TRF, also known as intermittent fasting, has been widely reported to protect against HFD-induced obesity and related metabolic disorders [1, 43, 44]. However, the underlying mechanism of how TRF improves DIO-related disorders is not completely understood. Therefore, the present study aimed to investigate the effects of TRF on the circadian rhythmicity of hepatic lipid metabolism and gut microbiota in mice.
The HFD-induced obesity mice model was well established in the study. In the present study, FA mice showed more weight gain, significant liver steatosis and elevated hepatic levels of TG compared to NA mice under HFD feeding. And implementation of TRF in mice fed HFD protected against obesity and hepatic lipid accumulation, presented with decreased weight gain, severity of liver steatosis and hepatic TG level compared to FA mice, which was consistent with previous studies in rodent animals [5, 38, 43, 45, 46]. Therefore, it was reasonable to further investigate the underlying mechanism of TRF using the well-established HFD-TRF mice model in the present study. And we mainly focused on changes on gut microbiota and hepatic lipid metabolism.
Changes in gut microbiota, characterized by increased Firmicutes and Actinobacteria abundance and decreased Bacteroidetes abundance, has been shown to be associated with DIO in rodent animals [36, 47, 48]. Our study reported that TRF resulted in an increase in sample richness, an increase in Bacteroidetes abundance and a decrease in Firmicutes abundance, which was consistent with previous knowledge. Some studies suggested that the gut microbiota composition oscillated in a diurnal pattern in wildtype mice, though the exact oscillation regularities of specific phyla were not well studied [37, 38]. Our study depicted the oscillation pattern of abundance of Bacteroidetes, Firmicutes, Actinobacteria and Proteobacteria using differences between four distinct ZT timepoints and found TRF led to clear oscillation patterns in Bacteroidetes and Firmicutes. In our study, the circadian rhythm in FT mice is characterized by a prominent increase of Bacteroidetes and decrease of Firmicutes at ZT20, which was not observed in the other two groups. Considering the feeding window of FT mice was restricted between ZT16 and ZT24, ZT20 basically represented the feeding phase in TRF. Therefore, such oscillation patterns indicated a distinct difference triggered by TRF regimen between feeding and fasting phases.
We then tested the hepatic expression of core circadian clock genes (Per1, Cry1 and Bmal1) and proteins related to lipid metabolism (SIRT1, SREBP and PPARα) to further understand the molecular mechanism of TRF. The core circadian feedback circuits composed of CLOCK, BMAL1, PER, and CRY maintain the cell-autonomous circadian rhythm, and further regulate cellular metabolism through intermediate proteins including SIRT1, SREBP and PPARα [18, 23, 24, 49–51]. Previous knowledge regarding the oscillation pattern of mRNA Per1, Cry1 and Bmal1 all manifested clear diurnal rhythms with one peak and one bottom, though the specific patterns were not completely consistent [5, 24, 45, 52]. In our study, the circadian rhythmicity in NA mice was comparable to previous studies with similar peak and bottom timepoints for all three mRNAs of interest [5, 45]. Comparison between the three experimental groups revealed that FT and FA mice had similar oscillation patterns for Per1, Cry1 and Bmal1, though the relative amounts were slightly different. Furthermore, both FT and FA mice had distinct oscillation patterns for Cry1 and Bmal1 compared to NA mice. Therefore, the TRF regimen in our study did not remodel the hepatic core circadian rhythm altered by HFD to a natural rhythmicity, which indicated that the core circadian clock was possibly influenced by the feeding content rather than feeding schedule.
In peripheral organs, SIRT1 regulates the oscillatory rhythms and metabolic pathways as a metabolic rheostat [23, 24]. The present study showed similar circadian expression of SIRT1 in NA and FT mice, but not in FA mice, indicating TRF restored the circadian rhythm of SIRT1 expression (Figure.6A). The protein level of SIRT1 was decreased under calorie restriction conditions (FA and FT mice), and it was consistent with previous studies suggesting TRF with calorie restriction can prevent liver lipid accumulation and alleviate liver inflammation by decreasing hepatic SIRT1 level [26, 53, 54]. Accumulated evidences showed SIRT1 negatively regulate SREBP and PPAR levels in the liver [26, 28, 55, 56]. Our study also presented a consistent increase in both SREBP and PPARα regardless of ZT timepoints, which was possibly sequential to the changes of SIRT1. SREBP and PPAR are involved in energy metabolism and liver lipid synthesis and accumulation, and elevations in SREBP and PPAR contribute to the development of obesity [57–60]. Furthermore, the hepatic circadian rhythm of PPARα is characterized as peak at ZT8 and bottom at ZT20 in NA mice and the rhythm disappeared in FA mice, which was exactly the situation in our study [61]. And our FT mice also exhibited a similar circadian pattern of PPARα with NA mice, suggesting TRF restored the hepatic rhythm of PPARα (Figure.6C). Regarding the hepatic rhythm of SREBP, no clear pattern was found in NA mice (Figure.6B), while FA mice had a profoundly higher level at ZT20. Though FT mice also exhibited significant differences between ZT timepoints, its SREBP level at ZT20 was lower than that of FA mice. It indicated that TRF reduced the HFD-induced elevation of SREBP at ZT20, but was not able to restore it to a natural condition.
Previous details about the underlying mechanisms of TRF on DIO-related disorders were limited. Herein, our study further demonstrated the beneficial effects of TRF, especially its effects on restoring the circadian rhythm of gut microbiota and hepatic lipid metabolism. It suggested this feeding regimen might improve metabolism and restructure circadian rhythms as a non-pharmacological intervention to prevent obesity by modulating the circadian rhythm.