Admistration of Exogenous Melatonin Improves the Diurnal Rhythms of Gut Microbiota in High Fat Diet-Fed Mice

Melatonin, a circadian hormone, has been reported to improve host lipid metabolism by reprogramming gut microbiota, which also exhibits rhythmicity in a light/dark cycle. However, the effect of admistartion of exogenous melatonin on the diurnal variation in gut microbiota in high fat diet (HFD)-fed mice is obscure. Here, we further confirmed the anti-obesogenic effect of melatonin on in mice feed with HFD for two weeks. Samples were collected every 4 h within a 24-h period and diurnal rhythms of clock genes expression (Clock, Cry1, Cry2, Per1, and Per2) and serum lipid indexes varied with diurnal time. Notably, Clock and triglycerides (TG) showed a marked rhythm only in the control and melatonin treated mice, but not in the HFD-fed mice. Rhythmicity of these parameters were similar between control and melatonin treated HFD mice compared with the HFD group, indicating an improvement of melatonin in the diurnal clock of host metabolism in HFD-fed mice. 16S rDNA sequencing showed that most microbiota exhibited a daily rhythmicity and the trends differentiated at different groups and different time points. We also identified several specific microbiota correlating with the circadian clock genes and serum lipid indexes, which might contribute the potential mechanism of melatonin in HFD-fed mice. Interestingly, administration of exogenous melatonin only at daytime exhibited higher resistance to HFD-induced lipid dysmetabolism than nighttime treatment companying with altered gut microbiota (Lactobacillus, Intestinimonas, and Oscillibacter). Importantly, the responses of microbiota transplanted mice to HFD feeding also varied at different transplanting times (8:00 and 16:00) and different microbiota donors. In summary, daily oscillations in the expression of circadian clock genes, serum lipid indexes, and gut microbiota, appears to be driven by a short-time feeding of an HFD. Administration of exogenous melatonin improved the compositions and diurnal rhythmicity of gut microbiota, which might be linked to host diurnal rhythm and metabolism. Importance Previous studies show that a circadian hormone, melatonin, involves in host lipid metabolism by reprogramming gut microbiota, which also exhibits rhythmicity in a light/dark cycle. However, the effect of melatonin drinking on the diurnal variation in gut microbiota in high fat diet-fed mice is obscure. Here, we found that 24-h oscillations were widely occurred in circadian clock genes, serum lipid indexes, and gut microbiota. Melatonin drinking improved the compositions and circadian rhythmicity of gut microbiota, which might be linked to host circadian rhythm and metabolism.


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Melatonin is a natural hormone mainly secreted by the pineal gland where its 61 synthesis is driven by the master circadian clock located in the suprachiasmatic 62 nucleous of the hypothalamus [1,2]. Melatonin synthesis is activated by darkness and 63 inhibited by light and thus this hormone is a key regulator of the circadian network 64 [3][4][5][6][7]. In addition, melatonin also exerts various physiological functions (i.e., 65 antioxidant function, bone formation, reproduction, cardiovascular, and immune 66 regulation) and has been confirmed the therapeutic effects on gastrointestinal diseases, 67 psychiatric disorders, cardiovascular diseases, and cancers [8][9][10]. More recently, a 68 few studies have reported that melatonin receptor 1 knock-out mice show insulin and 69 leptin resistance [11,12], indicating a role of melatonin and its downstream signals in 70 energy metabolism. Also, melatonin injection in lipopolysaccharide induced 71 endotoxemia markedly improves energy metabolism by enhancing ATP production 72 [13]. Similar potential of melatonin is also noticed in diabetes that lower melatonin 73 secretion is independently associated with a higher risk of developing type 2 diabetes 74 [14,15]. These findings indicate an interation between melatonin signal and 75 metabolic diseases. Indeed, Xu et al. also identified the anti-obese effect of melatonin 76 in high fat diet (HFD)-induced obesity in a murine model by improving liver steatosis, 77 low-grade inflammation, insulin resistance, and gut microbiota diversity and 78 compositions [16]. We further confirmed the underlying mechanism of melatonin in 79 HFD-induced lipid dysmetabolism which may be associated with reprogramming gut 80 microbiota, especially, Bacteroides and Alistipes-mediated acetic acid production 81 [17]. 82 Gut microbiota is highly shaped by dietary HFD and obese human and animals are 83 characterized by lower diversity and impaired gut microbiota compositions, especially 84 for Firmicutes and Bacteroidetes abundances [18][19][20][21]. Interestingly, several reports 85 have revealed that the gut microbiota and its metabolites exhibit circadian rhythm, 86 which are driven by HFD [22][23][24][25]. Also, some microbe has been reported to be 87 sensitive to melatonin [26], but the role of melatonin in the regulation of the diurnal 88 patterns of gut microbial structure and function, and whether gut microbiota 89 oscillations are associated with the anti-obese effect of melatonin is not yet known. 90 In this study we further analyzed the short-time effect of HFD feeding on diurnal 91 variations in gut microbiota and the relationship between gut microbiota oscillations 92 and the expression of circadian clock genes and serum lipids.

