Intestinal microbiota dysbiosis is related to many metabolic diseases in human health. Meanwhile, as an irregular environmental light–dark (LD) cycle, short day (SD) may induce host circadian rhythm disturbances and worsen the risks of gut dysbiosis. Herein, we investigated how LD cycles regulate intestinal metabolism upon the destruction of gut microbes with antibiotic treatments. The growth indices, serum parameters, concentrations of short-chain fatty acids (SCFAs), and relative abundance of intestinal microbes were measured after euthanasia; intestinal contents, epithelial metabolomics, and hepatic transcriptome sequencing were also assessed. Compared with a normal LD cycle (NLD), SD increased the body weight, spleen weight, and serum concentration of aspartate aminotransferase, while it decreased high-density lipoprotein. Meanwhile, SD increased the relative abundance of the Bacteroidetes phylum while it decreased the Firmicutes phylum in the gut of ABX mice, thus leading to a disorder of SCFA metabolism. Metabolomics data revealed that SD exposure altered gut microbial metabolism in ABX mice, which also displayed more serious alterations in the gut epithelium. In addition, most differentially expressed metabolites were decreased, especially the purine metabolism pathway in epithelial tissue. This response was mainly due to the down-regulation of adenine, inosine, deoxyguanosine, adenylsuccinic acid, hypoxanthine, GDP, IMP, GMP, and AMP. Finally, the transcriptome data also indicated that SD has some negative effects on hepatic metabolism and endocrine, digestive, and disease processes. Overall, SD induced an epithelial and hepatic purine metabolism pathway imbalance in ABX mice, as well as the gut microbes and their metabolites, all of which could contribute to host metabolism and digestion, endocrine system disorders, and may even cause diseases in the host.

PER2, a circadian clock gene, is associated with mammary gland development and lipid synthesis in rodents, partly via regulating peroxisome proliferator-activated receptor gamma (PPARG). Whether such a type of molecular link existed in bovines was unclear. We hypothesized that PER2 was associated with lipid metabolism and regulated cell cycles and apoptosis in bovine mammary epithelial cells (BMECs). To test this hypothesis, BMECs isolated from three mid-lactation (average 110 d postpartum) cows were used. The transient transfection of small interfering RNA (siRNA) was used to inhibit PER2 transcription in primary BMECs. The silencing of PER2 led to lower concentrations of cellular lipid droplets and triacylglycerol along with the downregulation of lipogenic-related genes such as ACACA, FASN, LPIN1, and SCD, suggesting an overall inhibition of lipogenesis and desaturation. The downregulation of PPARG and SREBF1 in response to PER2 silencing underscored the importance of circadian clock signaling and the transcriptional regulation of lipogenesis. Although the proliferation of BMECs was not influenced by PER2 silencing, the number of cells in the G2/GM phase was upregulated. PER2 silencing did not affect cell apoptosis. Overall, the data provided evidence that PER2 participated in the coordination of mammary lipid metabolism and was potentially a component of the control of lipid droplets and TAG synthesis in ruminant mammary cells. The present data suggested that such an effect could occur through direct effects on transcriptional regulators.

The core clock gene Period2 (PER2) is associated with mammary gland development and lipid synthesis in rodents and has recently been found to have a diurnal variation in the process of lactation, but has not yet been demonstrated in bovine mammary epithelial cells (BMECs). To explore the regulatory function of PER2 on milk fat synthesis in bovine mammary epithelial cells, we initially assessed the expression of clock genes and milk fat metabolism genes for 24 h using real-time quantitative PCR and fitted the data to a cosine function curve. Subsequently, we overexpressed the PER2 in BMECs using plasmid vector (pcDNA3.1-PER2), with empty vector pcDNA3.1-myc as the control. After transfecting BMECs for 48 h, we assessed the protein abundance related to milk fat synthesis by Western blot, the expression of genes coding for these proteins using real time-quantitative PCR, the production of triacylglycerol, and the fatty acid profile. The findings indicated that a total of nine clock genes (PER1/2, CRY1/2, REV-ERBα, BMAL1, NCOR1, NR2F2, FBXW11), seven fatty acid metabolism genes (CD36, ACSS2, ACACA, SCD, FADS1, DGAT1, ADFP), and six nuclear receptor-related genes (INSIG1, SCAP, SREBF1, C/EBP, PPARG, LXR) exhibited oscillation with a period close to 24 h in non-transfected BMECs (R² ≥ 0.7). Compared to the control group (transfected with empty pcDNA3.1-myc), the triglyceride content significantly increased in the PER2 overexpression group (p < 0.05). The lipogenic genes for fatty acid transport and triglyceride synthesis (ACACA, SCD, LPIN1, DGAT1, and SREBF1) were upregulated after PER2 overexpression, along with the upregulation of related protein abundance (p < 0.05). The contents and ratios of palmitic acid (C16:0), oleic acid (C18:1n9c), and trans-oleic acid (C18:1n9t) were significantly increased in the overexpression group (p < 0.05). Overall, the data supported that PER2 participated in the process of milk fat metabolism and is potentially involved in the de novo synthesis and desaturation of fatty acid in bovine mammary epithelial cells.