Time-of-day-dependent effects of exercise on the hepatic clock and proteome in high-fat diet-fed mice.

Abstract

Circadian disruption exacerbates high-fat diet-induced metabolic disease, but whether the time-of-day exercise differentially remodels the hepatic clockwork and downstream metabolic circuits remains unclear. Male C57BL/6J mice were fed normal diet or a high-fat diet (HFD) and then underwent 8 weeks of morning or afternoon treadmill training. We evaluated glucose-lipid phenotypes and hepatic core clock proteins and conducted quantitative liver proteomics with trend-based clustering, followed by immunoblot validation. Both morning and afternoon exercise mitigated high-fat diet induced weight gain, improved glucose tolerance and insulin sensitivity, and reduced hepatic lipid accumulation relative to sedentary HFD controls, with greater benefits in the morning. HFD increased hepatic CLOCK, BMAL1, and CRY1 and suppressed REV-ERBα. Morning training preferentially normalized CLOCK, BMAL1, and CRY1, whereas afternoon training more prominently restored REV-ERBα. Proteomics identified two dynamic modules. Cluster 1 consists of proteins induced by diet and suppressed by exercise, including enzymes of bile acid synthesis such as CYP7A1 and components of protein N-glycosylation and endoplasmic reticulum proteostasis such as DAD1 and DPAGT1, which shifted toward control levels with exercise intervention. Cluster 7 comprises proteins reduced by diet and enhanced by exercise, including IDNK involved in nucleotide metabolism and ATP6V1G1 associated with autophagy and lysosomal function, with a stronger recovery after morning training. Immunoblotting corroborated these protein level changes. Exercise acts as a time-of-day-dependent modulator of the hepatic proteome. Morning exercise produced a more pronounced normalization of proteins implicated in bile acid metabolism, ER proteostasis, and autophagy-lysosome pathways, thereby nominating candidate mechanisms that may contribute to improved metabolic control under HFD.