Effects of High-Intensity Interval Training and Ketone Ester Supplementation on Autophagy-Related Protein Signaling in Visceral Adipose Tissue of Western-Diet-Fed Mice

Abstract

Background: Western-diet (WD) exposure is associated with metabolic dysfunction and altered cellular quality-control pathways. Objective: This study examined whether high-intensity interval training (HIIT) and ketone ester (KE) supplementation modify autophagy-related protein signaling in visceral adipose tissue (VAT) of Western-diet-fed mice. Methods: Thirty male C57BL/6J mice were randomly assigned to five groups (n=6/group): Control, WD, WD+HIIT, WD+KE, and WD+HIIT+KE. WD was administered for 8 weeks and continued during a subsequent 4-week intervention period. HIIT was performed three times weekly for 4 weeks, consisting of repeated 2-min bouts at 100% of maximal running speed interspersed with 2-min active recovery. R, S-1,3-butanediol acetoacetate diester (BD-AcAc2) was administered by oral gavage at 5 g/kg/day. VAT was collected 24 h after the final intervention. Western blotting assessed the LC3-II/LC3-I ratio, p62/SQSTM1, and p-AMPK/total AMPK. Results: Compared with Control, WD increased LC3-II/LC3-I and p62/SQSTM1 and decreased p-AMPK/total AMPK. HIIT, KE, and their combination shifted these markers toward the Control profile. Welch’s one-way ANOVA demonstrated significant overall group effects for LC3-II/LC3-I (p=0.0036), p-AMPK/total AMPK (p=0.0395), and p62/SQSTM1 (p=0.0083). However, factorial analysis of the WD-based groups revealed no significant HIIT×KE interaction for LC3-II/LC3-I (p=0.580), p-AMPK/total AMPK (p=0.839), or p62/SQSTM1 (p=0.644). Conclusion: HIIT and KE supplementation were associated with alterations in autophagy-related protein signaling in VAT of Western-diet-fed mice, with the combined intervention producing a profile closer to Control. However, the absence of a significant HIIT×KE interaction does not support a statistical claim of synergy. Because autophagic flux, lysosomal function, and lipid-droplet-specific lipophagy were not directly assessed, these findings should be interpreted as changes in autophagy-related signaling rather than definitive evidence of restored lipophagy.