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All animals had free access to food and drinking water (temperature, 25±2°C; relative 98 humidity, 45-60%; lighting cycle, 12 h/d) during the experiment. Diets used in this 99 study were according to our previous study [17]. As sex produces difference in 100 melatoin profile [27], only female mice were used in this study to rule out the gender 101 interference.  Mice (26.89 ± 0.15 g) were randomly grouped to a control and three HFD groups 117 (n=12). One group of HFD mice were received melatonin at daytime (8:00-16:00) and 118 control water at night (16:00-8:00) (MelD) and another received melatonin at 119 nighttime (16:00-8:00) and control water at daytime (8:00-16:00). All mice were 120 killed at 8:00 am after 2-week feeding and samples were collected for further 121 analyses. Mice were treated with antibiotics (1g/L streptomycin, 0.5g/L ampicillin, 1g/L gentamicin, and 0.5g/L vancomycin) to clear gut microbiota [17]. After 1-week of 126 antibiotics treatment, the antibiotics-containing water was replaced with the regular 127 water and the microbiota-depleted mice were transplanted with donor microbiota.

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Fecal supernatants from control (MT-Cont), HFD (MT-HF), and MelHF (MT-MelHF) 129 (treated for 14 days) were transplanted into the microbiota-depleted mice at 8:00 and      Gut microbiota has been identified as a key element involving in host circadian 207 rhythms and itself also undergoes circadian oscillation, which is disturbed in HFD-fed 208 mice or obesity model [23,24,42]. Our previous study demonstrated that melatonin 209 treatment improved lipid metabolism by reprogramming gut microbiota in HFD-fed 210 mice [17], thus we hypothesized that administration of exogenous melatonin will 211 improve the daily rhythm of gut microbiota.

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Mice were sacrificed every 4 h within 24 h period and metagenomic DNA was 213 extracted from the cecal contents. Gut microbiota was tested by 16S rDNA 214 sequencing and the compositions were similar to our previous study [17] that the 215 largest phyla Bacteroidetes was reduced and Firmicutes abundance was increased in 216 HFD-fed mice, while melatonin reversed these alterations ( Figure 3A). HFD groups (P<0.05) ( Figure 3C; Table 3). Firmicutes relative abundance peaked at 220 4:00 in the HFD group, but at 8:00 in the control and MelHF groups ( Figure 3C).

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However, Proteobacteria and Actinobacteria failed to show a diurnal variation at the 222 phylum ( Figure 3C; Table 3).

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At the genus level, 18 genera were mainly analyzed and most of them exhibited a  258 We then investigate whether gut microbiota also show an association with clock genes 259 expression and serum lipid levels ( Figure 5A). Four genera (Alloprevotella, Anaerotruncus (P<0.01) were found to be correlated to Cry2 mRNA ( Figure 5F).

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Together, fourteen genera were found to correlate with clock genes expression,  306 We next transplanted fecal microbiota at two different time points (8:00 and 16:00) 307 from control, HFD, and MelHF groups into antibiotics-treated mice to investigate the 308 response to HFD feeding. Body weight was recorded and no significant difference  shown that the most gut microbiota exhibit daily cyclical variation in a variety of      (1)