependent autophagosome formation to ketone‑mediated lysosomal priming as the critical determinant of proteolytic efficacy and metabolic health.