A cacao by-product ingredient prevents locomotor circadian disruption due to high-fat diet administration by regulating clock genes

Cacao by-products are frequently discarded as waste despite representing a sustainable source of bioactive compounds, including dietary fiber and phenolic constituents. A cacao by-product ingredient (CBI), designed based on in silico predictions, has been proposed to contain components capable of modulating core clock proteins. Although high-fat diets (HFD) are known to disrupt circadian organization, the potential of CBI to counteract HFD-induced circadian alterations remains unexplored. This study aimed to evaluate the effects of CBI on circadian disruption induced by HFD consumption. Four experimental diets (STD, STD +6% CBI, HFD, and HFD +6% CBI) were formulated and characterized for their chemical, nutraceutical, and bioactive composition. Mice were fed ad libitum and maintained under a 12:12 h light-dark cycle (LD 12:12 h) for ten weeks, followed by two weeks in constant darkness (DD) and subsequent re-entrainment to LD 12:12 h. At the end of the protocol, animals were euthanized at ZT6 and ZT18 to assess the expression of the core clock genes Bmal1 and Per2 in the hypothalamus, liver, adipose tissue, and ileum; hepatic PPARα expression was also evaluated. Nutritional analyses showed that CBI enrichment significantly increased dietary fiber content and enhanced the phenolic profile. Behaviorally, HFD increased locomotor activity during the mid-day, shortened ultradian cycles, delayed activity onset, and lengthened the free-running circadian period under DD conditions. HFD also significantly altered the day/night expression profiles of clock genes across the tissues analyzed. Notably, CBI supplementation partially reversed these alterations in an organ-dependent manner, restoring free-running period and clock-gene expression patterns toward STD-like levels. These findings suggest for the first time that CBI, a functional ingredient designed from discarded cacao by-products, effectively may be mitigating HFD-induced circadian desynchronization This work highlights both the potential valorization of agricultural waste and a promising nutritional strategy to support metabolic and circadian health through modulation of central and peripheral clocks.